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Biology and Engineering of Stem Cell Niches [1st Edition]
 9780128027561, 9780128027349

Table of contents :
Content:
Front Matter,Copyright,List of Contributors,ForewordEntitled to full textPart I: Biology of Stem Cell Niches and Molecular MechanismsChapter 1 - The Need to Study, Mimic, and Target Stem Cell Niches, Pages 3-13, Ajaykumar Vishwakarma, Jeroen Rouwkema, Peter Anthony Jones, Jeffrey M. Karp
Chapter 2 - Harnessing the Biology of Stem Cells' Niche, Pages 15-31, Borja Saez, Rushdia Z. Yusuf, David T. Scadden
Chapter 3 - Pluripotent Stem Cell Microenvironment, Pages 33-49, Mio Nakanishi, Mickie Bhatia
Chapter 4 - Regulation of Hematopoietic Stem Cell Dynamics by Molecular Niche Signaling, Pages 51-61, Aparna Venkatraman, Meng Zhao, John Perry, Xi C. He, Linheng Li
Chapter 5 - HSC Niche: Regulation of Mobilization and Homing, Pages 63-73, Samiksha Wasnik, Wanqiu Chen, Abu S.I. Ahmed, Xiao-Bing Zhang, Xiaolei Tang, David J. Baylink
Chapter 6 - Neuronal Stem Cell Niches of the Brain, Pages 75-91, Joanne C. Conover, Krysti L. Todd
Chapter 7 - Cardiovascular Stem Cell Niche, Pages 93-109, Annarosa Leri, Marcello Rota, Polina Goichberg, Toru Hosoda, Tiziano Moccetti, Piero Anversa
Chapter 8 - Intestinal Epithelial Lgr5+ Stem Cell Niche and Organoids, Pages 111-125, Nobuo Sasaki, Toshiro Sato, Hans Clevers
Chapter 9 - The Epithelial Stem Cell Niche in Skin, Pages 127-143, Géraldine Guasch
Chapter 10 - The Satellite Cell Niche in Skeletal Muscle, Pages 145-166, Caroline E. Brun, Fabien P. Chevalier, Nicolas A. Dumont, Michael A. Rudnicki
Chapter 11 - The Cancer Stem Cell Niche, Pages 167-184, Naniye Malli Cetinbas, Jatin Roper, Ömer H. Yılmaz
Chapter 12 - Cellular Senescence and Stem Cell Niche, Pages 185-192, Arthur Krause, Michael J. Conboy, Irina M. Conboy
Chapter 13 - Matrix Chemistry Controlling Stem Cell Behavior, Pages 195-213, Christina Klecker, Lakshmi S. Nair
Chapter 14 - Matrix Growth Factor and Surface Ligand Presentation, Pages 215-231, Eike Müller, Tilo Pompe, Uwe Freudenberg, Carsten Werner
Chapter 15 - Effect of Matrix Mechanical Forces and Geometry on Stem Cell Behavior, Pages 233-243, Dekel Rosenfeld, Shulamit Levenberg
Chapter 16 - Wettability Effect on Stem Cell Behavior, Pages 245-255, Yingying Li, Shutao Wang, Lei Jiang
Chapter 17 - Fluid Flow Control of Stem Cells With Investigation of Mechanotransduction Pathways, Pages 257-272, Brandon D. Riehl, Henry J. Donahue, Jung Yul Lim
Chapter 18 - Hypoxia Regulation of Stem Cell: Mechanisms, Biological Properties, and Applications, Pages 273-291, Yijun Liu, Ang-Chen Tsai, Xuegang Yuan, Yan Li, Teng Ma
Chapter 19 - Polymer Design and Development, Pages 295-314, Christopher K. Arakawa, Cole A. DeForest
Chapter 20 - Design and Development of Ceramics and Glasses, Pages 315-329, Jie Huang
Chapter 21 - Surface Functionalization of Biomaterials, Pages 331-343, Deepti Rana, Keerthana Ramasamy, Maria Leena, Renu Pasricha, Geetha Manivasagam, Murugan Ramalingam
Chapter 22 - Biofunctional Hydrogels for Three-Dimensional Stem Cell Culture, Pages 345-362, Jenna L. Wilson, Todd C. McDevitt
Chapter 23 - Technologies to Engineer Cell Substrate Mechanics in Hydrogels, Pages 363-373, Makoto Funaki, Paul A. Janmey
Chapter 24 - Micro- and Nanosurface Patterning Technologies, Pages 375-390, Jane Wang, Jeffrey T. Borenstein
Chapter 25 - Self-Assembled Nanostructures (SANs), Pages 391-409, Mina Mekhail, Laila Benameur, Maryam Tabrizian
Chapter 26 - Biomimetic Nanofibers as Artificial Stem Cell Niche, Pages 411-427, Xiaowei Li, Yu-Hao Cheng, Jose Roman, Hai-Quan Mao
Chapter 27 - Employing Microfluidic Devices to Induce Concentration Gradients, Pages 431-444, Nathalie Brandenberg, Matthias P. Lutolf
Chapter 28 - Engineering Niches for Embryonic and Induced Pluripotent Stem Cells, Pages 445-457, Hongli Mao, Yoshihiro Ito
Chapter 29 - Engineering Niches for Cardiovascular Tissue Regeneration, Pages 459-478, Kay Maeda, Erik J. Suuronen, Marc Ruel
Chapter 30 - Engineering Niches for Blood Vessel Regeneration, Pages 479-497, Quinton Smith, Michael Blatchley, Sharon Gerecht
Chapter 31 - Engineering Niches for Bone Tissue Regeneration, Pages 499-516, Angad Malhotra, Clemens van Blitterswijk, Pamela Habibovic
Chapter 32 - Engineering Vascular Niche for Bone Tissue Regeneration, Pages 517-529, Johnathan Ng, Kara Spiller, Jonathan Bernhard, Gordana Vunjak-Novakovic
Chapter 33 - Engineering Niches for Cartilage Tissue Regeneration∗, Pages 531-546, Ting Guo, Kimberly M. Ferlin, David S. Kaplan, John P. Fisher
Chapter 34 - Engineering Niches for Stem and Progenitor Cell Differentiation Into Immune Cells, Pages 547-558, Sanaya N. Shroff, Fnu Apoorva, Ankur Singh
Chapter 35 - Engineering Niches for Skin and Wound Healing, Pages 559-579, Michael W. Findlay, Geoffrey C. Gurtner
Chapter 36 - Designing Stem Cell Niche for Liver Development and Regeneration, Pages 581-600, Amranul Haque, Joshua Guild, Alexander Revzin
Chapter 37 - Engineering the Niche for Intestinal Regeneration, Pages 601-615, Victor Hernandez-Gordillo, Abigail N. Koppes, Linda G. Griffith, David T. Breault, Rebecca L. Carrier
Index, Pages 617-625

Citation preview

BIOLOGY AND ENGINEERING OF STEM CELL NICHES Edited by

AJAYKUMAR VISHWAKARMA Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States

JEFFREY M. KARP Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; Harvard Stem Cell Institute, Broad Institute of MIT and Harvard, Cambridge, MA, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-128-02734-9 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Mica Haley Editorial Project Manager: Lisa Eppich Production Project Manager: Lucı´a Pe´rez Designer: Greg Harris Typeset by TNQ Books and Journals

List of Contributors Loma Linda University, Loma Linda, CA,

Abu S.I. Ahmed United States

Fnu Apoorva

Henry J. Donahue Virginia Commonwealth University, Richmond, VA, United States

Harvard Medical School, Boston, MA, United

Piero Anversa States

Nicolas A. Dumont Ottawa Hospital Research Institute, Ottawa, ON, Canada; University of Ottawa, Ottawa, ON, Canada

Cornell University, Ithaca, NY, United States

Christopher K. Arakawa WA, United States

Laila Benameur

University of Washington, Seattle,

Kimberly M. Ferlin University of Maryland, College Park, MD, United States

Loma Linda University, Loma Linda, CA,

David J. Baylink United States

Michael W. Findlay Stanford University, Stanford, CA, United States; The University of Melbourne, Royal Melbourne Hospital, Parkville, VIC, Australia

McGill University, Montreal, QC, Canada

Jonathan Bernhard United States

Columbia University, New York, NY,

John P. Fisher University of Maryland, College Park, MD, United States

Mickie Bhatia McMaster University, Hamilton, ON, Canada Michael Blatchley Johns Hopkins University, Baltimore, MD, United States Jeffrey T. Borenstein

Uwe Freudenberg Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Dresden, Germany

Draper, Cambridge, MA, United States

Nathalie Brandenberg Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland David T. Breault Children’s Hospital Boston, Harvard Medical School, Boston, MA, United States; Harvard Stem Cell Institute, Cambridge, MA, United States

Makoto Funaki Japan

Tokushima University Hospital, Tokushima,

Sharon Gerecht United States

Johns Hopkins University, Baltimore, MD,

Polina Goichberg Harvard Medical School, Boston, MA, United States

Caroline E. Brun Ottawa Hospital Research Institute, Ottawa, ON, Canada; University of Ottawa, Ottawa, ON, Canada Rebecca L. Carrier United States

University of Washington, Seattle, WA,

Cole A. DeForest United States

Linda G. Griffith Massachusetts Institute of Technology, Cambridge, MA, United States Ge´raldine Guasch Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Aix-Marseille University, Marseille, France

Northeastern University, Boston, MA,

Naniye Malli Cetinbas Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, United States

Joshua Guild University of California at San Francisco (UCSF), San Francisco, CA, United States Ting Guo University of Maryland, College Park, MD, United States

Wanqiu Chen Loma Linda University, Loma Linda, CA, United States

Geoffrey C. Gurtner United States

Yu-Hao Cheng Johns Hopkins University, Baltimore, MD, United States

Pamela Habibovic Netherlands

Fabien P. Chevalier Ottawa Hospital Research Institute, Ottawa, ON, Canada; University of Ottawa, Ottawa, ON, Canada

Amranul Haque States

Hans Clevers Hubrecht Institute and University Medical Centre, Utrecht, The Netherlands

Stanford University, Stanford, CA, University of Maastricht, Maastricht, The

University of California, Davis, CA, United

Xi C. He Stowers Institute for Medical Research, Kansas City, MO, United States

Michael J. Conboy University of California at Berkeley, Berkeley, CA, United States

Victor Hernandez-Gordillo Massachusetts Institute of Technology, Cambridge, MA, United States

Irina M. Conboy University of California at Berkeley, Berkeley, CA, United States

Toru Hosoda Tokai University, Isehara, Japan Jie Huang University College London, London, United Kingdom

Joanne C. Conover University of Connecticut, Storrs, CT, United States

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xii

LIST OF CONTRIBUTORS

Yoshihiro Ito

RIKEN Brain Science Institute, Saitama, Japan

Hongli Mao RIKEN, Saitama, Japan

Paul A. Janmey University of Pennsylvania, Philadelphia, PA, United States

Hai-Quan Mao Johns Hopkins University, Baltimore, MD, United States

Lei Jiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, People’s Republic of China

Todd C. McDevitt University of California at San Francisco (UCSF), San Francisco, CA, United States

Peter Anthony Jones Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; Harvard Stem Cell Institute, Cambridge, MA, United States FDA, Silver Spring, MD, United States

David S. Kaplan

Jeffrey M. Karp Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; HarvardMIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; Harvard Stem Cell Institute, Broad Institute of MIT and Harvard, Cambridge, MA, United States Christina Klecker University of Connecticut Health, Farmington, CT, United States Abigail N. Koppes United States Arthur Krause Maria Leena Annarosa Leri States

Northeastern University, Boston, MA,

Ulm University, Ulm, Germany Karunya University, Coimbatore, India Harvard Medical School, Boston, MA, United

Shulamit Levenberg Technion, Israel Institute of Technology, Haifa, Israel Xiaowei Li Johns Hopkins University, Baltimore, MD, United States Yingying Li Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, People’s Republic of China Yan Li Florida State University, Tallahassee, FL, United States Linheng Li Stowers Institute for Medical Research, Kansas City, MO, United States; University of Kansas Medical Center, Kansas City, KS, United States Jung Yul Lim University of Nebraska-Lincoln, Lincoln, NE, United States Yijun Liu States

Florida State University, Tallahassee, FL, United

Matthias P. Lutolf Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland Teng Ma Florida State University, Tallahassee, FL, United States Kay Maeda University of Ottawa Heart Institute, Ottawa, ON, Canada Angad Malhotra University of Maastricht, Maastricht, The Netherlands Geetha Manivasagam

VIT University, Vellore, India

Mina Mekhail McGill University, Montreal, QC, Canada; Shriners Hospitals for Children-Canada, Montreal, QC, Canada Tiziano Moccetti University of Zurich, Lugano, Switzerland Eike Mu¨ller Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Dresden, Germany Lakshmi S. Nair University of Connecticut Health, Farmington, CT, United States; University of Connecticut, Storrs, CT, United States McMaster University, Hamilton, ON,

Mio Nakanishi Canada Johnathan Ng States

Columbia University, New York, NY, United

Renu Pasricha National Centre for Biological Sciences, Bangalore, India John Perry Stowers Institute for Medical Research, Kansas City, MO, United States Tilo Pompe Universita¨t Leipzig, Leipzig, Germany Murugan Ramalingam Centre for Stem Cell Research (CSCR), Vellore, India; Tohoku University, Sendai, Japan Keerthana Ramasamy Centre for Stem Cell Research (CSCR), Vellore, India Deepti Rana India

Centre for Stem Cell Research (CSCR), Vellore,

Alexander Revzin United States

University of California, Davis, CA,

Brandon D. Riehl University of Nebraska-Lincoln, Lincoln, NE, United States Jose Roman Johns Hopkins University, Baltimore, MD, United States Jatin Roper Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, United States; Tufts Medical Center, Boston, MA, United States Dekel Rosenfeld Haifa, Israel Marcello Rota States

Technion, Israel Institute of Technology,

Harvard Medical School, Boston, MA, United

Jeroen Rouwkema Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; University of Twente, Enschede, The Netherlands Michael A. Rudnicki Ottawa Hospital Research Institute, Ottawa, ON, Canada; University of Ottawa, Ottawa, ON, Canada

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LIST OF CONTRIBUTORS

Marc Ruel University of Ottawa Heart Institute, Ottawa, ON, Canada Borja Saez Harvard University, Cambridge, MA, United States; Massachusetts General Hospital, Boston, MA, United States Nobuo Sasaki Keio University School of Medicine, Tokyo, Japan; Hubrecht Institute and University Medical Centre, Utrecht, The Netherlands Toshiro Sato Japan

Keio University School of Medicine, Tokyo,

David T. Scadden Harvard University, Cambridge, MA, United States; Massachusetts General Hospital, Boston, MA, United States Sanaya N. Shroff Cornell University, Ithaca, NY, United States Ankur Singh

Cornell University, Ithaca, NY, United States

Quinton Smith Johns Hopkins University, Baltimore, MD, United States Kara Spiller Columbia University, New York, NY, United States; Drexel University, Philadelphia, PA, United States Erik J. Suuronen University of Ottawa Heart Institute, Ottawa, ON, Canada Maryam Tabrizian Canada

McGill University, Montreal, QC,

Xiaolei Tang Loma Linda University, Loma Linda, CA, United States Krysti L. Todd States

University of Connecticut, Storrs, CT, United

Ajaykumar Vishwakarma Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States Gordana Vunjak-Novakovic York, NY, United States Jane Wang

Columbia University, New

National Tsing Hua University, Hsinchu, Taiwan

Shutao Wang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, People’s Republic of China Samiksha Wasnik United States

Loma Linda University, Loma Linda, CA,

Carsten Werner Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Dresden, Germany; Technische Universita¨t Dresden, Dresden, Germany Jenna L. Wilson Georgia Tech Bioengineering, Atlanta, GA, United States ¨ mer H. Yılmaz Koch Institute for Integrative Cancer O Research, Massachusetts Institute of Technology, Cambridge, MA, United States; Massachusetts Institute of Technology, Cambridge, MA, United States; Massachussetts General Hospital, Boston, MA, United States; Broad Institute of MIT and Harvard, Cambridge, MA, United States Xuegang Yuan Florida State University, Tallahassee, FL, United States

Ang-Chen Tsai Florida State University, Tallahassee, FL, United States

Rushdia Z. Yusuf Harvard University, Cambridge, MA, United States; Massachusetts General Hospital, Boston, MA, United States

Clemens van Blitterswijk University of Maastricht, Maastricht, The Netherlands

Xiao-Bing Zhang Loma Linda University, Loma Linda, CA, United States

Aparna Venkatraman Stowers Institute for Medical Research, Kansas City, MO, United States; Christian Medical College Vellore, India

Meng Zhao Stowers Institute for Medical Research, Kansas City, MO, United States

Foreword Four decades ago, James Rheinwald and Howard Green described the first long-term culture method for normal human cells. They combined freshly isolated human skin cells with irradiated mouse fibroblasts. Gradual improvements allowed them to generate large confluent sheets of epidermis, starting from relatively small numbers of primary proliferative skin progenitor/stem cells. In 1980, Green and his colleagues performed the first successful therapy of two third-degree burn patients with cultured autologous keratinocyte sheets. In a dramatic demonstration during the summer of 1983, they exhibited that large-scale use of the method was life-saving for two brothers: five-year-old Jamie Selby and six-year-old Glen; both had sustained burns over >95% of their body surface. Later studies accomplished similar spectacular results in the lab and in the clinic with a related tissue, the cornea. Despite these early successes, it has long been held that healthy mammalian cells cannot be maintained (let alone expanded) outside the body, in a dish. This is now rapidly changing. The stem cell field has gone through a period of prolonged expansion. Many new stem cell types have been identified and characterized. However, the ways by which stem cells are nurtured by their niches still remains uncovered. Based on the new insights in understanding stem cell niches, it is now possible to culture stem cells representing virtually any tissue type in a dish. Under the right conditions, these stem cells not only simply increase in their numbers but also self-organize into organoids: miniature versions of real organs, like mini-brains, kidneys, or guts. Organoids are great experimental tools to ask basic science questions. Yet, the ease of organoid production from stem cells and their resemblance to human organs in health and disease holds great appeal for translational research and invites their almost immediate application into the clinic. This book is written by scientists who have contributed to many of the recent stem cell discoveries. It touches on all aspects of stem cell niche research, basic and applied. It contains a wealth of information for anyone with a scientific interest in learning about newest approaches to engineer stem cells and their niches. Enjoy a good read! Hans Clevers

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C H A P T E R

1 The Need to Study, Mimic, and Target Stem Cell Niches Ajaykumar Vishwakarma1,2, Jeroen Rouwkema1,2,3, Peter Anthony Jones1,2,4, Jeffrey M. Karp1,2,4 1

Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; 2Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; 3University of Twente, Enschede, The Netherlands; 4Harvard Stem Cell Institute, Cambridge, MA, United States

O U T L I N E 1. Introduction 1.1 The Stem Cell Niche in Health and Disease 1.2 Components of Stem Cell Niche

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2. Biology of the Stem Cell Niche 2.1 Behavior of Stem Cells: Hierarchical Versus Stochastic Model 2.2 Embryonic and Adult Stem Cell Niches

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3. Biochemical and Biophysical Regulation of Stem Cell Behavior 3.1 Extracellular Matrix and Biochemical Cues 3.2 Soluble Growth Factors and Ligands

3.3 Physical Cues and Matrix Mechanics

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3.4 Oxygen and Metabolism

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3.5 Immune Cells, Inflammation, and Immunomodulation

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4. Mimicking the Stem Cell Niche: Bioengineering Tools and Techniques

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5. Bioengineering Specialized Artificial Stem Cell Niches for Clinical Therapies

12

References

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1. INTRODUCTION is composed of extracellular matrix (ECM) components for attachment/anchorage, diffusible biomolecules for cell signaling, cell surface ligands for signal transduction, and essential cellecell interactions. Studies of cell populations during embryonic development have led to the identification of stem cells that possess the capacity to produce a full organism from a fertilized egg.1 Stem cells are functionally defined as undifferentiated embryonic or adult cells, which can self-renew and generate differentiated cell types with varying degrees of potency. The fundamental replicative feature of stem cells, along with their generation of differentiated progeny, accounts for the origin of the

1.1 The Stem Cell Niche in Health and Disease As opposed to single-celled organisms, cells in complex multicellular organisms are associated with a tissue-specific physiological environment. Different cell types differ in morphology and function; yet, they are genetically identical. This variation, caused by differential gene expression, is controlled by intrinsic mechanisms and by extrinsic signals from the local environment, thereby controlling distinct cellular behavior, or “phenotype.” The local physiological microenvironment supporting the cell and driving extrinsic cues from outside the cell is known as the “cell niche,” which Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00001-9

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Copyright © 2017 Elsevier Inc. All rights reserved.

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1. THE NEED TO STUDY, MIMIC, AND TARGET STEM CELL NICHES

word “stemness.” However, whether stem cells need a special environment that controls stem cell renewal, maintenance, and survival, and what is the nature of such microenvironment are pertinent questions many researchers continue to explore. With growing evidence, there is a growing consensus that in vivo function and the fate of stem and progenitor cells are regulated by the interplay of various extrinsic signals of tissue-specific microenvironments, often referred to as “stem cell niches.” The concept of a stem cell niche was first proposed by Schofield in the late 1970s as a physiologically restricted microenvironment that supports stem cells.2 The initial concept of anatomically distinct sites that regulate hematopoietic stem cell (HSC) activity and selfrenewal was later extended to acknowledge the discovery of stem cells and their niches in multiple tissues.3 Stem cells are often linked with asymmetrical cell division, and the niche maintains a stable number of stem cells during homeostasis, and removal of the niche induces differentiation. Extrinsic signals interact and integrate to ensure that one cell remains in the niche, while another escapes it by receiving a differentiation signal. It is now clear that in high-turnover systems, such as in the gut and blood, the behavior of stem cells is not uniformly quiescent, and the various niche components may govern their relative proliferative activity.4e6 Also, it is emerging that stem cell performance is not

only dependent on factors promoting stemness but is also a result of factors inhibiting differentiation pathways. Hence, in homeostasis, the underlying relationship between stem cell and niche accommodates nuances and involves various elements influencing the stem cell functional parameters: replicative capacity and potency. However, when tissue is injured or diseased, the niche actively engages stem cells; guides their proliferation, migration, and differentiation; and regulates their participation in tissue regeneration and repair. Therefore, the niche should be regarded as a dynamic participant controlling stem cell number, fate, and behavior in the health and disease of the tissue and the organism.

1.2 Components of Stem Cell Niche The stem cell niche is a complex, heterotypic, and dynamic structure, which includes supporting ECM, neighboring niche cells, secreted soluble signaling factors (such as growth factors and cytokines), physical parameters (such as shear stress, tissue stiffness, and topography), and environmental signals (metabolites, hypoxia, inflammation, etc.) (Fig. 1.1).7,8 Stem cell niches are highly innervated and densely vascularized, thus are directly or indirectly influenced by vascular and neural inputs.

NICHE

Stem cell Selfrenewal

Progenitor cell Differentiation

FIGURE 1.1 Components of stem cell niche. Adapted from Lane SW, Williams DA, Watt FM. Modulating the stem cell niche for tissue regeneration. Nat Biotechnol 2014;32(8):795e803.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

2. BIOLOGY OF THE STEM CELL NICHE

In addition to matrix and cell signaling elements mentioned above, niche cells form functional units within the stem cell niche. These are neighboring tissue-specific stem or somatic cell populations that interact with resident stem cells to regulate cell fate. For example, mesenchymal stromal/stem cells in the HSC niche or parenchymal hepatocytes in liver. In addition to stem cell themselves, niche cells provide a source of physical and biochemical signals within the niche microenvironment by building extracellular matrix and producing cell surface or soluble signaling factors. Importantly therefore, stem cell microenvironments are highly dynamic and display temporal variations. Such variations in direct cellecell contacts and ECM components, as well as their interaction with regulatory molecules secreted by stem or niche cells and the spatial organization of niche components, ultimately enable the regulation of stem cells to render tissue homeostasis and regeneration.9

2. BIOLOGY OF THE STEM CELL NICHE 2.1 Behavior of Stem Cells: Hierarchical Versus Stochastic Model Understanding developmental biology is an important approach to fully comprehend the structure and function of the human body developed from a single totipotent stem cell, the zygote. The potency of a given cell to differentiate into many specialized cells is defined by the degree of its plasticity and versatility at various stages. Totipotent stem cells are those with the greatest Hierarchical Model

5

differentiation potential and can differentiate into any and all cells in an organism, plus the extraembryonic or placental cells. Pluripotent stem cells can differentiate into any cell within the three germ layers (endoderm, mesoderm, and ectoderm). Embryonic stem cells (ESCs) are pluripotent and can divide and differentiate into cells of various types found in the body. Multipotent stem cells are progenitor cells that can differentiate into numerous cell types but within a similar “family” or lineage. Lastly, unipotent stem cells, the most restricted precursor, can only result in one cell fate. Unlike ESCs, stem cells from adult tissues are multipotent or unipotent. During development and in the healthy body, stem cells can divide to produce new cells. This is a carefully controlled process that allows the body to grow and to replace lost or damaged cells during adult life. For the body to maintain homeostasis, stem cells proliferate before differentiating into a specific lineage, such that the generation of differentiated cells and the maintenance of stem/progenitor pools are balanced. Two distinct models have been proposed to explain the lineage choices of stem cells (Fig. 1.2). The hierarchical model suggests a discrete arrangement of cells consisting of slow-cycling stem cells that can self-renew extensively, which also give rise to short-lived transit amplifying progenitor cells that then further differentiate into committed nondividing cells. The stochastic model suggests that each stem cell chooses at random between self-renewal and differentiation. In this model, each individual clone will vary in size. Recent lineage tracing studies have supported the findings of the hierarchical model of stem cell behavior,

Stochastic Model

FIGURE 1.2 Hierarchial versus stochastic model for behavior of stem cells.

Stem cell Self renewal Transit amplifying cells

Differentiated cells

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

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1. THE NEED TO STUDY, MIMIC, AND TARGET STEM CELL NICHES

yet also support that progenitors can behave in a stochastic manner.10,11 Different types of stem cells at various body sites with distinct niches may work in different ways, and it is possible that both these theories are correct. In addition, based on recent discoveries, it has been proposed that the stem cell niche has a bicompartmental organization12: one compartment that engages in immediate, rapid new growth and one that contributes later to long-term growth. In this way, stem cells might work cooperatively with their progeny to sustain tissue regeneration. Although the precise mechanisms mediating these phenotypic changes may be complex, these lineage tracing studies demonstrate that the niche is a critical component for the stem cells.

2.2 Embryonic and Adult Stem Cell Niches The first example of a stem cell niche was demonstrated by Kimble and White in 1981, who showed that the mesenchymal distal tip cell provides the essential microenvironment for the maintenance of the germ line stem cell in Caenorhabditis elegans.13 Later, Drosophila melanogaster became the best characterized system to analyze the relationship between stem cells and their niche through studies in Drosophila germline stem cells.14 These studies have provided an extensive understanding of the molecular basis of stem cell niche regulation. Meanwhile, several niches of mammalian adult tissue stem cells were discovered, for example, the HSC niche in bone marrow, hair follicle stem cells at the follicle bulge, and skin interfollicular epidermis stem cells located in clusters at the tops of rete ridges in the basal layer,15e17 among others. This book covers in-depth many of the studied examples of embryonic and adult stem cell niches, along with molecular mechanisms, in Section 1. Schofield’s 1978 mammalian stem cell niche hypothesis was based on the observation that colony-forming stem cells were less capable of replacing blood cells compared with bone marrow cells when injected in vivo into irradiated animals.2 Since his discovery, the field of bone marrow HSCs has exploded. Schofield’s study postulated that HSCs reside in specific loci of bone marrow and receive support from multiple cellular components of their microenvironment. Since then, there have been several studies that aimed at recognizing such components and determining interactive signaling mechanisms of the HSC niche.18e20 Chapters 4 and 5 comprehensively review how microenvironmental cues from the bone marrow and intrinsic signals from the HSC dictate its fate to remain quiescent, become active, differentiate, migrate, or participate in regeneration. These two chapters detail advances in genetically modified mouse models and high-resolution, real-time imaging to identify HSC niche components and the

molecular signaling emanating from them. They discuss recent findings on how the niche network maintains different states of HSCs by providing multiple signaling inputs from different cellular sources during homeostasis and mobilization/homing. Compared with other somatic stem cell microenvironments described since the late 1970’s, the microenvironment of pluripotent cells had not been characterized until relatively recently. This is largely because of the transient nature of pluripotent cells in vivo and the characterization of their regulation being restricted to in vitro methods. Also, pluripotent cells seem to be mutually interacting with their neighboring cells, which are dynamically changing in a short period of time relative to somatic stem cells. Chapter 3 describes the microenvironment in early embryogenesis, which provides robust information about cellular interactions required for the acquisition of a pluripotent state. It also describes the transcriptional and functional heterogeneities observed within established ESC cultures and their emerging significance for self-renewal and maintenance of differentiation potentials of ESCs. Mesenchymal stem cells (MSCs) are multipotent stem cells found in stromal or connective tissue and are distinct from stem cells found in parenchymal or “functional” tissue. The MSC niche will not be discussed in this edition of the book, since our understanding of what constitutes an MSC niche at different tissue sites is limited due to few in vivo studies and heterogenous population variation of MSCs in vitro. Further chapters in this section will discuss stem cell niches in specific organs or organ systems. In the adult mammalian brain, the two major stem cell niches that support neurogenesis are the ventricularesubventricular zone that lies along the lateral walls of the lateral ventricles and the dentate gyrus subgranular zone found within the hippocampus. Chapter 6 focuses on the development of the brain’s neuronal stem cell niches and further reviews key characteristics and molecular regulators, including molecular pathways that help support the mammalian brain’s two neurogenic stem cell niches. Stem cell niches play a critical role in cardiac homeostasis and myocardial repair after injury by replication of a self-sustained pool of preexisting immature myocytes. In the myocardium, cardiac progenitor cells are clustered in interstitial microdomains with an architectural organization distinct from the surrounding nonestem cell tissue. The structural and molecular properties of this niche condition the response of the tissue to physiologic and pathologic cues by creating a favorable environment for the interaction of stem cells with the surrounding cells and the interstitial space. Chapter 7 emphasizes the relevance of cardiac progenitor cells and their niches to cardiomyogenesis. Also, the chapter discusses alternative stem celleindependent

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mechanisms that may account for the global replenishment of lost cardiomyocytes. The adult intestinal epithelium represents one of the most suitable models to study tissue stem cells in vitro. It is a rapidly self-renewing tissue, and the design of its basic unit, the cryptevillus, is highly stereotypical. Since the discovery of Lgr5þ stem cells at the base of intestinal crypts, it has become possible to unveil the molecular mechanisms that control the homeostasis of these stem cells, including the extrinsic signaling cues from the crypt niche. Based on these insights, in vitro propagation of murine and human intestinal stem cells in the form of ever-expanding organoids has also become possible.20a Chapter 8 discusses such recent developments in the field of the intestinal stem cell niche. Additionally, the chapter focuses on a three-dimensional (3D) organoid culture system, originally established for murine small intestine that has now been adapted for other types of tissue stem cells by adding additional small molecules and/or growth factors. Organoids offer numerous possibilities for the study of basic research questions on tissue development and maintenance, stem cell characteristics, the detailed description of endogenous niche factors, gene function, etc. Furthermore, in vitro organoids have the potential to contribute to drug development and hold promise for regenerative medicine, through the possibility to transplant these organoids back into their original organs. Skin is a primary protective barrier and is being constantly renewed. As such, skin stem cells play a key role in maintaining epithelial homeostasis, similar to the intestinal epithelium. Likewise, Lgr5þ cells in hair follicles has been show to comprise an actively proliferating and multipotent stem cell population.20b Chapter 9 describes the location and cellular hierarchy of each epithelial stem cell of the skin within the epidermis, hair follicle, sebaceous gland, and sweat gland and highlights the intrinsic regulators, which maintain their stemness. The chapter reviews the extrinsic regulators of epithelial stem cell function and sheds light on recent findings that introduce new actors in the epithelial stem cell niche in the skin. Also, it discusses how the niche components in the skin may vary depending on the body location. In the skeletal muscle, a specialized population known as satellite stem cells contribute to its regenerative capacity. Owing to their ability to generate both stem cells and committed myogenic progenitors, satellite stem cells allow self-renewal of the satellite cell reservoir and provide myogenic progenitor cells to repair the muscle tissue. Chapter 10 highlights the indispensable role of the satellite cell niche in the regulation of the stem cell functions. This niche maintains the muscle stem cell in a quiescent state; however, in response to muscle injury, the niche actively generates signals for

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satellite cell activation, proliferation, and differentiation. The chapter describes the cross talk between satellite cells and their niche along with regulation of satellite cell functions, namely commitment and self-renewal, in resting, injured, and pathologic muscle. Cancer stem cells (CSCs) reside in a microenvironment that comprises various other cell types such as tumor-associated endothelial cells, mesenchymal cells, and immune cells. In addition to these nonmalignant cells, ECM, metabolites, endocrine signals, waste products, and other secreted factors contribute to the proliferation, survival, and dissemination of tumor cells.21 It is clear from recent reports that the niche plays an important role in triggering stem cellelike programs in subpopulations of cancer cells and at each different stage of tumorigenesis such as tumor initiation, progression, metastasis, and drug resistance. Chapter 11 reviews the current knowledge on the CSC niche and its contribution to tumorigenesis. Understanding stem celleniche interactions requires the elucidation of a complex microenvironment with possibly hundreds of biochemical and biophysical cues acting in concert, perhaps synergistically, to control stem cell fate. The chapters in the following section focus on key niche factors that regulate stem cell behavior.

3. BIOCHEMICAL AND BIOPHYSICAL REGULATION OF STEM CELL BEHAVIOR 3.1 Extracellular Matrix and Biochemical Cues ECM is a natural substrate manufactured by the cells themselves providing multiple biochemical and biophysical cues to stem cells residing on or in it, thus maintaining stem cell pools. It is a mixture of long biopolymers consisting of proteins (e.g., collagen, fibronectin, vitronectin, elastin, and laminin) and glycosaminoglycans (e.g., heparin, chondroitin sulfate, keratin sulfate, and hyaluronic acid). ECM was once considered an inert support structure, but research has revealed it to be a signaling core, with a critical role in the niche for developing and maturing stem cells. Matrix provides the necessary chemical and mechanical signals to modulate cellular processes such as cell renewal, morphogenesis, differentiation, repair, and homeostasis. Stem cells bind to matrix adhesion ligands by integrins and other receptors localized in their plasma membrane. Integrins are heterodimeric proteins consisting of a form and b form, and the combination of their subtypes, such as a1b1, specifies the type of ECM proteins they bind (collagen or laminin in case of a1b1, for instance) and provides intracellular signals, leading to cellular responses.22 In addition to the effect of cellematrix adhesion on directing the behavior of cells, cells are also able to degrade and remodel matrix by providing enzymes into their surroundings, such as

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metalloproteinases.23 Thus, the communication between cells and matrix is bidirectional and dynamic. Furthermore, matrix is able to provide biochemical cues to cells effectively, because it sequesters various biomacromolecules, such as growth factors, through specific and nonspecific bindings, and makes residing cells more accessible to these molecules.24 Cells recognize and respond to secreted signaling factors through multiple pathways: for instance, cells express receptors to their agonists in their microenvironment and initiate intracellular signal transduction once binding between a receptor and an agonist is established. The chemical composition of the ECM can vary widely among tissues. Fig. 1.3 shows four different stem cell niches, together with their cellular and ECM components. ECM molecules playing major roles in

the different niches are indicated. Looking closely at the matrix, it is possible to identify the major structural and chemical entities as patterns and combinations of functional groups, present in proteins, proteoglycans, and peptide sequences. To date, many studies have examined the relationship between stem cells and matrix in vitro using biomaterial scaffolds that mimic the natural ECM.26 In a particularly interesting approach, researchers applied printing techniques using automatic pipettes or robots to generate a library of artificial niches in a single experiment.27 Another group created microarrays of artificial niche components comprising ECM proteins for probing single stem cell fates in high throughput.28 These studies clearly demonstrated a robust proof-of-concept that adding known ECM molecules or functional groups to synthetic

FIGURE 1.3

Extracellular matrix composition of four different stem cell niches. Adapted from Gattazzo F, Urciuolo A, Bonaldo P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta (BBA) 2014;1840:2506e19.

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constructs enables the development of controlled environments regulating stem cell behavior. It is critical to further understand such relationships to fully appreciate the effect of chemical functional groups in the stem cell niche on cell behavior. The small molecular patterns found throughout the body can have a significant influence on stem cell behavior when found in high numbers within a niche. Studying each unit in isolation helps to delineate their individual effects on the stem cells. Chapter 13 discusses some of the chemical functional groups that have been extensively investigated to understand their role in modulating stem cell behavior. In addition to matrix in the extracellular surrounding, niches may consist of cells, either a single type or a range of interacting multiple cell types. Celle cell communication can be mediated by interactions via direct contact between cells carrying distinct functional group, such as cadherin-based adherens junctions in epithelial cells and endothelial cells, affecting their behavior. The prospect of replicating the complex ECM chemistry using engineered natural or synthetic biomaterial substrates would be highly valuable, not only to understand complex synergies guiding stem cell behavior but also to develop novel translational technologies, such as regenerative scaffolds.

3.2 Soluble Growth Factors and Ligands Niches contain secreted or cell surface factors that can be secreted by stem cells themselves or by other cells that reside in the niche. Cellular communication employs different sets of secreted soluble hormones, growth factors, and cytokines to regulate cellular functions, including proliferation, differentiation, or migration. A few key biomolecules that control stem cell renewal, maintenance, or survival include Notch, Wnt, fibroblast growth factor, epidermal growth factor, transforming growth factor-b, stem cell factor, and chemokine families. A vast majority of the currently applied methods for ex vivo stem cell expansion, maintenance, and differentiation rely on the addition of soluble microenvironmental components, namely cytokines and growth factors. The action of signaling molecules is highly interconnected and activation steps downstream of receptor binding can depend on the mode of factor presentation. Hence, it is important to consider their pleiotropic actions in the design of engineered stem cell microenvironments. Contrary to the conditions of standard in vitro cell culture, growth factors do not randomly diffuse in vivo but are spatially and temporally organized by the neighboring cells and the ECM within the niche. Also, many ligands that influence stem cell fate decisions in vivo are tethered to the cell membrane or reversibly bound to the ECM. Thus, the

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control of the spatiotemporal presentation of soluble signaling molecules and insoluble, matrix-bound cues is essential for effective stem cell bioengineering schemes and critically depends on the development of advanced, multi-biofunctional biomaterials. Chapter 14 summarizes biomaterials-based approaches for the spatial and temporal control of factor and ligand presentation. The chapter focuses on the presentation of growth factors from versatile 3D polymeric matrix platforms for the biomimetic presentation of multiple molecular effectors and for the adjustment of physical constraints.

3.3 Physical Cues and Matrix Mechanics It is now well understood that not only the biochemical properties of the ECM but also the biophysical properties of the microenvironmentdstiffness and elasticity, electrostatic charges, wettability, and structural informationdregulate cellular functions. One of the biophysical properties that has been intensively studied is the stiffness of the microenvironment. A variety of cells recognize the stiffness of their surroundings and respond to this. For example, MSCs become quiescent on polyacrylamide gels with a stiffness of approximately 200 Pa, which is comparable with the stiffness of bone marrow.29 Myocytes and cardiomyocytes cultured ex vivo exhibit striation, when they contact polyacrylamide gels of which the stiffness mimics the stiffness of skeletal muscle or heart tissue, respectively.30,31 The lineage of MSC differentiation is also affected by the stiffness of the microenvironment: MSCs are able to differentiate into adipocytes most effectively on approximately 200-Pa polyacrylamide gels, which is the stiffness of adipose tissues,29 whereas osteogenic differentiation becomes more effective as the stiffness of polyacrylamide gels increases.32,33 Thus, mimicking the stiffness of the microenvironment, represented by the stiffness of tissues in many cases, may be a prerequisite for stem cells to reenact in vivo cellular functions. Along with stiffness, diverse substrate patterns generated using innovative fabrication processes have also been shown to alter stem cell behavior by applying mechanical constraints on cells. They can be regulated by careful design of the microscale and nanoscale features of the substrate geometry. Chapter 15 focuses on the recent advances in exploitation of mechanical stimulations to differentiate stem cells. It also discusses several mechanisms that underlie the stem cell’s response to mechanical stimuli, including changes in the cell cytoskeleton, nuclear alterations affecting gene expression, and cell adhesion site reconfigurations. Each of these biophysical elements mediates mechanical forces, and together guide cell behavior, organization, and differentiation.

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Interfacial wettability and its effect on cellular behaviors were first proposed in a study by Lampin in 1997, which established a correlation between matrix wettability and cell adhesion and migration.34 Over the years, many wettability-mediated matrices have been fabricated to modulate a wide range of cellular functions from cell adhesion and proliferation35 to cell pattern.36 The matrix wettability effect on stem cell behaviors can be extremely dependent on the cell type. To better understand and evaluate the interfacial wettability stem cells need according to their types, Chapter 16 discusses how interfacial wettability affects stem cell adhesion, proliferation, and differentiation. Stem cells, like several other cell types, are subjected to fluid flow in the body. In particular, stem cells in vivo experience shear and chemotransport from fluid flow within mechanically active tissues and while migrating from niches to homing targets in the body. Chapter 17 introduces this concept describing fluid flow devices used for cell stimulation. It further discusses fluid flow mechanical stimulus applied to stem cells as a regulator of proliferation and quiescence, differentiation into various lineages (osteogenic, cardiovascular, neural, etc.), migration, and tissue remodeling, with a brief discussion of mechanically active pathways.

3.4 Oxygen and Metabolism Of the many metabolic factors influencing cell behavior, oxygen tension in the cellular microenvironment plays a pivotal role, serving as both metabolic substrate and a signaling molecule regulating stem cell fate. In vivo, low oxygen tension or hypoxia is a common feature of stem cell niche shared among different types of stem cells and linked to their plasticity. Chapter 18 summarizes the physiological relevance of hypoxia in regulating stem cell metabolism and biological properties, including self-renewal, multipotency in differentiation, ischemic resistance, cellular senescence, and paracrine secretion. It also discusses hypoxia preconditioning as a therapeutic strategy to enhance efficacy during stem cell transplantation under the context of disease treatment, including stroke, ischemic heart injury, and kidney injury.

3.5 Immune Cells, Inflammation, and Immunomodulation Immunological cells provide dynamic biochemical regulation of the stem cell niche during homeostasis. The presence of innate and adaptive immune cells to help maintain the integrity of the stem cell

microenvironment is best characterized in the bone marrow. The HSC niche is laden with immune cells implicated in complex cell, ECM, and cytokine interactions, which is vital for the development of the lymphohematopoietic system.37e39 Researchers are also now able to reveal specific immune regulatory elements involved in regulating marrow function in concert with stromal cells. For example, macrophages have been implicated in HSC mobilization through the production of granulocyte-colony stimulating factor,40 whereas neutrophils are seen to indirectly influence the MSC niche through macrophages.41 Furthermore, inflammation in response to injury causes a transient increase of immune cells in tissues to protect against pathogens and promote tissue healing. The transient stem celleimmune niche interactions mediate endogenous tissue repair and regenerative mechanisms.42 The function of immune cells can be modulated to promote stem cell function in cases of continuous tissue injury and scarring. Application of immunomodulation remains an interesting aspect of tissue regeneration strategies.43 Designing and controlling for this is a current challenge, and greater appreciation is needed between the interactions of the immune system, the cells involved in tissue healing, and biomaterials.

4. MIMICKING THE STEM CELL NICHE: BIOENGINEERING TOOLS AND TECHNIQUES The stem cell niche is a complex, dynamic microenvironment, which is best characterized in an in vivo model. However, it is this complexity that makes understanding the specific function of individual niche components difficult. At the same time, decoding the effect of specific niche components at the single-cell level is challenging. Studying stem cells clonally, as individual cellular entities able to self-renew and differentiate, is emerging as a major focus to understand their origin and key features. Population-based analyses of stem cell behavior often fall short in defining mechanisms that may be unique to these specialized cells. Similarly, niche interactions at an individual cell level can reveal significant information on a cell’s microenvironment and behavior at specific locations when compared with studies performed with cell aggregates. Although the implementation of clonal assays for the analysis of stem cells and its niche interaction is experimentally challenging, it is crucial for understanding the complex mechanisms that govern stemness. Although traditional two-dimensional (2D) culture systems provide a simple means to study stem cell niche interactions, they suffer from inherent limitations to replicate the complexity of the native niche and in

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4. MIMICKING THE STEM CELL NICHE: BIOENGINEERING TOOLS AND TECHNIQUES

providing greater insights into in vivo cellecell and cellematrix interactions. This has motivated the need to develop 3D platforms for stem cell culture and for engineering artificial stem cell niches. The 3D culture technology opens new avenues for studying fundamental questions regarding stem cells and their niches. Interestingly, it has also allowed several advanced applications in personalized medicine,44,45 such as tissue-engineered constructs, biomanufacturing approaches, and platforms for drug discovery and toxicity testing. To recapitulate the in vivo niche environment and further examine the roles of the extrinsic cues in controlling the behavior of a stem cell, artificial niches with tunable physical, biochemical, and cellular parameters have been prepared.9 Bioengineering tools along with novel fabrication techniques provide useful ways to tune niche properties of these stem cells. Bioengineering tools include synthesizing novel natural, synthetic, or hybrid biomaterials and micro- or nanofabrication of niche components in 2D or 3D. Together with the application of sophisticated bioengineering techniques and analysis methods, manufacturing synthetic stem cell niches will allow us to (1) culture stem cells under defined conditions, thereby improving reproducibility; (2) facilitate mechanistic studies to reveal specific roles of various niche cues in regulation of stem cell fate; and (3) develop novel strategies to engineer stem cell fates in vivo for tissue regeneration.46 A range of synthetic and natural polymers is currently used to fabricate scaffolds. Broadly, bioactive materials can be classified into natural and synthetic biomaterials. Some of the commonly used natural materials used to mimic ECM include biopolymers like collagen, hyaluronic acid, chondroitin sulfate, fibronectin, alginate, chitosan, and silk fibroin. Synthetic polymers, such as poly(ethylene glycol), poly(lactic acid), poly(glycolic acid), and copolymer poly(lactic-glycolic acid) are some of the most commonly employed synthetically engineered scaffolds. Chapter 19 highlights recent progress in polymer design and development for 3D stem cell culture, comparing the advantages of both natural- and syntheticbased precursors. Additionally, special attention is given to smart synthetic polymer systems that exhibit responsiveness to environmental stimuli (e.g., electricity, temperature, enzyme, light, heat, pH). Although various natural and synthetic bioactive polymers have been engineered to improve the environmental conditions of a cell, artificially, the choice of the material depends on the selected application, the cell type used, and the tissue type. For example, bioceramics have been considered as one of the most suitable materials for the repair and reconstruction of diseased or damaged parts of the skeletal system. Ceramic materials are inorganic, nonmetallic solids, which include

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crystalline ceramic and amorphous glass compounds. Chapter 20 overviews the development of the most commonly used bioactive ceramics for tissue repair and regeneration. It highlights understanding in the relationship between their structure and biocompatibility toward designing next-generation bioceramic materials for stem cell niche applications. Stem cells can be regulated or controlled by manipulating their microenvironment, and therefore, controlling the interactions between biomaterials and stem cells is a critical factor for exploring the complete potential of biomaterials. There are a variety of methods and technologies available to fabricate and modify biomaterials according to the choice of the cell or tissue properties that needs to be regenerated. For example, surface functionalization or modification is one of the approaches that can create biomimetic microenvironments, which are able to control stem cell fate and functions. Chapter 21 highlights the different processes involved in modifying the surface of a biomaterial by attaching molecules or substances via physical or chemical methods, or both. It focuses on the biological relevance of surface-functionalized biomaterials in the context of stem cell research. Fabricating biomaterial scaffolds in 3D with controllable topographies and stiffnesses and conjugating signaling factors to scaffolds to regulate stem cell fates are critical to simulating in vivo conditions.9 Hydrogels, which are hydrated polymer networks, share many key physical properties with native tissues and can be designed to include elements to control cell fate and function. Chapter 22 comprehensively describes hydrogel design criteria, including source material, degradation, topography, adhesion, and growth factor presentation, in the context of 3D stem cell culture. To complement, Chapter 23 explicitly discusses the structural features that control the mechanical properties within hydrogel networks of different types. It also discusses how gels with different mechanical properties can be used clinically to control cell differentiation and function. Recent advancement in fabrication technologies like nano- and microfabrication has provided opportunities to design biomaterials with intricate topographical structures. In addition to functional tissue formation, some of the in vitro applications include (but are not limited to) identifying suitable ECM candidates as substrates for stem cell culture through micropatterning of ECM in 2D; high-throughput ECM microarrays; and synthesizing novel biomaterials. Chapter 24 reviews the principal techniques used to generate micro- and nanotopographical features in substrates suitable for stem cell culture and niche engineering. It describes use of micro- and nanotopographic patterning as a means to probe the stem cell niche, and ultimately to

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guide stem cell fate through mechanical cues that govern processes such as adhesion, proliferation, and differentiation. Self-assembly in particular is one of the most promising fields in building materials and structures at the nanoscale. It takes a bottom-up approach for fabrication, where small molecular components interact with each other under specific conditions to spontaneously organize into more complex 2D or 3D structures.47,48 Chapter 25 provides a comprehensive background on self-assembly nanofabrication techniques and their applications in bioengineering stem cell niches. The in vivo ECM in particular possesses a nanoscale fibrous topography. Chapter 26 discusses the role of this topography in modulating stem cell fates in vitro and details nanofiber fabrication techniques to produce matrices that have a morphological resemblance to fibers naturally found in the ECM. Their similar characteristics suggest that nanofibers could be used as a supportive matrix for creating artificial niches for stem cells, upon which additional functionalities could be incorporated to further modulate stem cell fates.49 Stem cells are exposed to a multitude of biochemical gradients across a niche, subjecting them to different signals in different locations in the niche, which shapes the way the cells divide. Hence, engineering a modulated niche with spatial complexity would better reflect the in vivo situation. Microfluidics allows for the controlled delivery of signals to specific locations within the niche, thus modulating the properties of an artificial stem cell niche. It provides ways to manipulate single stem cells to better understand behavior across a diverse population of stem cells. In addition, fabricating microfluidic channels within biomaterials has been shown to be a promising tool to mimic nutrient and gas transport for stem cell niche engineering.50 Chapter 27 reviews microfluidic concepts used to generate a wide variety of biomolecule gradients and presents the most successful applications of microfluidic deviceeinduced gradients for pluripotent stem cell patterning.

5. BIOENGINEERING SPECIALIZED ARTIFICIAL STEM CELL NICHES FOR CLINICAL THERAPIES Stem cells are promising cell source candidates for use in regenerative medicine. With the recent discovery of induced pluripotent stem cells (iPSCs), emerging combinations of biomaterials and iPSCs are bringing unprecedented opportunities for treating debilitating human diseases.51 Potential knowledge from the biomaterials-mediated approaches for enhancing stem cellebased tissue repair is being applied for achieving many therapeutic and nontherapeutic goals. Engineering

specialized artificial stem cell niches helps researchers to elucidate the mechanisms by which stem cells receive information from the multifactorial microenvironment. Promoting desirable stem cell phenotypes allows for the exploitation of their unique property of stemness and their ability to home and differentiate, thus contributing to tissue repair. Additionally, in vitro engineered niches can be used to screen for molecules that can regulate niche biology and thus avoid the need for exogenous cell therapy. The currently available therapeutic strategies for endstage debilitating disease such as congestive heart or liver failure are all invasive surgical approaches, including implantable devices and, ultimately, organ transplantation. However, these surgical treatments are unable to completely restore damaged tissue. For example, all current therapeutic procedures for heart failure to date only modulate hemodynamics and none are available to regenerate heart tissue. Regenerative therapies based on stem cells hold promise as a treatment to overcome this limitation, but to date have achieved only modest outcomes in clinical trials. To enhance potential stem cell behavior and exploit its maximum functional effect, numerous studies have taken great effort to elucidate the tissue-specific stem cell and progenitor cell niches. Chapters 28e37 demonstrate such current advances in the construction of biomimetic niches to modulate cellematrix interactions or cellecell interactions for enhanced repair or regeneration in different tissue types, namely cardiac, bone, cartilage, skin, and liver. For vascular tissues and organs, de novo blood vessel formation, also known as vasculogenesis, and the subsequent expansion of the nascent vascular network via angiogenesis, constitutes a complex vascular niche essential to tissue repair. Coordinating blood vessel generation along with parenchymal tissue development is critical so that an adequate supply of nutrients and oxygen is maintained along with the removal of waste metabolites. Harnessing niche biology and in vitro model systems to engineer a regenerative therapeutic is the ultimate goal.

References 1. Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function? Cell 2001;105:829e41. 2. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7e25. 3. Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 2008;132: 598e611. 4. Carlone DL, Breault DT. Slowly cycling versus rapidly cycling intestinal stem cells: distinct roles or redundancy. Cell Cycle 2011; 10:723e4. 5. Glauche I, et al. Stem cell proliferation and quiescenceetwo sides of the same coin. PLoS Comput Biol 2009;5:e1000447.

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REFERENCES

6. Wilson A, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 2008;135:1118e29. 7. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science 2009;324:1673e7. 8. Lane SW, Williams DA, Watt FM. Modulating the stem cell niche for tissue regeneration. Nat Biotechnol 2014;32:795e803. 9. Peerani R, Zandstra PW. Enabling stem cell therapies through synthetic stem cell-niche engineering. J Clin Invest 2010;120: 60e70. 10. Mascre G, et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 2012;489:257e62. 11. Doupe DP, Klein AM, Simons BD, Jones PH. The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate. Dev Cell 2010;18:317e23. 12. Greco V, Guo S. Compartmentalized organization: a common and required feature of stem cell niches? Development 2010;137:1586e94. 13. Kimble JE, White JG. On the control of germ cell development in Caenorhabditis elegans. Dev Biol 1981;81:208e19. 14. Xie T, Spradling AC. A niche maintaining germ line stem cells in the Drosophila ovary. Science 2000;290:328e30. 15. Driskell RR, Giangreco A, Jensen KB, Mulder KW, Watt FM. Sox2positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 2009;136:2815e23. 16. Hsu YC, Pasolli HA, Fuchs E. Dynamics between stem cells, niche, and progeny in the hair follicle. Cell 2011;144:92e105. 17. Trumpp A, Essers M, Wilson A. Awakening dormant haematopoietic stem cells. Nat Rev Immunol 2010;10:201e9. 18. Taichman RS. Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche. Blood 2005; 105:2631e9. 19. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006;116: 1195e201. 20. Boulais PE, Frenette PS. Making sense of hematopoietic stem cell niches. Blood 2015;125:2621e9. 20a. Yin X, Farin HF, van Es JH, Clevers H, Langer R, Karp JM. Nicheindependent high-purity cultures of Lgr5þ intestinal stem cells and their progeny. Nat Methods 2014;11:106e12. 20b. Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftga˚rd R. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 2008;40:1291e9. 21. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell Stem Cell 2014;14:275e91. 22. Reichardt LF, Tomaselli KJ. Extracellular matrix molecules and their receptors: functions in neural development. Annu Rev Neurosci 1991;14:531e70. 23. Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol 2011;3. 24. Vishwakarma A, Sharpe P, Shi S, Ramalingam M. Stem cell biology and tissue engineering in dental sciences. Academic Press; 2014. 25. Gattazzo F, Urciuolo A, Bonaldo P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta 2014; 1840:2506e19. 26. Gattazzo F, Urciuolo A, Bonaldo P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta 2014; 1840:2506e19. 27. Flaim CJ, Chien S, Bhatia SN. An extracellular matrix microarray for probing cellular differentiation. Nat Methods 2005;2:119e25. 28. Gobaa S, et al. Artificial niche microarrays for probing single stem cell fate in high throughput. Nat Methods 2011;8:949e55.

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29. Winer JP, Janmey PA, McCormick ME, Funaki M. Bone marrowderived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue Eng Part A 2009;15:147e54. 30. Engler AJ, et al. Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J Cell Biol 2004;166:877e87. 31. Chopra A, Tabdanov E, Patel H, Janmey PA, Kresh JY. Cardiac myocyte remodeling mediated by N-cadherin-dependent mechanosensing. Am J Physiol Heart Circ Physiol 2011;300: H1252e66. 32. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677e89. 33. Huebsch N, et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat Mater 2010; 9:518e26. 34. Lampin M, Warocquier-Cle´rout R, Legris C, Degrange M, SigotLuizard MF. Correlation between substratum roughness and wettability, cell adhesion, and cell migration. J Biomed Mater Res 1997;36:99e108. 35. Oliveira SM, Song W, Alves NM, Mano JF. Chemical modification of bioinspired superhydrophobic polystyrene surfaces to control cell attachment/proliferation. Soft Matter 2011;7:8932e41. 36. Piret G, et al. Culture of mammalian cells on patterned superhydrophilic/superhydrophobic silicon nanowire arrays. Soft Matter 2011;7:8642e9. 37. Golde DW, Gasson JC. Hormones that stimulate the growth of blood cells. Sci Am 1988;259:62e71. 38. Gordon MR. Superpowers seek accord on visiting mental hospitals. NY Times Web 1988;1:12. 39. Torok-Storb B. Cellular interactions. Blood 1988;72:373e85. 40. Chow A, et al. Bone marrow CD169þ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med 2011;208:261e71. 41. Kohler A, et al. G-CSF-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands. Blood 2011;117:4349e57. 42. Kizil C, Kyritsis N, Brand M. Effects of inflammation on stem cells: together they strive? EMBO Rep 2015;16:416e26. 43. Vishwakarma A, et al. Engineering immunomodulatory biomaterials to tune the inflammatory response. Trends Biotechnol 2016;34: 470e82. 44. Yin X, et al. Engineering stem cell organoids. Cell Stem Cell 2016;18: 25e38. 45. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol 2006;7:211e24. 46. Song H, et al. Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue. Proc Natl Acad Sci USA 2010;107:3329e34. 47. Ariga K, Hill JP, Ji Q. Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application. Phys Chem Chem Phys 2007;9:2319e40. 48. Borges J, Mano JF. Molecular interactions driving the layer-bylayer assembly of multilayers. Chem Rev 2014;114:8883e942. 49. Lim SH, Mao HQ. Electrospun scaffolds for stem cell engineering. Adv Drug Deliv Rev 2009;61:1084e96. 50. Cuchiara MP, Allen AC, Chen TM, Miller JS, West JL. Multilayer microfluidic PEGDA hydrogels. Biomaterials 2010;31:5491e7. 51. Tong Z, et al. Application of biomaterials to advance induced pluripotent stem cell research and therapy. EMBO J 2015;34: 987e1008.

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C H A P T E R

2 Harnessing the Biology of Stem Cells’ Niche Borja Saez1,2, Rushdia Z. Yusuf1,2, David T. Scadden1,2 1

Harvard University, Cambridge, MA, United States; 2Massachusetts General Hospital, Boston, MA, United States

O U T L I N E 1. Introduction

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2. Components of the Stem Cell Niche 2.1 Cellular Components 2.2 Noncellular Components

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3. Integrative Networks Regulating the Niche

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4. Distinct Niches for Different Stages of Differentiation

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1. INTRODUCTION

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6. Conclusion

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Abbreviations and Acronyms

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Glossary

26

References

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Intrinsic regulatory networks are important but there is undeniable evidence that stem cell fate is also controlled by signals received from the microenvironment or niche.13 The concept of the stem cell niche was first hypothesized by Schofield in the late 1970s.14 In his seminal work, Schofield postulated that the true stem cell, in this case, hematopoietic in origin, hematopoietic stem cell (HSC), exists only in the context of the tissue where it lives in close association with other cells, and that the stem cell properties are preserved by this intimate relationship, linking, for the first time, the fate of a stem cell to the fate of its neighboring cell14 (Fig. 2.1). In Schofield’s own words: “The cellular environment which retains the stem cell I shall call a stem cell ‘niche.’ As long as the stem cell remains fixed its further maturation is prevented and it continues indefinitely to replicate as a stem cell.although no direct evidence of this actually exists.”14 This ecologic view of tissue biology not only explains the long-term repopulation ability of the HSCs (or its immortality, according to Schofield) but it also explains how the number of mutations accumulated in those cells is minimized throughout life and therefore how tissues are protected against the emergence of dysplastic or neoplastic features.

Pluripotent cells that give rise to all somatic tissues and adult tissue resident stem cells sustain tissue homeostasis and contribute to repair after injury. Tissue formation, maintenance, and repair are achieved by sustaining a tight regulatory control over the ability of these stem cells to self-renew and differentiate. Regulatory networks controlling self-renewal and differentiation are cell-autonomous but also controlled by homo- and heterotypic interactions with other cell types in the tissue microenvironment, by response to chemical molecules secreted locally or systemically and by the biophysical properties of the environment in which these cells exist. Extensive knowledge of the intrinsic regulation of stem cells has been generated in the last decade, mainly through the use of gene-targeted mouse models. Most studies have been devoted to understanding how processes like transcription regulation, cell cycle control, and apoptosis operate in these populations.1,2 Oxygen levels,3,4 reactive oxygen species and oxidative stress,5,6 epigenetic regulation,7,8 noncoding RNAs,9,10 and metabolic changes11,12 have been studied and promise to unravel new layers of regulation of stem cells.

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00002-0

5. Neoplastic Transformation and Disturbance of the Tissue Ecosystem

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Copyright © 2017 Elsevier Inc. All rights reserved.

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Stem cell niche as postulated by Schofield: A specific anatomical location

o

A site that provides maintenance and self-renewal cues to stem cells

o

A site where differentiation is inhibited

o

A site limited in space, hence controlling the stem cell pool size

o

A site that allows reprograming of a more mature cell to a stem cell state

Stem Cell Niche

Stem Cell

o

Transiently amplifying cell

Stem Cell

2.1 Cellular Components The cellular complexity of the niche may reflect different needs of stem cells during tissue homeostasis and in response to different stress stimuli; hence, the niche may be understood to be a dynamic cellular ecosystem, rather than a static collection of cells, and their heterotypic interactions maintaining tissue homeostasis. Experimentally defining niche components has required both anatomic proximity and function.13 In vivo microscopic observation, as well as, genetic manipulation of cell populations or molecules expressed by specific cell types has led to the description of a number of components of stem cell niches.

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2015 2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 1996 1995 1994 1993 1992 1991 1990 1989 1983 1978 0

The concept of the stem niche remained a hypothesis for the next 20 years until Alan Spradling empirically demonstrated the existence of a niche, as postulated by Schofield, in the Drosophila ovary.15 These studies in an invertebrate model paved the way for an explosion in the field of stem cell niche during the time in which a number of tissue resident stem cells were described in mammalian tissues. Generation of knowledge in the stem cell niche field has been exponential as shown by the increase in the number of publications between 1990 and 2000 (Fig. 2.2). Since Schofield’s postulation of the existence of the stem cell niche, multiple studies have demonstrated its importance in a number of mammalian tissues like the hematopoietic system,16 the skin,17 the intestine,18e20 and the nervous system.21,22 In the present chapter we aim to present an overview of the complexity of the cellular, biochemical, and biophysical signals that shape stem cell niches in mammals and how they not only participate in tissue organization but also contribute to neoplastic transformation.

Year

FIGURE 2.1 Summary of Schofield stem cell niche hypothesis and graphical display of its components. Adapted from Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4(1e2):7e25.

Number of Manuscripts

FIGURE 2.2 Evolution in the number of publications on the stem cell niche per year since the introduction of Schofield’s hypothesis showing the exponential growth in knowledge generated in the years 1978e2015. Data obtained from the PubMed Website.

For example, in the bone marrow (BM), arguably the first and best characterized mammalian niche, an array of cell types have been shown to participate in tissue homeostasis such as osteolineage cells,23 vascular endothelial cells,24 mesenchymal stem cells,25,26 sympathetic neurons27 and glial cells,28 hematopoietic cells such as neutrophils,29 macrophages,30 megakaryocytes,31 T cells32 and osteoclasts,33 as well as adipocytes,34 among others. Here we briefly highlight the

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2. COMPONENTS OF THE STEM CELL NICHE

most commonly accepted components of stem cell niches with a focus on the best characterized mammalian niche, the BM microenvironment (summarized in Fig. 2.3). Chapter 5 in this book presents a comprehensive review on the HSC niche in the BM. Osteoblasts: The role of osteoblasts, bone forming cells, in regulating HSCs was first suggested upon observation that primitive hematopoietic cells reside in close proximity to the endosteal surface.35,36 This observation was soon followed by empirical evidence demonstrating that osteoblasts produce positive and negative regulators of hematopoietic growth such as G-colony stimulating factor (CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Interleukin (IL)-1, IL-6, and TGF-b.37e40 But it wasn’t until 2003 an in vivo demonstration was provided showing that genetic manipulation of osteolineage cells functionally altered hematopoiesis.23,41 These

17

independent studies showed a simultaneous increase in osteoblastic and hematopoietic primitive cells when the parathyroid hormone receptor (PTHR) was constitutively activated23 or the bone morphogenetic protein (BMP) receptor 1a was deleted41 in specific osteolineage populations. These studies were the first in vivo experimental confirmation of Schofield’s niche hypothesis in mammals. This was followed by studies in which systemic administration of parathyroid hormone (PTH) in WT mice expanded HSCs in vivo and facilitated survival of animals transplanted with limited numbers of HSCs after myeloablative conditioning.42 In 2004, Visnjic and colleagues, using a model of osteoblast ablation in vivo (thymidine kinase expression under the col2.3 promoter control), reported a severe decrease in the number of hematopoietic progenitor cells and multi-lineage cytopenia upon ganciclovir administration. Furthermore,

FIGURE 2.3 Simplified representation of the adult hematopoietic stem cell niche in the bone marrow. The major cellular sources of hematopoietic stem cell (HSC) support are depicted. HSCs are located adjacent to vascular structures, sinusoids, and arterioles, where they receive cues for their maintenance by vascular endothelial cells and perivascular mesenchymal cells (Nestin, LepR, Prx-1, or Mx-1) with osteolineage potential. Additional cell types regulating the HSC niche are sympathetic nerves enclosed in a sheath of nonmyelinating Schwann cells, macrophages and megakaryocytes, and osteoclasts among others. From Kfoury Y, Mercier F, Scadden DT. SnapShot: the hematopoietic stem cell niche. Cell 2014;158(1):228-e1. I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

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these defects were reversible, and upon ganciclovir discontinuation, osteoblast and hematopoietic progenitors recovered.43 Thrombopoietin (TPO), a glycoprotein, implicated in megakaryocyte activation and platelet production was also described to be a mediator of osteoblast-HSC interaction regulating HSC quiescence and retention in the niche.44 In addition, multiple other findings supported the regulatory role of the osteoblast implicating a variety of molecules and signaling pathways mediating this effect.45e47 Multiple subsequent studies have caused refinement in understanding of the role of the osteolineage cell in regulating HSCs and hematopoiesis. With the latest immunophenotyping and fine intravital imaging technologies it has become apparent that few HSCs reside in close proximity to osteoblastic cells under homeostatic conditions, though they do posttransplant.3,48,49 Moreover, we and others have demonstrated that acute depletion of osteolineage cells in vivo by means of diphtheria toxin injection, as well as biglycan deficiency, although drastically depletes the number of osteoblasts, has no short-term effect on the number or functionality of HSCs.50,51 Conversely, Lymperi and colleagues noted that increase of osteoblast numbers by administration of strontium had no apparent effect on HSCs.52 Furthermore, two independent studies have shown that the genetic deletion of key HSC regulatory molecules like CXCR12 and SCF in osteolineage cells do not alter the number or function of HSCs in vivo.24,26,53 Overall, growing evidence suggests that although the contribution of osteolineage cells to the regulation of hematopoiesis is indisputable, it may in fact be an indirect effect, at least under homeostatic conditions (Fig. 2.3). Nonetheless, niches may be more dynamic than previously anticipated and under stress conditions osteoblasts may directly influence HSC responses such as mobilization in response to G-CSF challenge.54 Much akin to the hypothesis that osteoblasts comprise a critical part of the HSC niche because of their proximity to the stem cell in question is the widely accepted and empirically borne out hypothesis that Paneth cells, secretory cells present in contact with Lrg5þ intestinal stem cells (ISCs), in the small intestine are part of the ISC niche. The strength of the evidence depends on the experimental models used as described below. The ensuing discussion highlights that in almost all cases the cellular components that are integral parts of stem cell niches need to be confirmed by multiple, exclusive experimental models. In 2007, ISCs long thought to reside in the crypt55,56 were identified to be Lrg5þ.57 Lrg5þ cells are now divided into central cells present at the base of the intestinal crypt and border cells located higher up. Both populations of Lrg5þ cells are endowed with self-renewal potential.58 Anatomically interspersed amongst the

Lrg5þ stem cells, Paneth cells have long been known to secrete enzymes that protect the integrity of the intestinal epithelial layer against the microbiota in the gut. Since the identification of Lrg5 as a marker of ISCs it has been shown that co-culture with Paneth cells greatly increases the efficiency of these cells to make organoids in culture and to be serially replated.20 Wnt 3A can mimic the effect of Paneth cells in early stages of culture and is thought to be the chemical effector of these cells in the intestinal niche. The Wnt pathway is one of several others through which the Paneth celleintestinal stem cell interaction occurs; other pathways involved being EGF, BMP/TGF-b, and Notch.59 Despite the rich selection of molecules the Paneth cells secrete, they have been shown to require direct contact with the stem cells to effectively perform their supporting function. Some key studies investigating the role of Paneth cells in the intestinal niche are worth noting only because they highlight some key assays and their limitations used in such studies. Deletion of the Paneth cells as a whole using expression of the diphtheria toxin receptor in these cells eliminated nearly 90% of this cell population in intestinal crypts with an increase in intestinal epithelial cells in one study60 and a decrease in Lrg5þ cells in another.20 The reason for this discrepancy was different definitions used for cells being measured. Whereas depletion of Paneth cells was incomplete in both studies, the read-out of intestinal crypt cells was by electron microscopy in the former study and by Lrg5 positivity in the latter. Using Lrg5 positivity as a marker for ISCs, the conclusion that a diminished number of Paneth cells is accompanied by a decrease in ISCs as well has been borne out by other studies in which mice had the Paneth cell population depleted by knock-out of the Gfi-1 transcription repressor,20 and the growth factor receptor Csfr161 or Sox9,20 showing that at least in the case of depletion the deletion of stem cells was concomitant with deletion of Lrg5þ stem cells. In two subsequent studies in which the transcription factor Math1 was inducibly deleted and Paneth cells were obliterated, Lrg5þ cells continued to function as normal.62,63 The former study noted that exogenous Wnt was needed for growth of organoids in vitro. In vivo maintenance of stem cells and in vitro dependence on exogenous Wnt were also seen in a knockout mouse model of Wnt 3.64 Hence depletion (even up to 90% of Paneth cells) will deplete Lrg5þ cells though complete obliteration will not deplete Lrg5þ, likely by activating other factors such as Wnt production by stroma.65 Mesenchymal stromal cells: It is now becoming apparent that more immature osteolineage cells, including multipotent mesenchymal cells, are hematopoietic stem cell regulators. Most of these mesenchymal stromal cells are perivascular and share microanatomical location with

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2. COMPONENTS OF THE STEM CELL NICHE

HSCs in the BM.48,49,66 This anatomical colocalization suggests that HSCs are maintained in perivascular niches by endothelial (discussed further below) or perivascular mesenchymal cells (Fig. 2.3). Mesenchymal populations in the BM are heterogeneous and their lineage relationships are yet to be elucidated. Nevertheless, they have been identified and are nowadays defined by the expression of a CXCL12, Nestin, Leptin receptor (LepR), Prx1, Osterix (Sp7), and Mx1 in the mouse.24e26,53,67e69 Without exception, these stromal cell populations give rise to osteoblastic cells and produce factors implicated in HSC maintenance. CXCL12-abundant reticular cells (CAR cells) were the first population of perivascular stromal cells implicated in the regulation of HSC due to their colocalization throughout the BM.69 Subsequent functional validation showed that ablation of CAR cells in the marrow not only impairs hematopoiesis but also adipogenesis and osteogenesis.70 Further evidence implicating mesenchymal populations in the regulation of HSCs was provided upon description of a population of perivascular cells in the BM that express high levels of Nestin. Nestinþ cells are neighboring HSCs in the marrow and produce high levels of stem cell key regulatory factors (CXCL12, VCAM1, SCF1 among others).25 More recently two studies have postulated the existence of a periarteriolar and a perisinusoidal niche for quiescent HSCs.48,49 These seemingly contradictory observations may be explained by the use of alternative experimental systems and warrant further investigation. Briefly, periarteriolar niches were characterized by the presence of NestinbrightNG2þ perivascular cells. These periarteriolar cells express high levels of CXCL12 and stem cell factor (SCF) and their acute depletion leads to HSC proliferation and loss of stemness.48 Contrarily, perisinusoidal niches have been recently suggested to maintain dividing and nondividing HSCs. These perisinusoidal niches are composed of LepR  CXCL12 expressing cells within 10 mm of sinusoidal blood vessels. Of note, this study also described that the distribution of HSCs in the BM is more common in the central marrow compared to the bone surface and in the diaphysis relative to the metaphysis49 in contrast to what was previously thought. Additional studies have used other markers such as LepR or Prx1 in order to define perivascular mesenchymal populations in the BM.24,26,53,68 LepRþ cells have been shown to be mesenchymal in origin and with multipotent repopulation ability in vivo. Furthermore, these cells do show the highest levels of expression of SCF and CXCL12 in the adult marrow and deletion of those key factors leads to a dramatic reduction of the number of functional HSCs in the marrow.24,53 BM perivascular Prx1þ cells have in vitro osteogenic and

19

adipogenic potential71 and deletion of CXCL12 from these cells in vivo leads to a profound depletion and loss of long-term repopulation ability of HSCs.26 Furthermore, perivascular niches also contribute to HSC regulation in extramedullary sites such as the spleen, at least under stress conditions.72 All these studies provide evidence demonstrating that HSCs reside in a perivascular location in close proximity to mesenchymal stromal cells that synthesize and secrete necessary factors to maintain stemness and modulate their localization. Nonetheless, it should be noted that the lineage relationships in between these perivascular mesenchymal cells is not well understood. It is most likely that an overlap exists in between the populations defined by the expression of the abovedescribed promoters. In fact, our understanding of the perivascular niche has been limited by the tools to visualize and genetically modify different cellular populations (reviewed elsewhere).73 Hence, further studies are warranted in order to better understand the characteristics of those populations and their functions in HSC regulation. The stromal populations in the BM as described above are fractionated by virtue of promoters into several groups, each of which have been postulated to be components of the niche. In other tissues, such as the intestine or skin, stromal cells are less well-defined but known to be equally important components of the respective stem cell niches. The niche in the gut is composed of both elements within and outside the crypt. Stromal cells in the lamina propria underlying the crypt can produce BMP4 and promote stem cell differentiation74 whereas myofibroblasts and myoblasts underlining the crypt counteract this signal and prevent differentiation by secreting BMP inhibitors.75 A parallel hedgehog pathway originating from the stromal cells causes increases in myofibroblasts and myoblasts, while reducing the formation of differentiated intestinal epithelial cells76 but attenuation of this pathway has the exact opposite effect on all cell populations concerned.77 Epidermal stem cells are present in the basal layer of the epidermis,78,79 which undergo self-renewal, as well as differentiation, into the three superior layers of the epidermis. IGF, FGF, EGFR ligands, FGF-7, and FGF 10 are all capable of causing colony formation from isolated stem cells in vitro and seem to comprise the chemical components of the epidermal stem cell niche.80e82 These elements are produced at least in part by the dermal fibroblasts. Similar to the support provided to epidermal stem cells, dermal fibroblasts support hair follicle stem cells as well. These stem cells undergo regular cycles of division (anagen) and quiescence (telogen) in the mouse. Hair follicle stem cells are located in the bulge or in

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the primed germ cell population in the sub-bulge area. Both these populations of stem cells give rise to transient amplifying cells at the beginning of the division or anagen phase. The germ cells give rise to the core of the hair shaft and the inner root lining whereas the bulge cells form the outer shaft lining.83,84 Because in the mouse the hair follicles alternate between telogen and anagen which mark quiescence versus division in the stem cells, the chemical signals and supporting cellular components that determine these two cell phases are fairly well-defined. BMPs are produced by dermal fibroblasts (in addition to fat and inner bulge cells) and cause both the bulge cells and the sub-bulge prime cells to be inhibited from differentiation, whereas cells in the dermal papillae and adipose tissue precursors secrete factors that inhibit telogen. Indeed removal of dermal papillary tissue results in inability to exit telogen.85,86 Endothelial cells: Together with mesenchymal cells, endothelial cells constitute the perivascular niche in the adult BM (Fig. 2.3). Endothelial cells were first postulated as integral part of the HSC niche by their colocalization in histological studies. The description of the SLAM markers (CD41, CD48, and CD150) allowed for the visualization of highly purified stem cells in close proximity to the sinusoidal network rather than the endosteum.66 Indirect evidence soon followed suggesting a regulatory role of endothelial cells in the HSC niche but that evidence was ambiguous and did not demonstrate the direct or indirect regulation of HSCs in homeostasis in vivo.87e89 Formal identification of the endothelial involvement in HSC regulation came from studies demonstrating that the deletion of SCF from Tie2 endothelial cells greatly reduces the number and long-term reconstitution ability of HSCs.53 This study was soon followed by two reports demonstrating that another endothelial factor CXCL12 is critical for HSC maintenance in the niche.24,26 Endothelial cells are also part of extramedullary niches controlling HSC responses under stress conditions.72 Adipocytes: Another mesenchymal population implicated in HSC regulation is adipocytes. Once believed to just fill space in the BM in response to aging or cytotoxic insult,90 adipocytes are now recognized as negative regulators of HSC function.34 However, adipocytes and osteoblasts are derived from the same mesenchymal primitive cells and their relative abundance in the marrow may be negatively correlated making it complicated to discern the specific contribution of each cell type.91 Adipocytes are also involved in the regulation of hair follicle stem cells as mentioned in the section on mesenchymal stromal cells. Cellular Progeny: Stem cell cellular descendants have been shown to become niche cells in various tissues.

The implication of mature blood cells in the regulation of hematopoietic stem cells has been postulated for quite some time although a direct role has only been demonstrated recently (Fig. 2.3). Early studies elegantly demonstrated that HSCs “sense” the loss of erythrocytes and proliferate in order to maintain tissue homeostasis.92 This effect is mediated indirectly and through the production of erythropoietin in the renal cortex. Macrophages have been implicated in the control of stem and progenitor cell egress from the BM and have been shown to enhance G-CSF induced mobilization.30,93 These studies postulated that BM resident macrophages cross talk with mesenchymal perivascular and osteolineage cells helping maintain the integrity of the HSC niche.30,93 T cells, discussed below, also play a role in HSC maintenance by creating immune sanctuaries for HSCs in the BM.32 Now megakaryocytes have been shown to be components of the niche regulating quiescence and function.94,95 Acute depletion of megakaryocytes in these studies resulted in an increase in the number of HSCs and their cycling, potentially due to a deregulation of CXCL4 and TGF-b signaling.94,95 Anatomically, megakaryocytes are physically associated with sinusoidal endothelium and mesenchymal pericytes, all of which compose the HSC niche. Neutrophils, albeit indirectly through macrophages, control the size and function of the niche and modulate the HSC egress into circulation.29 In the skin, the epidermal stem cells respond to a large number of growth factors, which are produced by dermal fibroblasts but also by the progeny epidermal cells at least in in vivo models.80e82 Moreover sub-basal cells which arise from division of epidermal basal cells (epidermal stem cells) are critically involved in fate determination of the stem cells in terms of whether the division produces two stem cells or one stem cell and a differentiated daughter eventually. Delamination or detachment of the stem cell from the extracellular matrix (ECM) is one key regulator of epidermal stem cell fate. The other is an axis of division perpendicular to the basement membrane allowing cell division to be asymmetric leading to the formation of differentiated spinous cells. Activation of the notch pathway is important for this differentiation.96e98 As described above, the progenitor cells can be part of the niche itself and it appears that the sub-basal layer of cells which are formed as a result of the perpendicular and hence asymmetric division described produce the notch ligands.98

2.2 Noncellular Components The hematopoietic stem cell niche is also regulated by a number of noncellular factors such as oxygen tension, calcium ions, and tissue stiffness and tension. It has been

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3. INTEGRATIVE NETWORKS REGULATING THE NICHE

long postulated that the BM may be compartmentalized into regions with different oxygenation and that HSCs reside in hypoxic areas in the marrow.3,99 These studies however relied on the staining with probes that reflects the metabolic state of the cell rather than the ambient oxygenation. Direct measurement of pO2 in the marrow space revealed that despite the high vascular density, pO2 in the marrow is relatively low and heterogeneous. Perisinusoidal regions showed the lowest pO2 levels in contrast to nestin-rich periarteriolar regions and the endosteum.100 Calcium is a by-product of bone catabolism and deletion of the calcium-sensing receptor from HSCs impairs their engraftment to the BM.101 Contrarily, pharmacological activation of the receptor improves HSC homing to the BM.102 It has also become apparent in the last few years that stem cells rely on physical and mechanical inputs from their surroundings.103 For example, circulatory shear stress in the dorsal aorta during HSC development induces the transcriptional program involved in definitive hematopoiesis.104 In the adult, hematopoietic stem and progenitor cell engraftment and niche sensing depend on contractile forces on a cell-autonomous fashion.105 Another example of a noncellular component regulating stem cells is found in the epidermis. Laminin 5, a major component of the ECM which is secreted both by basal epidermal cells and by dermal cells interacts with b1-integrin in basal cells which cycle less than those with sparse b1-integrin expression.106 Interaction of epidermal stem cells in the basal layer with laminin is controlled at least in part by epigenetic modifiers such as H3K27 methyltransferases which may promote selfrenewal and H3K27 demethylases, which may promote differentiation enabling response to wound healing.107 In order to function as appropriate stem cells the epidermal stem cells in the basal layer of the epidermis are required to self-renew and differentiate, and it is their interaction with the ECM which is critical in determining which of these two processes occurs. Delamination mediated by decreased laminineintegrin interaction is one of the two steps critical in promoting differentiation, an axis of division perpendicular to the basement membrane being the other one which has been discussed above.107

3. INTEGRATIVE NETWORKS REGULATING THE NICHE The regulatory role of the niche includes integrating external cues reflective of the tissue or the organismal state. Those external cues comprise neural transmission, the immune system, the endocrine system, and perhaps even the microbiome (Fig. 2.4).

Integration of Tissue and Organism Needs: ------Neural input, vascularization, immunity, endocrine etc

Niche cell

Stem Cell

OUTPUT: ------Self-renewal, proliferation, differentiation, localization

FIGURE 2.4 The stem cell niche integrates local and systemic signals. Tissue maintenance and repair depends on the ability of the niche to “sense” and integrate signals from the organism to subsequently regulate cell production to accommodate the organism needs. Adapted from Scadden DT. Nice neighborhood: emerging concepts of the stem cell niche. Cell 2014;157(1):41e50.

Signals from the sympathetic nervous system contribute to HSC regulation and have been implicated in the retention and egress of HSCs from the marrow. In particular, mice showing aberrant nerve conduction or adrenergic neurotransmission ablation (by pharmacologically or genetically means) show CXCL12 dysregulation and failed in induction of mobilization in response to G-CSF.27 Similarly, diabetic neuropathy in the marrow is associated with deficient mobilization in response to G-CSF.54 Furthermore, the sympathetic nervous system regulation of marrow HSC localization has been linked to central nervous circadian rhythms.108 Neural adrenergic signals are transmitted to perivascular stromal cells through the beta3-adrenoceptor leading to CXCL12 downregulation and HSC egress from the BM.108 Furthermore, conditions of chronic stress have also been shown to induce HSC activation, monocytosis, and neutrophilia through neurogenic norepinephrine release and beta3 adrenergic activation in the BM.109 Lastly, nonmyelinating Schwann cells that enclose nerves in the marrow produce TGF-b and regulate HSC quiescence as shown by the rapid loss of HSCs upon autonomic nerve denervation.28 The circadian clock has also been implicated in the regulation of the epidermal stem cells. The hair-follicle niche contains populations of stem cells in different phases of the clock that correspond with quiescent and active populations of stem cells allowing for the rapid

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response to stress stimuli without the depletion of stem cells.110 Disruption in the clock results in altered aging and predisposition to tumorigenesis.110 Likewise, alterations in the circadian clock in other tissues like the pancreas, retina, liver, and esophagus result in profound defects in tissue function.111e114 Signals from the immune system also participate in stem cell regulation. As previously mentioned, cells from the innate immune system such as neutrophils and macrophages participate in the regulation of the HSC niche.29,30,115 Regarding the adaptive immune system, T cells have been shown to promote HSC engraftment, and their depletion leads to engraftment failure.116,117 T cells, and in particular T-regulatory cells, also play a role in HSC maintenance by creating immune sanctuaries for HSCs in the BM.32 Cytotoxic CD8 cells also contribute to HSC activation and they do so by activating mesenchymal stromal cell production of interleukin-6 that in turn induces a strong myeloid differentiation from primitive hematopoietic cells.118 HSCs directly, and through their niche, also respond to systemic administration of immune triggers like interferon.118e120 Toll-like receptors (TLRs) are noncatalytic receptors that recognize structurally conserved ligands produced upon exposure to pathogen components such as lipopolysaccharide (LPS), single-stranded RNA, and peptidoglycans. TLRs are expressed in HSCs and cells of the immune system, as well as in endothelial cells and mesenchymal stromal cell populations in the marrow and are implicated in the regulation of HSCs. TLR4 activation in endothelial cells leads to a G-CSF-dependent increase in myelopoiesis.121 Moreover, in a model of systemic infection with Escherichia coli, HSCs mobilize and localize to the spleen, a phenomena dependent on TLR4 in radio-resistant stromal cells.122 These studies demonstrate acute responses of the HSC niche to organismal demand in the event of an infection. Although the evidence may be preliminary and circumstantial, it is worth hypothesizing that the niche also responds to commensal bacteria. The importance of the microbiome in organismal organization has become apparent.123 In the hematopoietic system, evidence suggests that neutrophil aging is regulated by the microbiome since depletion of the microbiota significantly decreases the number of circulating aged neutrophils.124 In turn, neutrophils have been shown to modulate HSC retention in the marrow29 and depletion of aged neutrophils in vivo leads to an accumulation of primitive hematopoietic cells in the BM and subsequent decrease in circulation. Luo and colleagues have provided the first experimental evidence suggesting that changes in the gut microbiota affect osteoblast-adipocyte homeostasis in the BM leading to alterations in the hematopoietic

stem cell compartment and its differentiation ability.125 These changes are, at least, partially explained by a metabolic stress induced by a high-fat diet. Caloric restriction also induces an adaptive response that affects stem cell niches. Experiments in the mouse gut suggest that reduced caloric intake modulate Paneth cells production of paracrine factors and the subsequent expansion of stem cells.126 Other systemic inputs also influence stem cell behavior. We have discussed elsewhere in this chapter the role of the parathyroid hormone and erythropoietin in the regulation of HSCs and their niche.23,92 Sex hormone regulation of mammary gland stem cells is well-documented. For example, progesterone indirectly induces expansion of mammary stem cells modulating the production of paracrine signals produced by their niche.127,128 A direct regulatory effect of estrogen on HSCs has been reported. Surprisingly, HSCs show gender differences and respond to estrogen levels in female mice resulting in enhanced cell division and stem cell frequency, as well as changes in cellularity and erythropoiesis.129 Besides this direct effect on the HSCs, estrogen deficiency expands primitive hematopoietic cells through regulation of T cells and stromal cells in the BM. In particular, estrogen deficiency was associated with an increase of CD40L, which in turn induced T cells and stromal cells in the marrow to produce hematopoietic cytokines with subsequent HSC expansion.130 Other hormones such as thrombopoietin are also required for HSC maintenance.44,131 Similar to the BM, hormones produced during pregnancy control stem cell behavior in the hair follicle. In the mouse, hair growth is stalled during pregnancy and lactation; a phenomenon explained by the fact that prolactin production during pregnancy blocks the progression of hair growth impeding anagen entry.132 In summary, a growing body of evidence suggests that systemic signals regulate stem cells and their niche function by incorporating information that may indicate the general status and necessities of the tissue and organism (Fig. 2.3).

4. DISTINCT NICHES FOR DIFFERENT STAGES OF DIFFERENTIATION Niches may also exist for transiently amplifying cells. Recent findings suggest that hematopoiesis is mainly maintained through the expansion of different clones of hematopoietic progenitors, rather than stem cells, throughout the life of an organism.133 These transient amplifying pools of progenitors, responsible for the maintenance of tissue homeostasis, are also regulated through heterotypic interactions with their microenvironment.

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5. NEOPLASTIC TRANSFORMATION AND DISTURBANCE OF THE TISSUE ECOSYSTEM

Mature osteoblasts, defined by the expression of osteocalcin, regulate a very specific lymphoid precursor in the BM with restricted T cell potential.50 Ablation of osteocalcin þ osteolineage cells leads to a marked reduction in T-biased lymphoid progenitors and intrathymic T cell precursors with a subsequent reduction in mature T cell numbers. This osteo-lymphoid interaction is at least partially orchestrated through the notch signaling pathway.50 Osteolineage cells not only control T cell maturation but also B-lymphopoiesis. Depletion of Col2.3osteolineage cells results in a rapid reduction in the numbers of B-lymphocytes at the pre-, pro, and pro-B differentiation stages.134 This regulation is in part dependent on the expression of SDF1, since its conditional deletion in Col2.3-osteolineage cells leads to a marked reduction in the number of lymphoid progenitors in the BM without an impact on HSC numbers.26,53,135 Moreover, deletion of Gs-alpha from osteolineage-committed progenitors, defined by the expression of Sp7, leads to an IL-7-dependent B-lymphoid defect.136 In addition, other niches have been proposed to regulate different stages of lymphopoiesis from naı¨ve immune cells to antibody secreting plasma cells, as well as memory cells (reviewed in Mercier et al.137) (Fig. 2.3). Perhaps niches for myeloid cells also exist. Under stress conditions, mesenchymal stromal cells produce inflammatory cytokines that promote myelopoiesis.118 Neutrophil release in inflammatory settings is in part controlled by CXCR2 ligands produced by megakaryocytes.138,139 Macrophages on the other hand are implicated in aged neutrophil clearance that in turn leads to changes in the HSC niche size and HSC localization.29 Macrophages also control RBC production by promoting late erythroid maturation.115 These and other studies reveal the true complexity of the marrow niche, which is not limited to the regulatory role on the HSCs. Potentially; tissue organization in the marrow is accomplished by the coexistence of a number of different anatomical and functional niches that may control serial stages in differentiation of the hematopoietic lineage. This phenomenon is not limited to the BM niche. In the airway epithelium, for example, basal stem cells contribute to the maintenance of their daughter cells through a notchdependent feed-forward mechanism suggesting that stem cells function as niche for their progeny.140

5. NEOPLASTIC TRANSFORMATION AND DISTURBANCE OF THE TISSUE ECOSYSTEM Understanding the dynamics of the stem cell niche becomes particularly relevant upon neoplastic transformation. Similar to normal tissue maintenance, tumors

23

are hierarchically organized. Cumulative evidence suggests that multiple tumor types develop from a rare population of cancer initiating cells (CICs).141e148 These CICs originate either from transformed tissue resident stem cells with self-renewal capacity or from more committed progenitors that acquire self-renewal-like properties following oncogenic transformation.145e149 Like their normal counterpart, CICs self-renew in order to replenish themselves and also differentiate to give rise to the cells that represent the tumor bulk. While tumor morbidity is likely produced by the tumor bulk, CICs provide the reservoir for disease maintenance, resistance to treatment and relapse.150 Akin to its normal counterpart, the BM is perhaps the best characterized neoplastic niche. Leukemia initiating cells (LICs) are thought to reside in specialized microanatomical locations that contribute to their maintenance to drug resistance. It is therefore valid to hypothesize that successful therapeutic eradication of the disease would require combinatorial targeting of both intrinsic leukemia pathways and the protective signals provided by the microenvironment consistent with initial evidence provided by Krause and colleagues.151 LIC dependency on the microenvironment has been demonstrated in recent years. Xenograft models of leukemia have demonstrated that LICs engraft in close proximity to osteolineage and vascular endothelial cells in the trabecular bone where they receive quiescence and antiapoptotic signals that protect them from chemotherapy152e154 (Fig. 2.5). Furthermore, the leukemiaemicroenvironment interactions are bidirectional. LICs in models of lymphoblastic leukemia and stem cells in myeloproliferative disorder (MPD), which can progress to Acute myeloid leukemia (AML) have been shown to “reprogram” their microenvironment to preferentially support LICs by severely compromising the ability of the microenvironment to maintain normal HSCs152,155 (Fig. 2.5). In xenograft models patient-derived leukemic cells create artificial niches that sequester normal HSCs, modulating their self-renewal abilities partially through the modulation of the SCF-cKit signaling pathway.152 Furthermore, myeloid cells in a model of murine myeloproliferative disease have been shown to affect mesenchymal cell differentiation toward the osteolineage, as well as the supportive capacities of those osteolineage cells. In particular, malignant myeloid cells modulate the supportive capacities of the osteolineage cells, which switch from an HSC maintenance to a leukemia-nurturing niche155 (Fig. 2.5). These observations may explain why normal hematopoiesis is frequently suppressed in leukemia patients. Two independent studies have demonstrated the implication of the sympathetic nervous system in the pathogenesis of AML and MPD.156,157 As mentioned before, sympathetic

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FIGURE 2.5 Niches in the context of neoplastic transformation and their contribution to pathogenesis. (A) Adhesion molecules expressed in tumor initiating cells facilitate their engaging in niches that nurture tumor maintenance and survival. (B) Tumor cells and tumor initiating cells hijack the normal stem cell niche in order to facilitate neoplastic progression at the expense of normal stem cells. (C) The malignant niche protects tumor initiating cells from the cytotoxic effects of conventional chemotherapy and provides an immune sanctuary for tumor initiating cells to escape immune surveillance and (D) Disruption of the niche contributes to neoplastic transformation of the stem cells, for example, by increasing the stem cell pool size or allowing clonal expansion of genetically aberrant precancerous cells. From Kfoury Y, Mercier F, Scadden DT. SnapShot: the hematopoietic stem cell niche. Cell 2014;158(1):228-e1.

innervation of perivascular mesenchymal cells regulate HSC. Importantly, JAK2-mutant HSCs disturb the normal HSC supportive perivascular mesenchymal cells and their sympathetic innervation.156 Engraftment of LICs in the BM in part depends on adhesion to the stromal compartment (Fig. 2.5). The integrin alpha4-beta1 (ITGA4) is frequently expressed in leukemic blasts and its expression is inversely correlated to the response to chemotherapy.158 Engaging of the integrin signal triggers the activation of a PI3KAKT-dependent pro-survival cascade promoting survival of the LICs and resistance to chemotherapy.159 In fact, targeting of the ITGA4 in combination with chemotherapy prevents the development of leukemia in a xenograft model of AML.159 Furthermore, CD44, a molecule implicated in cellecell and cellematrix interactions has been shown to be critical for homing and engraftment of chronic myelogenous leukemia (CML) and AML LICs in xenograft models.160,161 Interaction of CXCL12 and its receptor CXCR4 is critical for the maintenance of normal HSCs in the adult BM. Notably, CXCL12eCXCR4 interactions are also important for LICs homing to the BM and potentially to other extramedullary sites.162 Targeting of CXCR4 has been shown to sensitize leukemic cells to chemotherapy in models of CML, AML, and Acute lymphoblastic leukemia (ALL) preventing the protective effect of leukemiaestromal interaction.163,164 Stromal cells produce and secrete cytokines and morphogens such as SCF, GM-CSF, IL3, and BMPs among others that control leukemic survival, response to treatment, and fate decisions arguing the influence of

environmental cues for LICs lineage differentiation and expansion.165,166 Intriguingly, donor-derived hematopoietic malignancies have been reported in patients receiving an allogeneic BM transplant from healthy individuals167 suggesting that the environment in the recipient bone marrow may be contributing to the development of these malignancies (Fig. 2.5). In addition, nonhematopoietic stromal cells obtained from human patients have been shown to harbor genetic abnormalities168 again suggesting that BM stromal cell populations may be involved in the pathophysiology of leukemic disease. Similar observations have been made using murine models.125,169,170 Walkley and colleagues demonstrated that mice lacking the retinoic acid receptor gamma (RARgamma) in the BM microenvironment develop a myeloproliferative disease.170 Furthermore, selective deletion of the microRNA processing enzyme, Dicer1, in osteolineage progenitors but not in mature osteoblast induces a myelodysplastic-like syndrome with rare cases of secondary leukemia169 (Fig. 2.5). Understanding the cell intrinsic regulation, as well as the physical and molecular interactions of leukemic cells and their niches, provides potential opportunities to improve therapeutic targeting of LICs. It is plausible to predict that a number of novel therapeutic approaches will be developed in the next years targeting not only the leukemic clone but also its complex heterotypic interactions with cells in the BM. Designing effective combinatorial approaches will require novel preclinical model systems that accurately mimic the complexity of the tumor-microenvironment and the

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

ABBREVIATIONS AND ACRONYMS

dependence of LICs on this microenvironment and have the ability to allow efficacy assessment in highthroughput.171 Besides the BM, implication of tissue-specific niches in the regulation of neoplastic events has just become apparent. In the mammary epithelium, extended exposure to BMPs secreted by endothelial and stromal cells can initiate the malignant transformation and the establishment of luminal tumors.172 In the brain, glioblastoma-initiating cells seems to depend on the same anatomical niches as neural stem cells. Endothelial cells in the brain interact with CICs and promote tumor formation in vivo while their depletion eradicates tumor cells.173 Furthermore, this relationship is bidirectional, and glioblastoma-initiating cells protect their endothelial niche modulating survival pathways and protect them from genotoxic stresses.174 Analogous to the intestinal stem cell niches, in colorectal cancer, myofibroblasts regulate cancer stemness throughout the secretion of hepatocyte growth factor and the subsequent activation of a Wnt-transcriptional program and CIC clonogenicity. Furthermore, myofibroblasts-secreted factors are able to reprogram a stem program in differentiated cells of the tumor, suggesting that, at least partially, CICs in colon carcinomas are regulated by their microenvironment.175 Histological studies in squamous cell carcinomas in the skin have shown that CICs localize at the tumore stroma interface and that the tumor microenvironment tightly modulates CIC proliferation. Furthermore, CICs depend on vascular endothelial growth factor (VEGF) in order to stimulate tumor vascularization but also to modulate proliferation.107 One could also argue the existence of metastatic niches where tumor cells from primary sites are attracted. It has been long observed that tumors of different origins show some degree of tissue tropism in their dissemination and metastatic growth.176 Recently, two studies have shed some light into this process. In human and mouse models of lung, liver, and brain, tumor cell exosomes pave the way for the metastatic colonization by modulating organ-specific cells. They do so by expressing specific exosomal integrins that modulate the uptake by specific cells at the site of metastasis.177 Metastatic sites in the brain, on the other hand, facilitate colonization by epigenetically modifying transcriptional networks in disseminating cells through exosomal transfer of microRNAs.178

6. CONCLUSION Despite intense research during the last decades our understanding of the different layers of organization of the stem cell niche remains limited. Reductionist

25

approaches have been proven highly efficient dissecting the complexity of stem cell niches in a unidirectional fashion. For example, depletion of a given cellular compartment in a tissue and the subsequent evaluation of its consequence in the stem cell compartment have allowed the description of a number of functional niche populations. This approach, however, fails to recognize the tridimensional complexity of tissue organization. A lot has been said about the role of the osteolineage and endothelial cells in the HSC niche but it’s now becoming clear that the processes of osteogenesis and angiogenesis in the BM, previously thought to be independent, are tightly interconnected.179,180 Bone vasculature contains specialized endothelial cells that support bone maturation and regeneration and in return osteolineage cells produce and secrete paracrine factors that support angiogenesis.179,180 One step further, this process seems to be controlled at least in part by preosteoclasts through the production of platelet-derived growth factor-BB, linking bone catabolism to osteogenesis and angiogenesis.181 Perhaps then we should be looking at the components of stem cell niches as a network of cell types whose function as a whole, rather than individually, maintain tissue homeostasis. In fact, further studies should systematically evaluate the consequences of cell depletion strategies, or the deletion of specific molecules from different cell types, in all cell types on a given tissue. Furthermore, a systems biology approach such as single cell RNA sequencing may help establish not only the molecular landscape of cells that comprise the stem cell niche but also the lineage relationships that may exist between cells in the stroma. Perhaps then we could start to appreciate the elegant complexity of the ecosystem that the stem cell and its niche share.

ABBREVIATIONS AND ACRONYMS AKT Protein kinase B ALL Acute lymphoblastic leukemia AML Acute myeloid leukemia BM Bone marrow BMP Bone morphogenetic protein CAR cells Cxcl12 abundant reticular cells CD40L CD40 ligand CIC Cancer initiating cell CML Chronic myelogenous leukemia Col2.3 Type I collagen promoter Csfr1 Colony stimulating factor 1 receptor CXCL12 or SDF1 C-X-C motif chemokine 12 or stromal cell-derived factor 1 CXCR4 Chemokine (C-X-C Motif) receptor 4 ECM Extracellular matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor FGF Fibroblast growth factor

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G-CSF Granulocyte-colony stimulating factor Gfi-1 Growth factor independent 1 transcription repressor GM-CSF Granulocyte macrophage colony-stimulating factor Gs-alpha G protein subunit H3K27 Histone H3 lysine 27 HSC Hematopoietic stem cell IGF Insulin-like growth factor IL Interleukin ISC Intestinal stem cell ITGA4 Integrin, alpha 4 JAK2 Janus kinase 2 LepR Leptin receptor LIC Leukemia initiating cell LPS Lipopolysaccharide Lrg5 Leucine-rich repeat containing G protein-coupled receptor 5 MPD Myeloproliferative disorders Mx1 Myxovirus (influenza) resistance 1 NG2 Neural/glial antigen 2 PI3K Phosphoinositide 3-kinase pO2 Partial pressure of oxygen Prx1 Paired related homeobox 1 PTH Parathyroid hormone PTHR Parathyroid hormone 1 receptor RARgamma Retinoic acid receptor, gamma RNA Ribonucleic acid SCF Stem cell factor SLAM Signaling lymphocytic activation molecule Sox9 SRY (sex determining region Y)-box 9 TGF-b Transforming growth factor beta Tie2 Angiopoietin-1 receptor TLR Toll-like receptor TPO Thrombopoietin VCAM1 Vascular cell adhesion molecule 1 VEGF Vascular endothelial growth factor WT Wild Type

Glossary Cancer initiating cell A subset of cells within a cancer that are selfrenewing and can produce more differentiated progeny. They are defined experimentally and are thought to be the basis for metastasis, and perhaps disease relapse after therapy. Differentiation The process by which cells acquire more specialized functions and characteristics. It is how stem cells give rise to specialized cells of a given tissue. Endosteal niche Microenvironment within the bone marrow that is located adjacent to calcified bone or bony trabeculae and affects hematopoiesis. Mesenchymal stromal cells Stromal cells without the features of specialized mesenchymal cells such as cartilage cells (chondrocytes), bone cells (osteoblasts), or fat cells (adipocytes). Paneth cells Secretory cells present in contact with Lrg5þ intestinal stem cells that constitute their niche. Perivascular or vascular niche Microenvironment at a distance from the endosteal surface but near blood vessels where hematopoiesis is regulated. Progenitor Cells with limited self-renewal potential, but which are generally rapidly dividing and can differentiate to terminally differentiated cells. Self-renewal The process by which a stem cell divides giving rise to daughter cells with identical properties to the mother cell. Selfrenewal divisions can be symmetrical or asymmetrical. Stem cell A cell with the dual ability to self-renew, giving rise to more stem cells, and to differentiate into cells with more restricted characteristics.

Stem cell niche or microenvironment Specialized microanatomical locations where tissue stem cells reside conveying and receiving signals for their maintenance and function. Tissue stem cell or adult stem cell Tissue resident stem cells with potency limited to those cells of the tissue where they reside. These cells serve to create, maintain, and repair the tissue throughout life.

References 1. Zon LI. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature 2008;453(7193):306e13. 2. Orford KW, Scadden DT. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat Rev Genet 2008; 9(2):115e28. 3. Nombela-Arrieta C, Pivarnik G, Winkel B, Canty KJ, Harley B, Mahoney JE, et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat Cell Biol 2013;15(5):533e43. 4. Suda T, Takubo K, Semenza GL. Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell 2011;9(4): 298e310. 5. Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH, Cullen DE, et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 2007; 128(2):325e39. 6. Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, Miyamoto K, et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med 2006;12(4):446e51. 7. Trowbridge JJ, Snow JW, Kim J, Orkin SH. DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell 2009;5(4):442e9. 8. Challen GA, Sun D, Jeong M, Luo M, Jelinek J, Berg JS, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012;44(1):23e31. 9. Lechman ER, Gentner B, van Galen P, Giustacchini A, Saini M, Boccalatte FE, et al. Attenuation of miR-126 activity expands HSC in vivo without exhaustion. Cell Stem Cell 2012;11(6):799e811. 10. Yildirim E, Kirby JE, Brown DE, Mercier FE, Sadreyev RI, Scadden DT, et al. Xist RNA is a potent suppressor of hematologic cancer in mice. Cell 2013;152(4):727e42. 11. Simsek T, Kocabas F, Zheng J, Deberardinis RJ, Mahmoud AI, Olson EN, et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell 2010;7(3):380e90. 12. Gurumurthy S, Xie SZ, Alagesan B, Kim J, Yusuf RZ, Saez B, et al. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 2010;468(7324):659e63. 13. Scadden DT. The stem-cell niche as an entity of action. Nature 2006;441(7097):1075e9. 14. Schofield R. The relationship between the spleen colonyforming cell and the haemopoietic stem cell. Blood Cells 1978; 4(1e2):7e25. 15. Xie T, Spradling AC. Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 1998;94(2):251e60. 16. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature 2014;505(7483):327e34. 17. Fuchs E. Finding one’s niche in the skin. Cell Stem Cell 2009;4(6): 499e502. 18. Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 2014;15(1): 19e33. 19. van Es JH, Sato T, van de Wetering M, Lyubimova A, Nee AN, Gregorieff A, et al. Dll1þ secretory progenitor cells revert to stem cells upon crypt damage. Nat Cell Biol 2012;14(10):1099e104.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

27

REFERENCES

20. Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 2011;469(7330):415e8. 21. Conover JC, Notti RQ. The neural stem cell niche. Cell Tissue Res 2008;331(1):211e24. 22. Miller FD, Gauthier-Fisher A. Home at last: neural stem cell niches defined. Cell Stem Cell 2009;4(6):507e10. 23. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425(6960):841e6. 24. Ding L, Saunders TL, Enikolopov G, Morrison SJ. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 2012;481(7382):457e62. 25. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466(7308):829e34. 26. Greenbaum A, Hsu YM, Day RB, Schuettpelz LG, Christopher MJ, Borgerding JN, et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 2013; 495(7440):227e30. 27. Katayama Y, Battista M, Kao WM, Hidalgo A, Peired AJ, Thomas SA, et al. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 2006;124(2):407e21. 28. Yamazaki S, Ema H, Karlsson G, Yamaguchi T, Miyoshi H, Shioda S, et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 2011; 147(5):1146e58. 29. Casanova-Acebes M, Pitaval C, Weiss LA, Nombela-Arrieta C, Chevre R, A-Gonza´lez N, et al. Rhythmic modulation of the hematopoietic niche through neutrophil clearance. Cell 2013;153(5): 1025e35. 30. Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood 2010;116(23):4815e28. 31. Kacena MA, Gundberg CM, Horowitz MC. A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells. Bone 2006;39(5):978e84. 32. Fujisaki J, Wu J, Carlson AL, Silberstein L, Putheti P, Larocca R, et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature 2011;474(7350):216e9. 33. Lymperi S, Ersek A, Ferraro F, Dazzi F, Horwood NJ. Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo. Blood 2011;117(5):1540e9. 34. Naveiras O, Nardi V, Wenzel PL, Hauschka PV, Fahey F, Daley GQ. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature 2009;460(7252):259e63. 35. Gong JK. Endosteal marrow: a rich source of hematopoietic stem cells. Science 1978;199(4336):1443e5. 36. Lord BI, Testa NG, Hendry JH. The relative spatial distributions of CFUs and CFUc in the normal mouse femur. Blood 1975;46(1): 65e72. 37. Felix R, Elford PR, Stoerckle C, Cecchini M, Wetterwald A, Trechsel U, et al. Production of hemopoietic growth factors by bone tissue and bone cells in culture. J Bone Miner Res 1988;3(1): 27e36. 38. Horowitz MC, Einhorn TA, Philbrick W, Jilka RL. Functional and molecular changes in colony stimulating factor secretion by osteoblasts. Connect Tissue Res 1989;20(1e4):159e68. 39. Ishimi Y, Miyaura C, Jin CH, Akatsu T, Abe E, Nakamura Y, et al. IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 1990;145(10):3297e303. 40. Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, et al. Osteoblasts synthesize and respond to

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59. 60.

transforming growth factor-type beta (TGF-beta) in vitro. J Cell Biol 1987;105(1):457e63. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425(6960):836e41. Adams GB, Martin RP, Alley IR, Chabner KT, Cohen KS, Calvi LM, et al. Therapeutic targeting of a stem cell niche. Nat Biotechnol 2007;25(2):238e43. Visnjic D, Kalajzic Z, Rowe DW, Katavic V, Lorenzo J, Aguila HL. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 2004;103(9):3258e64. Yoshihara H, Arai F, Hosokawa K, Hagiwara T, Takubo K, Nakamura Y, et al. Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell Stem Cell 2007;1(6):685e97. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118(2):149e61. Fleming HE, Janzen V, Lo Celso C, Guo J, Leahy KM, Kronenberg HM, et al. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2008;2(3):274e83. Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, Grunewald E, et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med 2005;201(11): 1781e91. Kunisaki Y, Bruns I, Scheiermann C, Ahmed J, Pinho S, Zhang D, et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 2013;502(7473):637e43. Acar M, Kocherlakota KS, Murphy MM, Peyer JG, Oguro H, Inra CN, et al. Deep imaging of bone marrow shows nondividing stem cells are mainly perisinusoidal. Nature 2015; 526(7571):126e30. Yu VW, Saez B, Cook C, Lotinun S, Pardo-Saganta A, Wang YH, et al. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. J Exp Med 2015;212(5):759e74. Kiel MJ, Radice GL, Morrison SJ. Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell Stem Cell 2007;1(2):204e17. Lymperi S, Horwood N, Marley S, Gordon MY, Cope AP, Dazzi F. Strontium can increase some osteoblasts without increasing hematopoietic stem cells. Blood 2008;111(3):1173e81. Ding L, Morrison SJ. Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature 2013;495(7440):231e5. Ferraro F, Lymperi S, Mendez-Ferrer S, Saez B, Spencer JA, Yeap BY, et al. Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Sci Transl Med 2011;3(104):104ra1. Cairnie AB, Lamerton LF, Steel GG. Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic parameters. Exp Cell Res 1965;39(2):528e38. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. III. Entero-endocrine cells. Am J Anat 1974;141(4):503e19. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;449(7165):1003e7. Ritsma L, Ellenbroek SI, Zomer A, Snippert HJ, de Sauvage FJ, Simons BD, et al. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 2014; 507(7492):362e5. Sato T, Clevers H. SnapShot: growing organoids from stem cells. Cell 2015;161(7):1700-e1. Garabedian EM, Roberts LJ, McNevin MS, Gordon JI. Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice. J Biol Chem 1997;272(38):23729e40.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

28

2. STEM CELL NICHE IN MAMMALS

61. Akcora D, Huynh D, Lightowler S, Germann M, Robine S, de May JR, et al. The CSF-1 receptor fashions the intestinal stem cell niche. Stem Cell Res 2013;10(2):203e12. 62. Durand A, Donahue B, Peignon G, Letourneur F, Cagnard N, Slomianny C, et al. Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1). Proc Natl Acad Sci USA 2012;109(23):8965e70. 63. Kim TH, Escudero S, Shivdasani RA. Intact function of Lgr5 receptor-expressing intestinal stem cells in the absence of Paneth cells. Proc Natl Acad Sci USA 2012;109(10):3932e7. 64. Farin HF, Van Es JH, Clevers H. Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 2012;143(6):1518e1529.e7. 65. Kabiri Z, Greicius G, Madan B, Biechele S, Zhong Z, Zaribafzadeh H, et al. Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts. Development 2014; 141(11):2206e15. 66. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121(7):1109e21. 67. Park D, Spencer JA, Koh BI, Kobayashi T, Fujisaki J, Clemens TL, et al. Endogenous bone marrow MSCs are dynamic, faterestricted participants in bone maintenance and regeneration. Cell Stem Cell 2012;10(3):259e72. 68. Zhou BO, Yue R, Murphy MM, Peyer JG, Morrison SJ. Leptinreceptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 2014;15(2):154e68. 69. Sugiyama T, Kohara H, Noda M, Nagasawa T. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 2006; 25(6):977e88. 70. Omatsu Y, Sugiyama T, Kohara H, Kondoh G, Fujii N, Kohno K, et al. The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche. Immunity 2010;33(3):387e99. 71. Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 2002;33(2):77e80. 72. Inra CN, Zhou BO, Acar M, Murphy MM, Richardson J, Zhao Z, et al. A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature November 26, 2015;527(7579):466e71. 73. Kfoury Y, Scadden DT. Mesenchymal cell contributions to the stem cell niche. Cell Stem Cell 2015;16(3):239e53. 74. Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. J Biochem 2010;147(1): 35e51. 75. Kosinski C, Li VS, Chan AS, Zhang J, Ho C, Tsui WY, et al. Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl Acad Sci USA 2007;104(39):15418e23. 76. van Dop WA, Uhmann A, Wijgerde M, Sleddens-Linkels E, Heijmans J, Offerhaus GJ, et al. Depletion of the colonic epithelial precursor cell compartment upon conditional activation of the hedgehog pathway. Gastroenterology 2009;136(7):2195e2203 e1e7. 77. Kosinski C, Stange DE, Xu C, Chan AS, Ho C, Yuen ST, et al. Indian hedgehog regulates intestinal stem cell fate through epithelialemesenchymal interactions during development. Gastroenterology 2010;139(3):893e903. 78. Clayton E, Doupe DP, Klein AM, Winton DJ, Simons BD, Jones PH. A single type of progenitor cell maintains normal epidermis. Nature 2007;446(7132):185e9. 79. Mascre G, Dekoninck S, Drogat B, Youssef KK, Brohee S, Sotiropoulou PA, et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 2012;489(7415):257e62.

80. Sadagurski M, Yakar S, Weingarten G, Holzenberger M, Rhodes CJ, Breitkreutz D, et al. Insulin-like growth factor 1 receptor signaling regulates skin development and inhibits skin keratinocyte differentiation. Mol Cell Biol 2006;26(7):2675e87. 81. Guo L, Yu QC, Fuchs E. Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice. EMBO J 1993;12(3):973e86. 82. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 1977; 265(5593):421e4. 83. Rompolas P, Mesa KR, Greco V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 2013;502(7472): 513e8. 84. Greco V, Chen T, Rendl M, Schober M, Pasolli HA, Stokes N, et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 2009;4(2):155e69. 85. Plikus MV, Mayer JA, de la Cruz D, Baker RE, Maini PK, Maxson R, et al. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature 2008;451(7176): 340e4. 86. Rompolas P, Deschene ER, Zito G, Gonzalez DG, Saotome I, Haberman AM, et al. Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 2012;487(7408):496e9. 87. Yao L, Yokota T, Xia L, Kincade PW, McEver RP. Bone marrow dysfunction in mice lacking the cytokine receptor gp130 in endothelial cells. Blood 2005;106(13):4093e101. 88. Butler JM, Nolan DJ, Vertes EL, Varnum-Finney B, Kobayashi H, Hooper AT, et al. Endothelial cells are essential for the selfrenewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 2010;6(3):251e64. 89. Poulos MG, Guo P, Kofler NM, Pinho S, Gutkin MC, Tikhonova A, et al. Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis. Cell Rep 2013;4(5):1022e34. 90. Rosen CJ, Ackert-Bicknell C, Rodriguez JP, Pino AM. Marrow fat and the bone microenvironment: developmental, functional, and pathological implications. Crit Rev Eukaryot Gene Expr 2009;19(2): 109e24. 91. Wang LD, Wagers AJ. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat Rev Mol Cell Biol 2011;12(10):643e55. 92. Cheshier SH, Prohaska SS, Weissman IL. The effect of bleeding on hematopoietic stem cell cycling and self-renewal. Stem Cells Dev 2007;16(5):707e17. 93. Chow A, Lucas D, Hidalgo A, Mendez-Ferrer S, Hashimoto D, Scheiermann C, et al. Bone marrow CD169þ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med 2011;208(2):261e71. 94. Bruns I, Lucas D, Pinho S, Ahmed J, Lambert MP, Kunisaki Y, et al. Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion. Nat Med 2014;20(11):1315e20. 95. Zhao M, Perry JM, Marshall H, Venkatraman A, Qian P, He XC, et al. Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells. Nat Med 2014;20(11):1321e6. 96. Watt FM, Green H. Stratification and terminal differentiation of cultured epidermal cells. Nature 1982;295(5848):434e6. 97. Lechler T, Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 2005; 437(7056):275e80. 98. Ezratty EJ, Stokes N, Chai S, Shah AS, Williams SE, Fuchs E. A role for the primary cilium in Notch signaling and epidermal differentiation during skin development. Cell 2011;145(7):1129e41. 99. Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc Natl Acad Sci USA 2007;104(13):5431e6.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

REFERENCES

100. Spencer JA, Ferraro F, Roussakis E, Klein A, Wu J, Runnels JM, et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 2014;508(7495):269e73. 101. Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 2006;439(7076):599e603. 102. Lam BS, Cunningham C, Adams GB. Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow. Blood 2011;117(4):1167e75. 103. Holst J, Watson S, Lord MS, Eamegdool SS, Bax DV, NivisonSmith LB, et al. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat Biotechnol 2010;28(10):1123e8. 104. Adamo L, Naveiras O, Wenzel PL, McKinney-Freeman S, Mack PJ, Gracia-Sancho J, et al. Biomechanical forces promote embryonic haematopoiesis. Nature 2009;459(7250):1131e5. 105. Shin JW, Buxboim A, Spinler KR, Swift J, Christian DA, Hunter CA, et al. Contractile forces sustain and polarize hematopoiesis from stem and progenitor cells. Cell Stem Cell 2014;14(1): 81e93. 106. Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell 1995;80(1):83e93. 107. Hsu YC, Li L, Fuchs E. Emerging interactions between skin stem cells and their niches. Nat Med 2014;20(8):847e56. 108. Mendez-Ferrer S, Lucas D, Battista M, Frenette PS. Haematopoietic stem cell release is regulated by circadian oscillations. Nature 2008;452(7186):442e7. 109. Heidt T, Sager HB, Courties G, Dutta P, Iwamoto Y, Zaltsman A, et al. Chronic variable stress activates hematopoietic stem cells. Nat Med 2014;20(7):754e8. 110. Janich P, Pascual G, Merlos-Suarez A, Batlle E, Ripperger J, Albrecht U, et al. The circadian molecular clock creates epidermal stem cell heterogeneity. Nature 2011;480(7376):209e14. 111. Marcheva B, Ramsey KM, Buhr ED, Kobayashi Y, Su H, Ko CH, et al. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes. Nature 2010;466(7306):627e31. 112. Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheral tissue circadian clock. Proc Natl Acad Sci USA 2008; 105(39):15172e7. 113. Storch KF, Paz C, Signorovitch J, Raviola E, Pawlyk B, Li T, et al. Intrinsic circadian clock of the mammalian retina: importance for retinal processing of visual information. Cell 2007;130(4):730e41. 114. Burns ER, Scheving LE, Fawcett DF, Gibbs WM, Galatzan RE. Circadian influence on the frequency of labeled mitoses method in the stratified squamous epithelium of the mouse esophagus and tongue. Anat Rec 1976;184(3):265e73. 115. Chow A, Huggins M, Ahmed J, Hashimoto D, Lucas D, Kunisaki Y, et al. CD169(þ) macrophages provide a niche promoting erythropoiesis under homeostasis and stress. Nat Med 2013; 19(4):429e36. 116. Adams GB, Chabner KT, Foxall RB, Weibrecht KW, Rodrigues NP, Dombkowski D, et al. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood 2003;101(1): 45e51. 117. Ash RC, Horowitz MM, Gale RP, van Bekkum DW, Casper JT, Gordon-Smith EC, et al. Bone marrow transplantation from related donors other than HLA-identical siblings: effect of T cell depletion. Bone Marrow Transplant 1991;7(6):443e52. 118. Schurch CM, Riether C, Ochsenbein AF. Cytotoxic CD8þ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells. Cell Stem Cell 2014;14(4):460e72. 119. Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duchosal MA, et al. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature 2009;458(7240):904e8.

29

120. Baldridge MT, King KY, Boles NC, Weksberg DC, Goodell MA. Quiescent haematopoietic stem cells are activated by IFNgamma in response to chronic infection. Nature 2010;465(7299): 793e7. 121. Boettcher S, Gerosa RC, Radpour R, Bauer J, Ampenberger F, Heikenwalder M, et al. Endothelial cells translate pathogen signals into G-CSF-driven emergency granulopoiesis. Blood 2014; 124(9):1393e403. 122. Burberry A, Zeng MY, Ding L, Wicks I, Inohara N, Morrison SJ, et al. Infection mobilizes hematopoietic stem cells through cooperative NOD-like receptor and Toll-like receptor signaling. Cell Host Microbe 2014;15(6):779e91. 123. Sommer F, Backhed F. The gut microbiota e masters of host development and physiology. Nat Rev Microbiol 2013;11(4): 227e38. 124. Zhang D, Chen G, Manwani D, Mortha A, Xu C, Faith JJ, et al. Neutrophil ageing is regulated by the microbiome. Nature 2015; 525(7570):528e32. 125. Luo Y, Chen GL, Hannemann N, Ipseiz N, Kronke G, Bauerle T, et al. Microbiota from obese mice regulate hematopoietic stem cell differentiation by altering the bone niche. Cell Metab 2015; 22(5):886e94. 126. Yilmaz OH, Katajisto P, Lamming DW, Gultekin Y, BauerRowe KE, Sengupta S, et al. mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake. Nature 2012; 486(7404):490e5. 127. Joshi PA, Jackson HW, Beristain AG, Di Grappa MA, Mote PA, Clarke CL, et al. Progesterone induces adult mammary stem cell expansion. Nature 2010;465(7299):803e7. 128. Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, et al. Control of mammary stem cell function by steroid hormone signalling. Nature 2010;465(7299):798e802. 129. Nakada D, Oguro H, Levi BP, Ryan N, Kitano A, Saitoh Y, et al. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature 2014;505(7484):555e8. 130. Li JY, Adams J, Calvi LM, Lane TF, Weitzmann MN, Pacifici R. Ovariectomy expands murine short-term hemopoietic stem cell function through T cell expressed CD40L and Wnt10B. Blood 2013;122(14):2346e57. 131. Qian H, Buza-Vidas N, Hyland CD, Jensen CT, Antonchuk J, Mansson R, et al. Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells. Cell Stem Cell 2007; 1(6):671e84. 132. Goldstein J, Fletcher S, Roth E, Wu C, Chun A, Horsley V. Calcineurin/Nfatc1 signaling links skin stem cell quiescence to hormonal signaling during pregnancy and lactation. Genes Dev 2014;28(9):983e94. 133. Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, et al. Clonal dynamics of native haematopoiesis. Nature 2014; 514(7522):322e7. 134. Zhu J, Garrett R, Jung Y, Zhang Y, Kim N, Wang J, et al. Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells. Blood 2007;109(9):3706e12. 135. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, et al. Defects of B-cell lymphopoiesis and bonemarrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996;382(6592):635e8. 136. Wu JY, Purton LE, Rodda SJ, Chen M, Weinstein LS, McMahon AP, et al. Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways. Proc Natl Acad Sci USA 2008;105(44):16976e81. 137. Mercier FE, Ragu C, Scadden DT. The bone marrow at the crossroads of blood and immunity. Nat Rev Immunol 2012;12(1):49e60. 138. Eash KJ, Greenbaum AM, Gopalan PK, Link DC. CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J Clin Invest 2010;120(7):2423e31.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

30

2. STEM CELL NICHE IN MAMMALS

139. Kohler A, De Filippo K, Hasenberg M, van den Brandt C, Nye E, Hosking MP, et al. G-CSF-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands. Blood 2011;117(16):4349e57. 140. Pardo-Saganta A, Tata PR, Law BM, Saez B, Chow R, Prabhu M, et al. Parent stem cells can serve as niches for their daughter cells. Nature 2015;523(7562):597e601. 141. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, CaceresCortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994;367(6464):645e8. 142. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3(7):730e7. 143. Kikushige Y, Ishikawa F, Miyamoto T, Shima T, Urata S, Yoshimoto G, et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell 2011;20(2):246e59. 144. Michor F, Hughes TP, Iwasa Y, Branford S, Shah NP, Sawyers CL, et al. Dynamics of chronic myeloid leukaemia. Nature 2005; 435(7046):1267e70. 145. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007;1(5):555e67. 146. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature 2004; 432(7015):396e401. 147. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, et al. Significance of CD90þ cancer stem cells in human liver cancer. Cancer Cell 2008;13(2):153e66. 148. O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445(7123):106e10. 149. Passegue E. Hematopoietic stem cells, leukemic stem cells and chronic myelogenous leukemia. Cell Cycle 2005;4(2):266e8. 150. Savona M, Talpaz M. Getting to the stem of chronic myeloid leukaemia. Nat Rev Cancer 2008;8(5):341e50. 151. Krause DS, Fulzele K, Catic A, Sun CC, Dombkowski D, Hurley MP, et al. Differential regulation of myeloid leukemias by the bone marrow microenvironment. Nat Med 2013;19(11): 1513e7. 152. Colmone A, Amorim M, Pontier AL, Wang S, Jablonski E, Sipkins DA. Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells. Science 2008;322(5909):1861e5. 153. Sipkins DA, Wei X, Wu JW, Runnels JM, Cote D, Means TK, et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 2005;435(7044):969e73. 154. Saito Y, Uchida N, Tanaka S, Suzuki N, Tomizawa-Murasawa M, Sone A, et al. Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML. Nat Biotechnol 2010; 28(3):275e80. 155. Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, et al. Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell 2013;13(3):285e99. 156. Arranz L, Sanchez-Aguilera A, Martin-Perez D, Isern J, Langa X, Tzankov A, et al. Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. Nature 2014;512(7512): 78e81. 157. Hanoun M, Zhang D, Mizoguchi T, Pinho S, Pierce H, Kunisaki Y, et al. Acute myelogenous leukemia-induced sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell niche. Cell Stem Cell 2014;15(3):365e75. 158. Matsunaga T, Takemoto N, Sato T, Takimoto R, Tanaka I, Fujimi A, et al. Interaction between leukemic-cell VLA-4 and stromal

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

173.

174.

175.

176.

fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat Med 2003;9(9):1158e65. Tabe Y, Jin L, Tsutsumi-Ishii Y, Xu Y, McQueen T, Priebe W, et al. Activation of integrin-linked kinase is a critical prosurvival pathway induced in leukemic cells by bone marrow-derived stromal cells. Cancer Res 2007;67(2):684e94. Krause DS, Lazarides K, von Andrian UH, Van Etten RA. Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. Nat Med 2006;12(10):1175e80. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med 2006;12(10):1167e74. Kato I, Niwa A, Heike T, Fujino H, Saito MK, Umeda K, et al. Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis. PLoS One 2011;6(11): e27042. Tavor S, Petit I, Porozov S, Avigdor A, Dar A, Leider-Trejo L, et al. CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice. Cancer Res 2004;64(8):2817e24. Agarwal A, Fleischman AG, Petersen CL, MacKenzie R, Luty S, Loriaux M, et al. Effects of plerixafor in combination with BCRABL kinase inhibition in a murine model of CML. Blood 2012; 120(13):2658e68. Wei J, Wunderlich M, Fox C, Alvarez S, Cigudosa JC, Wilhelm JS, et al. Microenvironment determines lineage fate in a human model of MLL-AF9 leukemia. Cancer Cell 2008;13(6):483e95. Laperrousaz B, Jeanpierre S, Sagorny K, Voeltzel T, Ramas S, Kaniewski B, et al. Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and BMPRIb overexpression. Blood November 28, 2013;122(23):3767e77. Sala-Torra O, Hanna C, Loken MR, Flowers ME, Maris M, Ladne PA, et al. Evidence of donor-derived hematologic malignancies after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2006;12(5):511e7. Santamaria C, Muntion S, Roson B, Blanco B, Lopez-Villar O, Carrancio S, et al. Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients. Haematologica 2012;97(8): 1218e24. Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, et al. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature 2010;464(7290):852e7. Walkley CR, Olsen GH, Dworkin S, Fabb SA, Swann J, McArthur GA, et al. A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell 2007;129(6):1097e110. Junttila MR, de Sauvage FJ. Influence of tumour microenvironment heterogeneity on therapeutic response. Nature 2013;501(7467):346e54. Chapellier M, Bachelard-Cascales E, Schmidt X, Clement F, Treilleux I, Delay E, et al. Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response. Stem Cell Rep 2015;4(2):239e54. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B, et al. A perivascular niche for brain tumor stem cells. Cancer Cell 2007;11(1):69e82. Ezhilarasan R, Mohanam I, Govindarajan K, Mohanam S. Glioma cells suppress hypoxia-induced endothelial cell apoptosis and promote the angiogenic process. Int J Oncol 2007;30(3):701e7. Vermeulen L, De Sousa EMF, van der Heijden M, Cameron K, de Jong JH, Borovski T, et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol 2010; 12(5):468e76. Paget S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev 1989;8(2):98e101.

I. BIOLOGY OF STEM CELL NICHES AND MOLECULAR MECHANISMS

REFERENCES

177. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature 2015;527(7578):329e35. 178. Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, Huang WC, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 2015;527(7576): 100e4. 179. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 2014;507(7492):323e8.

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180. Ramasamy SK, Kusumbe AP, Wang L, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 2014;507(7492):376e80. 181. Xie H, Cui Z, Wang L, Xia Z, Hu Y, Xian L, et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nat Med 2014;20(11):1270e8. 182. Kfoury Y, Mercier F, Scadden DT. SnapShot: the hematopoietic stem cell niche. Cell 2014;158(1):228-e1. 183. Scadden DT. Nice neighborhood: emerging concepts of the stem cell niche. Cell 2014;157(1):41e50.

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3 Pluripotent Stem Cell Microenvironment Mio Nakanishi, Mickie Bhatia McMaster University, Hamilton, ON, Canada

O U T L I N E 1. Introduction 2. Pluripotent Cell Microenvironment in the Early Mammalian Embryo 2.1 Lineage Specification in the Preimplantation Embryo 2.2 Role of Extraembryonic Cells in Development of the Pluripotent Cells 2.2.1 Interactions Between Inner and Outer Blastomeres During the ICM Versus TE Specification and Associated Signals 2.2.2 Cellular Interplay Between the PrE and the Epiblast 3. Microenvironment of Pluripotent Stem Cells In Vitro 3.1 Derivation of Embryonic Stem Cells

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3.2 Cellular Heterogeneity in Mouse ESC Cultures 3.3 Epigenetic Mechanisms Underlying Metastable States of Mouse ESCs 3.4 Heterogeneity of Human ESC Culture 3.5 Functional and Epigenetic Distinctions Between Human ESC Subfractions 3.6 Regulatory Niche in Human ESC Culture

34 34 35

35 37

38 38

1. INTRODUCTION

40 41 42 44

4. Conclusion

46

Abbreviations and Acronyms

47

Glossary

47

Acknowledgment

47

References

47

potential. Furthermore, specific changes in the stem cell microenvironment is thought to be one of the critical events contributing to disordered stem cell homeostasis that can result in the emergence of neoplastic growth, underscoring the importance of understanding the stem cell-niche interactions and their mechanisms.2 Pluripotent cells are defined as cells that are able to give rise to all somatic tissues. Experimentally, pluripotency of the cells can be assessed through generation of the chimeric animals by injecting the cells into blastocysts. In vivo, epiblasts within the preimplantation embryo are considered to be pluripotent3 although the pluripotent state is very transient during embryogenesis and is distinct from the long-term multipotency of other somatic cells. Compared with other somatic stem cell microenvironments described since the late 1970s, the

The concept of the stem cell niche, defined as the tissue microenvironment that maintains and regulates stem cells, was initially proposed through studies in Drosophila germline stem cells1 and then in mammals using hematopoietic stem cells that reside in specific loci of bone marrow with support from multiple cellular components of their microenvironment. Since then, numerous observations of regulatory and supportive cellular interplays between somatic stem cells and their neighboring cells have been described in various tissues and organs including brain, muscle, intestine, and hair follicle. The support from these regulatory niches had been reported to be indispensable for somatic stem cells to maintain their self-renewal, as well as differentiation

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00003-2

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microenvironment of pluripotent cells has not been characterized until recently. This is largely due to the transient nature of pluripotent cells in vivo, and the characterization of their regulation being restricted to in vitro methods. Based on current knowledge, the relationship between pluripotent cells and their neighboring cells may be different from that between somatic stem cells and their niche. During the generation of pluripotent epiblasts within the preimplantation embryo, specification of extraembryonic lineages proceeds at the same time (described in Section 2.1). Therefore pluripotent cells appear to be mutually interacting with their neighboring cells that are dynamically changing in a short period of time relative to somatic stem cells. In fact, a list of studies revealed a critical role of the neighboring extraembryonic cells in epiblast development, indicating that cellular interplays between pluripotent cells and their neighbors are necessary for the normal development (described in Section 2.2). Derivation of preimplantation-embryo-derived stem cell lines (embryonic stem cells, ESCs) has enabled long-term maintenance of pluripotency in vitro. Strikingly, ESCs have shown significant heterogeneity and are composed of many interconvertible and interdependent subpopulations within the culture, which may represent cellular interactions between embryonic and extraembryonic lineages within early embryos (described in Section 3). In this chapter, we first describe the epiblast microenvironment in early embryogenesis, which provides robust information about cellular interactions required for the acquisition of pluripotent state. In the second half of this chapter, we will describe the transcriptional

and functional heterogeneities observed within established ESC cultures, followed by recent findings on the cellular interactions between autonomously generated ESC subfractions. These interactions have been shown to be essential for the self-renewal and maintenance of differentiation potentials of ESCs.

2. PLURIPOTENT CELL MICROENVIRONMENT IN THE EARLY MAMMALIAN EMBRYO 2.1 Lineage Specification in the Preimplantation Embryo During mammalian development, pluripotency is believed to be obtained through a formation of epiblast cells in the inner cell mass (ICM) of the blastocyst.3 After fertilization, mammalian embryos start their development with cleavage divisions generating a cell cluster known as morulae (Fig. 3.1). In mice, the first asymmetric division within the embryo can be observed as early as at the 8-cell stage with the onset of polarization of the outside cells. At this stage, these outside cells, precursors of extraembryonic trophectoderm (TE), obtain microvilli on the apical surface4 and become distinguishable from the inside cells, which are predominantly destined to be epiblasts.5,6 The first instances of cellular differentiation are thought to begin after the polarization events occur at the 8-cell stage although some recent experiments suggest that individual cells (blastomeres) at the 4-cell stage might have different biases toward TE or ICM (reviewed

FIGURE 3.1 Schematic representation of mouse preimplantation embryogenesis. The first cell fate choice between embryonic inner cell mass (ICM) and extraembryonic trophectoderm (TE) begins as late as the 16-cell stage with formation of polarized outer cells and apolar inner cells, which predominantly are fated to be TE and ICM, respectively. After generation of fluid-filled cavity called as blastocoel, the second wave of specification segregates the ICM into epiblast or primitive endoderm (PrE). The epiblast and PrE appear mixed when the blastocoel forms, followed by physical rearrangement to form two distinct layers. A monolayer of PrE on the blastocoelic surface of the ICM encloses the pluripotent epiblast in the mature blastocyst, which is ready to implant to the uterine wall. From Lanner F. Lineage specification in the early mouse embryo. Exp Cell Res 2014;321(1):32e9.

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in Ref. [7]). However, single-cell gene expression analysis revealed no distinguishing characteristics among individual cells at the 2-, 4-, or 8-cell stages,8 reinforcing the idea that differentiation occurs after these steps. Subsequent cell divisions result in the internalization of nonpolar cells, whereas the polar cells remain on the outside and develop into an epithelial layer of TE cells, the trophoblasts. Trophoblasts transfer fluid into the blastocyst to form a cavity, called blastocoel by the 32-cell stage [embryonic day 3.5 (E3.5) in mice]. After the cavitation, a second wave of specification segregates the ICM into two lineages: embryonic epiblast and extraembryonic PrE (also known as the hypoblast). The epiblast and PrE initially appear mixed as “salt and pepper” when the blastocyst cavity forms,9 followed by rearrangement in an actin-dependent process to form two distinct layers.5,10,11 In the mature blastocyst (by E4.5 in mice), PrE is morphologically distinguishable as an epithelium overlying the blastocoelic surface of the ICM. At this end, the blastocyst is ready to implant and then it adheres to the uterine wall after hatching out of a glycoprotein layer called zona pellucida. It is noteworthy that these lineage specifications in the preimplantation embryo occur progressively and the cells primed toward a specific lineage often can ultimately contribute to other lineages. For example, in the first lineage segregation between TE and ICM, the outer cells from 16-cell and even 32-cell stage embryos can contribute exclusively to the ICM lineages if incorporated with tetraploid cells (the cells made by electrofusing the cells at the 2-cell stage morula that can efficiently form the extraembryonic tissue but cannot contribute to the embryo). Indeed, a single blastomere of the 32-cell embryo can give rise to a whole E9.5 embryo,12 highlighting the plasticity of any lineage biases that have emerged by this stage. Correspondingly, single-cell transcriptome analyses demonstrate that the gene expression signature of individual blastomeres cannot be fully resolved into TE nor ICM lineages by the 32-cell stage, whereas essentially all cells are well resolved into one of the three cell types (TE, epiblast, or PrE) by the 64-cell stage.8 Moreover, many of the transcription factors that subsequently become cell-type restricted (e.g., Cdx2 and Gata3 in TE, Nanog and Klf2 in epiblast, Oct4 in epiblast and PrE, and Gata6 in PrE) are coexpressed at high levels in the majority of individual 16-cell blastomeres, and the expression levels are comparable to what are later found in the lineagerestricted individual cells at the 64-cell stage. These observations highlight regulatory nature of the early embryo and suggest that initial cell fate choices entail the downregulation of transcription factors specific to rival lineages, rather than the upregulation of appropriate factors for the respective lineages.

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During the transition from morulae to blastocysts, the epiblast microenvironment drastically changes along with dynamic morphogenesis of the embryos and cellular differentiation into the three distinct lineages (epiblasts, TE, and PrE). Although it has not been fully clarified how epiblast development is influenced by the preimplantation microenvironment of the embryo, there is increasing evidence showing a critical role for these cellular interactions within the embryo. In the following Section 2.2, we will describe the environmental cues provided within the microenvironment of pluripotent epiblast and its progenitor (the inner cell of ICM) largely by neighboring extraembryonic TE and PrE.

2.2 Role of Extraembryonic Cells in Development of the Pluripotent Cells 2.2.1 Interactions Between Inner and Outer Blastomeres During the ICM Versus TE Specification and Associated Signals One of the environmental cues affecting the epiblast’s development can be observed in the 16-cell stage morula. At this stage, inner blastomeres, which are predominantly fated to be epiblasts, are internalized by the polar outer cells that will generate trophoblasts at later stages. This inside environment within the morula appears to be essential for the inner blastomere to give rise to ICM as evidenced by embryo-manipulation experiments performed by Andrzej Tarkowski’s lab.13 In their studies, Tarkowski and his colleagues demonstrated that changing a cell’s position at cleavage stages can affect subsequent fate suggesting that instructive cues for epiblast commitment are provided in the inside microenvironment. Recent studies have described at least a part of the underlying molecular mechanisms mediating this positional information (Fig. 3.2). For example, a study using mouse embryos lacking both maternal and zygotic E-cadherin showed disrupted inner and outer cells organization due to failed E-cadherin-dependent polar epithelium formation, which ultimately caused abnormal inactivation of Hippo signaling in the entire embryo.14 In these mice, very few cells were observed to have exclusion of the Yap transcription factor from the nucleus (representing activated Hippo signaling), in contrast to normal embryos where Yap is translocalized to the nucleus only in the TE. Correspondingly, Cdx2, a key transcription factor of TE differentiation whose expression is dependent on Yap1,15 is upregulated in the larger population of the resulting aggregates, suggesting that generation of an organized outer epithelium ensures that inner cells are maintained in a stable environment to activate Hippo signaling and

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3. PSC MICROENVIRONMENT

(A) Inner cell / Inner Cell Mass

Outer cell / Trophectoderm P

Lats1/2

Lats1/2

Delta Notch

CDX2

NICD

NICD

Tead4 Yap

Yap

CDX2

Rbpj Tead4

P

HIPPO: ON NOTCH: OFF

HIPPO: OFF NOTCH: ON

(B) Epiblast

Primitive Endoderm

Nanog

Nanog Grb2 FGFR

Gata6

Erk

P Gata6

FGF4 FGF4

Dab2,Sox17,Gata4

FIGURE 3.2 Model for ICM/trophectoderm and epiblast/primitive endoderm interactions. (A) In the outer cells (progenitors of trophoblasts) within the 16-cell stage morula, Yap is not phosphorylated and can translocate into the nucleus, where it interacts with Tead4 and upregulates Cdx2 expression. Notch signaling is also responsible for the TE-specific expression of Cdx2 in cooperation with Yap/Tead4. In contrast, unknown cell-contact dependent factors promote activation of Lats kinase and Yap phosphorylation in the inner cells, which results in blockade of Cdx2 expression. (B) The ICM at the 64-cell stage blastocysts is a mixture of epiblast- and primitive endoderm (PrE) progenitors. In PrE progenitors at this stage, Gata6 potentiates the upregulation of FGFR2 that stimulates the Erk signaling pathway activated by FGF4. Increased Erk signaling leads to repression of Nanog and inhibits Gata6 repression. This feedback loop reinforces specification toward the PrE and enhances rearrangement of PrE progenitors probably through Gata6-target genes such as Laminin and Dab2. In epiblast progenitors, basal level of the Erk signaling activity allows Nanog expression, which results in FGF4 expression and Gata6 repression. Lines with arrowheads and lines with blocks indicate positive and negative regulation, respectively. (A) Adapted from Stephenson RO, Rossant J, Tam PP. Intercellular interactions, position, and polarity in establishing blastocyst cell lineages and embryonic axes. Cold Spring Harb Perspect Biol 2012;4(11); Rayon T, Menchero S, Nieto A, et al. Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the mouse blastocyst. Dev Cell 2014;30(4):410e22. (B) Adapted from Chazaud C, Yamanaka Y, Pawson T, Rossant J. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 2006;10(5):615e24; Schrode N, Saiz N, Di Talia S, Hadjantonakis AK. GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst. Dev Cell 2014;29(4):454e67; Frankenberg S, Gerbe F, Bessonnard S, et al. Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling. Dev Cell 2011;21(6):1005e13; Ohnishi Y, Huber W, Tsumura A, et al. Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages. Nat Cell Biol 2014;16(1):27e37.

block Cdx2 expression. A study described outer cellspecific activity of Notch signaling as another signaling pathway regulating TE-specific expression of Cdx2 in cooperation with Tead4/Yap.16 Interestingly, Notch does not only regulate Cdx2 expression but also can influence the fate of blastomeres by driving them to outer positions. The forced activation of the Notch pathway, achieved by overexpressing intracellular domain of Notch1, drives cells to relocate to the outside and adopt a TE fate. Although it remains controversial whether or not Notch1 directs the cell relocation through elevating Cdx2 expression,17,18 all of these findings exemplified the essential role of the cell position-dependent microenvironment to activate appropriate cellular signaling

(e.g., Notch activation and Hippo inactivation in TE), allowing for correct specification through lineagespecific transcription factor expression at the precise time and position. These findings also implicate a close association between cell polarity, cell position-dependent microenvironments, cell signaling activity, and cell fate; however, causal relationships between these factors remain to be resolved. The relocation of the blastomeres to outside positions by forced Notch activation demonstrates a possibility that the microenvironment does not definitively define the blastomere but can be adjusted by changing the cell position within the morula based on the cell signaling activities. This possible flexibility

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may result in the regulatory nature of early embryos. Future studies should resolve additional outstanding questions: what is the molecular cue provided within the inner cell’s microenvironment to activate Hippo signaling? How are Notch pathway components differentially localized (e.g., Notch ligands on the inner cells and receptors on the outer cells)? Furthermore, it remains unresolved which environmental cues are provided by the outer cells that permit the inner cells to commit to the pluripotent ICM fate. 2.2.2 Cellular Interplay Between the PrE and the Epiblast Unlike the first cell-fate decision between ICM and TE, the second wave of lineage specification of epiblast versus PrE seems to start in a stochastic manner rather than a positionally regulated manner (Fig. 3.1). Nanog and Gata6, key transcription factors to determine epiblast and PrE, respectively, begin to be expressed at the 8-cell stage with widespread co-expression in identical blastomeres until the 32-cell stage.11,19,20 Next, Gata6 and Nanog expression display mutually exclusive “salt and pepper” distribution at around the 64-cell stage. Then the Gata6-positive cells segregate from Nanog-positive ICM cells to localize adjacent to the cavity where they further undergo PrE specifications along with expression of other determinants including Sox7 and Pdgfra.21 Although the initial expression of key transcription factors begins stochastically, the epiblast and PrE precursors appear to require each other as their respective microenvironmental components supporting the specifications into each lineage (Fig. 3.2). This idea is evidenced by Nanog null mouse embryos in which the PrE is unexpectedly lacking although Nanog is not expressed by the PrE. Notably, Nanog null PrE cells can be rescued by the incorporation of wild-type blastomeres into Nanog null blastocysts, indicating that the PrE development is depending on support from the epiblast.22,23 Multiple studies have identified FGF4 as a paracrine factor secreted from epiblasts to allow for PrE specification.24 Embryo culture with inhibitors of FGF/Erk signaling result in blastocysts with excess Nanog þ epiblasts,3,25 by contrast, exogenous highdose FGF4 can block epiblast development.25 In mouse embryos lacking Grb2, an adaptor protein linking FGF receptors to the Erk pathway, ICM cell number is normal but all cells expressing epiblast markers including Nanog and Gata6 expression cannot be observed.9 Moreover, in FGF4 mutants, Gata6 expression can be initiated but cannot be maintained, resulting in the defect of PrE formation.11,20,21 All of these evidences indicate that the segregation of the two lineages is dependent on FGF/Erk signaling based on the reciprocal expression of FGF receptor in the PrE and FGF4

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in the epiblast.3 The derivation of mosaicism in FGF4 and FGF receptors remains unclear5,7,25; however, a study based on single-cell transcriptome analyses of blastomeres suggests that the initial transcriptional variability of individual epiblast and PrE determinants may be generated possibly stochastically and independently from one another. This is followed by the “signalreinforcement phase” in which a correlation of gene expression (e.g., Nanog/FGF4 co-expression in epiblast precursors and Gata6/FGFR co-expression in PrE precursors) emerges probably owing to the activation of lineage-specific FGF signaling.26 Collectively, these findings indicate the importance of cellular interplays via paracrine signaling in the segregation of two distinct precursor populations primed toward PrE or epiblast lineages. Given that Nanog expression in epiblast precursors is noncell autonomously required for the specification of the Gata6-positive PrE precursors probably through FGF signaling, do the PrE or its precursors also play an essential role in epiblast generation? Indeed, multiple mutant mice lacking PrE-specific genes do not only harbor abnormalities in PrE generation but also display severe defects in epiblast development resulting in lethality around implantation. For example, Gata6 null mouse embryos, lack PrE and its derivatives, show abnormal epiblast formation, and are lethal by E5.5 shortly after implantation.27 Gata6 null ES cells injected to wild-type blastocysts contribute mainly to the epiblast but cannot give rise to extraembryonic TE and PrE, resulting in a number of normal highly chimeric embryos. These observations validate that Gata6 is normally required in extraembryonic lineages and indicate an essential requirement of extraembryonic supportive tissues for epiblast development. Moreover, mouse embryos lacking signal transduction adapter protein Disabled-2 (Dab2), whose expression is first observed specifically in the PrE at E4.5, are also embryonic lethal earlier than E6.5 due to defects in the formation of PrE and its derivatives.28 Interestingly, in contrast to wildtype embryos in which the PrE cells relocate to the vicinity of blastocore cavity by E5.5, the PrE-like cells in these mutants remain scattered within the ICM even at this stage, emphasizing the importance of orderly PrE formation in normal epiblast development. These findings suggest an essential role of the neighboring PrE as essential supportive cellular components within epiblast’s microenvironment; however, the developmental stage at which the critical interaction between PrE (or its derivatives such as visceral endoderm) and epiblast take place is yet to be determined. Future work including further analyses of mutant embryos using fluorescentbased imaging techniques would further clarify the role of PrE in normal epiblast development besides a role of PrE-derivative in epiblast differentiation and

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embryonic axis patterning after implantation (reviewed by Rossant et al.29). Corresponding with the observations in mouse embryos, studies in other mammals demonstrated the Gata6 and Nanog expression patterns that initially overlap with each other within the ICM and then adopt mutually exclusive distributions.30e32 Surprisingly, these studies showed that modulation of FGF/Erk signaling did not clearly affect lineage commitment of ICMs in human and bovine blastocysts, sharply contrasting to mouse embryos. These findings suggest that speciesdependent discrepancies between rodents and primates exist even in a basic role of cell signaling mediating critical cell-fate decisions during early embryogenesis. Given restricted availability of human blastocysts due to significant ethical concerns, future work using human pluripotent stem cells (PSCs) in culture as a model of human early development may allow us to answer further questions about underlying mechanisms of the early lineage segregation. What is the signaling pathway responsible for the segregation between the PrE and epiblast in human embryos instead of FGF signaling in mice? How can the PrE support neighboring epiblast development? Does the PrE retain flexibility to convert to the epiblast lineage? Can such information allow efficient development of human embryos in in vitro fertilization (IVF)? In fact, as we will describe in the following sections, recent studies on human PSCs in vitro have revealed heterogeneity within PSC cultures representing early embryonic- and extraembryonic lineages. Cellular interactions between distinct PSC subfractions underscore the utility of PSC cultures as a model where we can observe interactions between pluripotent cells and their microenvironment within the embryo.

3. MICROENVIRONMENT OF PLURIPOTENT STEM CELLS IN VITRO 3.1 Derivation of Embryonic Stem Cells In parallel with the progress of mammal embryological studies described in the previous sections, extensive studies have been performed attempting to establish stem cell lines that can maintain pluripotent state for a prolonged time. Derivation of such PSC lines was expected to provide a unique resource for the functional analysis of early human development, as well as an unlimited source of any cell type in the body. After pioneering work to identify culturing conditions for pluripotent cells using embryonic carcinoma (EC) cells,33 ESCs were first established in 1981 by explanting mouse blastocysts in culture conditions using mitotically inactivated mouse embryonic fibroblasts (MEFs) as feeder cells.34,35 When transplanted into

adult mice, ESCs can form teratomas containing tissues resembling derivatives of three representative germ layers (ectoderm, mesoderm, and endoderm). The pluripotency of ESCs was further and conclusively revealed by blastocyst injection. By injecting ESCs into preimplantation embryos, the introduced ESCs were incorporated into normal development, resulting in chimeric mice with extensive contribution from the injected ESC progeny. Importantly, the ESC progenies are competent to be colonized to the germline (germline-transmission) that enables the generation of heterozygous offspring arising from the ESC-derived gamete of the chimeric animals. Using a tetraploid embryo complementation method whereby ESCs are introduced into tetraploid morulae to generate hybrid aggregation of the ESCs and tetraploid blastomeres, it is now possible to derive mice entirely composed of ESC derivatives that appear to be normal, viable, and fertile. ESCs thus show an unprecedented developmental capacity to contribute to a whole organism after prolonged culture in vitro.36,37 Primate ESCs were first derived from Rhesus macaque38 and then in 1998, the first human ESC lines were obtained using donated supernumerary embryos from IVF treatment.39 Although blastocyst injection and tetraploid embryo complementation, which are now considered to be gold standards in assessing pluripotency, are not applicable to the human cells because of ethical concerns, the established cell lines were designated as PSCs based on their derivation from the blastocysts and capability to produce teratomas after transplantation to adult mice. However, subsequent studies revealed numerous divergent properties of human ESCs compared with the mouse ESCs. For example, the human cells form larger flattened colonies compared with compact dome-shaped colonies of mouse ESCs. Human ESCs cannot be passaged as single cells and require addition of Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor40 or support from autologously produced fibroblast-like cells41 (see Section 3.6) for efficient cloning. Moreover, human ESCs do not respond functionally to the leukemia inhibitory factor (LIF)/STAT signaling pathway, which has a central role in the maintenance of mouse ESC pluripotency. Instead, human ESCs self-renew in response to FGF and activin/nodal signaling pathways in contrast to the mouse cells. These discrepancies had been commonly recognized as species-specific differences until other stem cell lines were established from postimplantation mouse epiblasts in 2007. These stem cell lines, namely postimplanation epiblast-derived stem cells (EpiSCs), prompted the alternative idea that human and mouse ESCs represent different developmental stages.42,43 Although EpiSCs can generate teratomas, they do not contribute

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efficiently to blastocyst chimeras and do not incorporate when aggregated with morulae. EpiSCs are distinct in gene expression and epigenetic states compared with mouse ESCs, showing reduced expression of genes expressed by ICM (e.g., Rex1 and Tbx3) and higher expression of epiblast- (Otx2) and PrE-associated genes (Gata6 and Cer1). EpiSCs and human ESCs share not only these gene expression and epigenetic signatures but also a number of common properties including two-dimensional and epithelial morphology of the colonies, low survival efficiency after passaging as single cells, and the failure of female-derived stem cells to reactivate the silenced X chromosome. Strikingly, unlike mouse ESCs, EpiSCs do not depend on the LIF/STAT signaling pathway to maintain the undifferentiated state and instead rely on basic FGF (bFGF) and the activin/ Nodal pathway that is sufficient for long-term maintenance of human ESCs. These properties support a widespread notion that human pluripotent cells maintained in the conventional culture conditions using bFGF are more similar to EpiSCs rather than mouse ESCs. These notions would raise a question whether the process of human PSC derivation from embryos (e.g., propagation in the explant culture) involve the equivalent of a transition into postimplantation state. In fact, a study describes a transient epiblast-like intermediate during human ESC derivation (4e5 days after blastocyst plating) that is morphologically and transcriptionally distinct from both ICM and established ESCs.44 The concept that human ESCs represent a later (postimplantation) developmental stage compared with mouse ESCs prompts an intriguing question as to whether the cellular heterogeneity observed more robustly in the postimplantation embryos can be observed in and be responsible for sustaining the self-renewing human ESC culture. Indeed, studies have shown that ESC cultures consist of multiple distinct and interconvertible subfractions which form autologous ESC microenvironments (described in following sections). Furthermore, human ESCs are reported to be more heterogeneous than mouse ESCs (described in Section 3.5) and differ markedly between cell lines showing different differentiation biases45,46 like EpiSCs.47

3.2 Cellular Heterogeneity in Mouse ESC Cultures Cellular heterogeneity in undifferentiated ESC cultures was first reported in mouse ESCs. In 2007, Austin Smith and his colleagues reported that the expression of Nanog, one of the transcription factors contributing to the core transcriptional circuits of PSCs, is fluctuating in the undifferentiated mouse ESC culture.48 Although downregulation of Nanog is transient, reversible, and appears to be a separable event from commitment to

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differentiation,48 the loss of Nanog compromises the self-sustaining feedback in the ESC regulatory network that can be followed by consolidation to committed fate decisions in the prolonged absence of Nanog.49 A series of subsequent studies revealed that the expression levels of other core elements of the pluripotency transcriptional circuits such as Oct4,50 Klf4,51 Rex1,52,53 Hes,54 Prdm14,55 pluripotency-associated genes (e.g., Stella56), and genes involved in early lineage specification (e.g., Hex52,57 and Sox1758) also fluctuate over time within mouse ESC cultures, implying multiple reversible states and distinct subpopulations of mouse ESCs that vary in their capacity for self-renewal and differentiation. Interestingly, some of these studies on mouse ESC heterogeneity reported the presence of mouse ESC subfractions, although interconverting, which are transcriptionally and functionally biased toward specific lineages within blastocysts such as PrE and epiblast (Fig. 3.3).52,57 In these studies, the authors separated the subfractions depending on the expression levels of key transcription factors visualized with fluorescent protein-based reporter systems. As a result, Toyooka et al. reported that the Rex1/Oct4þ mouse ESC subfraction showed higher expression levels of early differentiationassociated genes (e.g., Brachyury, Eomes) and poor ability to contribute to chimeric embryos in spite of efficient differentiation into somatic lineages in vitro as EpiSCs. In contrast, the Rex1þ/Oct4þ population showed higher ICM-specific genes (e.g., Tbx3) and Nanog expression levels, a predominant contribution to chimera formation, and an ability to differentiate into PrE, suggesting its ICM-like properties. This model of mouse ESC culture, consisting of two distinct subfractions possibly representing ICM and epiblast counterparts, was further developed by Canham et al.52,57 By using a reporter for another gene Hex, which is a marker for PrE and early definitive endoderm, they described that Hexþ/SSEA1þ/Oct4þ mouse ESCs appeared to be biased to the PrE lineage. This subpopulation representing PrE transcriptional signatures do not effectively contribute to the epiblast when introduced back into morulae or blastocysts, however, it can contribute to visceral and parietal endoderm derived from PrE. Moreover, the Hexþ population is significantly reduced by Nanog expression and inhibition of FGF signaling with a chemical inhibitor corresponding with the shift between the Nanog-positive ICM-like state and PrE regulation via FGF signaling. Another group independently reported a presence of the PrE-like subpopulation within the mouse ESC culture characterized by intense expression of Sox17.58 This subpopulation expresses the PrE marker proteins (e.g., Sox17, Dab2, Gata4), can exclusively contribute to the PrE and its derivatives (and not to somatic cells) when injected to blastocysts, and is diminished by homozygotic knockdown of Sox17 or Nanog

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Mouse ESC cultures ICM-like Nanog+ Rex1+ HexOct4+

Epi-like

?

NanogRex1HexOct4+

Epiblast

PrE-like NanogRex1Hex+ Oct4+

PrE

FIGURE 3.3 A model for the dynamic equilibrium within mouse ESC cultures. Multiple studies indicate that the self-renewing mouse embryonic stem cell (ESC) cultures can be considered as a dynamic equilibrium of distinct and interconverting subfractions, which may represent progenitors biased toward specific lineages within preimplantation embryos. At least three subpopulations have been suggested: ICM-like cells (Nanogþ Rex1þ Hex), cells primed for primitive endoderm (PrE) (Nanog Rex1 Hexþ), and epiblast (Epi)-like cells primed for somatic lineages. A hypothetical interconversion between PrE-like cells and Epi-like cells is indicated by the dashed line. Adapted from Lanner F, Rossant J. The role of FGF/Erk signaling in pluripotent cells. Development 2010;137(20):3351e60; Hayashi K, Lopes SM, Tang F, Surani MA. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 2008;3(4):391e401; Canham MA, Sharov AA, Ko MS, Brickman JM. Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript. PLoS Biol 2010;8(5):e1000379.

overexpression in part through a competitive occupation between Sox17 and Nanog for common DNA-binding sites. It is worthy to emphasize that all of the subfractions examined in these reports appear to be transient and interconvertible under self-renewing conditions, and distinguishable from stable and irreversible differentiation. For example, clonally isolated Rex1þ or Rex1 cells can give rise to the same proportion of Rex1þ cells after several weeks of culture, which are crucially different from EpiSCs that maintain their differentiation status consistently.52 Taken together, these results allow us to consider selfrenewing mouse ESC cultures as a dynamic equilibrium of multiple distinct and dynamically interconverting subfractions, which may represent intermediate progenitors biased toward specific lineages within blastocysts (e.g., epiblasts and PrE), rather than a stable culture of an uniform cell population. This idea is supported by the multiple studies described above evidencing at least two equivalently self-renewing and interconverting populations within the mouse ESC culture; this exhibits evidence of transcriptional and functional lineage priming toward either the epiblast or PrE. This idea raises a question whether or not the transcriptional heterogeneities observed in the mouse ESC cultures are capturing

those in embryos. As described previously, recent studies applying single-cell expression analysis to mouse morulae demonstrated considerably heterogenous expression of epiblast- (e.g., Sox2 and Nanog) and PrE markers (Gata4 and Gata6) observed as late as 32-cell ICM cells.8 Moreover, comparison of single-cell gene expression profiles between mouse ESCs and mechanically or immunosurgically isolated ICM, epiblasts, and PrE59 remarkably showed that Rex1 mouse ESCs were transcriptionally more close to PrE-derived tissue (visceral endoderm) isolated from E5.5 embryos in contrast to Rex1þ ESCs that are more similar to E5.5 epiblasts. These results confirm the idea that the heterogeneity of mouse ESC cultures reflects heterogeneity within ICM, which contains progenitors primed to specific lineages such as epiblast and PrE.

3.3 Epigenetic Mechanisms Underlying Metastable States of Mouse ESCs Although the fluctuating expression of key transcription factors such as Nanog seems to have a critical influence on the regulation of heterogeneity and dynamic equilibriums in mouse ESC culture, the underlying molecular mechanisms governing the transitions largely

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remains unclear. Recently, a series of studies have been done attempting to gain insights into these mechanisms. The intermittent expression of key pluripotent factor genes in ESC culture is thought to be reflected by the epigenetic states (e.g., histone modifications, gene silencing via microRNA, and DNA methylation) at the genomic loci of genes. Indeed, Azim Surani’s group56 identified Stella as a faithful marker of the ICM-like mouse ESC subfractions, where other previously reported markers (e.g., Rex1) are enriched, and then found that epigenetic modification at the Stella locus differs between Stella-positive and negative populations. For example, acetylated histone H3 lysine 9 (H3K9ac) and trimethylated histone H3 lysine 4 (H3K4me3), active gene markers, are relatively enriched in the Stella-positive ESCs whereas the level of repressive gene markers, trimethylated histone 3 lysine 27 (H2K27me3), and DNA methylation at the Stella locus did not show significant changes. Notably, mouse ESCs lacking either Dicer, which cleaves pre-microRNAs, or methyl-CpG-binding domain protein 3 (MBD3), a component of the nucleosome remodeling and histone deacetylation (NuRD) complex which preferentially binds to 5-hydroxymethyl cytosine (5-hmC),60 grow independently of LIF and fail to differentiate properly because of their inability to repress genetic programs associated with pluripotency.61e63 Recent studies revealed that the NuRD complex also modulates transcriptional heterogeneity and restricts the dynamic range of a number of pluripotency genes including Rex1, Tbx3, and Klf4 in mouse ESCs.53 In the MBD3 null ESCs, these genes showed excessive levels of expression in the self-renewing condition and misexpression in the absence of LIF, resulting in LIFindependent self-renewal and the differentiation defects. The transcriptional repression by the NuRD complex appears to be regulated by other signaling pathways such as FGF/Erk signaling. As described in Section 2.2.2, the FGF/Erk signaling is specifically activated in the PrE cells within preimplantation embryos. Correspondingly, Erk signaling is preferentially activated in the PrE-like subfractions (Hexþ/SSEA-1þ or Rex1-low populations) of mouse ESCs.53,57 Strikingly, FGF/Erk signaling inhibition by chemical inhibitors induces rapid and genomewide demethylation involving oxidation of 5-mC to 5hmC, suggesting a possibility that the transcriptional heterogeneities of the NuRD target genes reflect different DNA hydroxymethylation levels on these loci64 regulated by the Erk signaling pathway. The critical role of DNA methylation in regulating mouse ESC heterogeneity has been independently indicated by multiple whole-genome DNA methylome studies describing dynamic switching between two metastable transcriptional states reflecting DNA methylation changes.65,66 Using single-molecule RNA-FISH,

41

Michael B. Elowitz’s group clearly showed that the Rex1 expression level in each cell is positively and negatively correlated with Tet1- and Dnmt3b expression, respectively. Rex1-high and Rex1-low ESCs exhibited differential genome-wide promoter methylation levels whereby the highly methylated promoters shifted to even higher methylation levels in Rex1-low cells. Notably, Rex1-low cells that subsequently reverted to the Rex1-high state recovered the methylation levels of Rex1-high cells, supporting a view that the change in promoter methylation occurs during interconversion between the Rex1-high and Rex1-low states.

3.4 Heterogeneity of Human ESC Culture Numerous studies on the heterogeneity of PSC culture have been performed using mouse ESCs in part because of the ease of generation of knock-in or transgenic reporter lines, which allows efficient separation of the subfractions depending on the expression of key pluripotency-associated transcription factors. Nevertheless, reports revealed vast heterogeneity observed within human PSC cultures, in which equivalent genetic modifications are not possible.67 For example, a study using single-cell transcriptional profiling techniques reported remarkable heterogeneity in transcript levels even among pluripotent marker-positive (Tra-1-60þ/ SSEA4þ) human PSCs. In this study, human PSCs at the periphery of colonies were reported to exhibit lower levels of pluripotency-related transcripts although another group observed opposite spatial expression patterns of identical pluripotency-associated genes (e.g., Oct4 and Sox2 enriched in the periphery) through immunofluorescence staining of human ESCs in micropatterned culture.68 Regardless of why the results were different, these studies would suggest an intriguing question of whether functionally and transcriptionally distinctive subfractions of human PSCs are spatially localized (e.g., colony center vs. colony periphery) within the culture, which has never been confirmed in mouse PSCs. The notions that human ESCs share a number of common properties with mouse EpiSCs rather than mouse ESCs and more closely represent postimplantation embryos (as described in Section 3.1) also support the idea of possible human ESC subfractions corresponding to lineages within postimplantation embryos. Hans Scho¨ler’s group reported that EpiSCs comprise at least two transcriptionally and epigenetically distinct subfractions distinguished by the expression of an exogenous Oct4-GFP reporter.69 Oct4-GFP negative EpiSCs exhibit features of late mouse epiblasts and cannot contribute to chimeras, while Oct4-GFP positive EpiSCs (occupying only 7] in comparison to an acidic protein (pI < 7).27 The biomoleculeeHA affinity provides a potentially universal tool to achieve efficient incorporation and delivery of proteins, peptides and nucleic acids. The protein release rates from HA have been varied and controlled by doping with Zn and Mg.28 SiHA is significantly more negatively charged in comparison to pure HA29; there is a higher level of protein absorption on the SiHA surface due to the formation of a silicate network on the surface. The surface layer interacts with integrins, which triggers a signaling cascade and leads to the attachment of osteogenic cells, thus leads to enhanced biological responses in vitro and in vivo (Fig. 20.3). Synthetic apatites have been produced by various methods, such as aqueous reactions, solid-state reactions,

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Cell Recruitment

Cell Death

Cell Adhesion

Molecule expression

Cell Phenotype & metabolism

Division

Cell Migration

Signalling Cascade Protein/GF synthesis

Local pH Modulation

Protein-ion Complex

Protein Adhesion

Soluble Ionic Species

Bioceramic Surface

FIGURE 20.3 Schematic diagram of the principal cellebioceramic “communication” pathways. substitutes: influence of porosity and chemistry. Inter J Appl Ceram Tech 2005;2:184e99.

and hydrothermal reactions. Chemical precipitation is the most commonly used method, where phosphate solution is added into a calcium solution with Ca/P molar ratio maintained at 1.67 for stoichiometric HA. 10Ca(OH)2 þ 6H3(PO4) / Ca10(PO4)6(OH)2 þ 18H2O 10Ca(NO3)2 þ 6(NH4)2H(PO4) þ 2H2O / Ca10(PO4)6(OH)2 þ 12NH4NO3 þ 8HNO3 The calcium and phosphate concentrations must be adjusted accordingly for the preparation of substituted HA, e.g., to keep Ca/(Si þ P) ratio of 1.67 for SiHA. The HA produced is subjected to sintering (at the temperature of 950 to 1300 C) when a high strength ceramic is required. 3.1.2 Tricalcium Phosphates TCP is a biodegradable bioceramic with the chemical formula Ca3(PO4)2. The b-TCP is stable at room

From Hing KA. Bioceramic bone graft

temperature, and transforms to a-TCP at w1125 C. b-TCP is the most commonly used degradable bone graft, a-TCP is more reactive and soluble, often used in calcium phosphate bone cements. Neither can precipitate from aqueous solutions, and are usually prepared by a high temperature process, such as quenching calcium-deficient HA (CDHA) at 800 and 1125 C to make b- and a-TCP, respectively.31 Similar to HA ceramics, b-TCP has been doped with MgO, ZnO, SrO, and SiO2. These dopants were able to modify the physicochemical, mechanical, and biological properties of b-TCP.32 MgO/SrO-doped b-TCP was found to promote osteogenesis than pure b-TCP in vivo.33 TCP with a Ca/P ratio of 1.5 is more rapidly resorbed than HA. A biodegradable material is highly attractive for implants; with time it will be replaced by natural tissue. However, the resorption or degradation rate needs to be carefully designed, and closely matched with bone regeneration rate. If the solubility of calcium phosphate is too high, they will not be of use for cavity fillings.

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A mixture of HA and b-TCP, known as biphasic calcium phosphate (BCP), has been formulated for bone replacements.34 It has the advantage of tailor-making its chemical properties, such as varying the ratio of HA/b-TCP. The higher the TCP content in BCP, the higher the dissolution rate. The resorption rate of BCP can then be monitored and controlled. BCP ceramics are able to support the growth and differentiation of stem cells and maintained osteoblastic phenotypic properties,35 and also are commercially available as Triosite. 3.1.3 Calcium Phosphate Cements Calcium phosphate cement (CPC) is another widely used bioceramic,36 which are formed from a paste made by mixing CPC powder and a liquid (e.g., an aqueous solution). The powder is usually a mixture of two or more different calcium phosphates, and the hydrolysis reactions set the cements. The possible reaction products of CPCs are brushite (DCPD), HA, or CDHA. A paste can be injected and fitted perfectly in defect or cavity; solidified surface can be transformed to a carbonate apatite. Thus the cements shape and cure in situ, are gradually resorbed, and replaced by the newly formed bone.

3.2 Bioactive Glasses Glass is an amorphous material that has a disordered arrangement of atoms due to the rapid cooling of the molten ceramic. A number of bioactive glasses have been developed, including silicate-based, phosphatebased, and borate-based ones. 3.2.1 Bioglass Bioglass is a series of specially designed silica-based glasses, where a three-dimensional SiO2 network is modified by the incorporation of Na2O, CaO, and P2O5. Three key compositional features distinguished them from traditional soda-limeesilica glasses1: (1) lower than 60% SiO2, (2) higher Na2O and CaO content, TABLE 20.4

and (3) higher CaO:P2O5 ratio. These compositional features make the surface highly reactive when exposed to an aqueous medium, and the silica content of Bioglass influences the bioactivity of the glass. Bioglass consisting of 42e53% SiO2 (melt-derived glass) forms a bond to bone very rapidly (within days), and also forms an adherent bond with soft tissues. Bioglass of 54e60% SiO2 requires 2e4 weeks to bond to bone, and does not bond with soft tissues. Bioglass of more than 60% SiO2 does not bond to any living bone tissue.1 However, with sol-gel processing (as discussed later), the compositional range of bioactive glass can be extended up to 100% SiO2. The compositions of various types of bioactive glasses are shown in Table 20.4. The biologically active, soluble Si and Ca ions, which are gradually released from Bioglass 45S5, are able to increase the expression of an osteoblast mitogenic growth factor, and promotes the gene upregulation in osteogenic cells.37,38 The ions cause osteo-stimulation when present at a certain ratio of ions and a critical concentration range of 15e30 ppm Si and 60e90 ppm Ca.39 The stimulatory effect in osteoblast proliferation and differentiation leads to bone regenerative properties. Bioglass 45S5 was also able to stimulate the secretion of angiogenic growth factors from human stromal cells and to promote angiogenesis, thus bioactive glasses can provide a novel alternative approach for stimulating neovascularization.40 The glass composition can be further formulated to control the structure and properties of bioactive glasses. Some specific therapeutic ions, such as boron, copper, cobalt, silver, zinc, and strontium, in the form of metal oxides, have been incorporated into the glass composition in bone tissue engineering applications.41,42 These therapeutic ions can be used for controlled release for osteogenesis and angiogenesis, as well as sustained drug delivery for cancer treatments. Traditionally, Bioglass 45S5 is made by the meltquenching method, a precursor mixture was melted at high temperature (e.g., 1300 C) and quenched. Sol-gel synthesis involves the transformation of a sol

Composition of Some Bioactive Glasses

Component

45S5 (wt%)

45S5 (mol%)

58S (mol%)

58S (wt%)

70S30C (wt%)

13e93 (wt%)

SiO2

45.0

46.1

60.0

58.2

71.4

53.0

B2O3

13e93B3 (wt%)

P50C35N15 (wt%)

53.0

P2O5

6.0

2.6

Na2O

24.5

24.4

4.0

4.0

71.0

6.0

6.0

9.3

K2O

12.0

12.0

MgO

5.0

5.0

20.0

20.0

CaO

24.5

26.9

4.0

36

9.2

32.6

28.6

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19.7

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(suspension of colloidal particles) into a gel (threedimensional interconnected network). It is a relatively low temperature and wet chemistry approach to form an inorganic network of covalently bonded silica that could be dried and heated to produce glasses with high purity. Sol-gel-derived bioactive glasses have a porous texture in the nanometer range. Depending on the glass composition, the pH of the reaction and the precursors used, sol-gel glasses have mesopores ranging from 1 to 30 nm. Typical 58S and 70S30C glasses (e.g., with a size range of 1e32 mm) have specific surface areas of 70e130m2/g, which is at least an order of magnitude higher than that of melt-derived Bioglass 45S5 particles.42 As a result, the dissolution is more rapid for sol-gel glasses with similar compositions; then more silanol groups formed on sol-gel glasses surface act as nucleation sites for a rapid formation of apatite layer, thus leading to high bioactivity. Glass in the fiber form can be produced by the meltspinning approach; the fiber diameter is usually at micrometer-scale (e.g., tens to hundreds of micrometers). Electrospinning is the technique able to produce various polymer fibers in the range of 10e1000 nm, which has been applied to produce bioactive glass fibers in the micro-/nanoscale using the sol-gel glass. Nanoglass fibers in the forms of bundled filament, fibrous membranes, and 3D scaffolds have been produced.43 3.2.2 Mesoporous Silica Silica-based mesoporous materials (e.g., MCM-41) have unique structural characteristics, as an amorphous silica network constitutes the wall of well-ordered arrangement of pore system and cavities. A 2D hexagonal pore structure of silica was obtained using an evaporation-induced self-assembly method with the aid of a nonionic triblock copolymer (EO20PO70EO20), which showed an enhanced bioactivity.44 The mesoporous features of sol-gel bioactive glasses have led to further exploration of multifunctional bioactive glasses, which are not only osteocompatible but also can act as drug reservoir carriers for controlled delivery. The textural parameters, such as surface area, pore volume, and pore size were found to influence the loading and release rate of biologically active molecules (Fig. 20.4). By varying the synthesis parameter, the mesopore size can be tuned from 1.5 nm to several tens of nanometers, which in turn influences the loading capability of mesoporous materials, from small molecules to macromolecules (e.g., proteins) and their subsequent release. 3.2.3 Borate Glasses Borate-based glasses, such as 13-83B3, have been obtained by replacing the SiO2 in silicate glass with B2O3. Compared with silicate-based 45S5, it degrades rapidly

and demonstrates bioactivity.45 A concern with borate bioactive glasses is the toxicity related with the highlevel release of borate ions in a “static” in vitro culture, but the toxicity was diminished in “dynamic” culture conditions and in vivo.46,47 The degradation rate of bioactive glass can be controlled by its composition, such as partially replacing the SiO2 in silicate glass with B2O3. The compositional flexibility of glass can be further exploited by adding elements (e.g., Zn, Cu, Mn, Sr, and F) known to promote bone growth or angiogenesis.48 3.2.4 Phosphate Glasses An attractive feature of glass is that the properties of a glass system can be varied by adjusting its composition. Phosphate glasses are based on P2O5 as glass network former, and CaO and Na2O as network modifiers (e.g., P50C35N15). The chemical compositions of phosphate glasses are close to bone minerals. The degradation rate or the solubility of phosphate glasses can be controlled by altering the glass composition.49 Phosphate-based glasses have been used in both hard and soft tissue repair and regeneration procedures, such as phosphate glass fibers for muscle and ligament replacements.50

3.3 Bioactive Glass-Ceramics A glass-ceramic is a polycrystalline solid that is prepared by the controlled crystallization or devitrification of a parent glass. It generally consists of fine grain (with crystal sizes ranging from 0.1 to 10 mm) and has a small volume of residual glass located at the grain boundary. One advantage of glass-ceramics is that the crystallization and formation of the crystal phases can be designed and controlled to achieve a combination of special properties, such as bioactivity, machinability, and improved mechanical properties. A-W glass-ceramic, an assembly of small apatite particles reinforced by wollastonite, exhibits not only bioactivity, but also fairly high mechanical strength.51 The bending strength, fracture toughness, and Young’s modulus of A-W glass-ceramic are the highest among bioactive glasses and glass-ceramics (Table 20.5), enabling it to be used in some compression load bearing applications.52

3.4 Bioactive Ceramic Composites 3.4.1 Biocomposites In general, the advantages of bioactive glasses are the speed of their surface reactivity and the ability to bond with a variety of tissues by altering their chemical composition. The major disadvantage is their mechanical propertiesdrelatively low strength and toughness (Table 20.5). A way to further utilize the high bioactivity

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FIGURE 20.4 Parameters governing the loading and release rate of biologically active molecules into the silica-based ordered mesoporous materials. From Arcos D, Izquierdo-Barba I, Vallet-Regi M. Promising trends of bioceramics in the biomaterials field. J Mater Sci Mater Med 2009;20: 447e55.

TABLE 20.5

A Comparison of Mechanical Properties of Bioceramic Materials

Materials

Young’s Modulus (GPa)

Bending Strength (MPa)

Fracture Toughness KIC (MPa m1/2)

Bioglass 45S5

35

40e60

0.4e0.6

A-W glass-ceramic

118

215

2.0

Sintered HA

80e110

115e200

0.9e1.3

Alumina

365e400

595

3e5

Ti6Al4V

110

900

316 stainless steel

200

w80 a

540e1000

w100

a

Cancellous bone

0.1e1.5

1.5e3.8

Cortical bone

7e30

50e150a

2e12

a

Tensile strength.

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of bioactive glasses and ceramics is through the development of composites, such as bioactive coating on mechanically strong but bioinert metallic implants.53 This combines the strengths of both materials, namely, high bioactivity of bioceramic coating and excellent mechanical strength of metal implants. From a material point of view, bone is considered as an apatite/collagen composite. By mimicking the structure and properties of the tissue to be replaced, a new approach of designing a composite material has been proposed.54 Such biomimetic composites have been developed to eliminate the problems of conventional biomaterials, and to meet the challenge of a longer lifetime required for new generation implant materials.55 For tissue engineering applications, biodegradable composite scaffolds with high mechanical strength, enhanced bioactivity, and resorbability have been developed.56,57 The acidic degradation products from the most commonly used polylactide (PLA) and polyglycolide (PGA) polymers can have an adverse tissue response. The inorganiceorganic composite approach not only “mimics” the composite nature of bone, but by varying the alkali ceramic fillers, it can also neutralize the acidic autocatalytic degradation of polymers, hence improving the biocompatibility.58 3.4.2 Nanocomposites and Hybrids Materials containing components (e.g., crystallites or surface features) with at least one dimension less than 100 nm are considered as nanomaterials, which can be

in the form of nanoparticles and nanofibers. They have multiple applications in regenerative medicine (tissue engineering, cell therapy, diagnosis, and drug and gene delivery, etc.). Nano-scaled biomaterials have many advantages over micro-scaled ones, such as enhanced mechanical properties, bioactivity, and resorbability. There is a growing recognition that a nano-sized bioceramic component is likely to be more bioactive than a micro-sized one.59 To improve the mechanical properties of Bioglass, organiceinorganic hybrid materials, have been synthesized by combining inorganic and organic components with interactions at the molecular scale (Fig. 20.5). A hybrid is different from a composite (a combination of two or more distinguishable components); its components are indistinguishable at the submicrometer level. These bioactive and flexible materials may have a huge potential in a variety of biomedical applications.42,60

3.5 Nanomaterials and Nanostructure With the advances of novel processing and characterization methods in nanotechnology, the evolution of material manufacturing from the microscale to the nanoscale has been sped up. Nanostructured materials are able to interact specifically with nucleic acids, proteins, and other small-scale biological structures. Such highly specific interactions between small-scale biological structures and nanostructured materials can provide

FIGURE 20.5 Schematic of the interpenetrating inorganic and organic networks of a class II hybrid material. Three nanoparticles of the continuous silica network are highlighted. From Jones JR. Review of bioactive glass: from Hench to hybrids. Acta Biomater 2013;9(1):4457e86.

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nanostructured materials with unique functions, and lead to enhanced performance. Cells are sensitive to their surroundings and able to respond to the surface topography at the nanoscale. The surface topography is known to affect cell adhesion, intracellular signaling pathways, and gene expression. Nanostructured materials have significant effects on cellular responses.61,62 There is growing evidence indicating that nanofeatures are a compelling determinant of stem cell lineage commitment, and nanotopography can stimulate MSC differentiation in the absence of osteogenic supplements. The nanotopographical features were found to affect the differentiation of MSCs, increasing the expression of the bone-specific ECM protein osteopontin and promoting mineralization of bone. Bone is a complex tissue with hierarchical organization from the macro-scale to the nanostructured (extracellular matrix and bone mineral) components. The use of nanostructured biomaterials in bone regeneration is inspired by the bone architecture. Using different synthetic methods, nano-CaP crystals with diverse structures and morphology have been fabricated: spheres, rods, disks, whiskers, needles, wires, fibers, etc. Research showed that the incorporation of nano-CaP was able to promote the osteogenic differentiation, upregulation of osteogenic gene expression, and mineralization. HA-based nanocomposites were especially efficient at inducing rapid bone formation.63

4. ENGINEERING STEM CELL NICHES Tissue engineering is a rapidly growing field exploring synthetic solutions for tissue regeneration, and engineering of larger tissue constructs remains a great clinical challenge. The key elements in biomimetic bone regeneration strategies include (1) scaffolds, (2) growth factors to induce osteogenesis, and (3) cells that can undergo osteogenic differentiation.

4.1 Tissue Engineering Scaffolds Porous bioactive ceramics have attracted great interests as scaffolds for tissue engineering.64,65 A scaffold is required to provide cell anchorage sites, mechanical stability, and to serve as a structural guidance for tissue to grow in three dimensions. Therefore, scaffolds should be compatible with the host tissue mechanically, as well as biologically. The design and control of the internal architecture of the porous ceramic structure has an influence on tissue regeneration.66 The large (macropores, at least 100 mm in size) and interconnected pores are critical, which allows essential nutrients to reach the

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whole network and stimulate blood vessels to grow inside the pore network. Interconnected porosity not only provides pathways for micronutrients, but also provides higher surface area for improved mechanical interlocking between the scaffolds and surrounding host tissue. Many conventional techniques, such as impregnation and sintering, solvent casting, particulate leaching, gas foaming, fiber bonding, thermally induced phase separation, etc., have been used for scaffold fabrication. Now rapid prototyping has emerged as a new processing technique for making scaffolds, which allows highly complex structures to be built as a series of thin twodimensional slices using computer-aided design and computer-aided manufacturing programs. The properties, such as porosity, interconnectivity, and pore size, can be predefined. Among these layer-by-layer solid freeform manufacturing techniques, selective laser sintering, stereolithography, fused deposition modeling, and three-dimensional printing have been used to develop three-dimensional porous scaffolds for bone replacements, and the repair of osseous defects from trauma or disease. A three-dimensional HA structure with controlled patterns or porosity was built by direct-write assembly.67 A complex-shaped porous HA ceramic with fully interconnected channels was generated from HA powder.68 With 3D printing technology, it is possible to design and manufacture parts according to an individual patient’s anatomy, and patient-derived cells can then be seeded onto the scaffolds to promote integration of tissue grafts with the surrounding tissue, thus offering a potential for personalized medical implants. The development of osteoblast cells from bone marrow stromal cells is characterized by a sequence of events involving cell proliferation and differentiation, the expression of bone-related proteins, and synthesis and deposition of a collagenous extracellular matrix. Although bioactive ceramics are osteoconductive, unlike autografts, their ability to stimulate new bone growth is often limited; they are not osteoinductive in nature. For an engineered scaffold with both osteoconductivity and osteoinductivity, osteoinductive proteins and/or osteogenic cells have been incorporated with traditional bioactive materials. The growth factors are found to stimulate bone growth, collagen synthesis, and fracture repair both in vitro and in vivo. Bone morphogenetic proteins (BMPs) promote differentiation of MSCs toward an osteoblastic lineage; BMP-2 and BMP-7 have been approved by US Food and Drug Administration for use in specific treatment of nonunion fracture. The addition of BMPs can improve the biological properties of porous scaffolds further by inducing osteogenesis or angiogenesis. As osteogenesis is closely correlated and supported by angiogenesis,

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attempts have also been made to stimulate neovascularization, using recombinant protein or gene transfer of angiogenic growth factors.

4.2 Stem Cells Stem cells refer to cells that have the potential to differentiate into a variety of different lineages, which can be isolated from a variety of sources including embryos, umbilical cord blood, or from adult tissues.69,70 Embryonic stem cells (ESCs) are isolated from the inner cell mass of the blastocyst during embryological development; they have the unique property of indefinite self-renewal and can be cultured and maintained in an undifferentiated, pluripotent state. Adult tissues carry a variety of adult stem cells that are less pluripotent and are more committed than ESCs. These cells have been found in bone marrow, cord blood, adipose tissues, neural tissues, etc. Due to their relative ease in harvest, isolation, and expansion in vitro, and their ability to differentiate into variety of cell types, bone marrowderived progenitor cells MSCs are very attractive in tissue engineering. Bone has a vast capacity for regeneration from cells with stem cell characteristics, hBMSCs are the gold standard in stem cell-based bone regeneration. Bone marrow tissue is a rich source of progenitor stromal cells for potential osteogenic lineages.71,72 MSCs have shown great potential for bone regeneration, and are clinically available for the regeneration of various tissues due to their high proliferation/differentiation capabilities.73 The osteogenic differentiated MSCs from patients have been loaded into biomaterial scaffolds for clinical applications, such as the treatment of bone tumors and osteonecrosis of femoral head, alveolar augmentation for jaw bone defects, etc. However, the invasive procedure not only has the shortcomings of limited availability and donor site morbidity, but also the fact that aging and other diseases can cause the cells to lose potency. In addition to the harvest of pluripotent mesenchymal cells from bone marrow, the periosteum or adipose tissue has been considered.74,75 Adipose tissue contains more multipotent cells than bone marrow, but there are concerns of an inferior osteogenic ability of adipose MSCs, although some studies show no significant difference in osteogenic ability between these MSCs.63,76 The differentiation of stem cells can be controlled by the mechanics of the matrix material, which encourages the formation of specific tissue types. MSC differentiation into bone lineage requires materials with higher mechanical strength to closely mimic the tissue mechanical properties. Bone marrow-derived MSCs are highly sensitive and responsive to mechanical stimulation in vitro. It has been suggested that mechanical stimuli

may activate cell surface receptors and focal adhesion sites, which in turn triggers intracellular signaling cascades and leads to specific gene activation.70 Chemical and biological modifications to biomaterials can also directly influence stem cell behavior by altering substrate properties, surface interactions, scaffold architecture, and microenvironment, which ultimately manipulates the signal transduction pathways in stem cells. Biomaterials can be designed to fine-tune their properties to orchestrate the environment for stem cell niche. MSCs have osteogenic capability in both in vitro and in vivo conditions; bone tissue engineering using scaffolds and MSCs is highly promising, at least in animal studies. However, bone remodeling rates are different; there are many hurdles in translating in vivo outcomes to widespread clinical success. Further improvements in scaffold design are required to meet the challenge of lacking vascular supply in larger human defects.

5. SUMMARY AND FUTURE PERSPECTIVE The development of bioceramic materials has been shifted toward the design of deliberately “bioactive” materials that integrate with biological molecules or cells and regenerate tissues. The potential of bioceramics is immense, although the ultimate challenge remains to mimic the complex structure of native tissues. Engineering of bioactive materials in combination with stem cells technology will play a vital role in tissue repair. Scaffold, cells, and growth factors are the three main parts of tissue engineering. Special attention has been directed to the design of new scaffold by adding bioactive molecules, which promote smart interactions with seeded cells for better biological responses.77 The more we understand the fundamentals of cellular responses, the better we can design smart materials. The search for an ideal combination of scaffold, cells, and bioactive modules for tissue engineering is still going on. Scaffold should emulate the nanoscale surface topography and biochemistry of natural extracellular matrix to facilitate favorable cell binding and differentiation. Whilst considerable efforts have been focused on the materials and structures of scaffold, advanced manufacturing technology must also be further developed. Computer-aided scaffolding techniques provide significant advantages in terms of consistency, reproducibility of the designed scaffolds, and the capability of precise control over the architecture of the 3D scaffold. The anatomic structure of medical implants are routinely generated from patient data, a further optimized architectural designs of the scaffold structure should be closely followed, as well as the improvements

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REFERENCES

in the resolution of the techniques, the breadth of the materials selection, and the capabilities for printing biological factors and cells. In addition to the optimization of materials chemistry to promote desirable materialehost tissue interactions, considerable efforts have been made toward developing and engineering structures and surfaces that could elicit rapid and desired reactions with cells and proteins for specific applications.78 However, our knowledge of the physical and chemical functioning of biomaterials, and the interaction mechanism between the materials and the biological systems still need to be further understood. The emergence of the fields of biotechnology and nanotechnology and the advances in materials science, engineering, cell and molecular biology, and medicine will continue to offer new solutions in the further development of bioactive glasses and ceramics in regenerative medicine and tissue engineering.79,80 A paradigm in the developments of biomaterial engineering is shifting replacement to regeneration, and further toward the expansion of new generation materials to stimulate specific cellular and gene responses.39,81,82 There are further drives for cell therapy or organ printing to be combined with advanced diagnostic systems for personalized treatment in the future.83

References 1. Hench LL. Bioceramics: from concept to clinic. J Am Ceram Soc 1991; 74:1487e510. 2. Hench LL. The story of Bioglass. J Mater Sci Mater Med 2006;17: 967e78. 3. Hench LL, Best SM. Ceramics, glasses and glass-ceramics. In: Ratner BD, Schoen FJ, Hoffman AS, Lemons JE, editors. Biomaterials Science: An Introduction to Materials in Medicine. 2nd ed. San Diego (California, USA): Elsevier Academic Press; 2004. p. 153e70. 4. Nandi SK, Roy S, Mukherjee P, Kundu PB, De DK, Basu D. Orthopaedic application of bone graft & graft substitute: a review. Ind J Med Res 2010;132(4):15e30. 5. Hench LL, Wilson J. Surface-active biomaterials. Science 1984;226: 630e6. 6. Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res Symp 1971;2:117e41. 7. Hench LL. Bioceramics. J Am Ceram Soc 1998;81:1705e28. 8. Posner AS. Crystal chemistry of bone mineral. Physiol Rev 1969;49: 760e2. 9. Vallet-Regı´ M, Arcos D. Silicon substituted hydroxyapatites: a method to upgrade calcium phosphate based implants. J Mater Chem 2005;15:1509e16. 10. Shepherd JH, Shepherd DV, Best SM. Substituted hydroxyapatites for bone repair. J Mater Sci Mater Med 2012;23(10):2335e47. 11. LeGeros RZ, Trautz OR, LeGeros JP, Shirra WP. Apatite crystallites: effect of carbonate on morphology. Science 1967;155: 1409e11. 12. Carlisle EM. Silicon: a possible factor in bone calcification. Science 1970;167:279e80. 13. Carlisle EM. Silicon: an essential element for the chick. Science 1972; 178:619e21.

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14. Gibson IR, Best SM, Bonfield W. Chemical characterization of silicon-substituted hydroxyapatite. J Biomed Mater Res 1999;44: 422e8. 15. Hing KA, Revell PA, Smith N, Buckland T. Effect of silicon level on rate, quality and progression of bone healing within silicatesubstituted porous hydroxyapatite scaffolds. Biomaterials 2006;27(29): 5014e26. 16. Patel N, Best SM, Bonfield W, Gibson IR, Hing KA, Damien E, Revell PA. A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules. J Mater Sci Mater Med 2002;13:1199e206. 17. Patel N, Brooks RA, Clarke MT, Lee PMT, Rushton N, Gibson IR, Best SM, Bonfield W. In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model. J Mater Sci Mater Med 2005;16:429e40. 18. Gibson IR, Best SM, Bonfield W. Effect of silicon substitution on the sintering and microstructure of hydroxyapatite. J Am Ceram Soc 2002;85:2771e7. 19. Porter AE, Patel N, Skepper JN, Best SM, Bonfield W. Comparison of in vivo dissolution processes in hydroxyapatite and siliconsubstituted hydroxyapatite bioceramics. Biomaterials 2003;24: 4609e20. 20. Porter AE, Botelho CM, Lopes MA, Santos JD, Best SM, Bonfield W. Ultrastructural comparison of dissolution and apatite precipitation on hydroxyapatite and silicon-substituted hydroxyapatite in vitro and in vivo. J Biomed Mater Res 2004;69A:670e9. 21. Munir G, Edirisinghe MJ,G, Koller G, Di Silvio L, Bonfield W, Huang J. The pathway to intelligent implants: osteoblast response to nano silicon-doped hydroxyapatite patterning. J Royal Soc Interface 2011;8(58):678e88. 22. Huang J, Best SM, Bonfield W, Buckland T. Development and characterization of titanium-containing hydroxyapatite for medical applications. Acta Biomater 2010;6:241e9. 23. Huang J, Li X, Koller GP, Di Silvio L, Vargas-Reus MA, Allaker RP. Electrohydrodynamic deposition of nanotitanium doped hydroxyapatite coating for medical and dental applications. J Mater Sci Mater Med 2011;22(3):491e6. 24. Landi E, Uggeri J, Medri V, Guizzardi S. Sr, Mg cosubstituted HA porous macro-granules: potentialities as resorbable bone filler with antiosteoporotic functions. J Biomed Mater Res Part A 2013;101: 2481e90. 25. Webster TJ, Ergun C, Doremus RH, Bizios R. Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion. J Biomed Mater Res 2002;59: 312e7. 26. Thian ES, Konishi T, Kawanobe Y, Lim PN, Choong C, Ho B, Aizawa M. Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties. J Mater Sci Mater Med 2013;24(2):437e45. 27. Zhu XD, Zhang HJ, Fan HS, Li W, Zhang XD. Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption. Acta Biomater 2010;6:1536e41. 28. Dasgupta S, Banerjee SS, Bandyopadhyay A, Bose S. Zn- and Mgdoped hydroxyapatite nanoparticles for controlled release of protein. Langmuir 2010;26:4958e64. 29. Botelho CM, Lopes MA, Gibson IR, Best SM, Santos JD. Structural analysis of Si-substituted hydroxyapatite: zeta potential and X-ray photoelectron spectroscopy (XPS). J Mater Sci Mater Med 2002;13: 1123e7. 30. Hing KA. Bioceramic bone graft substitutes: influence of porosity and chemistry. Inter J Appl Ceram Tech 2005;2:184e99. 31. Dorozhkin S. Calcium orthophosphates. J Mater Sci 2007;42: 1061e95. 32. Bandyopadhyay A, Bernard S, Xue W, Bose S. Calcium phosphatebased resorbable ceramics: influence of MgO, ZnO, and SiO2 dopants. J Am Ceram Soc 2006;89:2675e88.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

328

20. DESIGN AND DEVELOPMENT OF CERAMICS AND GLASSES

33. Banerjee SS, Tarafder S, Davies NM, Bandyopadhyay A, Bose S. Understanding the influence of MgO and SrO binary doping on the mechanical and biological properties of b-TCP ceramics. Acta Biomater 2010;6:4167e74. 34. Daculsi G, Laboux O, Malard O, Weiss P. Current state of the art of biphasic calcium phosphate bioceramics. J Mater Sci Mater Med 2003;14:195e200. 35. Toquet J, Rohanizadeh R, Guicheux J, Couillaud S, Passuti N, Daculsi G, Heymann D. Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic. J Biomed Mater Res 1999;44:98e108. 36. Bohner M, Galea L, Doebelin N. Calcium phosphate bone graft substitutes: failures and hopes. J Euro Ceram Soc 2012;32: 2663e71. 37. Xynos ID, Edgar AJ, Buttery LD, Hench LL, Polak JM. Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis. Biochem Biophys Res Commun 2000;276: 461e5. 38. Xynos ID, Edgar AJ, Buttery LDK, Hench LL, Polak JM. Gene expression profiling of human osteoblasts following treatment with the ionic dissolution products of Bioglass 45S5 dissolution. J Biomed Mater Res 2001;55:151e7. 39. Hench LL. Genetic design of bioactive glass. J Eur Ceram Soc 2009; 29(7):1257e65. 40. Day RM. Bioactive glass stimulates the secretion of angiogenic growth factors and angiogenesis in vitro. Tissue Eng 2005;11(5e6): 768e77. 41. Hoppe A, Mourin˜o V, Boccaccini AR. Therapeutic inorganic ions in bioactive glasses to enhance bone formation and beyond. Biomater Sci 2013;2013(1):254e6. 42. Jones JR. Review of bioactive glass: from Hench to hybrids. Acta Biomater 2013;9(1):4457e86. 43. Kim HW, Kim HE, Knowles JC. Production and potential of bioactive glass nanofibers as a next-generation biomaterial. Adv Func Mater 2006;16:1529e35. 44. Arcos D, Izquierdo-Barba I, Vallet-Regi M. Promising trends of bioceramics in the biomaterials field. J Mater Sci Mater Med 2009;20: 447e55. 45. Rahaman MN, Day DE, Bal BS, Fu Q, Jung SB, Bonewald LF, Tomsia AP. Bioactive glass in tissue engineering. Acta Biomater 2011;7(6):2355e73. 46. Brown RF, Rahaman MN, Dwilewicz AB, Huang W, Day DE, Li Y, Bal BS. Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells. J Biomed Mater Res A 2009;88(2):392e400. 47. Fu Q, Rahaman MN, Bal BS, Bonewald LF, Kuroki K, Brown RF. Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. II. In vitro and in vivo biological evaluation. J Biomed Mater Res A 2010; 95(1):172e9. 48. Hoppe A, Gu¨ldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32(11):2757e74. 49. Knowles JC. Phosphate based glasses for biomedical applications. J Mater Chem 2003;13:2395e401. 50. Abou Neel EA, Pickup DM, Valappil SP, Newport RJ, Knowles JC. Bioactive functional materials: a perspective on phosphate-based glasses. J Mater Chem 2009;19:690e701. 51. Kokubo T, Ito S, Sakka S, Yamamuro T. Formation of a highstrength bioactive glass-ceramic in the system MgO-CaO-SiO2P2O5. J Mater Sci 1986;21:536e40. 52. Kokubo T. Bioactive glass ceramics: properties and applications. Biomaterials 1991;12:155e63. 53. de Groot K, Wolke JGC, Jansen JA. Calcium phosphate coatings for medical implants. Proc Inst Mech Eng H 1998;212:137e47.

54. Bonfield W. Composites for bone replacement. J Biomed Eng 1988; 10:522e6. 55. Wang M. Developing bioactive composite materials for tissue replacement. Biomaterials 2003;24:2133e51. 56. El-Ghannam. Bone reconstruction: from bioceramics to tissue engineering. Expert Rev Med Devices 2005;2(1):87e101. 57. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006;27(18):3413e31. 58. Ehernfried L, Patel MH, Cameron RE. The effect of tri-calcium phosphate (TCP) addition on the degradation of polylactide-coglycolide (PLGA). J Mater Sci Mater Med 2008;19(1):459e66. 59. Yang Z, Best SM, Cameron RE. The influence of a-tricalcium phosphate nanoparticles and microparticles on the degradation of poly(D,L-lactide-co-glycolide). Adv Mater 2009;21(38e39):3900e4. 60. Dorozhkin SV. Calcium orthophosphate-based biocomposites and hybrid biomaterials. J Mater Sci 2009;44:2343e87. 61. Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, Wilkinson CDW, Oreffo ROC. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater 2007;6:997e1003. 62. Dalby MJ, Gadegaard N, Oreffo ROC. Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Nat Mater 2014;13(6):558e69. 63. Wang P, Zhao L, Liu J, Weir MD, Zhou X, Xu HHK. Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells. Bone Res 2014;2:14017. 64. Chen QZ, Thompson ID, Boccaccini AR. 45S5 Bioglass-derived glass-ceramic scaffolds for bone tissue engineering. Biomaterials 2006;27:2414e25. 65. Sepulveda P, Jones JR, Hench LL. Bioactive sol-gel foams for tissue repair. J Biomed Mater Res 2002;59A:340e8. 66. Chu TMG, Orton DG, Hollister SJ, Feinberg SE, Halloran JW. Mechanical and in vivo performance of hydroxyapatite implants with controlled architectures. Biomaterials 2002;23:1283e93. 67. Michna S, Wu W, Lewis JA. Concentrated hydroxyapatite inks for direct-write assembly of 3-D periodic scaffolds. Biomaterials 2005; 26:5632e9. 68. Seitz H, Rieder W, Irsen S, Leukers B, Tille C. Three-dimensional printing of porous ceramic scaffolds for bone tissue engineering. J Biomed Mater Res Part B: Appl Biomater 2005;74B:782e8. 69. Mimeault M, Batra SK. Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 2006;24:2319e45. 70. Dawson E, Mapili G, Erickson K, Taqvi S, Roy K. Biomaterials for stem cell differentiation. Adv Drug Deliv Rev January 14, 2008;60(2): 215e28. 71. Ohgushi H, Caplan AI. Stem cell technology and bioceramics: from cell to gene engineering. J Biomed Mater Res (Appl Biomater) 1999;48: 913e27. 72. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41e9. 73. Chai C, Leong KW. Biomaterials approach to expand and direct differentiation of stem cells. Mol Ther 2007;15:467e80. 74. Hattori H, Sato M, Masuoka K, Ishihara M, Kikuchi T, Matsui T. Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source. Cells Tissues Organs 2004;178:2e12. 75. Hicok KC, Du Laney TV, Zhou YS, Halvorsen YD, Hitt DC, Cooper LF. Human adipose-derived adult stem cells produce osteoid in vivo. Tissue Eng 2004;10:371e80. 76. Yuan N, Rezzadeh KS, Lee JC. Biomimetic scaffold for osteogenesis. Recept Clin Investig 2015;2(3):898. 77. Motamedian SR, Hosseinpour S, Ahsaie MG, Khojasteh A. Smart scaffolds in bone tissue engineering: a systematic review of literature. World J Stem Cells 2015;7(3):657e68.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

REFERENCES

78. Anderson JM. The future of biomedical materials. J Mater Sci Mater Med 2006;17:1025e8. 79. Hench LL, Polak JM. Third generation biomaterials. Science 2002; 295(5557):1014e7. 80. Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005;4:518e24. 81. Best SM, Porter AE, Thian ES, Huang J. Bioceramics: past, present and for the future. J Eur Ceram Soc 2008;28:1319e27.

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82. Dorozhkin SV. Nanosized and nanocrystalline calcium orthophosphates. Acta Biomater 2010;6:715e34. 83. Williams DF. On the nature of biomaterials. Biomaterials 2009;30: 5897e909. 84. Bose S, Tarafder S. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review. Acta Biomater 2012;8:1401e21.

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C H A P T E R

21 Surface Functionalization of Biomaterials Deepti Rana1, Keerthana Ramasamy1, Maria Leena2, Renu Pasricha3, Geetha Manivasagam4, Murugan Ramalingam1,5 1

Centre for Stem Cell Research (CSCR), Vellore, India; 2Karunya University, Coimbatore, India; 3National Centre for Biological Sciences, Bangalore, India; 4VIT University, Vellore, India; 5Tohoku University, Sendai, Japan

O U T L I N E 1. Introduction

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2. Surface Modifications of Biomaterials 2.1 Physical Approach 2.1.1 Physical Adsorption 2.1.2 LangmuireBlodgett Method 2.1.3 Pulsed-Laser Deposition 2.1.4 Ion Beam Deposition 2.1.5 Rapid Prototyping 2.1.6 Layer-by-Layer Deposition 2.2 Chemical Approach 2.2.1 Adsorption via Covalent Bonding 2.2.2 Alkali Acid Hydrolysis Method 2.2.3 Self-Assembled Monolayer 2.2.4 Plasma Treatment

332 332 334 334 335 335 335 336 336 336 336 337 337

1. INTRODUCTION As a result of their natural abilities of self-renewal and differentiation into multiple lineages, stem cells possess extensive potential in tissue engineering and regenerative medicine. The concept of stem celledriven tissue engineering involves isolation of the stem cells from their native microenvironment followed by the in vitro propagation of the cells and finally placing them into the engineered tissue constructs for the regeneration of the defective tissues.1,2 Stem cell microenvironment/niche facilitates the regulation of cellular attachments, proliferation, and synthesis and remodeling of extracellular matrix (ECM) proteins, cellematrix interactions, cellecell communications and continued

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00021-4

3. Biological Relevance of Surface Modification for Biomaterials 3.1 Cellular Responses of Patterned Self-Assembled Monolayers 3.2 Extracellular Protein-Absorbed Surfaces 3.3 Dynamic Surfaces 3.4 Surfaces With Specific Signaling Functions

338 338 338 339 340

4. Concluding Remarks

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Abbreviations and Acronyms

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Glossary

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Acknowledgments

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References

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functions. Stem cell response to the substrate largely depends on its surface properties, surface chemistry, and physical structure; hence, it is essential to modify these biomaterial substrates with suitable surface modification techniques. Surface treatment is an essential step in any cell cultureebased tissue engineering application.3 Surface functionalization or modification is the act of modifying the surface of the scaffold by attaching molecules or substances via physical or chemical methods or by a combination of both the methods. Surface functionalization or modification is one of the approaches that can create biomimetic microenvironments, which are able to control stem cell fate and functions and enhances the cellular functions. For instance, the impact of surface coating on poly-L-lactic

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FIGURE 21.1 Schematic diagram depicting various surface functionalization techniques.

acid (PLLA)/polycaprolactone (PCL) hybrid scaffold using gelatin was investigated to study the flexibility, plasticity, mechanical property, compatibility, and inflection of scaffolding systems using adipose-derived stem cells (ADSCs) as a model system. The results of this study elucidated significant increase of ADSCs proliferation and viability in gelatin-coated scaffolds as compared with uncoated PLLA/PCL blends.4 Keeping these points in view, the contents of the chapter have been arranged with the idea of reviewing current trends and progress that have been made in elucidating the significant role of different types of surface modification techniques for biomaterials in manipulating stem cell microenvironment suitable to enhance stem cell research and applications. For the benefit of readers, the commonly used substrate materials with detailed mechanism and distinctive examples have also been discussed for each technique. Additionally, authors have also discussed about the challenges related to surface functionalization and understanding the interactions between the implants and stem cell under the heading of biological relevance of these techniques in the context of stem celledriven tissue engineering.

2. SURFACE MODIFICATIONS OF BIOMATERIALS For mimicking the native microenvironment of the cultured cells, it is critical to perform appropriate

changes to the surface chemistry and physical topography of the biomaterials surface, which results in the improvement of biocompatibility, cell adhesion, and cellebiomaterial interactions.5,6 Surface functionalization or modification can be achieved either by altering the atom or molecules on the surface through physical or chemical approach or by coating the bioactive molecules over the surface of biomaterials. The development of surface modification technologies for the creation of spatially controlled substrates with suitable surface chemistry and topography is of great interest for maintaining the spatially organized functional cells over the substrates. Some of the commonly used surface modification techniques have been shown in Fig. 21.1. Cutting-edge techniques have been introduced in recent times to improve physical, chemical, and biological properties of biomaterials so as to meet the clinical and translational requirements for modified specific biomaterials for tissue engineering and regenerative medicine. This section covers the recent advances in chemical and physical approaches being used currently for the surface modification of different types of biomaterials (Table 21.1).

2.1 Physical Approach Physical approaches deal with the coating of a biomimetic material onto the biomaterial surface without altering the chemical characteristics of either. The coating of biomaterials with adhesive proteins [collagen,

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TABLE 21.1

Summary of Different Methods for Surface Functionalization of Biomaterials

Methods

Description

Advantage

Disadvantage

Application

References

SURFACE MODIFICATION BY PHYSICAL APPROACHES Monolayer of molecules absorbed homogenously onto the biomaterial surfaces by immersion

Modifiable LB parameters (surface pressure, temperature, barrier speed, dipping speed, and molar composition)

Possibility of mixing of subphases while transferring the monolayers

Structure-controlled thinfilm formation

37

Immobilization via adsorption

Adsorption of molecules through weak interactions

Requires minimum activation step Less disruptive in nature than chemicals

Sensitive to change in temperature, pH, and ionic strength

Biocompatible films formation

38

Electrophoretic deposition

Colloidal particles suspended in liquid migrate/deposit under the influence of electric field to the electrode

Uniform thickness, less porosity, high speed, controlled deposition

Decrease in deposition yield

Modification of thin/thick films, multilayered composites, functionally graded materials/hybrid

39

SURFACE MODIFICATION BY CHEMICAL APPROACHES Pulsed-laser deposition

High-power pulsed-laser beam strikes a target material that is to be deposited in vacuum chamber

Flexible, easy to implement, epitaxy at low temperature and greater control of growth

Uneven coverage and high defect concentration, mechanisms behind and dependence on parameters not well understood

Superconducting and insulating components for medical applications

40

Ion beam deposition

Create nanopatterns on surface by applying ion beams

Independent control over the energy and flux of ions

Lower spatial resolution

Bioactive treatment

41

Plasma treatment

Gas molecules in plasma state react with the surface to deposit biomolecules

Universal method for all types of organic compounds

No control over chemical composition of the surface after modification

For toughened coating

42

Covalent immobilization

Covalent interaction between molecules and material surfaces

Strong linkage to biomolecules and no desorption

Loss of functional conformation of biological molecules

Increased biocompatibility of the substrates

43

Photochemical modification

Selective surface modification using light and mask

Uniform modification and does not affect the bulk properties of the biomaterials

Expensive

Surface modification with nanoparticles

44

Acid etching

Etch anisotropically to form nanostructures

It renders the substrate with homogeneous roughening

Biocompatibility issues

Etching implant surface

45

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LangmuireBlodgett (LB) method

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laminin, integrin, and fibronectin (FN)]7 and ECMresembling molecules (chitosan and gelatin)8 has been one of the most common approach used for the surface modification of biomaterials. Surface coating of protein has showed enhanced cell adhesion and proliferation in comparison with bare surfaces.9 The surface coating of proteins is further influenced by surface topography, surface charge, and wettability.10 2.1.1 Physical Adsorption Physical adsorption is one of the simplest and widely applicable surface modification method, which involves dipping the substrate in solution consisting of the target molecules. The major force governing physical adsorption includes electrostatic interactions, van der Waals forces, hydrogen bond, or hydrophobic interactions. It is considered as a nondestructive and easily accessible method with only one major limitation of having limited control over the orientation of the adsorbed biomolecules. The biomolecules adsorbed onto the substrate can orient themselves randomly leading to conformational change and subsequent loss of functionality of the adsorbed biomolecules. In native cellular microenvironment/niche, ECM presents many physical, chemical, and biological cues to the cells for determining their fate and functions. It has been found that many ECM components (e.g., collagen, elastin, hyaluronic acid, FN, vitronectin, laminin, Integrin, growth factors, etc.)11 are involved in the celleECM interaction for regulating the cell attachment, survival, proliferation, growth, and differentiation of the stem cells.9 Reportedly, many bioresorbable polyesters such as poly-L-lactide (PLA),

poly(D,L-lactide-co-glycolide) (PLGA), and PCL have been used as scaffold materials for tissue regeneration, but have found to show negative influence on cell adhesion because of the surface’s hydrophobic nature. To nullify this effect, simple adsorption of biomolecules has been used to make the biomaterial surfaces more biocompatible and cell specific. For example, this technique has been used to deposit calcium phosphate on PCL nanofibers scaffold to improve the osteoblastic activity of the cultured cells.12 Similarly, ECM proteins such as FN have been deposited on the PLGA scaffold surface and have found to provide cell recognition by specific integrin-binding sites.13 Some of the commonly used ECM mimetic proteins to modulate the nanoscale organization of the stem cell microenvironment in vitro for various tissue engineering applications include poly-D-lysine (neurons and stem cells), laminin (neurons and stem cells), FN (endothelium), vitronectin (muscle), gelatin (cardiac), Matrigel (cardiac, epithelial, and stem cells), and collagen (bone and cartilage). 2.1.2 LangmuireBlodgett Method LangmuireBlodgett (LB) method is an elegant technique of depositing mono- and multi-molecular films of controlled thickness on any substrate. The film of amphiphilic compounds formed on the airewater interface is transferred by simple LB technique at controlled surface pressure, thus controlling the packing density of the molecules at the water surface. A simple schematic representation of LB monolayer formation and vertical dipping method is shown in Fig. 21.2. The underlying method involves adsorption

FIGURE 21.2 Schematic diagram of LangmuireBlodgett method for monolayer synthesis. III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

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of homogenous monolayers with each immersion and emersion cycle thereby resulting in accurate thickness of the total film.14 An added advantage of this technique is the freedom to choose the method of deposition depending on the surface properties (hydrophobic/hydrophilic) of the substrate. 2.1.3 Pulsed-Laser Deposition Pulsed-laser deposition (PLD), a physical vapor deposition method, is popular for fabricating calcium phosphate coating on metallic substrate. The technique is able to stoichiometrically transfer material from target to substrate and could obtain an ultrathin coating layer (thickness of several atoms).15 PLD has been proved as a suitable method for thin layer coating of hydroxyapatite (HAp) (200 and 400 nm) on coaxial PCL/PVA nanofibers, while preserving the fibrous morphology of the nanofibers. Coating of thin HAp layers has been shown to improve Young’s moduli of elasticity of the coated nanofibers in comparison with uncoated nanofibers.16 Additionally, these coated nanofibers were found to stimulate mesenchymal stem cell proliferation and differentiation toward osteogenic lineage. PLD can be considered as a potential technique for the development of artificial bone tissue and bone healing scaffolds/biomaterials. 2.1.4 Ion Beam Deposition Ion beam deposition (IBD) is another important method that comes within the broad umbrella of physical surface modification techniques/methods. Other available modifications of this method, which includes the employment of ion beams, are ion beameinduced deposition, ion beam sputtering deposition, and ion beameassisted deposition (IBAD). IBD is a direct beam deposition process that directly applies an ionized particle beam onto substrate surface to fabricate thinfilm coatings on substrate surface (Fig. 21.3). At low energy, molecular ion beams would deposit intact (soft landing), whereas at high deposition energy, molecular ions fragment and atomic ions penetrate further into the material (ion implantation). As a modification of IBD, IBAD combines ion implantation with simultaneous sputtering or other physical vapor deposition methods. IBAD is widely used to create gradual transitions between the substrate and the deposited film and depositing films with less internal strains. In addition to be able to independently control the process parameters (ion energy, temperature, and arrival rate of atomic species), films resulting from IBAD technique adhere more strongly to the substrate.17 Because of their superior advantage over other modifications of IBD, IBAD have been widely used for the surface modifications of the biomaterials to increase their bioactivity. For instance, Chen et al. have reported the preparation of

FIGURE 21.3 Schematic diagram of surface modification using ion beam deposition method.

calcium phosphate thin-film coatings on pure titanium by using IBAD and further created biomimetic apatite precipitation layers by immersing the coating in Dulbecco’s phosphate-buffered saline solutions containing calcium chloride and biomolecules to modulate precipitation processes and enhance its bioactivity.18 2.1.5 Rapid Prototyping Rapid prototyping (RP) comprises a chain of techniques using three-dimensional computer-aided design data to quickly fabricate a model or performing the replacement for some part. Some of the RP techniques were used to construct coating for biomaterials, especially for metallic biomaterials. Laser-engineered net shaping is an additive RP-manufacturing technique that uses a focused, high-energy laser beam to melt metallic powders directly injected on the focused laser beam spot to form a new layer.19 Tissue-engineering constructs can be customized according to the data acquired from the medical scans to match each patient’s individual needs.20 In addition, RP enables the control of the scaffold porosity making it possible to fabricate applications with desired structural integrity. For example, the electrospinning-based rapid prototyping (ESRP) technique unifies RP and electrospinning to create a controllable pattern and a continuous fine fiber. The technique follows RP process of fused deposition modeling, but instead of using extrusion process for fiber creation, electrospinning is applied to generate a continuous fiber from a liquid solution. A machine prototype has been constructed and used in the experiments to evaluate the technique. The important factors include machine vibrations that can influence the fiber size and the ability to control straightness and gap size of the electrospun nanofibers. Also, incomplete solidification of the fibers before deposition could obstruct the

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FIGURE 21.4

Surface modification by layer-by-layer deposition method.

control of layer thickness. Improvement on vibration suppression and fiber solidification will strengthen the capability of this ESRP technique.21 2.1.6 Layer-by-Layer Deposition Layer-by-layer (LbL) deposition is a thin-film fabrication technique that fabricates thin films by depositing alternating layers of oppositely charged materials (Fig. 21.4). LbL technique has been reported for the fabrication of a stable collagen hyaluronic acid polyelectrolyte multilayer (Col/HA PEM) film on titanium-based implants via covalent immobilization, and its efficiency was compared with the film fabricated using simple adsorption methods.22 The results of the study confirmed that the chemical stability of PEM film contributed to improved covalent cross-linking. Furthermore, cell culture analysis of these chemically stabled PEM films using human mesenchymal stem cells (hMSCs) as model stem cells resulted in enhanced cell functions such as attachment, spreading, proliferation, and differentiation in comparison with the simply absorbed Col/HA PEM scaffold. This observation supported the efficient behavior of covalently immobilized Col/HA PEM on titanium-based implants compared with the absorbed Col/HA PEM.23

2.2.1 Adsorption via Covalent Bonding Unlike physical adsorption, this method requires characteristic covalent bonding or at least electrostatic attraction between the substrate and the adsorbed molecules. Because of strong covalent or electrostatic forces governing the stability of chemically adsorbed biomaterials, this method shows higher surface stability than the physical adsorption of the biomaterials. Additionally, this method has been proven to be highly biocompatible in terms of cell growth and bodily fluid flow. Chemical adsorption technique has been widely used for immobilization of biomolecules on the surface of various polymeric biomaterials through covalent bonding (Fig. 21.5).27 The nature of covalent bonding (strong or weak interaction) between the reactive groups on the exposed surfaces is the prime determinant for the physical and chemical properties of the surface-modified biomaterial. For instance, in a study, adhesive peptides, arginylglycylaspartic acid (RGD) peptides, and peptides mapped on human vitronectin were reported to covalently adsorb onto the oxidized titanium substrate to improve the endogenous implant integration for bone tissue engineering.28 In another recent study, immobilization of osteogenic growth factor (bone morphogenetic protein2) via catechol chemistry on PLGA scaffolds precoated with polydopamine (pDA) were reported to facilitate the enhanced in vitro osteogenic differentiation and calcium mineralization of human ADSCs.29

2.2 Chemical Approach Different chemical surface modification techniques involve surface activation, that is, synthesis of functionalities on the surface of biomaterials. Some of the common chemical approaches for surface modifications are alkali hydrolysis, covalent adsorption, and wet chemical method. Other famous chemical surface modification techniques are photochemical modification,24 covalent interactions, ionic interactions, acid etching,25 and plasma treatment26 for the synthesis of biomaterials applicable in tissue engineering.

2.2.2 Alkali Acid Hydrolysis Method Alkali acid hydrolysis method includes diffusion of small protons between the polymer chains for cleaving ester bonds on the surface and performing surface hydrolysis, thereby resulting in the formation of carboxyl and hydroxyl functional groups. For example, a traditional alkali acid hydrolysis method has been used to enhance the hydrophilicity of PLLA-based scaffolds. With the addition of citric acid wash cycles, a significant decrease in the surface water contact angle of PLLA film

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FIGURE 21.5 Schematic diagram of chemical surface modification of biomaterial’s surface via covalent bonding for bone tissue regeneration.

FIGURE 21.6 Alkali and acid hydrolysis method for surface modification.

was observed while the surface roughness of the film increased dramatically (Fig. 21.6).30 2.2.3 Self-Assembled Monolayer Self-assembly is an energy-saving process to prepare patterns on the biomaterial surfaces in situ. At a critical point, intermolecular or interparticle forces make molecules or nanoparticles arrange themselves in a regular pattern to minimize total free energy of the entire surface. Molecules or nanoparticles can self-assemble in an area ranging from nanoscale to microscale. Many types of materials have already been used in selfassembly of patterns, such as block copolymers,31 nanospheres,32 nanoparticles,33 biomolecules,34 and so on (Fig. 21.7). For instance, studies have demonstrated that the immobilization of the aldehyde- or ketonefunctionalized ligand molecules for the attachment of peptides can be achieved via formation of SAMs of oxyamine- and oligo(ethylene glycol)-terminated thiols on

gold substrate. Furthermore, these ligand molecules were immobilized with the thrombopoietin mimetic peptide RILL for increasing the bioactivity of the substrate. Interestingly, this functionalized substrate was found to support the growth of hematopoietic CD34þ cells comparable with the standard thrombopoietinsupplemented culture.35 2.2.4 Plasma Treatment Plasma treatment facilitates the deposition of ultrathin adherent coatings in nanometer-thickness range by filling a low-pressure gas (e.g., argon, ammonia, or oxygen) in the plasma chamber. Several plasma-based technologies have been developed to contently immobilize proteins or biomolecules onto the biomaterial surface. Sorkia et al. reported surface modification of a thin porous poly(L-lactide-co-caprolactone) membrane by atmospheric plasma processing to enable the formation of functional human embryonic stem cellederived

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FIGURE 21.7 Schematic representation of surface modification by self-assembly method.

retinal pigment epithelial (hESC-RPE) monolayer followed by coating with collagen IV to enhance hESCRPE cell growth and maturation. It is considered as a potential tissue-engineered construct for regenerative retinal repair applications.36

3. BIOLOGICAL RELEVANCE OF SURFACE MODIFICATION FOR BIOMATERIALS Surface modification techniques of the biomaterials allow alteration of properties (biocompatibility) to enhance performance of a stem cell/biomaterial system in a biological environment while retaining bulk properties of the system. Being milestone in regenerative medicine, stem cells have been extensively studied because of their functional characteristics of self-renewal and multilineage differentiation potential. Stem cells are regulated by a combination of physical and chemical factors from the extracellular surrounding. To mimic the complexities of the stem cell natural microenvironment, surface-modified biomaterials have been actively investigated as artificial extracellular matrices. In this section, authors will discuss the highlights related to the application of design or different types of surfacemodified biomaterials in controlling stem cell/cell behavior, fate, and functions.

3.1 Cellular Responses of Patterned Self-Assembled Monolayers Emerging studies in stem cell biology and engineering are particularly reliant on well-defined model substrates and rapid, highly controllable fabrication

methods for characterizing the wide array of stem celle material interactions. Therefore, the patterning of SAMs has emerged as a thrust area for positioning the stem cells to control cell shape, growth, and function. A variety of techniques have been developed for generating the micropatterned and nanopatterned surfaces such as photolithography, microcontact printing, etc. Murphy et al. have developed SAMs cell culture substrates with multiple discrete regions of controlled peptide identity and density (Fig. 21.8).46 The method includes replacement of bioinert, hydroxyl-terminated oligo (ethylene glycol) alkanethiolate SAMs with mixed SAMs of hydroxyl- and carboxylic acideterminated oligo(ethylene glycol) alkanethiolates by using a NaBH4 solution. This substrate was then covalently linked with the cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) though the carboxylic acideterminated mixed SAM regions, which were further tested by culturing hMSCs. This unique assembly resulted in the substrate, with cell-adhesive microenvironments, within a bioinert background. The results indicated that immobilized RGDSP promoted spatially localized attachment of hMSCs within specified regions, while maintaining a stable, bioinert background in serum-containing cell culture conditions for up to 14 days. The hMSCs attachment to the patterned regions with a range of cell adhesion peptide densities confirmed that substrates contain discrete and well-defined microenvironments, which could ultimately enable high-throughput screening of the stem cells.46

3.2 Extracellular Protein-Absorbed Surfaces Surface topography can be used to guide different biological molecules and stem cells fate and function

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FIGURE 21.8 Self-assembled monolayers-based cell culture substrates with multiple discrete regions of controlled peptide identity and density cultured with human mesenchymal stem cells. From Koepsel JT, Murphy WL. Patterning Discrete Stem Cell Culture Environments via Localized Self-Assembled Monolayer Replacement. Langmuir 2009;25(21):12825e12834.

that can be applied to a wide research activity. One important approach is the attachment of various extracellular and plasma proteins or mimetics such as polyD-lysine, laminin, FN, vitronectin, gelatin, matrigel, and collagen to the biomaterial surfaces by means of different surface treatment protocols. These ECM proteinecoated surfaces have been optimized for different cell types to understand the scaffoldecell interactions. In 2007, Mayer et al. assessed the effect of different protein precoatings on elastomeric poly(glycerol sebacate) with endothelial progenitor cells.47 Scaffolds were precoated with laminin, FN, fibrin, collagen type I/III, or elastin. The results confirmed the increased cellularity and altered phenotypes of endothelial progenitor cells, which lead to changes in cellular behavior and ECM production.47 Therefore, extracellular proteinabsorbed surfaces hold promise for more precise “engineering” of cellular and phenotypic behaviors for stem cells. Though the extracellular protein absorption technique is simple and easy, a lack of control over the absorbed protein’s conformations, variability in source and composition of extracellular proteins, uncontrolled degradation and lack of flexibility in surface properties of the biomaterials are the few issues that can affect the reliability and reproducibility of the technique.

3.3 Dynamic Surfaces The surface interactions of biomaterials with cells are fundamental to determine critical issues such as host response and biocompatibility. Indeed, surface interactions can be directly related to the cell behavior. For instance, in a natural biological environment, cellular interactions are dynamic in nature, whereas in artificially synthesized engineered surfaces, signals are in the static form. It is very difficult to create a dynamic system that can change together

with the development and growth of the cells maintained in a defined feedback loop between stem cells and their extracellular environment. This has become one of the major problems in optimizing celle biomaterial systems for different applications. The versatility of the surface-engineered biomaterials enables the engineering of switchable surfaces, which can change the environmental parameters (temperature, pH, magnetic field, or electrical potential) in response to external stimulus.48,49 For example, Yousaf et al. reported a cell surface molecular engineering strategy via liposome fusion delivery to create a dual photoactive and bio-orthogonal cell surface for remote-controlled spatial and temporal manipulation of microtissue assembly and disassembly (Fig. 21.9).50 Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation.50 Similarly, Yui et al. synthesized ABA block copolymers composed of highly methylated polyrotaxane and hydrophobic anchoring terminal segments containing methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate [PMB (OMe-PRX-PMB)] as a platform of molecularly dynamic biomaterials.51 The results revealed that the OMe-PRX-PMB surface allowed low platelet adhesion and high fibroblast adhesion, emphasizing that molecular movement on biomaterial surfaces could be one of the important parameters in the regulation of a nonspecific biological response.51 Therefore, these smart surfaces offer a fundamentally new paradigm for the engineering of interfacial properties as they undergo reversible conformational transition at the molecular level. The controlled conformational reorientations of single-layered molecules induced observable changes in wettability, raising hope in implicating dynamic regulation of macroscopic properties.

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FIGURE 21.9

Schematic describing the molecular-level control of tissue assembly and disassembly via a chemoselective, bioorthogonal, and photo-switchable cell surface engineering approach. From Luo W, Pulsipher A, Dutta D, Lamb BM, Yousaf MN. Remote control of tissue interactions via engineered photo-switchable cell surfaces. Sci Rep 2014;4:6313.

3.4 Surfaces With Specific Signaling Functions Cell contacts with the biomaterial surface are major determinants of cell behavior and performance, and this fact has significantly shifted the focus of surface modifications for biomaterial designs toward the bioactive materials that can modulate and control cell behavior. The trends in biomaterial designs have focused on the attachment of signaling molecules into scaffold materials instead of diffusive or soluble solution forms. This emerging technology of cell typeespecific surfaces has high significance in tissue engineering and regenerative medicine. Some of the most studied multifunctional proteins include growth factors, cytokines, and small molecules such as neurotransmitters. For instance, Schmidt et al. have reported covalent immobilization of nerve growth factor (NGF) on patterned polydimethyl siloxane surfaces to induce neuronal responses.52 The results confirmed NGF as the major component in the enhancement of axon length (approximately 10%) of the cultured embryonic hippocampal neurons.52 Interestingly, Zandstra et al. have explored this approach for maintaining the stemness of

the used mouse embryonic stem cells (mESCs).53 Cytokines such as leukemia inhibitory factor (LIF) and stem cell factor were immobilized onto maleic anhydride copolymer thin-film coatings to investigate control over stem cell fate through spatially constrained ligand presentation that provides quantitative control of the signaling ligands at the cellebiomaterial interfaces. The results revealed that mESCs cultured on immobilized LIF surfaces retained pluripotency for 2 weeks in culture and were capable of activating LIF-specific cellular signaling pathways to support self-renewal of mESCs in a dose-dependent manner.53 Therefore, these approaches follow a trend toward the biomaterial designs where stem cell performance can be tuned through the control of dose and spatial distribution of signaling molecules.

4. CONCLUDING REMARKS Surface modification strategies and methodologies of biomaterials responsible for controlling stem cell fate

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and functions have been discussed in the context of tissue engineering and regenerative medicine. The experimental illustrations summarized highlight the application of various surface modification techniques such as absorption, covalent modification, surface patterning, etc. The result of these illustrations, along with other studies, clearly denotes the potential of surface-modified biomaterials to regulate stem cell growth and function. However, these surface modification methods shows great promise for the translation of biomaterials into clinical research, but it still needs to overcome different biocompatibility and postoperative complications. Challenges such as hemocompatibility, antibacterial, anticoagulant, and antiproliferative complications need to be addressed. The different approach of surface modifications (dynamic surfaces, for example) continues to be an exciting area of research for manipulating cellular assemblies in a controlled fashion. This is an exciting time to be involved in surface modification of biomaterials with new innovative microand nanotechnological techniques to develop them as a promising synthetic ECM for stem cell engineering, with great challenges and expectations lying ahead.

ABBREVIATIONS AND ACRONYMS ADSCs Adipose-derived stem cells BMP-2 Bone morphogenetic protein-2 Col Collagen ECM Extracellular matrix ESRP Electrospinning-based rapid prototyping FN Fibronectin HA Hyaluronic acid HAp Hydroxyapatite hESCs-RPE Human embryonic stem cellederived retinal pigment epithelial hMSCs Human mesenchymal stem cells IBAD Ion beameassisted deposition IBD Ion beam deposition LB LangmuireBlodgett LbL Layer-by-layer LIF Leukemia inhibitory factor mESCs Mouse embryonic stem cells MPC-2 Methacryloyloxyethyl phosphorylcholine NaBH4 Sodium borohydride NGF Nerve growth factor PCL Polycaprolactone pDA Polydopamine PEM Poly electrolyte multilayer PLA Poly-L-lactide PLD Pulsed-laser deposition PLGA Poly(D,L-lactide-co-glycolide) PLLA Poly-L-lactic acid PMB n-Butyl methacrylate PVA Polyvinyl alcohol RGD Arginylglycylaspartic acid RGDSP Arg-Gly-Asp-Ser-Pro RP Rapid prototyping SAMs Self-assembled monolayers

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Glossary Adsorption Adhesion of atoms, ions, or molecules from a gas, liquid, or dissolved solid to a surface. Amphiphilic A molecule or protein having both hydrophilic and hydrophobic parts. Bioinert Materials that do not initiate a response or interact when introduced to biological tissues. Biomaterial A biological or synthetic substance, which can be introduced into the human body for restoring or regenerating the defective/damaged tissues or organs. Biomolecules A molecule that is involved in the maintenance and metabolic processes of living organisms. Bioresorbable Materials that could be dissolved or absorbed in the human body. Conformational change It is a change in the shape of a macromolecule, often induced by environmental factors. Cytokines Any of a number of substances, such as interferon, interleukin, and growth factors, which are secreted by certain cells of the immune system and have an effect on other cells. ECM Collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. Microenvironment The immediate small-scale environment of an organism or a part of an organism. Patterning It is the act of decorating surfaces with a recurring design using cells or organic molecules. Plasticity The adaptability of stem cells to changes in its environment or differences between its various habitats. Scaffolds Templates made up of biomaterials to provide mechanical support to the residing cells and guide their growth. Self-assembly The process by which a complex macromolecule (as collagen) or a supramolecular system (as a virus) spontaneously assembles itself from its components. Stem Cells An undifferentiated cell of a multicellular organism that is capable of giving rise to indefinitely more cells of the same type and from which certain other kinds of cells arise by differentiation. Surface charge It is the electrical potential difference between the inner and outer surface of the dispersed phase in a colloid. Surface functionalization It is the act of modifying the surface properties of a material through physical, chemical, or biological ways. Surface roughness It is a component of surface texture. It is quantified by the deviations in the direction of the normal vector of a real surface from its ideal form. Tissue engineering It is the study of the growth of new connective tissues, or organs, from cells and a collagenous scaffold to produce a fully functional organ for implantation back into the donor host. Topography The arrangement of the natural and artificial physical features of an area. Wettability It is the interaction between fluid and solid phases. Wettability can be defined by the contact angle of the fluid with the solid phase.

Acknowledgments This work was supported by the Center for Stem Cell Research (CSCR). The authors Deepti Rana and Keerthana Ramasamy thank CSCR for the award of junior research fellowships and Maria Leena for awarding short-term project, respectively.

References 1. Rana D, Sampath Kumar TS, Ramalingam M. Cell-laden hydrogels for tissue engineering. J Biomater Tissue Eng 2014;4:507e35.

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342

21. SURFACE FUNCTIONALIZATION OF BIOMATERIALS

2. Rana D, Ramalingam M. Impact of nanotechnology in induced pluripotent stem cells-driven tissue engineering and regenerative medicine. J Bionanosci 2015;9:13e21. 3. Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Microscale technologies for tissue engineering and biology. Proc Natl Acad Sci USA 2006;103:2480e7. 4. Mashhadikhan M, Soleimani M, Parivar K, Yaghmaei P. ADSCs on PLLA/PCL hybrid nanoscaffold and gelatin modification: cytocompatibility and mechanical properties. Avicenna J Med Biotechnol 2015;7(1):32e8. 5. Jiao Y-P, Cui F-Z. Surface modification of polyester biomaterials for tissue engineering. Biomed Mater 2007;2:R24e37. 6. Murugan R, Molnar P, Rao KP, Hickman JJ. Biomaterial surface patterning of self assembled monolayers for controlling neuronal cell behavior. Int J Biomed Eng Technol 2009;2:104e34. 7. Ibara A, Miyaji H, Fugetsu B, Nishida E, Takita H, Tanaka S, Sugaya T, Kawanami M. Osteoconductivity and biodegradability of collagen scaffold coated with nano-b-TCP and fibroblast growth factor 2. J Nanomater 2013;201. 8. Yang L, Yaseen M, Zhao X, Coffey P, Pan F, Wang Y, Xu H, Webster J, Lu JR. Gelatin modified ultrathin silk fibroin films for enhanced proliferation of cells. Biomed Mater 2015;10(2). 9. Conway A, Schaffer DV. Biophysical regulation of stem cell behavior within the niche. Stem Cell Res Ther 2012;3:50. 10. Yang Z, Wu Y, Li C, Zhang T, Zou Y, Hui JH, et al. Improved mesenchymal stem cells attachment and in vitro cartilage tissue formation on chitosan-modified poly(L-lactide-co-epsilon-caprolactone) scaffold. Tissue Eng Part A 2012;18:242e51. 11. Wojak-Cwik IM, Hintze V, Schnabelrauch M, Moeller S, Dobrzynski P, Pamula E, et al. Poly(L-lactide-co-glycolide) scaffolds coated with collagen and glycosaminoglycans: impact on proliferation and osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res Part A 2013;101:3109e22. 12. Mavis B, Demirtas TT, Gu¨mu¨sderelio glu M, Gu¨ndu¨z G, Colak U. Synthesis, characterization and osteoblastic activity of polycaprolactone nanofibers coated with biomimetic calcium phosphate. Acta Biomater 2009;5:3098e111. 13. Campos DM, Gritsch K, Salles V, Attik GN, Grosgogeat B. Surface entrapment of fibronectin on electrospun PLGA scaffolds for periodontal tissue engineering. Biores Open Access 2014;3:117e26. 14. Gaines GL. An introduction to ultrathin organic films from LangmuireBlodgett to self-assembly. J Colloid Interface Sci 1991; 147:289. 15. Lo WJ, Grant DM, Ball MD, Welsh BS, Howdle SM, Antonov EN, et al. Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy. J Biomed Mater Res 2000;50:536e45. 16. Prosecka´ E, Buzgo M, Rampichova´ M, Kocourek T, Kochova´ P, Vyslou zilova´ L, et al. Thin-layer hydroxyapatite deposition on a nanofiber surface stimulates mesenchymal stem cell proliferation and their differentiation into osteoblasts. J Biomed Biotechnol 2012;2012. 17. Qiu Z-Y, Chen C, Wang X-M, Lee I-S. Advances in the surface modification techniques of bone-related implants for last 10 years. Regen Biomater 2014;1(1):67e79. 18. Chen C, Lee I-S, Zhang S-M, et al. Biomimetic fibronectin/mineral and osteogenic growth peptide/mineral composites synthesized on calcium phosphate thin films. Chem Commun 2011;47:11056e8. 19. Balla VK, Banerjee S, Bose S, Bandyopadhyay A. Direct laser processing of a tantalum coating on titanium for bone replacement structures. Acta Biomater 2010;6:2329e34. 20. Peltola SM, Melchels FPW, Grijpma DW, Kelloma¨ki M. A review of rapid prototyping techniques for tissue engineering purposes. Ann Med 2008;40:268e80. 21. Chanthakulchan A, Koomsap P, Auyson K, Supaphol P. Development of an electrospinning-based rapid prototyping for scaffold fabrication. Rapid Prototyp J 2015;21(3):329e39.

22. de Villiers MM, Otto DP, Strydom SJ, Lvov YM. Introduction to nanocoatings produced by layer-by-layer (LbL) self-assembly. Adv Drug Deliv Rev 2011;63:701e15. 23. Ao H, Xie Y, Tan H, Yang S, Li1 K, Wu X, et al. Fabrication and in vitro evaluation of stable collagen/hyaluronic acid biomimetic multilayer on titanium coatings. J R Soc Interface 2013;10(84). 24. Khan M, Yang J, Shi C, Lv J, Feng Y, Zhang W. Surface tailoring for selective endothelialization and platelet inhibition via a combination of SI-ATRP and click-chemistry using Cys-Ala-Gly-peptide. Acta Biomater 2015;20:69e81. 25. Orsini E, Giavaresi G, Trire` A, Ottani V, Salgarello S. Dental implant thread pitch and its influence on the osseointegration process: an in vivo comparison study. Int J Oral Maxillofac Implants 2012;27:383e92. 26. Liu W, Zhan J, Su Y, Wu T, Wu C, Ramakrishna S, et al. Effects of plasma treatment to nanofibers on initial cell adhesion and cell morphology. Colloids Surf B Biointerfaces 2014;113:101e6. 27. Rosellini E, Cristallini C, Guerra GD, Barbani N. Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering. J Biomater Sci Polym Ed 2015;21: 1e19. 28. Bagno A, Piovan A, Dettin M, Chiarion A, Brun P, Gambaretto R, et al. Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides. Bone 2007;40: 693e9. 29. Ko E, Yang K, Shin J, Cho SW. Polydopamine-assisted osteoinductive peptide immobilization of polymer scaffolds for enhanced bone regeneration by human adipose-derived stem cells. Biomacromolecules 2013;14:3202e13. 30. Guo C, Xiang M, Dong Y. Surface modification of poly (lactic acid) with an improved alkali-acid hydrolysis method. Mater Lett 2015; 140:144e7. 31. Mai Y, Eisenberg A. Self-assembly of block copolymers. Chem Soc Rev 2012;41:5969. 32. Yang Z, Xu F, Zhang W, Mei Z, Pei B, Zhu X. Controllable preparation of multishelled NiO hollow nanospheres via layer-by-layer self-assembly for supercapacitor application. J Power Sources 2014;246:24e31. 33. Ruan W, Zhou T, Cui Y, Dong Y, Liu Z, Dong F, et al. Submicron patterns obtained by thermal-induced reconstruction of selfassembled monolayer of Ag nanoparticles and their application in SERS. Appl Surf Sci 2014;309:295e9. 34. Bajaj P, Schweller RM, Khademhosseini A, West JL, Bashir R. 3D biofabrication strategies for tissue engineering and regenerative medicine. Annu Rev Biomed Eng 2014;16:247e76. 35. Lee E-J, Be CL, Vinson AR, Riches AG, Fehr F, Gardiner J, Gengenbach TR, Winkler DA, Haylock D. Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34þ cells. Biomaterials 2015;37:82e93. 36. Sorkio A, Porter PJ, Juuti-Uusitalo K, Meenan BJ, Skottman H, Burke GA. Surface modified biodegradable electrospun membranes as a carrier for human embryonic stem cell derived retinal pigment epithelial cells. Tissue Eng Part A 2015;21(17e18): 2301e14. 37. Caseli L, Cavalheiro RP, Nader HB, Lopes CC. Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: a Langmuir film study. Biochim Biophys Acta 2012;1818(5):1211e7. 38. Mas-Moruno C, Garrido B, Rodriguez D, Ruperez E, Gill FJ. Biofunctionalization strategies on tantalum-based materials for osseointegrative applications. J Mater Sci Mater Med 2015;26:109. 39. Oh B, Melchert RB, Lee CH. Biomimicking robust hydrogel for the mesenchymal stem cell carrier. Pharm Res 2015;32:3213e27. 40. Dusad A, Chakkalakal D. Titanium implant with nanostructured zirconia surface promotes maturation of peri-implant bone in osseointegration. Proc Inst Mech Eng Part H 2013;227:510e22.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

REFERENCES

41. Schwartzman D, Pasculle AW, Ceceris KD, Smith JD, Weiss LE, Campbell PG. An off-the-shelf plasma-based material to prevent pacemaker pocket infection. Biomaterials 2015;9:1e8. 42. Pop-Georgievski O, Kubies D, Zemek J, Neykova N,  Demianchuk R, Cha´nova´ EM, Slouf M, Houska M, Rypacek F. Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability. Beilstein J Nanotechnol 2015;2:617e31. 43. Kim SE, Wang J, Jordan AM, Korley LT, Baer E, Pokorsik JK. Surface modification of melt extruded poly(ε-caprolactone) nanofibers: toward a new scalable biomaterial scaffold. ACS Macro Lett 2014;3:585e9. 44. Lee C, Shin J, Lee JS, Byun E, Ryu JH, Um SH, et al. Bioinspired, calcium-free alginate hydrogels with tunable physical and mechanical properties and improved biocompatibility. Biomacromolecules 2013;14:2004e13. 45. Cao B, Qiu P, Mayo C. Mesoporous iron oxide nanoparticles prepared by polyacrylic acid etching and their application in gene delivery to mesenchymal stem cells. Microsc Res Tech 2013;76:936e41. 46. Koepsel JT, Murphy WL. Patterning discrete stem cell culture environments via localized self-assembled monolayer replacement. Langmuir 2009;25:12825e34. 47. Sales VL, Engelmayr GC, Johnson JA, Gao J, Wang Y, Sacks MS, et al. Protein precoating of elastomeric tissue-engineering scaffolds

48.

49.

50.

51.

52.

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increased cellularity, enhanced extracellular matrix protein production, and differentially regulated the phenotypes of circulating endothelial progenitor cells. Circulation 2007;116(Suppl. 11): I55e63. Ebara M, Uto K, Idota N, Hoffman JM, Aoyagi T. Shape-memory surface with dynamically tunable nano-geometry activated by body heat. Adv Mater 2012;24:273e8. Le DM, Kulangara K, Adler AF, Leong KW, Ashby VS. Dynamic topographical control of mesenchymal stem cells by culture on responsive poly(caprolactone) surfaces. Adv Mater 2011;23: 3278e83. Luo W, Pulsipher A, Dutta D, Lamb BM, Yousaf MN. Remote control of tissue interactions via engineered photo-switchable cell surfaces. Sci Rep 2014;4:6313. Seo Ji-H, Kakinoki S, Inoue Y, Yamaoka T, Ishihara K, Yui N. Designing dynamic surfaces for regulation of biological responses. Soft Matter 2012;8:5477e85. Gomez N, Lu Y, Chen S, Schmidt CE. Immobilized nerve growth factor and microtopography have distinct effects on polarization versus axon elongation in hippocampal cells in culture. Biomaterials 2007;28:271e84. Alberti K, Davey RE, Onishi K, George S, Salchert K, Seib FP, et al. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods 2008;5:645e50.

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22 Biofunctional Hydrogels for Three-Dimensional Stem Cell Culture Jenna L. Wilson1, Todd C. McDevitt2 1

Georgia Tech Bioengineering, Atlanta, GA, United States; 2University of California at San Francisco (UCSF), San Francisco, CA, United States

O U T L I N E 1. Introduction 1.1 Applications of 3D Stem Cell Culture 1.1.1 Studying Stem Cell Behavior and Niche Interactions 1.1.2 Creating Tissue-Engineered Constructs 1.1.3 Improving Cell Engraftment and Survival In Vivo 1.1.4 Stem Cell Expansion and Biomanufacturing 1.1.5 Drug Discovery and Toxicity Testing

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2. Hydrogel Materials 2.1 Naturally Derived Materials 2.1.1 Protein-Based Materials 2.1.2 Polysaccharide Materials 2.2 Synthetic Materials 2.3 Hybrid Materials

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3. Hydrogel Material Properties 3.1 Degradation 3.2 Topography 3.3 Matrix Mechanics 3.4 Mass Transport Properties 3.5 Quantitative Properties of Hydrogels

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1. INTRODUCTION There are significant differences between the native environment of stem cell populations and the artificial in vitro culture platforms used by the majority of scientists today. For example, pluripotent stem cells (PSCs) Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00022-6

4. Engineering Hydrogel Bioactivity 4.1 Cell Adhesion Motifs 4.2 Growth Factor Presentation 4.3 Stimuli-Responsive Materials 5. Technologies for Hydrogel Production and Assessment 5.1 Hydrogel Production Methods 5.1.1 Formation of Microparticles 5.1.2 Microencapsulation Methods 5.1.3 Microfabrication Techniques for Bulk Hydrogels 5.2 Translating 2D Assays to 3D Systems

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6. Challenges and Future Directions 6.1 Spatiotemporal Control Over Biophysical and Biochemical Hydrogel Properties 6.2 Promoting Endogenous Tissue Morphogenesis

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7. Conclusion

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Abbreviations and Acronyms

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References

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exist transiently in the preimplantation blastocyst constituting the inner cell mass, a tightly packed cluster of cells, which is encased in a layer of epithelial trophoblast and surrounded by the fluid of the blastocoel cavity.1 In contrast, bone marrow mesenchymal stem/ stromal cells (MSCs), which are multipotent cell

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Copyright © 2017 Elsevier Inc. All rights reserved.

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FIGURE 22.1 In a two-dimensional monolayer (left), stem cells are polarized by attachment to a stiff plastic surface on a single side and have limited interactions with other cells and 3D extracellular matrix components. In contrast, a 3D hydrogel environment (right) provides cues in three dimensions and enables cellecell and cellematrix interactions in multiple dimensions.

populations present throughout adult life, are encompassed in a semisolid matrix consisting of various collagens, proteoglycans (PGs), and glycosaminoglycans (GAGs).2,3 Despite these distinct yet similarly complex microenvironments, in vitro studies of embryonic stem cells (ESCs) and MSCs are generally conducted in the same manner: as two-dimensional (2D) cell monolayers on plastic surfaces. In typical 2D culture, cells are specifically polarized by attachment to a plastic surface on one side and contact with liquid culture medium on the other (Fig. 22.1). Interactions between neighboring cells occur laterally within a single plane, if at all, and the threedimensional (3D) physical cues presented by interstitial extracellular matrices are absent entirely. Because celle matrix and cellecell interactions dictate much of a cell’s behavior, it is logical that the attenuation of these cues can lead to cell dysfunction, and this has been observed in cell types ranging from liver hepatocytes to ovarian and colorectal cancer cells.4e6 Therefore, a greater emphasis on 3D culture platforms for stem cell culture may provide more accurate insights into in vivo cell physiological function and enable more advanced applications, such as tissue-engineered constructs, biomanufacturing approaches, and platforms for drug discovery and toxicity testing. There are several standard approaches for 3D culture of mammalian cells, including the formation of scaffoldfree microtissues (often in the form of spheroids or aggregates), which offer limited exogenous control over extracellular matrix (ECM) properties, or the seeding of cells onto porous scaffolds or decellularized ECM from native tissues.7 However, perhaps the most pervasive technique has been culture of cells within hydrogel scaffolds. Hydrogels, which are simply hydrated polymer networks, share many key physical properties with native tissues, including a high water content, a similar range of elasticities, and mass transport characteristics that permit the diffusion of external stimuli and internal autocrine and paracrine signals. Additionally, depending on the source or modification of the

hydrogel polymer, the gels can serve as a platform to integrate specific biological elements in a wellcontrolled manner and consequently offer superior control when compared with cell or tissue-derived ECM.8 Thus, this chapter will focus on the use of hydrogels as platforms for 3D culture of stem cell populations, with a specific emphasis on systems that provide biofunctionality as opposed to simple biocompatibility (Fig. 22.2).

1.1 Applications of 3D Stem Cell Culture 1.1.1 Studying Stem Cell Behavior and Niche Interactions The function of stem cell populations in vivo is still being actively investigated to provide insights into cell regulators, which can be translated to in vitro differentiation protocols, and using typical 2D culture methods may lead to an unintentional bias in the interpretations of stem cell phenotype and function. Unfortunately, performing studies of complex niche interactions in vivo is technically challenging because of limitations in imaging and real-time monitoring. Thus, engineering hydrogel properties to mimic physical and chemical elements of the native stem cell niche may enable the discovery of novel insights into cell physiology and interactions with the surrounding microenvironment, both in terms of matrix properties and the soluble milleu.9,10 Depending on the degree to which a stem cell niche has been previously characterized, hydrogels can be designed to specifically simulate known niche components. For example, culture of MSCs in hydrogels constructed with collagen, a major component of the bone marrow niche, enhanced MSC secretion of angiogenic factors (compared with 2D cultures) and augmented chondrogenic differentiation of MSCs, as evidenced by increased GAG and collagen production, when compared with noncollagen-based hydrogels.11,12 In addition to mimicking niche material composition,

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FIGURE 22.2 Culture of stem cells in 3D hydrogels enables many downstream applications, including investigation of the stem cell niche, the creation of tissue-engineered constructs, advanced drug screening platforms, biomanufacturing systems, and improved cell survival and function posttransplantation.

simulating the mechanical properties of the native niche can enhance stem cell function, as has been observed with skeletal muscle satellite cells cultured on substrates with mechanical properties similar to native muscle.13 In many cases though, specific niche components are not well defined and/or their functional effects are poorly understood, and thus design criteria for engineering hydrogel materials are lacking. Recent advances in material microarray technologies have attempted to address this dearth of information by allowing for highthroughput combinatorial analysis of cell response under many different conditions. In recent work seeking to investigate factors capable of promoting the self-renewal of murine ESCs, an array of more than 1000 variations of synthetic polyethylene glycol (PEG) hydrogels was created with varying mechanical properties (elastic modulus of 300e5400 Pa), sensitivity to matrix metalloproteinase (MMP) degradation, and inclusion of ECM (collagen, fibronectin, and laminin) and cellecell interacting (E-cadherin, Jagged, and epithelial cell adhesion molecule) proteins.14 The wide range of hydrogel properties in combination with differential cell densities and soluble factor [fibroblast growth factor 4 (FGF4), bone morphogenetic protein-4 (BMP-4), and leukemia inhibitory factor (LIF)] in addition provided a large data set in which systems biology approaches identified key regulators of ESC self-renewal. Similar combinatorial platforms of 3D microenvironments could be similarly applied to other

systems to determine the role of specific niche components in cell processes in a controlled manner. 1.1.2 Creating Tissue-Engineered Constructs Since the original principles of tissue engineering first emerged in the 1980s, the model of seeding cells onto/ within a scaffold has served as a paradigm for the entire field. However, the functional role of scaffold materials has shifted away from simple physical support structures and emerged as an essential contributor to the overall biophysical and biochemical properties of engineered tissues. The combination of stem cells or stem cellederived cell types with growth factors on a scaffold remains the iconic vision of a tissue-engineered product.15 However, tissue engineering and regenerative medicine applications have not yet attained the widespread clinical use that was predicted, primarily because of a continued lack of understanding about cellular processes in vitro and in vivo and the inability to recapitulate known processes in an ex vivo setting. Thus, it is critical for the field of regenerative medicine that 3D platforms for stem cell culture continue to be created and assessed as platforms for complex tissueengineered constructs. 1.1.3 Improving Cell Engraftment and Survival In Vivo While greater numbers of cell-based therapies demonstrate efficacy and thus seem to be a promising

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clinical approach, most are limited by the lack of long-term survival and functional engraftment of transplanted cell populations. For example, engraftment rates of cells delivered to the heart are typically below 10% and more often in the range of 1e2%, if not lower.8,16 Part of the lack of stable engraftment may be attributed to the dysfunctional environment the cells are transplanted into, as the injured tissue typically includes damaged matrix and tissue necrosis that can adversely affect stem cell fate and subsequent function.17 Hydrogel biomaterial carriers, which tend to be conveniently injectable, can be used to provide a controlled niche with instructive cues for transplanted stem cells (or stem cellederived cells) to improve their survival, engraftment, and long-term function. In addition to protecting transplanted cells, the hydrogel itself can promote tissue regeneration through the release of soluble cues or the recruitment of endogenous stem cell populations.17 Hydrogels composed of hyaluronic acid (HA), heparin, gelatin, and PEG are able to recruit bone marrow MSC populations when modified with hepatocyte growth factor as were gelatin hydrogels containing BMP-2 and stromal cellederived factor-1 (SDF-1).18,19 In this sense, incorporating cues within the material that target host cell populations could lead to synergistic regeneration with the transplanted stem cellederived populations and ultimately yield more effective cell therapy products. 1.1.4 Stem Cell Expansion and Biomanufacturing The clinical translation of autologous stem cell therapies is dependent on the development of biomanufacturing processes to expand stem cells to the appropriate numbers, often estimated at 107e1010 cells per patient depending on the application.20 Because of the amount of surface area required to culture such large quantities of cells in 2D, moving to 3D platforms is a necessity for economic reasons and for ease of monitoring because many online sensors for temperature, pH, dissolved oxygen, etc., are designed for use in suspension bioreactors. Encapsulation of stem cells within hydrogel microbeads or microcapsules can be used to provide a defined environment for adherent stem cell populations [i.e., ESCs, induced pluripotent stem cells (iPSCs), MSCs, neural stem cells (NSCs)] while enabling culture in large-scale bioreactor systems.21 Culture within hydrogels can also protect the enclosed cells from experiencing shear forces inherent to stirred bioreactor systems, which is particularly important in the case of stem cells because of their phenotypic sensitivity to hydrodynamic forces.22 1.1.5 Drug Discovery and Toxicity Testing The successful development of new pharmaceutical compounds and screening of existing chemical libraries

is highly dependent on the context in which cells are screened for toxicity and other cellular responses. Some of the reasons for the failure of late-stage drug candidates lies with inadequacies in the predictive ability of traditional 2D cell culture methods.23 The advent of iPSCs now allows for individual patient- and phenotype-specific drug assessments, and differentiating PSC populations into more mature tissue types of interest will enable a higher degree of functional testing than is currently possible.24 Specifically, drug testing may benefit from analyzing the response of functional “organoid” structures, such as intestinal organoids and cerebral brain-like organoids, which have been formed from PSC aggregates suspended in MatrigelÔ , a natural hydrogel product derived from mouse sarcoma cells and consisting of a mixture of laminin I, collagen IV, entactin, and various PGs and growth factors.25,26 To translate organoid cultures from the basic research environment to high-throughput industrial screening processes, it will be necessary to design well-defined hydrogel systems to ensure scalability and reproducible models.27

2. HYDROGEL MATERIALS As a class of materials, hydrogel polymers can vary greatly in source and structure, making them extremely versatile for applications in the 3D culture of stem cells. Although hydrogels can be classified in several ways, such as by their structural composition, charge, degradability, cross-linking chemistry, or responsiveness to different types of stimuli, distinguishing by them by material origin allows for distinction between naturally derived “promoting” polymers, which consist of some inherent bioactive components, and synthetic “permitting” polymers, which begin as more of a blank slate for directed feature design.7,28

2.1 Naturally Derived Materials Because hydrogels are often desired as ECM mimics, it is rational that researchers often turn to polymers consisting of natural ECM components or biomolecules with similar chemical structures. Naturally derived hydrogel materials are intrinsically biocompatible and bioactive, which often leads to high cell viability and proliferation rates without the need for significant material modification. However, the specific bioactive components have the potential to convolute results, as it can be challenging to isolate the effects of individual cues on cell fate.8 For example, increasing the mechanics of a collagen hydrogel will also lead to an increase in the concentration of adhesive sites. Additional drawbacks

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include the inherent batch-to-batch variability and potential economic cost that come with the isolation and purification of a naturally derived hydrogel material.29 Furthermore, it can be difficult to tune the system for the desired mechanical properties, degradability, and bioactivity because the molecules have their own intrinsic properties that are inherent to the polymer backbone and native cross-linking mechanisms. Naturally derived hydrogel polymers can be further subdivided into protein-based materials, such as collagen, fibrin, and MatrigelÔ , and polysaccharide-based materials, including agarose, alginate, chitosan, and GAGs, such as chondroitin sulfate, heparin/heparan sulfate, and HA. 2.1.1 Protein-Based Materials 2.1.1.1 Collagen Collagen is the most abundant protein found in mammals, and therefore, it has been extensively studied as a hydrogel for 3D cell culture applications. Specifically, collagen I, which contains several cell adhesion sequences including the integrin-binding sites GFOGER (glycinee phenylalanineehydroxyprolineeglycineeglutamatee arginine) and DGEA, is present in the highest abundance and is most commonly used in hydrogel applications. Collagen I is typically isolated from bovine skin or rat tail, solubilized in an acidic buffer (typically acetic acid), and formed into a hydrogel by neutralizing the acidic solution with sodium hydroxide and heating to 37 C to create a collagen matrix.29 The resultant gels are relatively soft, typically with an elastic modulus of less than 5 kPa and thus have limited use in applications that might require the use of stiffer hydrogel materials. As a result, collagen is often combined with other materials to form hybrid hydrogels, or alternatively, the collagen is modified to enable greater chemical crosslinking, such as with glutaraldehyde, formaldehyde, and succinic anhydride.30 Degradation of collagen is mediated by several classes of proteases, including MMPs and cathepsins, which are secreted by many cell types and can promote hydrogel degradation. MSCs are often encapsulated within collagen matrices because of the naturally occurring adhesive peptide sequences in the collagen chains, which lead to improved cell viability. Self-assembled microspheres of collagen and human MSCs have been formed by suspending the cells in liquid collagen solution and inducing gelation of the resulting droplets.31 The resulting constructs could be cultured in suspension, and the collagen matrix supported MSC viability, growth, and differentiation while improving the self-renewal capacity of the cells relative to monolayer controls. Encapsulation of embryoid bodies (EBs), cellular aggregates derived from ESCs, within collagen I hydrogels has

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also enabled the recapitulation of blood vessel formation and has been used as a model to understand the sprouting processes of angiogenesis.32 2.1.1.2 Fibrin The motivation for fibrin hydrogel formation can be traced to the natural process of coagulation in which blood is transformed from a liquid to a gel-like state in response to an injury that entraps cells. After the accumulation of platelets at the site of injury, thrombin cleaves soluble fibrinogen molecules into fibrin monomers, which in turn cross-link to form a dense fibrous matrix. The resulting fibrin gel plays a significant role in the healing and regeneration of the damaged tissue and is able to interact with the surrounding cells through binding domains for integrins, heparin, and fibrinogen, and many growth factors.33 To induce fibrin hydrogel formation ex vivo, a chilled fibrinogen solution (typically 2e4 mg/mL) is combined with thrombin and cross-linked by raising the temperature to 37 C. By varying the thrombin concentration from 0.001 to 1 units of thrombin/mg of fibrinogen, a range of mechanical properties can be achieved, with the elastic modulus generally ranging from 1 to 30 kPa.29,34 In the presence of cell-secreted enzymes such as plasmin, fibrin gels begin to exhibit decreased mechanical stability and eventually degrade completely, though the inclusion of enzymatic inhibitors such as ε-aminocaproic acid can be used to slow degradation.35 Thus, alternative cross-linking chemistries, such as with formaldehyde, gluteraldehyde, and succinic anhydride, have been similarly investigated for fibrin as they have for collagen.29 As with collagen, fibrin has also been commonly used as a hydrogel scaffold for MSCs. Early studies identified appropriate fibrinogen and thrombin concentrations, which promote MSC proliferation and permitted injection in vivo, after which MSC migration out of the fibrin gel was observed.36 Fibrin gels have also been used as scaffolds for ESC culture, particularly for differentiation toward neural lineages in which EBs are cultured in fibrin gels and exposed to neurotrophic factors.37 2.1.1.3 Matrigel Unlike the single-protein composition of collagen and fibrin hydrogels, Matrigel contains a complex mixture of basement membrane proteins derived from mouse sarcoma tumors. In addition to the ECM proteins laminin I, collagen IV, entactin, and heparan sulfate proteoglyclans, a number of growth factors are also commonly present in Matrigel, including transforming growth factor-bs (TGF-bs), FGFs, epidermal growth factors, platelet-derived growth factors, and insulin-like growth factors.38 Although this complex mixture of growth factors has been observed to readily support the culture of many cell types, there is significant

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batch-to-batch variability of Matrigel and the number of factors present can obfuscate specific biological mechanisms. A growth factorereduced Matrigel was developed by using a precipitation step to remove many of the growth factors in an attempt to address some of these issues, though the molecular complexity of the hydrogel material remains.39,40 Matrigel is a thermoreversible hydrogel that exists as a liquid below 10 C and is prone to degradation by cell-secreted proteases (primarily MMPs) over time. Matrigel has been used extensively as a surface coating for 2D cultures of human PSCs as a replacement for stromal feeder layers.41 More recently, Matrigel has been used as an essential 3D matrix to permit the selforganization of stem cells into organoid structures. The formation of intestinal crypts from Lgr5þ intestinal cells was performed in Matrigel because of the similar presence of laminin in the native crypt.25 In the formation of cerebral organoids, neuroectoderm-biased EBs formed from human ESCs were embedded in Matrigel droplets and cultured in a stirred bioreactor system to induce neural tissues with features unique to specific regions of the brain.26 2.1.2 Polysaccharide Materials 2.1.2.1 Marine Origin Agarose, alginate, and chitosan are biocompatible linear polysaccharides of marine origin, with agarose obtained from red algae, alginate from brown seaweed, and chitosan from the shells of crustaceans. Agarose consists of b-D-galactopyranosyl and 3,6-anhydro-a-Lgalactopyranosyl units and exhibits a temperaturedependent gelation below a threshold temperature (usually 15e30 C, based on the extent of methylation of the source agarose). Agarose is relatively stable because mammalian cells do not produce enzymes capable of agarose degradation, but degradation is observed over long time periods (25% over the course of 60 days) because of interruption of hydrogen bonding.42 Alginate comprises two anionic monomers, a-L-guluronic acid and b-D-mannuronic acid, and is ionically cross-linked by divalent cations, typically calcium or barium.43 As with agarose, alginate is not directly degraded by cells; however, the material can break down because of chelation of the divalent cations or displacement of divalent cations with monovalent cations present in high concentration.44 Chitosan consists of b(1e4) linked glucosamine and N-acetyl glucosamine and is ionically crosslinked with divalent cations as described with alginate. Unlike agarose and alginate, chitosan can be degraded by mammalian cells that express lysozyme, an enzyme with a role in the innate immune system.45 All three polysaccharide species lack specific biological moieties; thus, they are often used in combination with other

biological materials or as blank slate materials in situations in which extracellular biological stimuli are not desired. Agarose has been used in several instances to encapsulate PSC aggregates to prevent agglomeration while promoting differentiation.46,47 Similarly, alginate has also been used to culture embryonic stem cells, typically in a microbead configuration conducive to long-term bioreactor culture.48 Though alginate is often thought of as relatively inert for a naturally derived material, differences in ESC and MSC differentiation have been observed as a result of varying the physical properties of alginate hydrogels.49,50 A hybrid chitosan-alginate porous scaffold has also been developed for feederfree expansion and maintenance of pluripotency of human ESCs for up to 21 days.51 2.1.2.2 Glycosaminoglycans Unlike the relatively inert quality of other polysaccharide species, GAGs play critical roles in many biological processes, including attachment to PGs within the ECM, recruitment of water into connective tissues to increase osmotic pressure and allow tissues to withstand compressive loading, lubrication of joint areas, and sequestration and immobilization of growth factors.52 There are several classes of GAG molecules, all of which are negatively charged linear polysaccharides with varying degrees of sulfation. HA is the only nonsulfated GAG species and has been the most extensively studied for biomaterial applications because of its ease of chemical modification because GAGs do not have an inherent cross-linking ability. Unlike most other ECM components, HA does not have integrin-binding sites for cell adhesion, though it can reportedly bind to specific cell receptors (e.g., CD44) and intercellular adhesion molecules (e.g., ICAM-1).29,53 Other GAG species, including heparin/heparan sulfate and chondroitin sulfate, have a higher net negative charge than HA and thus have an even higher affinity for positively charged growth factors.54 Hydrogels of GAG species are typically created as hybrid materials with synthetic polymers, though pure materials of chemically modified heparin and chondroitin species have also been developed.55e57 HA is found in the greatest abundance during early embryogenesis and thus has been investigated for ESC culture. Indeed, hydrogels of HA were found to promote the feeder-free self-renewal of human ESCs, and human ESCs were determined to possess active HA-binding sites and receptors.58 Additionally, HA hydrogels have been investigated for tissue-engineered skeletal muscle constructs using MSCs and enhance chondrogenic differentiation of MSC in comparison with synthetic hydrogels.59 Hydrogels modified with chondroitin sulfate (PEG-based) and heparin (collagen-based) have also

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been used to promote osteogenic and chondrogenic differentiation of MSCs.60,61

2.2 Synthetic Materials Many of the inherent limitations of hydrogels made of natural materials can be overcome with the use of synthetic polymers. With chemically defined materials, there is a priori control over cross-linker density and spacing to promote uniform quality with the desired mechanical properties. Although synthetic materials may not intrinsically offer biological information, such as adhesive cues or presentation of growth factors, to cells, bioactive and/or stimulus-responsive epitopes can be engineered into the polymers to confer biological signals.7 Many synthetic polymers have been investigated for cell encapsulation, including PEG, polyvinyl alcohol, and poly N-isopropylacrylamide (PNIPAAm). The most widely used polymer is PEG, which exhibits high hydrophilicity with low protein adsorption and can be readily modified at its hydroxyl ends.8 Cross-linking conditions that are noncytotoxic are possible with Michael-type addition reactions in which PEG modified with amine or thiol groups is mixed with methacrylicmodified PEG. Alternatively, photocross-linkable systems can be used in which acrylic or methacrylicmodified PEG is mixed with a biocompatible photoinitiator and exposed to a specific, noncytotoxic wavelength of ultraviolet (UV) light.29 The mechanical properties of PEG hydrogels are typically modulated by varying the concentration and molecular weight of the polymers; however, these changes also alter pore size and permeability of the gels. PEG is often modified with adhesion proteins (e.g., fibronectin, collagen, laminin) or more commonly with short peptide sequences (i.e., ArgeGlyeAsp, typically referred to as RGD).62 Although PEG is nondegradable, PEG hydrogels can be copolymerized with degradable polymers, such as PLGA, in varying ratios to tune the degradation kinetics.63 Alternatively, protease-cleavable crosslinkers can be incorporated to permit cell-mediated degradation.64

2.3 Hybrid Materials To combine the biological functionality of natural materials with the improved control of synthetic materials, the design of novel hybrid or semisynthetic materials has been an active area of research. In hybrid biomaterials, the synthetic polymer imparts the key physical qualities of the material, such as the mechanics and molecular structure, whereas the natural protein or polysaccharide endows the material with bioactive

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features. Typically, hybrid materials are produced through copolymerization reactions between the synthetic polymer precursor and the natural species of interest, often using free-radicaleinitiated copolymerization or click chemistry.65 Often, the protein or ECM species is covalently modified with linear or branched, nonionic hydrophilic polymers, such as PEG, and then cross-linked using standard conditions. For example, a fibrinogenePEG hybrid material was created by PEGylating denatured fibrinogen fragments with PEG diacrylate (PEGDA) groups, subsequently mixing the polymers with a photoinitiator, and crosslinking with UV light exposure.66 Although there are advantages in using hybrid materials, there are also a number of challenges, including the dependence of downstream protein function on the specific chemical configuration. For example, the quantity, length, and specific architecture (linear vs. branched vs. dendrimeric) of the polymer chain in addition to the site of attachment have all been observed to influence the ultimate bioactivity.67

3. HYDROGEL MATERIAL PROPERTIES 3.1 Degradation In the body, cells often remodel their surrounding ECM via matrix production and degradation, particularly postinjury, and these processes allow cells to migrate and spread to form cellecell contacts and produce functional tissue structures.68 Local degradation of native ECM is typically mediated by cell-secreted proteases such as MMPs, a process which can be recapitulated to a certain extent when using hydrogels derived of natural materials.69 However, many synthetic hydrogels rely on dissolution or hydrolytic degradation mechanisms, which are typically based on the density of cross-linking or the quantity of hydrolytically unstable chemical moieties and are thus not dependent on celldriven mechanisms.7 This lack of control over material degradation has motivated a number of novel approaches, including the creation of specific biohybrid materials, the incorporation of protease-cleavable peptide cross-links, and the design of photodegradable networks (Fig. 22.3). In one classic example, the creation of hydrogels of PEG modified with an RGD cell adhesion peptide and a highly sensitive MMP cleavage site enabled greater rates of fibroblast invasion and a more functional spindle-like fibroblast morphology when compared with PEG hydrogels with less sensitive proteasecleavable sites.64 In the context of 3D stem cell culture, MSCs cultured within alginate hydrogels containing the MMP degradable peptide PVGLIG in addition to

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FIGURE 22.3 Many of the key design features of hydrogels, including degradation kinetics, fiber orientation and topography, matrix mechanics, and permeability, are critical to promote cell survival and function and can be specifically tuned to mimic the native stem cell niche. Adding additional bioactive components, such as sites for cell adhesion, presentation of specific biologic signaling molecules, and stimuliresponsive elements, can be used to further regulate the environment of the encapsulated cells.

the RGD adhesion peptide exhibited a more spread morphology and formed cellular networks and extensive cellematrix interactions. In contrast, MSCs cultured within alginate containing only the RGD peptide remained round and had fewer cellecell interactions.70 Although the incorporation of protease-cleavable sequences within hydrogels allows for cell-mediated degradation, there is little external control over the system. Thus, the advent of specific photodegradable hydrogels that allow for spatiotemporal control over degradation provides for enhanced control of stem cellehydrogel environments. Hydrogels constructed of PEG modified with a nitrobenzyl etherederived moiety permit hydrogel cleavage upon exposure to UV light (365 nm), leading to local decreases in crosslink density in a nontoxic and spatially controlled manner.71 In addition to changing physical properties of the gel, photodegradation can also release or remove bioactive components. Photolabile RGD peptides incorporated into PEG gels containing MSCs were removed via irradiation after 10 days in culture, at a time when MSCs differentiating to chondrocytes downregulate

fibronectin. The specific temporal removal of the fibronectin-binding RGD peptide led to a fourfold increase of GAG production by the MSCs, suggesting enhanced chondrogenic differentiation and that the MSCs were responding to the change in extracellular signaling.72

3.2 Topography In native cell microenvironments, oriented fibers of ECM molecules provide topographical cues that influence cell polarity, migration, and ECM interactions. In fiber networks, it has been observed that smaller fiber diameters enhance cell adhesion to multiple fibers and lead cells to adopt a round morphology, as they sense traction forces in multiple dimensions. In contrast, cells on fibers with larger diameters tend to adhere to a single fiber and thus adopt an elongated morphology along the axis of the fiber because of the unidirectional traction force.73,74 To create nanofibers with polymers, electrospinning techniques are typically used, in which the polymer is

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expelled from a charged needle and the resulting fibers are collected on a grounded surface and aligned. The relative diameter and porosity of the scaffold can be adjusted by changing the concentration of the polymer, the flow rate, or the voltage.75 Although most efforts have focused on semicrystalline polymers, recent work has translated electrospinning techniques to hydrogel fibers.69,76 By electrospinning hydrogel precursors and cross-linking them in a dry rather than a hydrated state, soft electrospun hydrogels can be formed by placing the materials in an aqueous environment and allowing them to swell.77 Additionally, one of the issues with electrospinning is that small pore size may limit the infiltration and migration of cells. A novel approach to overcome this problem is to electrospin photocross-linkable polymers such that photopatterned pores can be created. MSCs cultured in electrospun HA/polyethylene oxide hydrogels with photopatterned pores exhibited increased infiltration into the aligned scaffolds.78 In addition to electrospinning, there have been a number of alternative approaches to recapitulate the fibrillar structure observed in native tissues, including the use of self-organizing peptides, which form entangled nanofibers because of sequences of alternating hydrophilic and hydrophobic side groups.73,79,80

3.3 Matrix Mechanics The mechanical microenvironment for cells within the body can vary by orders of magnitude, from very soft (e.g., w0.1 kPa) in the brain to very stiff (e.g., w80 kPa) in precalcified bone.81 Thus, to properly mimic the mechanical aspects of tissue niches, hydrogels of varying composition and biological cues should be capable of different degrees of stiffness. A wide body of work has examined the influence of mechanical stiffness on stem cell differentiation and phenotype, with the majority of these studies conducted by culturing cells in 2D on different hydrogel substrates. To examine whether the findings of 2D studies translate to 3D environments, MSCs were encapsulated in RGD-modified alginate gels produced with a wide range of mechanical properties (elastic moduli from 2.5 to 110 kPa). Similar to previous results with 2D systems, softer environments (2.5e5 kPa) promoted adipogenic differentiation of MSCs, whereas materials of intermediate stiffness (11e30 kPa) promoted osteogenic differentiation. However, the previous correlation with 2D morphology was not observed in the 3D setting (i.e., greater spreading was not observed in stiffer hydrogels), and investigation into integrin binding indicated that the underlying mechanism for the osteogenic lineage induction may be because of changes in the presentation of adhesive ligands.49

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Mechanotransductive pathways that cause the observed phenotypic changes are not yet completely understood, though recent work indicates that the transcriptional coactivators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZbinding motif), both part of the Hippo pathway, may be key regulators that can sense cytoskeletal tension.82 The translocation of YAP to the nucleus indicates activation and seems to be “switched on” based on the surrounding mechanical environment; for example, YAP is localized to the cytoplasm when cells are cultured on “soft” substrates (w1 kPa) but localizes to the nucleus when cells are cultured on a “stiff” substrate (w40 kPa).83 This switch-like behavior has led to studies examining the mechanical “memory” of MSC populations. By using photodegradable PEG hydrogels that can be “softened” (10e2 kPa) upon exposure to UV light, it was observed that while YAP/TAZ activation is reversible when switched to a soft substrate after 1 day of culture on a stiff hydrogel, MSCs cultured on the stiff hydrogels for 10 days retained active YAP/ TAZ activity for at least 10 days even after the switch to the soft hydrogel.84 The results of this study indicate that stem cells not only have immediate downstream responses to their surrounding mechanical environment, but they in fact retain a mechanical memory of their past surroundings. Studies that continue to investigate the influence of three dimensionality may provide different clues to the mechanisms underlying celle matrix mechanical interactions. One of the primary challenges in deciphering the specific role(s) of matrix mechanics on cell fate is the difficulty in separating changes in mechanical stiffness from the associated changes in hydrogel porosity and solute permeability, as the properties are inherently linked. Efforts to decouple these variables have led to a number of novel strategies, including the design of hydrogels, which incorporate methacrylic alginate with PEG dimethacrylate. In this system, the elastic modulus can be adjusted by changing the concentration of methacrylic alginate and the degree of substitution of the methacrylic groups. However, these changes do not alter the bulk hydrogel permeability because of the hydrophilicity of the network.85 Another approach has been to use different polymer configurations that exhibit similar permeabilities but different mechanical properties, such as a structured a-helix [formed of poly(propargyl-L-glutamate) derived from g-propargyl-Lglutamate] versus a flexible random coil [formed of poly(propargyl-L-glutamate) derived from equal parts of gpropargyl-L-glutamate and g-propargyl-D-glutamate].86 In addition to the challenge of decoupling hydrogel permeability from matrix mechanics, hydrogels from natural materials often have the additional complication of separating the mechanical and biochemical factors.

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For example, if the concentration of the natural polymer is increased to augment the mechanical stiffness, the density of bioactive groups, such as adhesive peptides, is also increased. Varying the density of adhesive moieties can have downstream phenotypic effects, such as changing cell spreading, growth, and migration.73

3.4 Mass Transport Properties Stem cells cultured statically within hydrogels generally rely on diffusive mass transport to receive the necessary oxygen and nutrients required for cell survival, and thus, examining mass transport properties is a key design parameter, particularly for large scaffolds. The rate of diffusion of molecules through the hydrogel is dependent on a number of factors, including the pore size of the hydrogel network and the charge of the material and the diffusing species.7 Several approaches have been applied to increase hydrogel pore size, including electrospinning (see Section 3.2), gas foaming, lyophilization, solvent casting with particle leaching, and the creation of hydrophilice hydrophobic hybrid hydrogels.87 Increasing the pore size with these methods can also facilitate cellular processes, such as proliferation and migration, in addition to changing the system permeability; however, the isotropic porous environment created by these techniques is often distinct from the more fibrillar structure of ECM in native tissues. Instead of creating pores throughout the hydrogel system, an alternative approach based upon native vasculature has been to incorporate microchannels using microfabrication techniques such as molding with microneedles, soft lithography, bioprinting, photopatterning, and modular assembly.88,89

3.5 Quantitative Properties of Hydrogels To compare between different hydrogel systems, it is important to be able to assign quantitative parameters that describe material properties (Fig. 22.4). Many of these properties are interconnected, and several

theoretical and empirical correlations have been established to quantitatively relate them. For example, the cross-linking density (rx), which denotes the number of polymer chains present in a set volume of material, is directly associated with volumetric swelling ratio, the mesh size, and the shear modulus. Briefly, the volumetric swelling ratio (Q) is the proportion of hydrated gel volume to dry gel volume, the mesh size (x) describes the diffusivity of species through the gel, and the shear modulus (G) defines the mechanical properties of the gel in response to shear stress.8 The mechanical properties can also be defined by the Young’s or elastic modulus (E), which also describes the stiffness of the system but alternatively describes the material’s response to uniaxial stress and is typically defined as the ratio of tensile stress to extensional strain specifically in the linear elastic region of the stressestrain curve. Some of these parameters are easier to empirically determine than others; for example, the swelling ratio (Q) is easily measured and the shear modulus can be determined using standard rheometric techniques. Using these two values, the harder to measure crosslinking density (rx) can be derived using the following Eq. (22.1), based on elastic theory: G ¼ RTrx Q1=3

(22.1)

where R is the universal gas constant and T is the temperature.90 The mesh size (x) can be determined by first estimating the average molecular weight between crosslinking points (Mc), which is described by the following Eq. (22.2) relating the cross-linking density (rx) and the elastic modulus (E): Mc ¼ ð3rx RTÞ=E

(22.2)

From there, the mesh size is described by the following Eq. (22.3):  1 Mc 2 x ¼ 2a  2:21Q1=3 (22.3) Mr where a and Mr are dependent on the polymer species of interest.91 The diffusivity (D) of a species through a

FIGURE 22.4 Key quantitative properties of hydrogels, including the volumetric swelling ratio (Q), the mesh size (x), the shear modulus (G), the Young’s or elastic modulus (E), and the diffusivity (D), are interrelated and can be used to compare across hydrogel platforms.

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hydrogel network of a known mesh size can be determined if the diffusivity of the species through a pure solvent (Do) and the hydrodynamic radius of the species (rs) are known. This relationship is described by the Eq. (22.4) below:  D ¼ Do

 rs Y 1 e x

Q Q1

 (22.4)

where Y describes the ratio of the volume required for translational movement of the entrapped particle to the free volume per molecule of solvent (typically approximated as 1 because of empirical findings).92

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The sequence and density of adhesive peptides have been found to specifically impact stem cell phenotype. Although it has been generally observed that the presence of the RGD peptide in PEG gels leads to higher viability of encapsulated MSCs, it has been determined that the flexibility and spacing of the RGD peptide play a role in MSC survival. PEG gels with RGD attached via a single link as a pendant group promoted improved MSC survival (w80% viability) when compared with PEG gels with constrained RGD peptides (w60% viability), and the addition of a glycine spacer arm to the tethered RGD further augmented MSC viability to w88%.96 The results of this study indicate that the spatial configuration in which the RGD peptide is presented to the MSCs affects the cell’s ability to interact with it and that increasing accessibility can be achieved by slightly extending the peptide away from the PEG backbone.

4.1 Cell Adhesion Motifs A fundamental aspect to enable bioactivity in biofunctional hydrogels are adhesive ligands for cell attachment; without them, many of the key material properties, such as topography and matrix mechanics, cannot be accurately sensed by entrapped cells, and the cells will be unable to spread or migrate through the system. This is particularly important for stem cell populations, most of which are typically anchorage dependent. Depending on the cell type and its native microenvironment, different adhesive cues for specific cell surface receptors are more appropriate than others. Typically, cells bind to the surrounding ECM through transmembrane receptors known as integrins, which in turn bind to adhesive proteins found in the ECM such as fibronectin and laminin. Rather than incorporate full-length proteins into synthetic polymers, short peptide sequences have been identified which facilitate cell adhesion and can be more easily incorporated because of their small size, which is less disruptive to the overall hydrogel structure. The three-peptide sequence arginine-glycine-aspartic acid (RGD), which is found in many adhesion proteins, is by far the most commonly used because of its interactions with a broad range of integrins. Other common peptide adhesive sequences include isoleucineelysineevalineealaninee valine, which is derived from laminin and thought to bind a3b1, a4b1, and a6b1 integrins, and the collagenmimicking hexapeptide GFOGER, recognized by a2b1 and a11b1 integrin.93e95 Although many naturally derived hydrogels contain native adhesive sequences, synthetic polymers must be specifically modified to include them, and typical techniques include covalent grafting posthydrogel formation or chemical incorporation/physical entrapment during polymerization.73

4.2 Growth Factor Presentation In the native stem cell microenvironment, the ECM can bind and present a diverse array of growth factors that promote specialized cell functions and tissue morphogenesis. Thus, it can be important to include growth factors in hydrogel design schemes, either through direct incorporation and covalent tethering or through the addition of growth factorebinding motifs. Direct encapsulation of growth factors within the hydrogel has been the conventional approach to present localized soluble cues to entrapped cells and leads to sustained presentation when the hydrodynamic radius of the growth factor is larger than the hydrogel mesh size, as it will remain trapped such that it can provide signals to the cells in the vicinity. However, simple entrapment of growth factors has a number of limitations, including its restriction to large protein molecules and the general lack of control over subsequent release kinetics. Covalent tethering of growth factors to synthetic polymers, such as PEG, without adversely affecting bioactivity can be accomplished via a number of conjugation reactions, including succinimide or carboxylic acid with amine groups, maleimide with thiol groups, Michael type addition, or click reactions of azide with alkyne groups.97 Bioconjugated growth factors have been used in a variety of stem cellehydrogel combinations, including NSCs within 3D chitosan hydrogels modified with interferon-gamma, which improved differentiation toward neuronal lineages.98 Many of the studies performed in 2D, such as the immobilization of stem cell factor and SDF-1a on PEG hydrogels to promote HSC adhesion and spreading, will benefit from replication in 3D systems to see if the 2D functional results can be recapitulated in a configuration more

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similar to the in vivo environment.99 A general caveat of covalent tethering approaches is the potential attenuation or loss of protein bioactivity because of a lack of control over site-specific attachment and orientation of the ligand such that its associated receptor can properly bind. The entrapment of growth factoreladen microparticles or microspheres into larger bulk hydrogels or directly within cellular aggregates has been investigated to improve the control over growth factor release while preventing complications from covalent tethering. There is a plethora of materialeprotein combinations available because of the breadth of research in the drug delivery field regarding materials with controlled-release profiles. For example, modified PEG microspheres loaded with TGF-b3 and BMP-6 entrapped within chitosan hydrogels were found to enhance the chondrogenic differentiation of adipose-derived stem cells, with the local delivery from the microspheres leading to a higher ratio of collagen II to collagen I when compared with soluble growth factor delivery, which may be because of the manner in which the growth factors were present or the difference in local concentration.100 An advanced system has recently been developed for the simultaneous formation of coacervate microparticleeentrapped hydrogels using BMP-2eladen methacrylated alginate and MSC-containing methacrylated gelatin to promote osteogenic differentiation.101 Using photo-cross-linking, the constructs can be fashioned in situ, reducing the complexity of current systems in a noncytotoxic manner. An alternative approach to incorporating specific growth factors within hydrogels from the outset is to sequester growth factors, either from the media solution or from those produced by cells. Systems of this nature more closely mimic native tissue environments in which PGs and GAGs interact noncovalently with growth factors, thus many of the current sequestering approaches incorporate native GAG species or peptide fragments that bind GAGs.102 For example, the incorporation of a heparin-binding peptide sequence into fibrin gels facilitated binding and controlled release of soluble betanerve growth factor and other neurotrophic factors.103 The recent identification of a domain in placenta growth factor-2123e144, which binds with very high affinity to a number of ECM molecules suggests that protein engineering strategies in which this sequence is fused to a growth factor of interest, may be used to enhance growth factor sequestration.104 In addition to using ECM-based protein sequestration strategies, peptidebinding motifs for specific growth factors can be directly incorporated into engineered materials. For example, a peptide derived from vascular epidermal growth factor (VEGF) receptor-2 incorporated into PEG microspheres promoted specific binding of VEGF with high affinity, which has implications for angiogenic applications.105 A similar approach could be used for a variety of

situations in which increasing or decreasing growth factor activity is desired.

4.3 Stimuli-Responsive Materials Native biomacromolecules are dynamically regulated by their surrounding environment; thus, the design of hydrogel systems which exhibit a specific response to an external stimulus adds an extra dimension to hydrogel capabilities. Common external stimuli include altering the pH or temperature, treatment with light or electromagnetic fields, introduction of specific enzymes or peptides, or application of mechanical forces.106 A caveat is that the applied stimulus should not cause cytotoxic effects; thus, there is a small range of acceptable pH and temperatures and limitations in the wavelength and duration of UV exposure that are generally considered tolerable. The inclusion of photolabile groups such that the hydrogel structure can be dynamically modulated has already been discussed in the context of changing the degradation and mechanical properties (see Sections 3.1 and 3.3), and the recent incorporation of photo-activated caged RGD residues in PEGDA hydrogels enables spatiotemporal control of hydrogel adhesive properties in vivo through transdermal light exposure.107 Thus, photo-activated material systems continue to be a powerful tool for dynamic regulation of physical and biochemical material properties. Novel culture systems that take advantage of the native material’s sensitivity to temperature changes also offer possibilities for real-time control. Thermoreversible PNIPAAm-PEG hydrogels, which form gels at physiologic 37 C but remain liquid at 4 C, have recently been investigated as a 3D culture platform for human pluripotent stem cell (hPSC) culture.108 Cells were mixed with the polymer in its low-temperature liquid state, grown in the hydrogel at 37 C, and easily retrieved for further analysis or expansion by simply reducing the temperature. Using this fully defined system, significant expansion of multiple hPSC lines was observed (approximately 20-fold expansion per 5 day passage) while the cells retained their pluripotent qualities, indicating that thermoresponsive hydrogel systems can serve an important purpose in developing scalable bioprocesses for hPSC expansion.

5. TECHNOLOGIES FOR HYDROGEL PRODUCTION AND ASSESSMENT 5.1 Hydrogel Production Methods Hydrogels are useful tools in 3D stem cell culture across a range of scales, spanning from microparticles

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at the micrometer scale, to microcapsules at the submillimeter scale, and finally to bulk gels at the centimeter scale (Fig. 22.5). All three configurations can be used to encapsulate cells within a matrix, though microparticles are more typically used in scaffold-free cell aggregate configurations or in combination with larger hydrogels. The production of bulk hydrogels typically uses molds that are filled with the polymer solution before cross-linking, and more sophisticated shapes can be achieved by using microfabrication technologies such as rapid prototyping and soft lithography. In contrast to forming whole hydrogel systems, a modular bottom-up approach can also be used in which small units are fabricated for future assembly into larger constructs.109 This is similar to the way in which many native tissues are composed; for example, muscle is made up of many myofiber bundles. The construction of larger structures from smaller units can be performed manually or through the use of self-assembled materials. With hydrogels, one can take advantage of hydrophobicehydrophilic interactions at liquideliquid and aireliquid interfaces to drive self-assembly of microgels.110 5.1.1 Formation of Microparticles Hydrogel microparticles have been used for controlled-release drug delivery purposes for some time by engineering material properties to control release profiles.111 For the most part, emulsion polymerization techniques are used to produce droplets of the polymer phase within a surfactant-stabilized carrier solution before cross-linking of the hydrogel.112 Microparticles loaded with small molecules or growth factors can be incorporated into bulk hydrogels to facilitate controlled release of soluble cues to encapsulated stem cells.113 Alternatively, microparticles can be incorporated into

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scaffold-free 3D multicellular constructs and act as local reservoirs of soluble factors that may otherwise face diffusional barriers if delivered externally.114,115 Spatial control over microparticle position within cell constructs can also be used to promote morphogenic events stimulated by soluble factor gradients.116,117 5.1.2 Microencapsulation Methods The formation of spherical microcapsules or microbeads can be achieved by a number of available methods including the use of micromolds or microfluidic droplet generators.7 Specialized microencapsulation devices use a variety of methods to produce small, uniform hydrogel spheres, with most relying on electrostatic potential, coaxial flow, vibration, or water jet cutting to form single beads from a stream of liquid. In all cases, microencapsulation techniques rely on rapid gelation of the hydrogel material after bead formation. A distinction exists between microcapsules, which have a solid shell with a liquid, and microbeads, which are a solid hydrogel, and the two configurations may be suited for different applications.21 For example, liquid core capsules facilitate aggregation of enclosed cells into a single aggregate, whereas cells in solid microbeads are more constrained and often proliferate to form multiple smaller and distinct aggregates. 5.1.3 Microfabrication Techniques for Bulk Hydrogels Techniques for producing hydrogels with a high degree of spatial control often rely on microfabrication techniques that have a fine control over positioning. Many complex hydrogel structures are enabled through microfabrication technology, including the formation of custom, multilayer tissue-engineering constructs.118 Unfortunately, many of the standard fabrication methods

FIGURE 22.5

Hydrogels are useful across a range of scales, spanning from microparticles at the micrometer scale, to microcapsules at the submillimeter scale, and finally to bulk gels at the centimeter scale. Each of these types of hydrogels can play a key role in controlling stem cell function.

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used to produce highly defined 2D patterns are not easily translated to 3D systems, prompting the development of novel fabrication techniques. Soft lithography techniques often have the highest resolution (w100 nm) and include a range of methods that facilitate the design of micro-features from elastomeric materials, typically PDMS, which can then be used as master molds for bulk hydrogel formation. Stereolithography approaches offer less spatial resolution (w1 mm) than soft lithography, but can be used directly with photopolymerizable hydrogels to create structures in a layer-bylayer approach. The advent of 3D printing technologies has also been applied to create hydrogel systems, though the spatial resolution (w10 mm) is currently less than that obtained with other microfabrication techniques.73

5.2 Translating 2D Assays to 3D Systems Despite the advantages of culturing stem cells in hydrogels, many of the routine assays to monitor outcomes, including cell number, viability, metabolic activity, and phenotype, are not easily translated to 3D systems. Common assays that make use of cellular dyes can be limited by diffusion through the hydrogel construct, though often increasing the dye concentration and/or incubation time or introducing convective flow (i.e., mixing) into the system can overcome mass transport constraints. Even if mass transport is not an issue, which it typically is not for common small-molecule stains (1000 nm; oligolamellar vesicles (OLV): 100e500 nm; and Multilamellar vesicles (MLV): >500 nm], and the surface charge of vesicles (neutral, anionic, or cationic).87 Liposomal membranes are composed of natural and/or synthetic lipids which are relatively biocompatible, biodegradable, and nonimmunogenic. Because of their unique bilayer structure, liposomes are used as carrier systems for both lipophilic and hydrophilic molecules.88 There are many conventional methods of liposome preparation at the laboratory scale that have been developed and optimized, leading to the formation of vesicles of different sizes ranging from 10 to 20 nm to several microns in diameter and composed of one or more bilayers. Mainly, these methods involve three basic stages, which are: drying down of lipids from organic solvents, followed by dispersion of the lipids in an aqueous media, and finally purification and analysis of the final product.89 Hydration of the phospholipid layer is a technique where a mixture of phospholipids and cholesterol are dispersed in an organic solvent such as chloroform or ethanol, followed by the removal of the organic solvent using a rotary vacuum evaporator for a couple of hours at a reduced pressure. Finally, the dry phospholipid film deposited on the flask wall is hydrated by adding an aqueous buffer solution.90 The resulting MLVs suspension contains vesicles that are heterogeneous in size and lamellarity. Thus, in order to obtain more defined size and homogeneity in population of these vesicles, LUVs can be formed by sequential extrusion through polycarbonate membranes,91 and SUVs can be produced by sonication.88 The reverse phase evaporation technique involves the formation of large unilamellar and oligolamellar vesicles when an aqueous buffer is introduced into a mixture of phospholipids, and the organic solvent is subsequently removed by evaporation under reduced pressure. This method leads to the preparation of liposomes (from many lipid formulations) with a high aqueous space-to-lipid ratio and a capability to encapsulate large macromolecular assemblies such as drugs, proteins, nucleic acids, and other biochemical reagents with high efficiency.92 Injection of organic solvent with dissolved phospholipids into an aqueous phase involves the dissolution of the lipid into an organic phase (ethanol or ether), which is then injected into an aqueous buffer, leading to the formation of liposomes. The procedure is rapid, highly reproducible, and allows preparation of large amounts of liposomes without extrusion or sonication. However, the liposome suspension will contain the organic solvent that can inactivate

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biologically active macromolecules.93 There are other conventional techniques for the production of liposomes with a high encapsulation efficiency of drugs, enzymes, and other macromolecules, such as the detergent dialysis technique94 and the double emulsion method.95,96 Nonetheless, these methods involve use of either organic solvents or detergent agents, which can potentially induce toxic effects and have harmful consequences on human health. Moreover, the consumption of a large amount of energy and the need for many steps for size homogenization made these conventional techniques undesirable for the mass production of liposomes.88 In order to improve on some of the shortcomings of liposomes such as sensitivity to osmotic pressure and temperature, as well as limited stability in biological fluids over prolonged periods of time, solid lipid nanoparticles have been explored as an alternative. Solid lipid nanoparticles also provide several advantages, such as: (1) eliminating the need for organic solvents; (2) providing high encapsulation efficiency and controlled release kinetics; (3) ease of upscaling using currently available manufacturing techniques used by pharmaceutical companies (e.g., high pressure homogenization); (4) and long-term colloidal stability (up to 3 years).97 Some of the well-established methods for the fabrication of solid lipid nanoparticles are: high pressure homogenization (with either hot or cold techniques), the microemulsion technique, lipid nanopellet, lipospheres, and precipitated lipid particles.98 The readers are referred to an excellent review on these methodologies by Muller et al.98 Recently, other interesting methods have been explored. Yu et al. used a polymer-based microparticle template for lipid nanoparticle formation (Fig. 25.4C).99 They utilized a method called electrospraying to fabricate the polymeric microparticles. Electrospraying is a method which involves inducing an electric field between the tip of a needle ejecting the polymeric solution and a metal collector plate. At a certain voltage potential, a rapid ejection of the polymeric solution occurs and under optimized conditions will form microparticles that will travel toward the collector plate and dry in the process. Yu et al. prepared a mixture of a triglyceride (tristearin), an antiinflammatory hydrophobic drug (naproxen), and a polymer template (polyvinylpyrrolidone) dissolved in ethanol and electrospraying was used to create composite microparticles. Upon exposure to an aqueous environment, the polymeric microparticles rapidly disintegrated and prompted the self-assembly of solid lipid nanoparticles complexed with the hydrophobic drug.99 Lipidepolymer hybrid nanoparticles have also been explored to further enhance drug encapsulation, retention, and release of therapeutic agents. PLGA-lipidPEG nanoparticles have been fabricated in a single step and have been shown to be effective in

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encapsulating and releasing docetaxel, a hydrophobic anticancer drug.64 The major advantage of adding a lipid bilayer to the PLGA-PEG nanoparticles was providing a molecular barrier that increased drug retention and encapsulation efficiency (threefold more than PLGA-PEG nanoparticles).64 This system was further modified by introducing different functional groups on the surface of the PLGA-lipid-PEG nanoparticles, and it was shown that methoxyl groups induced lower levels of complement activation and coagulation as compared to carboxyl and amine groups.100 Furthermore, conjugating half-antibodies against carcinoembryonic antigen on the surface of PLGA-lipid-PEG nanoparticles has been shown to effectively target pancreatic cancer cells and deliver paclitaxel.101

8.3 Micelles Micelles by definition are self-assembled amphiphilic molecules that form core-shell structures in an aqueous environment, with the hydrophobic region forming the core (away from water) and the hydrophilic region forming the shell via hydrogen bonds with the water molecules. Micelles are different from liposomes in that they do not form a lipid bilayer, and can be fabricated using either amphiphilic polymers or lipids.102 Polymeric micelles have been gaining more interest due to the narrow range of sizes that can be achieved (10e100 nm), the versatility of modifying different segments of the polymers to efficiently encapsulate either hydrophobic or hydrophilic agents, ease of fabrication, and ease of storage through lyophilization without the need for cryoprotectants.102,103 Many of the fabrication methods used are similar to those of liposomes and polymer-based nanoparticles. For example, poly(ethylene glycol)-blockpoly(D,L-lactic acid) (PEG-b-PLA) copolymers were used to fabricate micelles for the delivery of anticancer drugs that have low solubility in water. The fabrication method involved dissolving the anticancer agent and PEG-b-PLA in acetonitrile and evaporating the solvent using a rotary evaporator at 60 C to form a thin film, which was then rehydrated and stirred gently to form drug-loaded micelles.104 In another modification of the solvent evaporation method, others have used a film sonication technique to form micelles. This method involves rehydrating the thin film containing the block copolymer and hydrophobic drugs and sonicating the solution to obtain drug-loaded micelles.105 Micelles could also be fabricated using dialysis; however, this is a more prolonged method compared to film sonication and solvent evaporation. In this method, the block copolymer and drug dissolved in an organic phase are dialyzed against distilled water for 72 h or more in order to form micelles.106 Also, similar to polymer-based nanoparticles, nanoprecipitation (oil-in-water) has been used to

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fabricate micelles. In this method, the polymers and hydrophobic drugs are dissolved in an organic solvent, which is then added to water under rapid stirring (w1000 rpm) in order to form micelles. The organic phase is then evaporated under vacuum, and the micelles are centrifuged and rehydrated.107 All the above fabrication methods depend on a critical parameter that determines the formation and stability of micelles called critical micelle concentration (CMC). Polymeric concentrations below CMC do not yield the formation of micelles, but rather the polymeric chains are concentrated at the airewater interface with the hydrophobic segment pointing away from the aqueous environment in order to reduce the interfacial free energy. At the CMC, the bulk solution and the interface are saturated with the block copolymers, and further addition of polymer causes the formation of micelles, ultimately as a means to reduce the interfacial free energy. Stability of the formed micelles depends not only on CMC but also on parameters related to the aqueous environment (temperature, pH, and salt concentrations) and polymeric parameters (molecular weight, hydrophobic and hydrophilic segment properties).108 Moreover, the core of micelles can be stabilized by increasing: hydrophobic interactions (via incorporating hydrophobic drugs), electrostatic interactions (via using polyion complexation between anionic and cationic polymers), and metal complexation.109,110 Nowadays, there is a huge therapeutic use of micelles, especially as drug delivery systems. Due to their structure, the outer shell of micelles allow the control of the in vivo pharmacokinetic behavior of drugs, while the inner core controls the drug loading, the stability, and drug release capacity.111 For example in oncology and cancer therapy, the micelles have been used as a system to eradicate tumor stem cells which are implicated in the recurrence of cancer after treatment and in the increased resistance to conventional chemotherapy.112 In that regard, a study conducted by Zhang et al.113, octreotide (Oct)-modified paclitaxel (PTX)-loaded PEGb-PCL polymeric micelles (Oct-M-PTX) and salinomycin (SAL)-loaded PEG-b-PCL polymeric micelles (M-SAL) have been used separately or synergistically to target both breast cancer cells and breast cancer stem cells (Fig. 25.4D). It is shown that the micelles have the capacity to target the somatostatin receptors, which are overexpressed in tumors in general including breast cancer. Likewise, the combination treatment of the micelles Oct-M-PTX and M-SAL led to a higher inhibitory effect to the tumors by eradication of both non-stem cells and cancer stem cells. Another example of breast cancer therapy by using micelles was performed by Ke et al.114 In this study, the micelles were self-assembled from a combination of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC)

and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC) and loaded with two type of drugs: thioridazine (THZ) (an anticancer drug specific to cancer stem cells) and doxorubicin (DOX). The codelivery of THZ and DOX loaded into the micelles led to a significant antitumor activity and selectively inhibit the proliferation of breast cancer stem cells both in vitro and in vivo. Another example of enhancing antitumor activity of cancer stem cells by using micelles was presented in the work of Wang et al.115 This team developed a formulation of nanosized micelles based on curcumin (a well-known anticancer substance) encapsulated in stearic acid-g-chitosan oligosaccharide (CSO-SA). The CSO-SA micelles showed a strong internalization capacity within the cells and antiproliferative effects on primary colorectal cancer cells in vitro. Moreover, these types of micelles had the capacity to inhibit the proliferation of putative colorectal cancer stem cell markers, which are subpopulations of CD44þ/CD24þ cells both in vitro and in vivo. In addition to their promising delivery carriers for anticancer drugs and to their potential strategy in oncology, micelles can be used to deliver functional biomolecules such as enzymes, proteins, and nucleic acids to cells.111,116,117

8.4 DNA Origami Controlling the monomeric sequence allowed polymer chemists to design the physicochemical properties of synthesized nanoparticles. Similarly, controlling the nucleotide sequence in DNA and taking advantage of the Watson-Crick base pairing phenomenon, allowed molecular biologists to create DNA-based nanoparticles with any shape or size.118 Nadrian C. Seeman,119 was first to propose using DNA as building blocks to create immobile junctions, which can then be arranged to form more complex 3D structures. The field of DNA nanotechnology has since exploded into existence, with investigators designing a myriad of DNA nanostructures, both 2D and 3D, that can be used in a range of applications from microelectronics to drug delivery.120,121 In this chapter, we will focus our attention on DNA origami and its application in the field of drug delivery. “Scaffolded DNA origami,” a term coined by Paul Rothemund,118 is a method of folding a long single strand of DNA (scaffold) into precise shapes by using multiple short oligonucleotide “staple strands” to stabilize the overall structure (Fig. 25.5A). First, the desired shape is drawn and filled with the scaffold strand in a raster fill arrangement. A computer program is then used to design and place the staple strands on the scaffold strand to fold it into the desired shape. The scaffold and staple strands are mixed, heated, and then cooled

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FIGURE 25.5

(A) Scaffolded DNA origami, a method that involves the use of a long single strand of DNA and shorter DNA sequences (staples) to form virtually any 2D or 3D shape. The images on top are the computer-produced folding sequence of the DNA strand and the bottom row is an atomic force microscopy image of the different DNA origami shapes; (B) It has been shown that different shapes can illicit different biological effects. The triangular-shaped origami provided the best accumulation at the breast tumor site as compared to square and tube-shaped origami. Doxorubicin was intercalated within the DNA and was readily released at the tumor site due to low pH; (C) (i) A barrel-shaped nanorobot used as a drug delivery system. The nanorobot has covalently stabilized hinges in the back, and an aptamer lock mechanism in the front. The aptamers dissociate in the presence of an antigen and releases the payload. (ii) TEM images of the nanorobot in closed and open configurations. Adapted from (A) Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature 2006;440(7082):297e302; (B) Zhang Q, Jiang Q, Li N, Dai LR, Liu Q, Song LL, et al. DNA origami as an in vivo drug delivery vehicle for cancer therapy. ACS Nano 2014;8(7):6633e43; and (C) Douglas SM, Bachelet I, Church GM. A logic-gated nanorobot for targeted transport of molecular payloads. Science 2012;335(6070):831e4.

down to allow the self-assembly of the DNA origami.118 This precise control over DNA nanoparticle fabrication prompted many researchers to apply DNA origami for drug delivery applications. In one study, it was shown that triangular DNA origami with side lengths of 120 nm was able to accumulate much more readily at the tumor site due to passive targeting as opposed to square and tube-like DNA origami (Fig. 25.5B).122 Moreover, the triangular DNA origami was able to retain (via intercalation) and release doxorubicin at the tumor site, and caused no systemic toxicity.122 Triangular and tubular DNA nanostructures have also been shown to effectively enhance doxorubicin uptake in doxorubicin-resistant MCF-7 cells.123 In another, more complex structure, a DNA origami nanorobot was designed for targeted drug delivery.124 The nanorobot, which resembles a hexagonal container with a lid, has an aptamer-based locking mechanism that interacts with target proteins to open and release the payload. The investigators fabricated one of those nanorobots (35  35  45 nm) using 196 oligonucleotide

staple strands and a 7308-base scaffold strand (Fig. 25.5C).124 There are more examples demonstrating the effectiveness of using DNA origami nanostructures in drug delivery applications, and the readers are referred to an excellent review on this subject matter.121

9. SELF-ASSEMBLED NANOFIBERS AND NANOTUBES Peptides that can undergo self-assembly into nanofibers or nanotubes have been discovered in the early 1990s by Zhang Shuguang and have since been widely explored in the field of biomedical engineering.125 The main principle behind peptide self-assembly is the presence of alternating hydrophobic and charged (cationic and anionic) hydrophilic amino acids in the peptide chain, which promotes hydrophobic, electrostatic, and hydrogen bonding between the peptides and the surrounding solution.125 There are various parameters

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that can affect the self-assembly process besides the amino acid sequence, which include: the molecular size, peptide concentration, solution pH, temperature, ionic strength, and presence of denaturation agents.125 Under optimized conditions, amphiphilic peptides undergo self-assembly to form stable secondary structures, namely a helices or b sheets. Zhang et al. reported the first 16 residue peptide sequence (w5 nm in size) composed of alternating polar and nonpolar amino acids that self-assembled into b sheets in physiological conditions leading to an interwoven nanofibrous macrostructure with nanofibers diameters between 10 and 20 nm.126 The mechanism of action was proposed to be due to the interaction between alternating charges on the polar side of the peptide, which formed a checkered-like structure due to ionic attractions. Moreover, the nonpolar section of the peptide was oriented inside the b sheets to avoid contact with water.126 These nanofibers have also been shown to retain large quantities of water and form hydrogels with >99.5% water content.127 The biomimetic properties of selfassembling amphiphilic peptides make them highly desirable for regenerative medicine applications. A self-assembled nanofibrous hydrogel has been shown to support neuronal attachment, neurite outgrowth, and synaptic formation.128 Peptides have also been modified by the addition of functional motifs such as RGD sequence (Arg-Gly-Asp) to improve cellular attachment, proliferation, and differentiation. A study demonstrated that peptides conjugated to a functional motif were able to support neural stem cell survival, attachment, proliferation, and differentiation over a period of 5 months.129 Moreover, functionalized amphiphilic peptides have been used to guide cartilage,130 bone,131 and adipose tissue132 regeneration. Peptide-amphiphiles have also been used to create nanotubes under physiological conditions. Using an alkyl tail with 16 carbons coupled to an ionic peptide region can self-assemble into nanotubular structures when placed in water, with the hydrophobic region oriented toward the inside and ionic peptide region on the outside of the tube.133 This tendency to form cylindrical structures with high aspect ratios (length: diameter) is due to the conical shape of the peptide-amphiphiles, with the peptide region being bulkier than the hydrophobic carbon chain.134 Moreover, the surfactant-like peptides can be functionalized to include moieties for improving cell attachment and for guiding certain phenomenon such as mineralization.133,134 Having a hollow cylindrical structure also provides the capability for entrapping hydrophobic drugs and releasing them in a controlled manner. The hydrophobic pyrene was used as a model molecule, and was successfully encapsulated within peptide-amphiphiles.135 Besides the entrapment of hydrophobic drugs, investigators have explored cell

encapsulation within the nanofibrous structure formed by peptide-amphiphiles.136 MC-3T3 osteoblastic cells were physically entrapped in the nanofibrous structure and were shown to survive and proliferate within the scaffold for up to 20 days.136 The readers are referred to an excellent review on the application of peptideamphiphiles in various biomedical applications.137 Besides peptide-based self-assembled nanofibers and nanotubes, other building blocks have also been explored. Chitin, which is acetylated chitosan, is the second most abundant polysaccharide found in nature after cellulose. Formation of chitin nanofibers using the “bottom-up” approach has been challenging, since it is insoluble in water and most organic solvents. However, in 2010, two methods were developed by Zhang et al. that allowed for the development of chitin nanofibers with diameters of 3 and 10 nm respectively.138 The two methods involve dissolving b-chitin (derived from pen squid) in hexafluooro-2-propanol and LiCl/N,N dimethylacetamide respectively. Drying the hexafluoro-2propanol led to the formation of chitin nanofibers (w3 nm), while the addition of ample amounts of water to the LiCl/N,N dimethylacetamide chitin solution, led to the self-assembly of thicker chitin nanofibers (w10 nm).138 It was also shown that the chitin nanofibers can be oriented using a micro-contact printing technique, and that NIH-3T3 cells survived and adhered on the chitin nanofibers.139 Since the discovery of multi-walled carbon nanotubes in 1991 by Sumio Iijima, there has been tremendous interest in using carbon nanotubes in a wide range of applications due to their useful electrical, biological, and mechanical properties.140 Furthermore, the ability to functionalize biomolecules on the surface of carbon nanotubes expanded the range of potential biomedical applications to include drug delivery and biosensing. Chen et al. developed a versatile, yet simple non-covalent method to allow for the functionalization of SWCNTs.141 A bi-functional molecule, 1-pyrenebutanoic acid succinimidyl ester, irreversibly adsorbed on the surface of SWCNTs in dimethylformamide or methanol, due to pp interactions between the aromatic rings. The presence of succinimidyl groups that are highly reactive to nucleophilic substitution in the presence of primary or secondary amines allows for protein adsorbtion on the surface of SWCNTs.141 The readers are referred to excellent reviews on the role functionalized carbon nanotubes played in a range of biomedical applications.142,143 One of the major challenges related to SWCNT fabrication has been to “un-tangle” randomly oriented SWCNTs and be able to align them. That is important for many reasons, such as providing anisotropic mechanical properties to scaffolds used in regenerative medicine, improving conductivity and creating more efficient circuitry, aligning cells for guided tissue engineering

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ABBREVIATIONS AND ACRONYMS

applications, and producing patterns for studying cells in vitro. Self-assembly of SWCNTs, has therefore, emerged as a viable solution for aligning SWCNTs. In one study, SWCNTs were cut into shorter segments, which were then oxidized to obtain carboxyl groups on both ends of the nanotube. The carboxyl groups were further functionalized to introduce a thiol group, which was utilized to self-assemble aligned SWCNTs on a gold surface using Au-S chemical bonding.144 In another interesting study, iron catalyst positioned at one end of carbon nanotubes was magnetically attracted to a nickel surface and microfluidic flow was used to align the free end of the tubes in a specific direction. This simple procedure, allowed the precise orientation of single carbon nanotubes.145 Park et al. have also produced self-assembled monolayer patterns of carbon nanotubes on a gold surface for guiding the orientation of mesenchymal stem cells.146 Patterns were formed by patterning nonpolar 1-octadecanethiol on a gold surface using either nanolithography or micro-contact printing. The carbon nanotubes were then added to the surface, and preferentially self-assembled on the bare gold pattern. The rest of the surface was passivated using 1-octadecanethiol leaving behind the carbon nanotube patterns. Mesenchymal stem cells adhered and were aligned on the pattern for up to 1 week.146 A comprehensive review is suggested for readers interested in learning more about applications of carbon nanotubes in stem cell differentiation.147 That being said, there are not many studies exploring self-assembled carbon nanotubes on guiding stem cell orientation and differentiation; more research is warranted in this topic.

10. CONCLUSION Self-assembly techniques have been used to develop a multitude of smart nanostructures with a tremendous range of applications in many fields especially in the area of regenerative medicine and stem cell technology. The utilization of these techniques led to new breakthroughs in the development of controlled delivery systems for drugs, proteins, and DNA and also substrates for improving cell attachment and for guiding proliferation and differentiation. All the overarching methodologies for the fabrication of SANs covered in this chapter are faced with the same challenges, namely, being able to control their stability and aggregation state in a complex biological environment, as well as their sterility, nonimmunogenic response, and biodegradability over prolonged periods of time and last but not least the concerns regarding biocompatibility and bioactivity at physiological pH. Thus, innovative ways of fabricating LbL self-assembly or SANs are continuously being improved in order to overcome these shortcomings.

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Currently from an industrial standpoint, much effort is put to provide a large-scale production and create alternative methods to replace conventional ones. The overall results of the studies published to date and presented in this chapter highlight the absence of significant cytotoxic effects of SANs on stem cells which is a promising step toward clinic applications. Moreover, the SANs can be judiciously used as biosensors for the early detection of cancer markers leading in turn to improve the diagnosis of early stage carcinogenesis and complex diseases. Finally, the recent developments of DNA nanostructures such as DNA origami nanorobot for drug delivery applications opens up many exciting opportunities toward the fields of nanomedicine and personalized medicine. Finally, enhancing interdisciplinary research and better understanding of SANs interaction mechanisms with the biological systems using either genomic or proteomic approaches can move the technology forward and lead to the conception of next-generation SANs in the near future.

ABBREVIATIONS AND ACRONYMS ASTM American Society of the International Association for Testing and Materials BMP Bone morphogenetic protein CMC Critical micelle concentration CSO-SA Stearic acid-g-chitosan oligosaccharide DNA Deoxyribonucleic acid DOX Doxorubicin GO Graphene oxide GUV Giant unilamellar vesicles H-LbL Hydrogen-bonded layer-by-layer ISO International Organization for Standardization LbL Layer-by-layer LNP Lipid-based nanoparticles LUV Large unilamellar vesicles MLV Multilamellar vesicles Oct-M-PTX Octreotide (Oct)-modified paclitaxel (PTX) OLV Oligolamellar vesicles PAH Poly(allylaminehydrochloride) PEG Poly(ethylene glycol) PEG-b-PCL Poly(ethylene glycol)-block-polycaprolactone PEG-b-PLA Poly(ethylene glycol)-block-poly(lactic acid) PEG-b-PLD Poly(ethylene glycol)-block-poly(L-aspartic acid) pEGFP-C3 Plasmids encoding enhanced green fluorescent protein PEG-PAC Poly(ethylene glycol)-acid-functionalized poly(carbonate) PEG-PUC Poly(ethylene glycol)-urea-functionalized poly(carbonate) PEI Polyethylenimine PLGA Poly(lactic-co-glycolic acid) PLGA-PEG Poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) PLL Poly-L-lysine PNIPAm Poly(N-isopropylacrylamide) PSA Prostate specific antigen PSS Poly(sodium 4-styrenesulfonate) RGD Arginine-glycine-aspartic acid RNA Ribonucleic acid SAL Salinomyocin SANs Self-assembled nanostructures siRNA Silencing ribonucleic acid

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SUV Small unilamellar vesicles SWCNTs Single-walled carbon nanotubes TGFb1 Transforming growth factor b1 THZ Thioridazine

References 1. Ariga K, Hill JP, Ji Q. Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application. Phys Chem Chem Phys 2007;9(19): 2319e40. 2. Borges J, Mano JF. Molecular interactions driving the layer-bylayer assembly of multilayers. Chem Rev 2014;114(18):8883e942. 3. Whitesides GM, Kriebel JK, Mayers BT. Self-assembly and nanostructured materials. In: Nanoscale assembly. Springer; 2005. p. 217e39. 4. Decher G, Hong J, Schmitt J. Buildup of ultrathin multilayer films by a self-assembly process: III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces. Thin Solid Films 1992;210:831e5. 5. Decher G. Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science 1997;277(5330):1232e7. 6. Ariga K, Ji Q, Nakanishi W, Hill JP. Thin film nanoarchitectonics. J Inorg Organomet Polym Mater 2015;25(3):466e79. 7. Ariga K, Yamauchi Y, Rydzek G, Ji Q, Yonamine Y, Wu KC-W, et al. Layer-by-layer nanoarchitectonics: invention, innovation, and evolution. Chem Lett 2014;43(1):36e68. 8. Zhang X, Chen H, Zhang H. Layer-by-layer assembly: from conventional to unconventional methods. Chem Commun 2007;(14): 1395e405. 9. Tang Z, Wang Y, Podsiadlo P, Kotov NA. Biomedical applications of layer-by-layer assembly: from biomimetics to tissue engineering. Adv Mater 2006;18(24):3203. 10. Richardson JJ, Bjornmalm M, Caruso F. Multilayer assembly. Technology-driven layer-by-layer assembly of nanofilms. Science 2015;348(6233):aaa2491. 11. Cai P, Xue Z, Qi W, Wang H. Adsorbed BMP-2 in polyelectrolyte multilayer films for enhanced early osteogenic differentiation of mesenchymal stem cells. Colloids Surf A 2013;434:110e7. 12. Crouzier T, Ren K, Nicolas C, Roy C, Picart C. Layer-by-layer films as a biomimetic reservoir for rhBMP-2 delivery: controlled differentiation of myoblasts to osteoblasts. Small 2009;5(5):598e608. 13. Jessel N, Oulad-Abdelghani M, Meyer F, Lavalle P, Haikel Y, Schaaf P, et al. Multiple and time-scheduled in situ DNA delivery mediated by b-cyclodextrin embedded in a polyelectrolyte multilayer. Proc Natl Acad Sci USA 2006;103(23):8618e21. 14. Dierich A, Le Guen E, Messaddeq N, Stoltz J-F, Netter P, Schaaf P, et al. Bone formation mediated by synergy-acting growth factors embedded in a polyelectrolyte multilayer film. Adv Mater 2007; 19(5):693. 15. Jessel N, Atalar F, Lavalle P, Mutterer J, Decher G, Schaaf P, et al. Bioactive coatings based on a polyelectrolyte multilayer architecture functionalized by embedded proteins. Adv Mater 2003; 15(9):692e5. 16. Benkirane-Jessel N, Lavalle P, Meyer F, Audouin F, Frisch B, Schaaf P, et al. Control of monocyte morphology on and response to model surfaces for implants equipped with anti-inflammatory agent. Adv Mater 2004;16(17):1507e11. 17. Gangloff SC, Ladam G, Dupray V, Fukase K, Brandenburg K, Guenounou M, et al. Biologically active lipid A antagonist embedded in a multilayered polyelectrolyte architecture. Biomaterials 2006;27(9):1771e7. 18. Picart C. Polyelectrolyte multilayer films: from physico-chemical properties to the control of cellular processes. Curr Med Chem 2008;15(7):685e97.

19. Stankovich S, Dikin DA, Dommett GHB, Kohlhaas KM, Zimney EJ, Stach EA, et al. Graphene-based composite materials. Nature 2006; 442(7100):282e6. 20. Geim AK, Novoselov KS. The rise of graphene. Nat Mater 2007; 6(3):183e91. 21. Liu KP, Zhang JJ, Cheng FF, Zheng TT, Wang CM, Zhu JJ. Green and facile synthesis of highly biocompatible graphene nanosheets and its application for cellular imaging and drug delivery. J Mater Chem 2011;21(32):12034e40. 22. Liu JQ, Cui L, Losic D. Graphene and graphene oxide as new nanocarriers for drug delivery applications. Acta Biomater 2013; 9(12):9243e57. 23. Alzhavan O, Ghaderi E, Shahsavar M. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon 2013;59:200e11. 24. Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, et al. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater 2011;23(36):H263e7. 25. Nayak TR, Andersen H, Makam VS, Khaw C, Bae S, Xu XF, et al. Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells. ACS Nano 2011;5(6):4670e8. 26. Lee T, Min SH, Gu M, Jung YK, Lee W, Lee JU, et al. Layer-bylayer assembly for graphene-based multilayer nanocomposites: synthesis and applications. Chem Mater 2015;27. 27. Dai K, Lu L, Liu Q, Zhu G, Wei X, Bai J, et al. Sonication assisted preparation of graphene oxide/graphitic-C(3)N(4) nanosheet hybrid with reinforced photocurrent for photocatalyst applications. Dalton Trans 2014;43(17):6295e9. 28. Hong J, Shah NJ, Drake AC, DeMuth PC, Lee JB, Chen JZ, et al. Graphene multilayers as gates for multi-week sequential release of proteins from surfaces. ACS Nano 2012;6(1):81e8. 29. Feng LZ, Zhang SA, Liu ZA. Graphene based gene transfection. Nanoscale 2011;3(3):1252e7. 30. Zhang B, Cui T. An ultrasensitive and low-cost graphene sensor based on layer-by-layer nano self-assembly. Appl Phys Lett 2011; 98(7):073116. 31. Kong BS, Geng J, Jung HT. Layer-by-layer assembly of graphene and gold nanoparticles by vacuum filtration and spontaneous reduction of gold ions. Chem Commun 2009;(16):2174e6. 32. Zeng G, Xing Y, Gao J, Wang Z, Zhang X. Unconventional layerby-layer assembly of graphene multilayer films for enzymebased glucose and maltose biosensing. Langmuir 2010;26(18): 15022e6. 33. Haidar ZS, Azari F, Hamdy RC, Tabrizian M. Modulated release of OP-1 and enhanced preosteoblast differentiation using a coreshell nanoparticulate system. J Biomed Mater Res A 2009;91(3): 919e28. 34. Haidar ZS, Tabrizian M, Hamdy RC. A hybrid rhOP-1 delivery system enhances new bone regeneration and consolidation in a rabbit model of distraction osteogenesis. Growth Factors 2010; 28(1):44e55. 35. Haidar ZS, Hamdy RC, Tabrizian M. Biocompatibility and safety of a hybrid core-shell nanoparticulate OP-1 delivery system intramuscularly administered in rats. Biomaterials 2010;31(10): 2746e54. 36. Nayef L, Rendon JS, Matthys R, Hamdy RC, Tabrizian M. Liposome encapsulated quantum dots show efficient in vivo retention of a nanoparticulate drug delivery system at its target in a rat model of distraction osteogenesis. J Nanopharm Drug Deliv 2014; 2(2):93e102. 37. Thierry B, Winnik FM, Merhi Y, Silver J, Tabrizian M. Bioactive coatings of endovascular stents based on polyelectrolyte multilayers. Biomacromolecules 2003;4(6):1564e71. 38. Hillberg AL, Tabrizian M. Biorecognition through layer-by-layer polyelectrolyte assembly: in-situ hybridization on living cells. Biomacromolecules 2006;7(10):2742e50.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

407

REFERENCES

39. Ramasamy T, Haidar ZS, Tran TH, Choi JY, Jeong JH, Shin BS, et al. Layer-by-layer assembly of liposomal nanoparticles with PEGylated polyelectrolytes enhances systemic delivery of multiple anticancer drugs. Acta Biomater 2014;10(12):5116e27. 40. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 2005;4(2):145e60. 41. Ariga K, Lvov YM, Kawakami K, Ji Q, Hill JP. Layer-by-layer selfassembled shells for drug delivery. Adv Drug Deliv Rev 2011;63(9): 762e71. 42. Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 2009;3(1):16e20. 43. Scientific basis for the definition of the term “nanomaterial” Brussels. 2010. Available from: http://ec.europa.eu/health/scientific_ committees/emerging/docs/scenihr_o_030.pdf. 44. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther 2008;83(5):761e9. 45. Black KCL, Wang YC, Luehmann HP, Cai X, Xing WX, Pang B, et al. Radioactive Au-198-doped nanostructures with different shapes for in vivo analyses of their biodistribution, tumor uptake, and intratumoral distribution. ACS Nano 2014;8(5):4385e94. 46. Poon Z, Lee JB, Morton SW, Hammond PT. Controlling in vivo stability and biodistribution in electrostatically assembled nanoparticles for systemic delivery. Nano Lett 2011;11(5):2096e103. 47. Shutava TG, Balkundi SS, Vangala P, Steffan JJ, Bigelow RL, Cardelli JA, et al. Layer-by-layer-coated gelatin nanoparticles as a vehicle for delivery of natural polyphenols. ACS Nano 2009; 3(7):1877e85. 48. Dreaden EC, Morton SW, Shopsowitz KE, Choi JH, Deng ZJ, Cho NJ, et al. Bimodal tumor-targeting from microenvironment responsive hyaluronan layer-by-layer (LbL) nanoparticles. ACS Nano 2014;8(8):8374e82. 49. Poon Z, Chang D, Zhao XY, Hammond PT. Layer-by-layer nanoparticles with a pH-sheddable layer for in vivo targeting of tumor hypoxia. ACS Nano 2011;5(6):4284e92. 50. Fan YF, Wang YN, Fan YG, Ma JB. Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-by-layer adsorption. Int J Pharm 2006; 324(2):158e67. 51. Morton SW, Shah NJ, Quadir MA, Deng ZJ, Poon Z, Hammond PT. Osteotropic therapy via targeted layer-by-layer nanoparticles. Adv Healthc Mater 2014;3(6):867e75. 52. Hujaya SD, Marchioli G, Roelofs K, van Apeldoorn AA, Moroni L, Karperien M, et al. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection. J Control Release 2015;205:181e9. 53. Holmes C, Daoud J, Bagnaninchi PO, Tabrizian M. Polyelectrolyte multilayer coating of 3D scaffolds enhances tissue growth and gene delivery: non-invasive and label-free assessment. Adv Healthc Mater 2014;3(4):572e80. 54. Easton CD, Bullock AJ, Gigliobianco G, McArthur SL, MacNeil S. Application of layer-by-layer coatings to tissue scaffolds - development of an angiogenic biomaterial. J Mater Chem B 2014;2(34): 5558e68. 55. Truong YB, Glattauer V, Briggs KL, Zappe S, Ramshaw JAM. Collagen-based layer-by-layer coating on electrospun polymer scaffolds. Biomaterials 2012;33(36):9198e204. 56. Monteiro IP, Shukla A, Marques AP, Reis RL, Hammond PT. Spray-assisted layer-by-layer assembly on hyaluronic acid scaffolds for skin tissue engineering. J Biomed Mater Res A 2015; 103(1):330e40. 57. Krogman KC, Zacharia NS, Schroeder S, Hammond PT. Automated process for improved uniformity and versatility of layerby-layer deposition. Langmuir 2007;23(6):3137e41. 58. Singh M, Holzinger M, Tabrizian M, Cosnier S. Layer-by-layer scaffold formation using magnetic attraction between HiPCO

59.

60.

61.

62. 63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

(R) single-walled carbon nanotubes and magnetic nanoparticles: application for high performance immunosensors. Carbon 2015; 81:731e8. Kharlampieva E, Kozlovskaya V, Sukhishvili SA. Layer-by-layer hydrogen-bonded polymer films: from fundamentals to applications. Adv Mater 2009;21(30):3053e65. Ono SS, Decher G. Preparation of ultrathin self-standing polyelectrolyte multilayer membranes at physiological conditions using pH-responsive film segments as sacrificial layers. Nano Lett 2006;6(4):592e8. Lynam DA, Shahriari D, Wolf KJ, Angart PA, Koffler J, Tuszynski MH, et al. Brain derived neurotrophic factor release from layer-by-layer coated agarose nerve guidance scaffolds. Acta Biomater 2015;18:128e31. Grzelczak M, Vermant J, Furst EM, Liz-Marzan LM. Directed selfassembly of nanoparticles. ACS Nano 2010;4(7):3591e605. Wang Y, Gao S, Ye WH, Yoon HS, Yang YY. Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer. Nat Mater 2006;5(10):791e6. Zhang L, Chan JM, Gu FX, Rhee JW, Wang AZ, RadovicMoreno AF, et al. Self-assembled lipidepolymer hybrid nanoparticles: a robust drug delivery platform. ACS Nano 2008;2(8): 1696e702. Lee H, Lytton-Jean AK, Chen Y, Love KT, Park AI, Karagiannis ED, et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat Nanotechnol 2012;7(6):389e93. Kim JH, Kim YS, Kim S, Park JH, Kim K, Choi K, et al. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. J Control Release 2006;111(1e2):228e34. Kim JH, Kim YS, Park K, Kang E, Lee S, Nam HY, et al. Selfassembled glycol chitosan nanoparticles for the sustained and prolonged delivery of antiangiogenic small peptide drugs in cancer therapy. Biomaterials 2008;29(12):1920e30. Min KH, Park K, Kim YS, Bae SM, Lee S, Jo HG, et al. Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy. J Control Release 2008;127(3):208e18. Mi FL, Wu YY, Lin YH, Sonaje K, Ho YC, Chen CT, et al. Oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid) and a chitosan derivative functionalized by trimethylation. Bioconjug Chem 2008;19(6):1248e55. Csaba N, Koping-Hoggard M, Alonso MJ. Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery. Int J Pharm 2009;382(1e2):205e14. Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, et al. Selfassembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010;31(1):106e14. Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, et al. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 2011;32(7):1880e9. Paques JP, van der Linden E, van Rijn CJ, Sagis LM. Preparation methods of alginate nanoparticles. Adv Colloid Interface Sci 2014; 209:163e71. Douglas KL, Tabrizian M. Effect of experimental parameters on the formation of alginateechitosan nanoparticles and evaluation of their potential application as DNA carrier. J Biomater Sci Polym Ed 2005;16(1):43e56. Gu F, Zhang L, Teply BA, Mann N, Wang A, Radovic-Moreno AF, et al. Precise engineering of targeted nanoparticles by using selfassembled biointegrated block copolymers. Proc Natl Acad Sci USA 2008;105(7):2586e91. Kolishetti N, Dhar S, Valencia PM, Lin LQ, Karnik R, Lippard SJ, et al. Engineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy. Proc Natl Acad Sci USA 2010;107(42):17939e44.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

408

25. SELF-ASSEMBLED NANOSTRUCTURES (SANs)

77. Soppimath KS. pH-triggered thermally responsive polymer coreshell nanoparticles for drug delivery. Adv Mater 2005;17(3):318. 78. Soppimath KS, Liu LH, Seow WY, Liu SQ, Powell R, Chan P, et al. Multifunctional core/shell nanoparticles self-assembled from pH-induced thermosensitive polymers for targeted intracellular anticancer drug delivery. Adv Funct Mater 2007;17(3):355e62. 79. Yadav AK, Mishra P, Mishra AK, Jain S, Agrawal GP. Development and characterization of hyaluronic acid-anchored PLGA nanoparticulate carriers of doxorubicin. Nanomedicine 2007;3(4): 246e57. 80. Huang J, Zhang H, Yu Y, Chen Y, Wang D, Zhang G, et al. Biodegradable self-assembled nanoparticles of poly (D,L-lactide-coglycolide)/hyaluronic acid block copolymers for target delivery of docetaxel to breast cancer. Biomaterials 2014;35(1):550e66. 81. Ganesh S, Iyer AK, Morrissey DV, Amiji MM. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials 2013;34(13): 3489e502. 82. Chalikwar SS, Mene BS, Pardeshi CV, Belgamwar VS, Surana SJ. Self-assembled, chitosan grafted PLGA nanoparticles for intranasal delivery: design, development and ex vivo characterization. Polym Plast Technol 2013;52(4):368e80. 83. Yang XQ, Lyer AK, Singh A, Choy E, Hornicek FJ, Amiji MM, et al. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer. Sci Rep 2015;5. 84. Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev 2009;109(2):259e302. 85. Gindy ME, DiFelice K, Kumar V, Prud’homme RK, Celano R, Haas RM, et al. Mechanism of macromolecular structure evolution in self-assembled lipid nanoparticles for siRNA delivery. Langmuir 2014;30(16):4613e22. 86. Mozafari MR. Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett 2005;10(4):711e9. 87. Patil YP, Jadhav S. Novel methods for liposome preparation. Chem Phys Lipids 2014;177:8e18. 88. Laouini A, Jaafar-Maalej C, Limayem-Blouza I, Sfar S, Charcosset C, Fessi H. Preparation, characterization and applications of liposomes: state of the art. J Colloid Sci Biotechnol 2012;1(2):147e68. 89. Torchilin V, Weissig V. Liposomes: a practical approach. Oxford University Press; 2003. 90. Bangham AD, De Gier J, Greville G. Osmotic properties and water permeability of phospholipid liquid crystals. Chem Phys Lipids 1967;1(3):225e46. 91. Olson F, Hunt CA, Szoka FC, Vail WJ, Papahadjopoulos D. Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta 1979; 557(1):9e23. 92. Szoka Jr F, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 1978;75(9): 4194e8. 93. Batzri S, Korn ED. Single bilayer liposomes prepared without sonication. Biochim Biophys Acta 1973;298(4):1015e9. 94. Zumbuehl O, Weder HG. Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid-detergent mixed micelles. Biochim Biophys Acta 1981;640(1):252e62. 95. Nii T, Ishii F. Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method. Int J Pharm 2005;298(1):198e205. 96. Shum HC, Lee D, Yoon I, Kodger T, Weitz DA. Double emulsion templated monodisperse phospholipid vesicles. Langmuir 2008; 24(15):7651e3. 97. Kaur IP, Bhandari R, Bhandari S, Kakkar V. Potential of solid lipid nanoparticles in brain targeting. J Control Release 2008;127(2): 97e109.

98. Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery e a review of the state of the art. Eur J Pharm Biopharm 2000;50(1):161e77. 99. Yu DG, Williams GR, Yang JH, Wang X, Yang JM, Li XY. Solid lipid nanoparticles self-assembled from electrosprayed polymerbased microparticles. J Mater Chem 2011;21(40):15957e61. 100. Salvador-Morales C, Zhang L, Langer R, Farokhzad OC. Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials 2009; 30(12):2231e40. 101. Hu CM, Kaushal S, Tran Cao HS, Aryal S, Sartor M, Esener S, et al. Half-antibody functionalized lipid-polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells. Mol Pharm 2010;7(3):914e20. 102. Banerjee A, Onyuksel H. Peptide delivery using phospholipid micelles. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2012;4(5): 562e74. 103. Thipparaboina R, Chavan RB, Kumar D, Modugula S, Shastri NR. Micellar carriers for the delivery of multiple therapeutic agents. Colloids Surfs B 2015;135:291e308. 104. Shin HC, Alani AW, Rao DA, Rockich NC, Kwon GS. Multi-drug loaded polymeric micelles for simultaneous delivery of poorly soluble anticancer drugs. J Control Release 2009;140(3):294e300. 105. Danquah M, Li F, Duke 3rd CB, Miller DD, Mahato RI. Micellar delivery of bicalutamide and embelin for treating prostate cancer. Pharm Res 2009;26(9):2081e92. 106. Wei L, Cai C, Lin J, Chen T. Dual-drug delivery system based on hydrogel/micelle composites. Biomaterials 2009;30(13):2606e13. 107. Chitkara D, Singh S, Kumar V, Danquah M, Behrman SW, Kumar N, et al. Micellar delivery of cyclopamine and gefitinib for treating pancreatic cancer. Mol Pharm 2012;9(8):2350e7. 108. Miller T, Rachel R, Besheer A, Uezguen S, Weigandt M, Goepferich A. Comparative investigations on in vitro serum stability of polymeric micelle formulations. Pharm Res 2012;29(2):448e59. 109. Kakizawa Y, Kataoka K. Block copolymer micelles for delivery of gene and related compounds. Adv Drug Deliv Rev 2002;54(2): 203e22. 110. Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev 2001;47(1):113e31. 111. Gong J, Chen M, Zheng Y, Wang S, Wang Y. Polymeric micelles drug delivery system in oncology. J Control Release 2012;159(3):312e23. 112. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 2008;100(9):672e9. 113. Zhang Y, Zhang H, Wang X, Wang J, Zhang X, Zhang Q. The eradication of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles. Biomaterials 2012;33(2):679e91. 114. Ke XY, Lin Ng VW, Gao SJ, Tong YW, Hedrick JL, Yang YY. Codelivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells. Biomaterials 2014;35(3):1096e108. 115. Wang K, Zhang T, Liu L, Wang X, Wu P, Chen Z, et al. Novel micelle formulation of curcumin for enhancing antitumor activity and inhibiting colorectal cancer stem cells. Int J Nanomedicine 2012; 7:4487e97. 116. Gunkel-Grabole G, Sigg S, Lomora M, Lorcher S, Palivan CG, Meier WP. Polymeric 3D nano-architectures for transport and delivery of therapeutically relevant biomacromolecules. Biomater Sci 2015;3(1):25e40. 117. Blanazs A, Armes SP, Ryan AJ. Self-assembled block copolymer aggregates: from micelles to vesicles and their biological applications. Macromol Rapid Commun 2009;30(4e5):267e77. 118. Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature 2006;440(7082):297e302.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

REFERENCES

119. Seeman NC. Nucleic acid junctions and lattices. J Theor Biol 1982; 99(2):237e47. 120. Pinheiro AV, Han D, Shih WM, Yan H. Challenges and opportunities for structural DNA nanotechnology. Nat Nanotechnol 2011; 6(12):763e72. 121. Li J, Fan C, Pei H, Shi J, Huang Q. Smart drug delivery nanocarriers with self-assembled DNA nanostructures. Adv Mater 2013; 25(32):4386e96. 122. Zhang Q, Jiang Q, Li N, Dai LR, Liu Q, Song LL, et al. DNA origami as an in vivo drug delivery vehicle for cancer therapy. ACS Nano 2014;8(7):6633e43. 123. Jiang Q, Song C, Nangreave J, Liu XW, Lin L, Qiu DL, et al. DNA origami as a carrier for circumvention of drug resistance. J Am Chem Soc 2012;134(32):13396e403. 124. Douglas SM, Bachelet I, Church GM. A logic-gated nanorobot for targeted transport of molecular payloads. Science 2012;335(6070): 831e4. 125. Luo ZL, Zhang SG. Designer nanomaterials using chiral selfassembling peptide systems and their emerging benefit for society. Chem Soc Rev 2012;41(13):4736e54. 126. Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci USA 1993;90(8):3334e8. 127. Zhang SG. Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol 2003;21(10):1171e8. 128. Holmes TC, de Lacalle S, Su X, Liu GS, Rich A, Zhang SG. Extensive neurite outgrowth and active synapse formation on selfassembling peptide scaffolds. Proc Natl Acad Sci USA 2000; 97(12):6728e33. 129. Koutsopoulos S, Zhang SG. Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I. Acta Biomater 2013;9(2): 5162e9. 130. Shah RN, Shah NA, Lim MMD, Hsieh C, Nuber G, Stupp SI. Supramolecular design of self-assembling nanofibers for cartilage regeneration. Proc Natl Acad Sci USA 2010;107(8):3293e8. 131. Mata A, Geng YB, Henrikson KJ, Aparicio C, Stock SR, Satcher RL, et al. Bone regeneration mediated by biomimetic mineralization of a nanofiber matrix. Biomaterials 2010;31(23):6004e12. 132. Liu X, Wang XM, Wang XJ, Ren H, He J, Qiao L, et al. Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro. Acta Biomater 2013;9(6):6798e805.

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133. Hartgerink JD, Beniash E, Stupp SI. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001;294(5547): 1684e8. 134. Hartgerink JD, Beniash E, Stupp SI. Peptide-amphiphile nanofibers: a versatile scaffold for the preparation of self-assembling materials. Proc Natl Acad Sci USA 2002;99(8):5133e8. 135. Guler MO, Claussen RC, Stupp SI. Encapsulation of pyrene within self-assembled peptide amphiphile nanofibers. J Mater Chem 2005;15(42):4507e12. 136. Beniash E, Hartgerink JD, Storrie H, Stendahl JC, Stupp SI. Selfassembling peptide amphiphile nanofiber matrices for cell entrapment. Acta Biomater 2005;1(4):387e97. 137. Cui H, Webber MJ, Stupp SI. Self-assembly of peptide amphiphiles: from molecules to nanostructures to biomaterials. Biopolymers 2010;94(1):1e18. 138. Zhong C, Cooper A, Kapetanovic A, Fang Z, Zhang M, Rolandi M. A facile bottom-up route to self-assembled biogenic chitin nanofibers. Soft Matter 2010;6(21):5298e301. 139. Hassanzadeh P, Kharaziha M, Nikkhah M, Shin SR, Jin J, He S, et al. Chitin nanofiber micropatterned flexible substrates for tissue engineering. J Mater Chem B 2013;1(34). 140. Iijima S. Helical microtubules of graphitic carbon. Nature 1991; 354(6348):56e8. 141. Chen RJ, Zhang Y, Wang D, Dai H. Noncovalent sidewall functionalization of single-walled carbon nanotubes for protein immobilization. J Am Chem Soc 2001;123(16):3838e9. 142. Yang W, Thordarson P, Gooding JJ, Ringer SP, Braet F. Carbon nanotubes for biological and biomedical applications. Nanotechnology 2007;18(41):412001. 143. Vardharajula S, Ali SZ, Tiwari PM, Eroglu E, Vig K, Dennis VA, et al. Functionalized carbon nanotubes: biomedical applications. Int J Nanomedicine 2012;7:5361e74. 144. Liu Z, Shen Z, Zhu T, Hou S, Ying L, Shi Z, et al. Organizing single-walled carbon nanotubes on gold using a wet chemical self-assembling technique. Langmuir 2000;16(8):3569e73. 145. Shim JS, Yun Y-H, Rust MJ, Do J, Shanov V, Schulz MJ, et al. The precise self-assembly of individual carbon nanotubes using magnetic capturing and fluidic alignment. Nanotechnology 2009;20(32):325607. 146. Park SY, Park SY, Namgung S, Kim B, Im J, Kim JY, et al. Carbon nanotube monolayer patterns for directed growth of mesenchymal stem cells. Adv Mater 2007;19(18):2530. 147. Stout DA, Webster TJ. Carbon nanotubes for stem cell control. Materials Today 2012;15(7):312e8.

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C H A P T E R

26 Biomimetic Nanofibers as Artificial Stem Cell Niche Xiaowei Li, Yu-Hao Cheng, Jose Roman, Hai-Quan Mao Johns Hopkins University, Baltimore, MD, United States

O U T L I N E 1. Introduction

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2. Methods for Preparing Polymeric Nanofibers 2.1 Electrospinning 2.2 Solution Phase Separation 2.3 Self-Assembly 2.4 Other Fibrous Scaffold Preparation Methods

413 415 415 416 417

3. Control Stem Cell Behaviors Through Biomimetic Nanofibers In Vitro 3.1 Adhesion 3.2 Alignment 3.3 Migration 3.4 Proliferation 3.5 Differentiation and Maturation

417 417 418 418 418 418

4. Delivery of Stem Cells With Biomimetic Nanofibers for Tissue Regeneration

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1. INTRODUCTION The concept of stem cell “niche” was first proposed by Schofield in the late 70’s to illustrate the physiologically restricted microenvironment that supports stem cells residing in normal tissues.1 Stem cell niche is a complex, heterotypic, and dynamic structure, which includes supporting neighboring cells, extracellular matrix (ECM), secreted signaling factors, physical parameters (such as shear stress, tissue stiffness, and topography), immunological factors (inflammation and scarring), and environmental signals (hypoxia) (Fig. 26.1 and Chapter 1, Fig. 1.1).2 This niche maintains a stable number of stem

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00026-3

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12

Brain Tissue Regeneration Spinal Cord Regeneration Peripheral Nerve Regeneration Cardiac Regeneration Blood Vessel Regeneration Liver Regeneration Bone Regeneration Cartilage Regeneration Ligament and Tendon Regeneration Skin Regeneration Corneal Regeneration Retinal Regeneration

420 422 422 422 422 423 423 424 424 424 424 425

5. Concluding Remarks

425

Abbreviations and Acronyms

425

References

425

cells during homeostasis. When tissue is injured or diseased, the niche actively engages stem cells, guides their proliferation, migration, differentiation, and regulates their participation in tissue regeneration and repair.3 Adult stem cells have been identified in different types of tissues, such as the brain,4 spinal cord,5 skin,6 intestine,7 adipose,8 muscle,9 and bone marrow.10 Table 26.1 summarizes several types of adult stem cells and the surrounding ECMs. It remains partially understood how the stem cell niche interprets a variety of cellular scale physical and biological signals and modulates tissue repair process based on physiological requirement and pathological

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Copyright © 2017 Elsevier Inc. All rights reserved.

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FIGURE 26.1 Stem cell niches are complex, heterotypic, and dynamic structures, which include different cellular components, secreted factors, immunological control, extracellular matrix (ECM), physical parameters, and metabolic control. The interactions between stem cells and their niches are bidirectional and reciprocal. Adapted from Lane SW, Williams DA, Watt FM. Modulating the stem cell niche for tissue regeneration. Nat Biotech 2014;32(8):795e803.

state.11 To address this question, artificial niches with tunable physical, biochemical, and cellular parameters have been prepared to examine the roles of the extrinsic cues in controlling the microenvironment of a stem cell in a reproducible manner.12 Some of the engineering approaches include, but are not limited to, identifying suitable ECM candidates as substrates for cell culture through micro-patterning ECM in two dimensions (2D) and high-throughput ECM microarrays; synthesizing novel biomaterials; fabricating biomaterial scaffolds in three dimensions (3D) with controllable topographies and stiffnesses; and conjugating signaling factors to scaffolds to regulate stem cell fates.12 There are several benefits derived from manufacturing synthetic stem cell niches: (1) culturing stem cells under defined conditions, thereby improving reproducibility; (2) facilitating mechanistic studies to reveal specific roles of various niche cues in regulation of stem cell fate; and (3) developing novel strategies to engineer stem cell fates in vivo for tissue regeneration.13

ECM as a major component of the stem cell niche not only serves as an anchor to support stem cell adhesion, but also mediates the presentation of various biochemical signals, and thereby collectively regulates stem cell fate decisions such as survival, proliferation, and differentiation.14 Cell binding to ECM molecules (Table 26.1) is mediated through integrin receptors that modulate cell adhesion specificity and strength, and transduce outside-in signals to cells. Since most stem cell niches are associated with basal lamina, basement membrane forming molecules, laminin, nidogen, collagen IV, etc., have been frequently examined as cell binding ligands in artificial matrices. Signaling cue presentation function of the niche is achieved through sequestration, enrichment, and clustering of soluble growth factors via ECM molecules or cell surface proteoglycans. Such a presentation mode can modulate the distribution and activity of the growth factors at even single cell level.2 The in vivo ECM, particularly basement membrane, possesses ubiquitous nanoscale fibrous topography.

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2. METHODS FOR PREPARING POLYMERIC NANOFIBERS

TABLE 26.1

Stem Cells Identified in Different Tissues and Their Surrounding Extracellular Matrices (ECMs)

Type of Stem Cells

Tissue Locations

ECM Components

References

Epithelial stem cells

Hair follicle bulge, sebaceous gland, basal layer of interfollicular epithelium

Collagen IV, nephronectin, laminin

6

Intestinal stem cells

Near the bottom of the intestinal crypt

Laminin

7

Neural stem cells

Brain: subventricular zone of the lateral ventricles and subgranular zone of the dentate gyrus in the hippocampus Spinal cord: white matter parenchyma or close to the central canal, either in the ependyma or subependyma

Collagen IV, chondroitin sulfate or heparan sulfate, lecticans, nidogen, hyaluronic acid, tenascin C and R, laminin

4

Hematopoietic stem cells

Endosteal surface of trabecular bone, close to osteoblasts and the endothelial cells that line blood vessels

Collagen I, IV, VI; fibronectin; tenascin-C

10

Muscle stem cell

Satellite cells on the surface of basal lamina

Collagen IV, VI; perlecan; nidogen; fibronectin; laminin

9

The role of this topography in modulating stem cell fates has been primarily derived in vitro.15,16 Nanofibers have a morphological resemblance to fibers naturally found in the ECM. This similar characteristic suggests that nanofibers could be used as a supportive matrix for creating artificial niche for stem cells, upon which additional functionalities could be incorporated to further modulate stem cell fates.17 In this chapter, three most common techniques for generating nanofibers both for in vitro stem cell culture and in vivo stem cell delivery are discussed in detail, together with major applications of these nanofibers in regulating stem cell fates in vitro on the topics of adhesion, morphology/alignment, migration, proliferation, differentiation, and maturation. Specifically, recent advances in nanofiber matrix-mediated stem cell transplantation for regenerating different types of tissues, including brain, spinal cord, peripheral nerve, heart, blood vessels, liver, bone, cartilage, tendon, ligament, skin, cornea, and retina are reviewed. In addition, key challenges and future directions are highlighted in the development of nanofibers as artificial niches for stem cell applications.

2. METHODS FOR PREPARING POLYMERIC NANOFIBERS There are three major methods for preparing polymeric nanofiber scaffolds: electrospinning, solution phase separation, and self-assembly. These methods have been applied to fabricate nanofibers from various types of polymers, including naturally derived polymers, such as collagen,18 gelatin,19 fibrin,20 laminin,21 keratin,22 chitosan,23 dextran,24 hyaluronic acid (HA),25 cellulose,26 and alginate.27 Additionally, nanofibers can

be derived from synthetic polymers such as polyglycolide (PGA),28 polylactide (PLA),29 poly(lactide-co-glycolide) (PLGA),30 poly(ε-caprolactone) (PCL),16,31,32 and polyethersulfone (PES).33e35 Table 26.2 summarizes a few examples of nanofiber matrices tested as artificial stem cell niches and compares their general physical and biochemical properties. The physical and biochemical properties of these polymer-derived nanofibers, including topography, structure, and biological modifications, greatly influence stem cell fates and their applications in tissue regeneration. Natural polymers are preferred choices of materials for nanofiber preparation due to their good biocompatibility and intrinsic bioactivity. Many of them are biodegradable by the naturally occurring enzymes, although not all of these are degradable in vivo. However, there are also several pitfalls associated with the use of biologically derived materials. They often contain residual signaling molecules and undefined constituents. These poorly characterized components make it difficult to control the quality of the products, and may generate high batch-to-batch variation. The donor tissuederived components may elicit strong inflammation and specific immune responses. In case there are residual growth factors, retaining their bioactivity during nanofiber fabrication process could be very challenging. More importantly, additional cross-linking step is frequently needed to render these nanofibers nonsoluble in aqueous environments, since many of them are watersoluble. In contrast, synthetic polymers have several notable advantages, including controllable chemical composition, degradation profile, and easily modifiable fiber diameter and orientation. However, these fibers are typically bioinert; additional modifications are needed to conjugate adhesion ligands for supporting stem cell adhesion and signaling. Various strategies have been

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TABLE 26.2

Examples of Polymer Nanofibers Tested as Artificial Stem Cell Niches

414

Physical and Biochemical Properties Strong/Weak Fibers (S/W)

Integrin Binding Sites (Y/N)

Collagen

E

W

Gelatin

E

Fibrin

Modification of Nanofibers: Stem Cell Fates

Applications

References

Y

Combined with PCL and PLLA fibers: hMSC differentiation

Bone, nerve

18

W

Y

Combined with poly(L-lactic acid)-copoly(e-caprolactone) (PLACL): hMSC osteogenic differentiation

Bone

19

E

W

Y

Combined with PLGA nanofibers: ASCs in vivo survival Fibrin and PCL blended nanofibers: hMSC chondrogenic differentiation

Tendon, cartilage

20

Laminin

E

W

Y

Coated on polystyrene nanofiber: NSC attachment and alignment

Nerve

21

Keratin

E

W

Y

Adjusting concentration of keratin to control fiber diameter Combined with PCL nanofibers

Skin

22

Chitosan

E

W

N

Combined with PCL fibers: hMSC differentiation

Skin, tendon

23

Dextran

E

W

N

Mixed with PLACL fibers: ASC survival

Skin

24

Hyaluronic acid (HA)

E

W

N

Mixed with PLLA fibers: hMSC chondrogenic differentiation

Cartilage

25

Cellulose

E

S

N

Nanofibrous cellulose with HA and gelatin hydrogel: liver progenitor cells differentiation into functional hepatocytes 3D culture of hiPSCs;

Liver

26

Alginate

E

W

N

Glycerol incorporated into alginate to obtain fibers Combined with PCL fibers: BMP2 release for in vivo bone regeneration

Bone

27

Polyglycolide (PGA)

H

W

N

Fiber diameter: hMSC chondrogenic differentiation

Cartilage

28

Polylactide (PLA)

H

W

N

Conjugation with YIGSR peptide: mESCs neural differentiation Fiber orientation: hMSC migration and differentiation

Nerve, bone

29

Poly(ε-caprolactone) (PCL)

H

S

N

Fiber diameter and orientation: aNSC differentiation Mesh stiffness: hiPSC morphology and proliferation

Nerve

16,31,32

Poly(lactic-co-glycolic acid) (PLGA)

H

W

N

As the substrate: hMSC chondrogenic and osteogenic differentiation

Bone, cartilage

30

Polyethersulfone (PES)

Nondegradable

S

N

Fiber diameter: aNSC differentiation Fiber surface modification with hydroxyl, carboxyl and amine groups: HSPC proliferation Fiber orientation: NCSC differentiation

Nerve, blood vessels, skin

33e35

26. BIOMIMETIC NANOFIBERS AS ARTIFICIAL STEM CELL NICHE

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

Polymers

Hydrolytic/Enzymatic Degradable (H/E)

2. METHODS FOR PREPARING POLYMERIC NANOFIBERS

developed to functionalize synthetic polymer fibers thus mimicking different bioactivities of natural ECM discussed above.36 Several considerations should be taken into account when designing nanofibers as artificial stem cell niche for tissue regeneration. First, the nanofiber matrix design should include integrated biochemical and topographical cues matching the specific cell type. The optimal configuration of topographical cues, such as fiber diameter, alignment, and mechanical property, and biochemical cues, including matrix-bound and supplemental factors, needs to be tailored for each stem cell type. More importantly, beyond supporting cell adhesion and survival, optimal cues guiding stem cell differentiation could be different from those favoring cell proliferation and expansion. Next, a nanofiber matrix should allow for cellular infiltration and migration by either initially being macroporous or becoming macroporous upon degradation over time. The structure of the nanofiber matrix should be similar to that of the native tissue in order to ensure that stem cells within the scaffold receive the appropriate cues to organize and integrate with host tissue. Aligned nanofibers are uniquely designed to induce uniaxial tissue formation for tissue types with cells that are organized in a highly aligned arrangement, such as nerve, tendon, ligament, bone, and skeletal and cardiac muscles. In this context, the role of nanofiber matrix is expanded beyond just maintaining stem cell homeostasis by providing cell adhesion and regulating cell fate specification, to serving as a 3D artificial niche facilitating tissuespecific differentiation, controlled organization, and integration when implanted in vivo.

2.1 Electrospinning Electrospinning is a reliable method to fabricate long continuous strands of fibers with a diameter ranging from tens of nanometers to several microns.37 A typical electrospinning setup includes a spinneret, a syringe pump, and a collecting plate. An extruded polymer solution or melt is stretched into a thin fiber under an applied high voltage with the evaporation of the solvent or cooling of melts, whipping in a random manner before depositing on a grounded collecting plate. When a rotating wheel or frame is used for collection, aligned fibers can be produced. Electrospun fiber size and microstructure can be precisely controlled by several processing parameters, such as solvent composition and vapor pressure, polymer solution viscosity, conductivity and feed rate, applied voltage, and capillary-to-collector distance. Electrospinning has been shown to efficiently generate nanofibers from a variety of synthetic and natural polymers. Although

415

devices or constructs with different geometries can be produced by electrospinning, it remains challenging to generate 3D scaffolds with these nanofibers. A new fiber spinning method has been developed for fabricating hydrogel fibers with uniaxial alignment in 3D through a combination of electrospinning and mechanical stretching.38 Hydrogel fibers were prepared from aqueous solutions of natural polymers such as alginate, fibrin, gelatin, and HA. The chain alignment of hydrogel fibers is induced by electrospinning of the aqueous polymer solution and then fixed by crosslinking in a rotating disk as the collecting plate. The unique internal alignment feature significantly improves the mechanical properties of these hydrogel fibers. Furthermore, the organic solvent-free processing conditions can be applied to the incorporation of live stem cells within the hydrogel fiber. This approach effectively induces cellular alignment in a 3D space. These hydrogel fibers are ideal scaffold candidates for generating aligned tissue structures to restore some functions of specific tissues, such as blood vessel, nerve, tendon, and muscle (Fig. 26.2).

2.2 Solution Phase Separation When a solution of natural or synthetic polymer is thermally induced to undergo phase separation under prescreened solvent condition and temperature that allow for the spinodal liquideliquid phase separation, a polymer-rich phase will gradually form followed by crystallization of the polymer-rich phase. This process will generate a 3D nanofibrous structure,39,40 following the steps of phase separation-gelation, solvent extraction, and lyophilization. The diameters of fibers generated using this method range from tens to hundreds of nanometers. The phase separation-gelation is the most critical step of this process and important for the control over the porosity and fibrous morphology.41 The success in developing an effective strategy relies on the ability to identify suitable polymeresolvent conditions for a given polymer. Several polymers have been used to generate nanofiber scaffolds using this phase separation technique. For example, Ma and Zhang have developed a unique solution phase separation procedure for the preparation of nanofibrous scaffolds using PLA and gelatin.39 Gelation temperature, polymer concentration, and freezing temperature before freeze-drying have shown to affect the structures of nanofibrous scaffolds. A low gelation temperature benefits the formation of a nanoscale fiber network. Lower initial polymer concentration results in smaller diameter fibers and a more porous network.41 In addition, porogen particles, comprised of salt or paraffin, can be incorporated into the nanofibrous scaffolds to generate macroscale

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FIGURE 26.2 Electrostretching setup and features of hydrogel microfibers. (A) Illustration of the electrostretching setup. To prepare a dual component microfiber, two syringes (a and b) are used (e.g., sodium alginate solution in syringe “a” and fibrinogen solution in “b” to mix the solutions prior to extrusion; typically only one syringe is needed for single solution spinning. (B) Effect of alginate solution feeding rate on the diameter of hydrogel microfibers. (CeK) SEM micrographs of hydrogel fibers prepared with simple extrusion and electrostretching. Fibrin (C), gelatin (D), and HA (E) hydrogels prepared by simple extrusion or mixing consist of randomly oriented nanofiber network. Electrostretched fibrin (F), gelatin (G), and HA (H) hydrogel fibers showing preferential alignment. Arrows indicate the orientation of the microfiber longitudinal axis. Fibrin (I), gelatin (J), and HA (K) hydrogel fibers following stretching and dehydration in air forming fiber bundles. Both fibrin and gelatin fibers preserved surface texture and grooves. Samples in (CeH) were prepared by the critical point drying technique; and samples in (IeK) were stretched and dried in air. Adapted from Zhang S, Liu X, Barreto-Ortiz SF, et al. Creating polymer hydrogel microfibres with internal alignment via electrical and mechanical stretching. Biomaterials 2014;35(10):3243e51.

pores.42,43 The porosity and macropore size of the scaffold can be controlled through adjusting the quantity and size of these porogen particles. This solution phase separation method provides a convenient way to produce polymer nanofiber scaffolds without any specific equipment and is easily scalable, although it is difficult to control the organization of the nanofibers in 3D.43

2.3 Self-Assembly Peptides with specific secondary structures could self-assemble into nanofibrous structures as a result of specific, local interactions including hydrogen bonding, hydrophobic interaction, and electrostatic reactions.44 By designing specific sequences, such an assembly

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process could be initiated upon external stimuli. For example, b-sheet forming peptides could assemble into nanofiber hydrogels when the ionic strength of the peptide solution in aqueous medium increases to physiological level. This peptide assembly method allows for the formation of a fibrous 3D hydrogel matrix under facile conditions without using organic solvents, thus amenable to maintaining high cell viability during the encapsulation process.45 For example, Zhang et al. 50 have developed a class of self-assembling nanofibers with short 8- to 16-residue peptides with alternating hydrophobic and hydrophilic amino acids, which form stable b-sheet structures in water upon addition of physiological concentrations of salt solutions, forming nanofiber hydrogels with a water content higher than 99.5%.46 These chemically designed self-assembling peptides have been made to incorporate specific ligands identified from the ECM proteins, such as lamininderived peptide sequences, isoleucine-lysine-valinealanine-valine (IKVAV) and arginine-glycine-aspartic acid (RGD).47 Peptide functionalization with these adhesion and differentiation motifs improved cell adhesion and survival, and directed neural stem cell differentiation, in comparison with unmodified peptide fibers. Another example of a self-assembled nanofiber matrix is prepared from water-soluble peptide amphiphiles (PAs) that consist of a hydrophobic tail and a hydrophilic short peptide sequence. The charge screening by counter ions in the cell culture medium, along with the collapse of hydrophobic sequence, as well as hydrogen bond formation between adjacent peptide residues, promotes self-assembly of the nanofibrous hydrogel. Similarly, IKVAV sequence has been incorporated into a peptide amphiphile, which could self-assemble into nanofibrous hydrogel. These IKVAVmodified nanofibers stimulated the rapid and selective differentiation of human neural stem cells (hNSCs) into neurons due to the high epitope density of IKVAV presented on the fiber surface. Furthermore, these nanofiber hydrogels have been shown to inhibit glial scar formation and promote axonal outgrowth, which resulted in behavioral improvement after implantation in a murine model of spinal cord injury.48,49 Molecular self-assembly generates biocompatible and degradable 3D fibrous matrices. However, these nanofibers have relatively low mechanical strength and limited physical guidance properties as a tissue engineering scaffold. Alignment of the nanofibers is generally difficult to achieve due to a lack of longrange order. Recent advances have shown that through a thermal induction process, long-range alignment of some PA-based self-assembled nanofibers can be obtained.50 Presentation of IKVAV or RGD epitopes to this aligned PA enhanced the growth of neurites from neurons encapsulated in a nanofibrous scaffold, while

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the aligned nanofiber guided these neurites along the direction of these nanofibers.51

2.4 Other Fibrous Scaffold Preparation Methods Besides discussed above, there are several other methods for producing nanofibers, including templating, drawing, extraction, and vapor-phase polymerization, which have been reviewed elsewhere.52 These methods however, suffer from low scalability and limited applicability to particular polymers. Many cell or tissue ECM-derived matrices preserve the nanofibrous nature of the ECM structure. These types of processed matrices can also be used for stem cell culture. However, they fall outside the scope of this chapter, which focuses only on the artificial matrices.

3. CONTROL STEM CELL BEHAVIORS THROUGH BIOMIMETIC NANOFIBERS IN VITRO Biomimetic nanofibers can modulate various aspects of cell activities, such as stem cell adhesion/survival, morphology/alignment, migration, proliferation, differentiation, and maturation. These nanofibers present cell adhesion signals, thus supporting cell spreading, morphology/alignment, and migration. In addition, topographical and biomechanical characteristics of the nanofibers significantly influence stem cell proliferation, differentiation, and maturation.

3.1 Adhesion Most synthetic polymer-derived nanofibers are bioinert and, therefore, need surface functionalization in order to support stem cell adhesion. Various strategies have been adopted to introduce adhesion ligands on the fiber surface, including physical adsorption and covalent conjugation of cell adhesion proteins or peptides. Introducing surface charge through plasma treatment and generating surface functional groups by grafting polymerization have been shown to be effective to modulate the activities of surface incorporated cell adhesion proteins.53 For example, PES nanofibers have been physically coated with positively charged poly-Lornithine (PLO) and laminin to facilitate NSC adhesion.33 PES nanofibers chemically grafted with amine groups mediated higher degree of adhesion and more efficiently supported the expansion of human umbilical cord blood derived hematopoietic stem cells than those modified with hydroxyl and carboxyl groups.34,54

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3.2 Alignment Nanofiber matrix offers opportunity to organize stem cells and their differentiated progenies. Several important cell types, neurons, tendinocytes, ligament cells, skeletal muscle cells, cardiomyocytes, endothelial cells, and smooth muscle cells, develop into uniaxial or circumferential alignment structure, which are crucial to their maturation and functions. Nanofiber matrices can be used to promote the regeneration of these tissues by mediating corresponding cell arrangement. It has been shown that cells adopt a linear orientation on uniaxially aligned nanofibers.35 For example, NSCs cultured on aligned PCL fibers extend along the fiber axis. With the induction of retinoic acid, the majority of NSCs cultured on aligned fibers differentiated into neuronal lineage in contrast to those grown on random fibers or un-patterned surfaces. The substrate-induced elongation alone was shown to upregulate the canonical Wnt signaling in NSCs, which was potentiated by retinoic acid treatment, thus contributing to neuronal differentiation program on aligned fiber substrates.16,31

tumor-derived matrix is widely used as an experimental standard substrate coating for stem cell culture. To eliminate the use of animal-derived products, nanofibrous substrates prepared from PCL and poly (ethylene terephthalate) (PET), have been developed as an alternative to Matrigel for long-term culture of hPSCs. The mechanical properties of the substrates have been shown to play a critical role in determining hiPSC colony morphology. Specifically, the stiffness of the substrate was inversely correlated with the spherical pattern of hiPSC colony, which, in turn, was correlated with hiPSC self-renewal and spontaneous differentiation.32 Recently, a well-defined and scalable nanofibrous cellulose hydrogel system for long-term culture of hPSCs with high expansion efficiency and purity in 3D has generated great interests.56 The pluripotency of hiPSCs cultured in this hydrogel was maintained for over 3 weeks as confirmed by the expression of stem cell markers, such as Oct4, Nanog, and SSEA-4, in in vitro embryoid body formation and in vivo teratoma formation.26 Nanofibrous scaffolds may promote the clinical application of hiPSCs in patient-specific cell therapies.

3.3 Migration Cell migration is a prerequisite for cell organization that ultimately determines the success of tissue regeneration.55 For example, random and aligned PLA fibers have been examined for their effects on the migration of transplanted hMSCs and bone growth in vivo in a critical-sized calvarial defect model. Human MSCs that were cultured on aligned fibers migrated approximately 10-fold faster along the parallel direction than along the perpendicular direction. After being implanted into the calvarial defect for 2 months, the regenerated bone area was larger with aligned fibers compared to random ones. More importantly, the orientation of collagen fibrils in regenerated bone tissue aligned with the direction of the implanted nanofibers. Ample evidence has demonstrated the effectiveness of aligned nanofibers as an instructive cue for guided cell migration and the stimulation of tissue regeneration in vivo.29

3.4 Proliferation Stem cell expansion presents a significant challenge for cell-based regenerative therapy. Substrates and techniques are needed for effective expansion of stem cells at various stages, from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), to tissue-specific stem/progenitor cells. Well-defined matrix and serum-free media have been developed aiming for scalable cell expansion under GMP conditions in future clinical translation. Matrigel, a mouse

3.5 Differentiation and Maturation Effective control over cell differentiation and maturation also remains a major challenge for stem cell-based therapy. Accumulative evidence suggests that nanofibrous matrices, partially mimicking the topographical features of the natural ECM, can significantly influence cell differentiation and maturation.31,33 As an example, NSC differentiation was enhanced by the topographical and biochemical cues presented by nanofiber matrices. First, the fiber diameter was shown to drastically influence the differentiation of rat adult NSCs when cultured on electrospun fiber matrix. As shown in Fig. 26.3, NSCs cultured on 283 nm- and 749 nm-diameter fibers differentiated preferentially into RIPþ oligodendrocytes and Tuj1þ neurons, respectively. Additionally, the adhesion and migratory activities of these cells may have effects on their differentiation kinetics and lineage specification. Cells cultured on small diameter fibers (i.e., 283 nm) are able to spread randomly along the fiber matrix and assume a dendritic cell morphology similar to the glial lineage. Cells on large diameter fibers (i.e., 749 nm) can extend on single fibers with the cell shape similar to that of differentiated neurons.33 Recently, nanofibers have also been used to support oligodendrocyte precursor cell (OPC) maturation in an axon-free model of myelination.57,58 In this study, electrospun polystyrene nanofibers of varying diameters (0.2e4.0 mm) have been developed as a substitute for axons to induce oligodendrocyte myelination, thereby determining a possible

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FIGURE 26.3 (A) Control over fiber alignment and geometry through electrospinning. Fiber alignment can be achieved by switching from a stationary collector to a rotating disk collector. Fiber diameter can be controlled via changing parameters such as polymer solution concentration or flow rate. (BeG) Immunofluorescence analysis of rat NSCs cultured on various substrates in the presence of 1 mM retinoic acid and 1% fetal bovine serum for 5 days. Cell nuclei (blue) are stained using DAPI (4,6-diamidino-2-phenylindole) as a counter staining. Quantification of staining results is shown (B) with corresponding representative images of cells on each substrate (CeE). Scale bar ¼ 100 mm. Circled cells on 283-nm fiber mesh are cells stained double positive for RIP and Tuj-1 (F). Example of statistically unquantifiable 1452-nm mesh is shown in (G). Adapted from Lim SH, Mao HQ. Electrospun scaffolds for stem cell engineering. Adv Drug Deliv Rev 2009;61(12):1084e96; Christopherson GT, Song H, Mao HQ. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 2009;30(4):556e64.

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mechanism for axonal-oligodendroglial interaction. OPCs displayed sensitivity to the biophysical properties of fiber diameter and initiated membrane ensheathment before differentiation. The minimum fiber diameter threshold was determined to be 0.4 mm.59 Secondly, various chemical and biological cues are well-known to regulate fate specification of stem cells once they are incorporated with or on nanofibers.17 For example, graphene oxide (GO) has been demonstrated to promote the growth and differentiation of various stem cell lines, including iPSCs, MSCs, and NSCs.60 GO has been coated on PCL nanofibers with varying concentrations (0.1 and 0.5 mg/mL) for the selective differentiation of rat NSCs. The higher concentration of GO (0.5 mg/mL) enhanced oligodendrocyte differentiation and maturation into myelin basic protein positive (MBPþ) cells. GO coating on the nanofibers has been shown to potentiate the overexpressing of a number of key integrin-related intracellular signaling molecules, such as focal adhesion kinases and tyrosine-protein kinases. These molecules mediate cytoskeletal remodeling and process extension during oligodendrocyte development and maturation.61 As another example, microRNAs (miRNAs) are a class of small noncoding RNAs that contain 21e23 nucleotide base pairs. They silence the expression of their target mRNAs to regulate stem cell differentiation. Many studies have indicated the involvement of miRNAs during different stages of OPC development.62 PCL nanofiber-mediated delivery of miRNA-219 and miRNA-338 not only efficiently knocked down oligodendrocyte differentiation inhibitory regulators but also promoted oligodendrocyte maturation by significantly increasing MBPþ cells significantly.63 In addition, the orientation of nanofibers has also been shown to affect oligodendrocyte differentiation.16 We have verified that aligned fiber substrates were less receptive to the attachment and continued survival of oligodendrocytes derived from NSCs than cells cultured on random fiber or un-patterned substrates. Recently, nanofiber membranes with different orientation have been used for epigenetic mechanomodulation. Fibroblasts transduced with four transcription factors (Oct4, Sox2, Klf4, and c-Myc) were seeded onto nanofiber surfaces to determine the effects of random and aligned fiber orientation on reprogramming efficiency. Indeed, these cells cultured on aligned nanofibers generated significantly more Nanogþ colonies than random fibers.64

4. DELIVERY OF STEM CELLS WITH BIOMIMETIC NANOFIBERS FOR TISSUE REGENERATION As shown in in vitro setting, nanofibers facilitate stem cell survival, proliferation, differentiation, and

maturation following transplantation. Specific nanofibers mediated stem cell transplantation for treating brain and spinal cord injury, stimulating peripheral nerve regeneration, accelerating cardiac repair, deriving liver-like tissue, promoting bone tissue formation, assisting cartilage regeneration, repairing tendon/ ligament tear, enhancing chronic skin wound healing, and supporting corneal and retinal regeneration. A few case studies covering biomimetic nanofibers reconstruct the regenerative niches for endogenous stem/ progenitor cells for tissue regeneration and will be discussed in detail.

4.1 Brain Tissue Regeneration Brain cortex has a very organized layered structure. Stroke and traumatic injury often lead to the loss of nerve tissue and the formation of a lesion cavity that is primarily located to the cortex. Furthermore, the ongoing inflammation at the lesion site and the lack of supportive tissue structure and vasculature within the cavity present a hostile environment that result in low cell survival, as well as poor control over differentiation and engraftment of transplanted stem cells. Radially aligned nanofibers, mimicking some of the physical characteristics of brain cortex, have been implanted into the injured lesion cavity to promote host brain tissue regrowth and regeneration after injury. Aligned, electrospun PLA nanofibers have induced robust and functional vascularization in the fiber orientation, neurogenesis, and integration of the newly generated neurons into a normal brain circuitry (Fig. 26.4).55 However, this nanofibrous scaffold failed to fill the lesion cavity cohesively. Another study has looked at implanting, self-assembled hydrogels with nanofibrous structure to deliver exogenous NSCs for brain tissue regeneration after injuries. The RADA16-IKVAV peptide solution, when injected into the injured brain lesion site, rapidly assembled into nanofibrous hydrogel in situ that filled the lesion cavity. The hydrogel not only created a permissive environment for axons to regenerate at the lesion site but also connected the brain tissue together.65 The hydrogel further enhanced the survival of encapsulated NSCs and reduced the formation of reactive glial cells.66 However, few premature neurons survived at the lesion site 6 weeks posttransplantation; and these cells also exhibited a disorganized structure with limited integration with host cortical tissue. Since the ordered and layered cortical structure is important for its function, nanofibrous scaffolds should be carefully designed for controlling cell organization and integration following transplantation into the lesion site, which is critical to the success of stem cell-based therapy after a brain injury or stroke.

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FIGURE 26.4 Extent of brain tissue regeneration and vascularization after 1 year. Macroscopic view of brains with (A) control lesions and (B) radial scaffolds 1 year after implantation. Coronal sections show GFAPþ astroglia around (C) a control lesion and (D) in the radial scaffold. (E) Bright-field coronal section shows the material remaining. (F) Magnification showing extensive colonization by Tuj-1þ neurons and (G) blood vessels labeled by DiI perfusion. (H) Mature neurons stained with MAP-2 in the middle of the scaffold. Adapted from Alvarez Z, Castano O, Castells AA, et al. Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold. Biomaterials 2014;35(17):4769e81.

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4.2 Spinal Cord Regeneration Traumatic spinal cord injury (SCI) interrupts nerve connections between the brain and the rest of the body resulting in paralysis and loss of sensation below the level of injury. The process of tissue remodeling after SCI leads to the formation of a cavity that is surrounded by a dense glial scar, which is additionally potentiated by endogenous factors that inhibit axon growth, such as myelin-associated inhibitors and chondroitin sulfate proteoglycans (CSPGs). A promising strategy for dealing with this inhibitory environment and cavity is to bridge the lesion with a growth-permissive scaffold that could support axonal regrowth and the reinnervation of the damaged connections.67 Uniaxially aligned nanofibers have been shown to provide topographical guidance cues to direct and improve axonal extension during regeneration. A tenascin-C derived peptide, when covalently modified on these nanofibers, improved the ability of the nanofibers to facilitate axonal regrowth.68 Rolipram, a small molecule able to suppress inflammatory responses, when immobilized onto aligned PLLA/PLGA nanofibers, decreased the astrocyte population and CSPG production at the SCI lesion.69 Liu et al. have applied PLGA-PEG nanofiber scaffolds to deliver induced NSCs (iNSCs), which were reprogrammed from mouse embryonic fibroblasts by Sox2-retrovirus, for spinal cord regeneration. The implanted iNSC-seeded scaffolds restored the continuity of the completely transected spinal cord, reduced cavity formation, and contributed to functional recovery of the spinal cord.70 These iNSCs hold great potential in stem cell-based therapy for SCI since they have no ethical controversy, and are less likely to cause teratomas compared to ESCs. However, the Sox2-retrovirus used to induce reprogramming may pose a safety risk with tumor formation through insertional mutagenesis, and the induction efficiency of iNSCs needs to be further improved in order to provide and maintain adequate cell number and quality. Other reprogramming methods, such as nonviral gene delivery vehicles, could be used and further investigated to improve differentiation efficiency from a reliable source.71

4.3 Peripheral Nerve Regeneration Severe injuries to the peripheral nervous system (PNS) often lead to life-long loss and/or disruption of functions mediated by the injured nerve. Most therapeutic applications have interposed grafts (natural or artificial) between the proximal and distal stumps to provide a growth-promoting scaffold and repair the injured nerve. Nerve autografts, the gold standard technique for the repair of a peripheral nerve lesion, must be surgically removed from the host and can cause morbidity

and functional loss at the harvested site. Synthetic nerve guidance conduits (NGCs) have been developed as an alternative to autografts for peripheral nerve regeneration.72 In most studies, uniaxially aligned nanofibers are collected as a rectangular sheet and then rolled up to obtain NGCs, with the direction of fiber alignment parallel to the longitudinal direction of the NGCs. The NGC, when transplanted with autologous Schwann cells, has shown to improve nerve fiber extension, as well as motor recovery in a sciatic nerve injury model.73 Recently aligned self-assembled PA hydrogels have been filled into a PLGA conduit to direct the axon growth inside the conduit (Fig. 26.5). Functional recovery in conduit/PA was comparable to the autograft group, and significantly faster than in animals treated with the empty PLGA conduit alone.51,74

4.4 Cardiac Regeneration Myocardial tissue cannot self-regenerate following infarction. Rebuilding the blood flow in the region of myocardial infarction (MI) represents a key approach for cardiac regeneration. Self-assembled nanofiber hydrogel has been applied to deliver vascular endothelial growth factor (VEGF) at the lesion site to create an intra-myocardial microenvironment suitable for recruitment, proliferation, and maturation of vascular cells.75 In addition, insulin-like growth factor 1 (IGF-1), promoting cardiomyocyte growth and differentiation, has been conjugated to self-assembling peptide hydrogels for prolonged delivery to the myocardium.76 Cardiac progenitor cells transplanted with this IGF-1-conjugated nanofiber hydrogel improved endogenous and exogenous myocardial regeneration in an MI mouse model.77 However, the low mechanical strength of these peptide hydrogels has limited its effectiveness in cardiac tissue regeneration. A nanofiber scaffold with matching mechanical strength and alignment feature of the cardiac tissue would be ideal to respond to the dynamic working conditions of heart muscle, induce alignment and transduce the mechanical stimulation to regenerating cardiac progenitors, thus promoting their differentiation and maturation into functional cardiomyocytes with appropriate orientation at the lesion site.78 For example, cardiac patches composed of elastic poly(glycerol sebacate) (PGS) nanofiber mesh grafted with gelatin has been shown to facilitate cell adhesion and retention, and promote the differentiation of cardiomyocyte progenitor cells.79 This PGS-gelatin nanofiber scaffold with good biocompatibility and mechanical properties holds great potential for myocardium restoration.

4.5 Blood Vessel Regeneration Synthetic vascular grafts have been developed to regenerate functional small diameter blood vessels but

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FIGURE 26.5 (A) The PA solution is loaded into the PLGA tubes by first passing through a 40-ı`m mesh screen. The liquid crystalline behavior of nanofibers allows them to align in response to the shear flow experienced as they pass through the mesh. Nanofiber size is exaggerated for graphical appeal. (B)w0.5 mm longitudinal section of the tube constructs is imaged using optical microscopy. (C) SEM showing the aligned PA nanofibers inside the PLGA tube constructs. Adapted from Li A, Hokugo A, Yalom A, et al. A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers. Biomaterials 2014;35(31):8780e90.

have had limited success. Current grafts are often highly thrombogenic and cannot simulate the complicated structural architecture and dynamic functions of blood vessels.80 Nanofibrous scaffolds are advantageous for vascular graft development since they can mimic the native ECM, and their physicochemical properties can be adjusted to match the native blood tissue. Furthermore, the high surface area to volume ratio of the nanofibers allows good cell proliferation and infiltration, which benefits vasculogenesis.81 Huang et al. 82 have generated aligned collagen fibrous scaffolds with high mechanical strength and woven-like helical and crimped configurations, which simulate the structure of collagen bundles in relaxed blood vessels. These aligned nanofibrous scaffolds, when compared to random fibrils, could efficiently guide cytoskeletal assembly and nuclear orientation of hiPSCs-derived endothelial cells and enhance their survival after implantation in normal and ischemic tissues in the femoral artery excision lesion.82 Vascular smooth muscle cells may be incorporated into the scaffold to further induce physiologically relevant cell alignment pattern mimicking the native blood vessel.

4.6 Liver Regeneration Liver is a highly metabolic, complex array of vasculature and hepatocytes, which perform many functions in the body, including plasma protein synthesis and

transport, glucose homeostasis, and urea synthesis. The major challenge in liver tissue regeneration involves generating functional hepatocytes that can effectively reproduce these characteristics. Nanofibrous scaffolds have been adopted to mediate stem cell differentiation into functional hepatocytes.83 For example, Kazemnejad et al. have demonstrated that the PCL/collagen/PES nanofibers, when compared to the conventional cell culture system, not only improved the differentiation of hMSCs into hepatocytes, but also enhanced hepatocyte-specific cell functions, such as synthesis and secretion of albumin, urea, and transferrin. An in vivo study of this fibrous scaffold seeded with hMSCs implanted into rat liver has shown liver growth with functional hMSC-derived hepatocytes. These hepatocytes were able to secrete human albumin, which was detected in the rat serum.84

4.7 Bone Regeneration In the bone tissue, collagen fibers overlap the adjacent fibers and hydroxyapatite crystals are arranged in layers within each fiber, resembling overlapping bricks. Nanofibrous scaffolds have been designed as highly porous and incorporating hydroxyapatite or growth factors for bone regeneration both in vitro and in vivo.85 For example, uniaxially aligned PCL nanofibers have been shown to induce osteogenesis of hMSCs and increase deposition of mineralized ECM along the aligned fiber

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direction in vitro.86 Gene delivery offers an attractive alternative to growth factor delivery, especially in instances where the signaling protein of interest is highly unstable or difficult to formulate in vitro. For example, liposomes encapsulated with plasmids encoding runt-related transcription factor 2 (Runx2), a key factor involved in osteoblast phenotype induction, have been immobilized on the surface of a PCL nanofiber mesh. Human MSCs cultured on these immobilized fibers presented enhanced levels of metabolic activity and total protein synthesis. This approach was effective in differentiating hMSCs with an early onset of osteoblastic marker expression compared to Runx2loaded liposomes supplied only in suspension.87

scaffolds to that of natural ligaments/tendons. Owing to their success in controlling alignment, nanofibrous scaffolds have been investigated in repairing and regenerating ligament and tendon tissues.23,90 For example, braided PCL fiber scaffolds with mechanical behavior similar to native tendons and ligaments during mechanical loading have been employed. Human iPSC-derived MSCs aligned parallel to the direction of these nanofibers and differentiated into the tenogenic lineage when coupled with the presence of tenogenic growth factors and upon stimulation with cyclic tensile strain.91

4.10 Skin Regeneration 4.8 Cartilage Regeneration In articular cartilage, chondrocytes, collagen, and elastic fibers are embedded in a stiff hydrogel-like matrix, rich in mucopolysaccharides, exhibiting flexibility and elasticity. Articular cartilage lacks selfregeneration potential due to the absence of vascular networks and progenitor cells, as well as the inability of chondrocytes to migrate in the dense ECM network. By mimicking physical and biological cues of native ECM, nanofibrous scaffolds hold potential utility for articular cartilage repair. For example, peptide amphiphile molecules have been designed for cartilage regeneration by displaying a high density of binding epitopes to transforming growth factor-b1 (TGF-b1). This nanofibrous hydrogel supported the survival and promoted the chondrogenic differentiation of hMSCs while also enhancing regeneration of articular cartilage in a full thickness chondral defect treated by microfracture in a rabbit model, even without the addition of exogenous growth factors.88 This injectable nanofiber hydrogel offers additional advantage in the minimally invasive delivery into the joint space in comparison with current clinical cartilage repair strategies.

4.9 Ligament and Tendon Regeneration Ligaments and tendons are dense connective tissues with packed collagen fiber bundles aligned parallel to the longitudinal axis and arranged in a hierarchical structure. This structure is crucial for ligaments to stabilize joints by connecting adjacent bones and for tendons to transmit forces between muscle and bone. High tensile forces transmitted by ligaments and tendons make them susceptible to tearing or complete rupture. Ligaments and tendons fail to heal because of their hypocellularity and hypovascularity.89 The major challenge in ligament and tendon regeneration involves mimicking the mechanical performance of artificial

For skin tissue regeneration, an ideal scaffold should mimic the native skin ECM structure and function, protect the wound from infection and mechanical irritation, be permeable to moisture and oxygen, and enable the removal of exudate; it should provide appropriate cell infiltration, adhesion, and proliferation. The large surface area and porosity of nanofibrous scaffolds enable good permeability for oxygen and water, as well as the adsorption of liquids, while concomitantly protecting the wound from bacterial penetration and dehydration.92 Incorporation of growth factors such as basic fibroblast growth factor (bFGF) into nanofibrous scaffolds stimulated skin stem cells to accelerate wound healing.93 The gradual release of bFGF from the fibrous mesh enhanced collagen deposition and ECM remodeling. Moreover, the arrangement and composition of regenerated collagen fibers were similar to the normal tissue. A layer-by-layer nanofiber scaffold has been developed to mimic the multilayered structure of the native skin.94

4.11 Corneal Regeneration The cornea stroma is composed of dense, regularly packed collagen fibrils arranged in orthogonal layers or lamellae. Limbal epithelial stem cells (LESCs) located in the limbus at the corneoscleral junction are responsible for renewing and regenerating the corneal epithelium. Nanofibrous scaffolds have been proposed as a substrate for LESC cultivation because of their extremely large surface area and their ability to mimic the structure to the underlying collagen fibrils. LESCs delivered using a polyamide nanofiber scaffold have been shown to enhance corneal healing and significantly prevent the local inflammatory reaction.95 Nanofibrous scaffolds represent a convenient strategy for growth and transplantation of LESCs to treat various ocular surface injuries.

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REFERENCES

4.12 Retinal Regeneration Bruch’s membrane is a thin, acellular, five-layered ECM located between the metabolically active retinal pigment epithelium (RPE) and choriocapillaris. It thus serves as the substratum of the RPE. Nanofibrous networks closely mimic the fibrous architecture of the native inner collagenous layer of Bruch’s membrane. Human RPE cells cultured on a PLGA nanofibrous membrane exhibited a properly oriented monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces, which displayed a striking resemblance to native retina.96 Nanofibrous membrane may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with degenerative eye disorders such as age-related macular degeneration and glaucoma.

5. CONCLUDING REMARKS Nanofibrous scaffolds provide a unique set of platforms to reconstruct ECM niches and mediate stem cell survival, morphology, proliferation, differentiation, and maturation in vitro and in vivo for a wide range of stem and progenitor cell types. Great progress has been made in demonstrating the utility of nanofibrous scaffolds to restore native tissue architectures and biological functions. The challenge will be, however, to integrate multiple biochemical, physical, and mechanical cues presented by nanofiber scaffolds, and to incorporate spatial and temporal control of these signaling cues into modular and scalable platforms for clinical translation. In addition, by incorporating appropriate signaling cues, nanofibrous scaffolds can be applied to recruit endogenous stem and progenitor cells to these injured and diseased sites and direct their proliferation and differentiation into the desired cell type. Such an in situ tissue regeneration approach takes full advantage of the body’s own regenerative capacity and offers a more convenient approach for tissue regeneration.97,98

ABBREVIATIONS AND ACRONYMS aNSC Adult neural stem cells bFGF Basic fibroblast growth factor CSPGs Chondroitin sulfate proteoglycans ECM Extracellular matrix GO Graphene oxide HA Hyaluronic acid hASCs Human adipose stem cells hESCs Human embryonic stem cells hiPSCs Human induced pluripotent stem cells hMSCs Human mesenchymal stem cells hNSCs Human neural stem cells hPSCs Human pluripotent stem cells

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HSPC Hematopoietic stem/progenitor cells IGF-1 Insulin-like growth factor 1 IKVAV Isoleucine-lysine-valine-alanine-valine LESCs Limbal epithelial stem cells MBP Myelin basic protein mESCs Mouse embryonic stem cells MI Myocardial infarction NCSC Neural crest stem cell NGCs Nerve guidance conduits OPCs Oligodendrocyte precursor cells PA Peptide amphiphile PCL Poly(ε-caprolactone) PES Polyethersulfone PET Poly(ethylene terephthalate) PGA Polyglycolide PLA Polylactide PLGA Poly (lactide-co-glycolide) PLO Poly-L-ornithine PNS Peripheral nervous system RGD Arginine-glycine-aspartic acid RPE Retinal pigment epithelial Runx2 Runt-related transcription factor 2 SCI Spinal cord injury TGF-b1 Transforming growth factor-b1 VEGF Vascular endothelial growth factor

References 1. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4(1e2):7e25. 2. Lane SW, Williams DA, Watt FM. Modulating the stem cell niche for tissue regeneration. Nat Biotech 2014;32(8):795e803. 3. Scadden DT. The stem-cell niche as an entity of action. Nature 2006; 441(7097):1075e9. 4. Doetsch F, Caille´ I, Lim DA, Garcı´a-Verdugo JM, Alvarez-Buylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 1999;97(6):703e16. 5. Thuret S, Moon LDF, Gage FH. Therapeutic interventions after spinal cord injury. Nat Rev Neurosci 2006;7(8):628e43. 6. Blanpain C, Fuchs E. Plasticity of epithelial stem cells in tissue regeneration. Science 2014;344(6189). 7. Sato T, van Es JH, Snippert HJ, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 2011; 469(7330):415e8. 8. Kaewsuwan S, Song SY, Kim JH, Sung J-H. Mimicking the functional niche of adipose-derived stem cells for regenerative medicine. Expert Opin on Biol Ther 2012;12(12):1575e88. 9. Kuang S, Gillespie MA, Rudnicki MA. Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell 2008; 2(1):22e31. 10. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature 2014;505(7483):327e34. 11. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science 2009;324(5935): 1673e7. 12. Peerani R, Zandstra PW. Enabling stem cell therapies through synthetic stem cell-niche engineering. J Clin Invest 2010;120(1):60e70. 13. Song H, Yoon C, Kattman SJ, et al. Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue. Proc Natl Acad Sci USA 2010;107(8): 3329e34. 14. Watt FM, Huck WTS. Role of the extracellular matrix in regulating stem cell fate. Nat Rev Mol Cell Biol 2013;14(8):467e73. 15. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126(4):677e89.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

426

26. BIOMIMETIC NANOFIBERS AS ARTIFICIAL STEM CELL NICHE

16. Lim SH, Liu XY, Song H, Yarema KJ, Mao HQ. The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells. Biomaterials 2010;31(34):9031e9. 17. Lim SH, Mao HQ. Electrospun scaffolds for stem cell engineering. Adv Drug Deliv Rev 2009;61(12):1084e96. 18. Prabhakaran MP, Venugopal JR, Ramakrishna S. Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials 2009; 30(28):4996e5003. 19. Rim NG, Lee JH, Jeong SI, Lee BK, Kim CH, Shin H. Modulation of osteogenic differentiation of human mesenchymal stem cells by poly[(L-lactide)-co-(epsilon-caprolactone)]/gelatin nanofibers. Macromol Biosci 2009;9(8):795e804. 20. Perumcherry SR, Chennazhi KP, Nair SV, Menon D, Afeesh R. A novel method for the fabrication of fibrin-based electrospun nanofibrous scaffold for tissue-engineering applications. Tissue Eng Part C: Methods 2011;17(11):1121e30. 21. Bakhru S, Nain AS, Highley C, et al. Direct and cell signalingbased, geometry-induced neuronal differentiation of neural stem cells. Integr Biol 2011;3(12):1207e14. 22. Edwards A, Jarvis D, Hopkins T, Pixley S, Bhattarai N. Poly(ε-caprolactone)/keratin-based composite nanofibers for biomedical applications. J Biomed Mater Res B: Appl Biomater 2015;103(1):21e30. 23. Leung M, Jana S, Tsao C-T, Zhang M. Tenogenic differentiation of human bone marrow stem cells via a combinatory effect of aligned chitosan-poly-caprolactone nanofibers and TGF-[small beta]3. J Mater Chem B 2013;1(47):6516e24. 24. Pan JF, Liu NH, Sun H, Xu F. Preparation and characterization of electrospun PLCL/Poloxamer nanofibers and dextran/gelatin hydrogels for skin tissue engineering. PLoS One 2014;9(11):e112885. 25. Nesti LJ, Li WJ, Shanti RM, et al. Intervertebral disc tissue engineering using a novel hyaluronic acid-nanofibrous scaffold (HANFS) amalgam. Tissue Eng Part A 2008;14(9):1527e37. 26. Lou Y-R, Kanninen L, Kuisma T, et al. The use of nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells. Stem Cells and Dev 2014;23(4): 380e92. 27. Kolambkar YM, Dupont KM, Boerckel JD, et al. An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. Biomaterials 2011;32(1):65e74. 28. Itani Y, Asamura S, Matsui M, Tabata Y, Isogai N. Evaluation of nanofiber-based polyglycolic acid scaffolds for improved chondrocyte retention and in vivo bioengineered cartilage regeneration. Plast Reconstr Surg 2014;133(6):805ee13e. 29. Lee JH, Lee YJ, Cho HJ, Shin H. Guidance of in vitro migration of human mesenchymal stem cells and in vivo guided bone regeneration using aligned electrospun fibers. Tissue Eng Part A 2014; 20(15e16):2031e42. 30. Liu Y, Zhang X, Xia Y, Yang H. Magnetic-field-assisted electrospinning of aligned straight and wavy polymeric nanofibers. Adv Mater 2010;22(22):2454e7. 31. Mahairaki V, Lim SH, Christopherson GT, et al. Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro. Tissue Eng Part A 2011; 17(5e6):855e63. 32. Maldonado M, Wong LY, Echeverria C, et al. The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells. Biomaterials 2015;50(0):10e9. 33. Christopherson GT, Song H, Mao HQ. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 2009;30(4):556e64. 34. Jiang X, Christopherson GT, Mao HQ. The effect of nanofibre surface amine density and conjugate structure on the adhesion and proliferation of human haematopoietic progenitor cells. Interface Focus 2011;1(5):725e33.

35. Ren YJ, Zhang S, Mi R, et al. Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix. Acta Biomater 2013;9(8):7727e36. 36. Li X, Katsanevakis E, Liu X, Zhang N, Wen X. Engineering neural stem cell fates with hydrogel design for central nervous system regeneration. Prog Polym Sci 2012;37(8):1105e29. 37. Lu T, Li Y, Chen T. Techniques for fabrication and construction of three-dimensional scaffolds for tissue engineering. Int J Nanomed 2013;8:337e50. 38. Zhang S, Liu X, Barreto-Ortiz SF, et al. Creating polymer hydrogel microfibres with internal alignment via electrical and mechanical stretching. Biomaterials 2014;35(10):3243e51. 39. Ma PX, Zhang RY. Synthetic nano-scale fibrous extracellular matrix. J Biomed Mater Res 1999;46(1):60e72. 40. Zhang R, Ma P. Processing of polymer scaffolds: phase separation. In: Atala A, Lanza RP, editors. Methods of tissue engineering. San Diego: Academic Press; 2002. p. 715e24. 41. Yang F, Murugan R, Ramakrishna S, Wang X, Ma YX, Wang S. Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering. Biomaterials 2004;25(10):1891e900. 42. Hu J, Feng K, Liu X, Ma PX. Chondrogenic and osteogenic differentiations of human bone marrow-derived mesenchymal stem cells on a nanofibrous scaffold with designed pore network. Biomaterials 2009;30(28):5061e7. 43. Hsu SH, Huang S, Wang YC, Kuo YC. Novel nanostructured biodegradable polymer matrices fabricated by phase separation techniques for tissue regeneration. Acta Biomater 2013;9(6):6915e27. 44. Whitesides GM, Grzybowski B. Self-assembly at all scales. Science 2002;295(5564):2418e21. 45. Luo Z, Zhang S. Designer nanomaterials using chiral selfassembling peptide systems and their emerging benefit for society. Chem Soc Rev 2012;41(13):4736e54. 46. Zhang S. Fabrication of novel biomaterials through molecular selfassembly. Nat Biotechnol 2003;21(10):1171e8. 47. Koutsopoulos S, Zhang S. Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I. Acta Biomater 2013;9(2): 5162e9. 48. Tysseling-Mattiace VM, Sahni V, Niece KL, et al. Self-assembling nanofibers inhibit glial scar formation and promote axon elongation after spinal cord injury. J Neurosci 2008;28(14):3814e23. 49. Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004;303(5662):1352e5. 50. Zhang S, Greenfield MA, Mata A, et al. A self-assembly pathway to aligned monodomain gels. Nat Mater 2010;9(7):594e601. 51. Berns EJ, Sur S, Pan L, et al. Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels. Biomaterials 2014;35(1):185e95. 52. Beachley V, Wen X. Polymer nanofibrous structures: fabrication, biofunctionalization, and cell interactions. Prog Polym Sci 2011; 35(7):868e92. 53. Jiang X-S, Chai C, Zhang Y, Zhuo R-X, Mao H-Q, Leong KW. Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34þ cells. Biomaterials 2006;27(13):2723e32. 54. Chua KN, Chai C, Lee PC, et al. Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells. Biomaterials 2006;27(36):6043e51. 55. Alvarez Z, Castano O, Castells AA, et al. Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold. Biomaterials 2014;35(17):4769e81. 56. Lei Y, Schaffer DV. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci USA 2013;110(52):E5039e48.

III. DESIGNING SMART BIOMATERIALS TO MIMIC AND CONTROL STEM CELL NICHE

REFERENCES

57. Emery B. Regulation of oligodendrocyte differentiation and myelination. Science 2010;330(6005):779e82. 58. Li Y, Ceylan M, Shrestha B, et al. Nanofibers support oligodendrocyte precursor cell growth and function as a neuron-free model for myelination study. Biomacromolecules 2014;15(1):319e26. 59. Lee S, Leach MK, Redmond SA, et al. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nat Methods 2012;9(9):917e22. 60. Solanki A, Chueng S-TD, Yin PT, Kappera R, Chhowalla M, Lee K-B. Axonal alignment and enhanced neuronal differentiation of neural stem cells on graphene-nanoparticle hybrid structures. Adv Mater 2013;25(38):5477e82. 61. Shah S, Yin PT, Uehara TM, Chueng ST, Yang L, Lee KB. Guiding stem cell differentiation into oligodendrocytes using graphenenanofiber hybrid scaffolds. Adv Mater 2014;26(22):3673e80. 62. Emery B. Transcriptional and post-transcriptional control of CNS myelination. Curr Opin Neurobiol 2010;20(5):601e7. 63. Diao HJ, Low WC, Milbreta U, Lu QR, Chew SY. Nanofibermediated microRNA delivery to enhance differentiation and maturation of oligodendroglial precursor cells. J Control Release 2015;(0). 64. Downing TL, Soto J, Morez C, et al. Biophysical regulation of epigenetic state and cell reprogramming. Nat Mater 2013;12(12):1154e62. 65. Ellis-Behnke RG, Liang YX, You SW, et al. Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc Natl Acad Sci USA 2006; 103(13):5054e9. 66. Cheng TY, Chen MH, Chang WH, Huang MY, Wang TW. Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering. Biomaterials 2013;34(8): 2005e16. 67. Hellal F, Hurtado A, Ruschel J, et al. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 2011;331(6019):928e31. 68. Meiners S, Ahmed I, Ponery AS, et al. Engineering electrospun nanofibrillar surfaces for spinal cord repair: a discussion. Polym Int 2007;56(11):1340e8. 69. Zhu Y, Wang A, Shen W, et al. Nanofibrous patches for spinal cord regeneration. Adv Funct Mater 2010;20(9):1433e40. 70. Liu C, Huang Y, Pang M, et al. Tissue-engineered regeneration of completely transected spinal cord using induced neural stem cells and gelatin-electrospun poly (lactide-co-glycolide)/polyethylene glycol scaffolds. PLoS One 2015;10(3):e0117709. 71. Bhise NS, Wahlin KJ, Zack DJ, Green JJ. Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells. Int J Nanomed 2013;8:4641e58. 72. Krick K, Tammia M, Martin R, Ho¨ke A, Mao H-Q. Signaling cue presentation and cell delivery to promote nerve regeneration. Curr Opin Biotechnol 2011;22(5):741e6. 73. Xie J, MacEwan MR, Liu W, et al. Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system. ACS Appl Mater Interfaces 2014; 6(12):9472e80. 74. Li A, Hokugo A, Yalom A, et al. A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers. Biomaterials 2014;35(31):8780e90. 75. Lin Y-D, Luo C-Y, Hu Y-N, et al. Instructive nanofiber scaffolds with VEGF create a microenvironment for arteriogenesis and cardiac repair. Sci Transl Med 2012;4(146):146ra109. 76. Davis ME, Hsieh PC, Takahashi T, et al. Local myocardial insulinlike growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci USA 2006;103(21):8155e60. 77. Padin-Iruegas ME, Misao Y, Davis ME, et al. Cardiac progenitor cells and biotinylated insulin-like growth factor-1 nanofibers improve endogenous and exogenous myocardial regeneration after infarction. Circulation 2009;120(10):876e87.

427

78. Coulombe KLK, Bajpai VK, Andreadis ST, Murry CE. Heart regeneration with engineered myocardial tissue. Annu Rev Biomed Eng 2014;16(1):1e28. 79. Kharaziha M, Nikkhah M, Shin SR, et al. PGS: Gelatin nanofibrous scaffolds with tunable mechanical and structural properties for engineering cardiac tissues. Biomaterials 2013;34(27):6355e66. 80. Cittadella G, de Mel A, Dee R, De Coppi P, Seifalian AM. Arterial tissue regeneration for pediatric applications: inspiration from upto-date tissue-engineered vascular bypass grafts. Artif Organs 2013; 37(5):423e34. 81. Hashi CK, Zhu Y, Yang G-Y, et al. Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts. Proc Natl Acad Sci USA 2007;104(29):11915e20. 82. Huang NF, Okogbaa J, Lee JC, et al. The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds. Biomaterials 2013;34(16):4038e47. 83. Yu Y-Q, Jiang X-S, Gao S, et al. Local delivery of vascular endothelial growth factor via nanofiber matrix improves liver regeneration after extensive hepatectomy in rats. J Biomed Nanotechnol 2014; 10(11):3407e15. 84. Tai BC, Du C, Gao S, Wan AC, Ying JY. The use of a polyelectrolyte fibrous scaffold to deliver differentiated hMSCs to the liver. Biomaterials 2010;31(1):48e57. 85. Ribeiro N, Sousa SR, van Blitterswijk CA, Moroni L, Monteiro FJ. A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regeneration. Biofabrication 2014;6(3):035015. 86. Martins A, Alves da Silva ML, Faria S, Marques AP, Reis RL, Neves NM. The influence of patterned nanofiber meshes on human mesenchymal stem cell osteogenesis. Macromol Biosci 2011;11(7):978e87. 87. Monteiro N, Ribeiro D, Martins A, et al. Instructive nanofibrous scaffold comprising runt-related transcription factor 2 gene delivery for bone tissue engineering. ACS Nano 2014;8(8):8082e94. 88. Shah RN, Shah NA, Del Rosario Lim MM, Hsieh C, Nuber G, Stupp SI. Supramolecular design of self-assembling nanofibers for cartilage regeneration. Proc Natl Acad Sci USA 2010;107(8):3293e8. 89. Strauss EJ, Ishak C, Jazrawi L, Sherman O, Rosen J. Operative treatment of acute Achilles tendon ruptures: an institutional review of clinical outcomes. Injury 2007;38(7):832e8. 90. Younesi M, Islam A, Kishore V, Anderson JM, Akkus O. Tenogenic induction of human MSCs by anisotropically aligned collagen biotextiles. Adv Funct Mater 2014;24(36):5762e70. 91. Czaplewski SK, Tsai TL, Duenwald-Kuehl SE, Vanderby Jr R, Li WJ. Tenogenic differentiation of human induced pluripotent stem cellderived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds. Biomaterials 2014;35(25):6907e17. 92. Kim HS, Yoo HS. Therapeutic application of electrospun nanofibrous meshes. Nanomedicine 2014;9(4):517e33. 93. Yang Y, Xia T, Zhi W, et al. Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor. Biomaterials 2011;32(18):4243e54. 94. Wang Y-f, Guo H-f, Ying D-j. Multilayer scaffold of electrospun PLAePCLecollagen nanofibers as a dural substitute. J Biomed Mater Res B: Appl Biomater 2013;101(8):1359e66. 95. Holan V, Javorkova E. Mesenchymal stem cells, nanofiber scaffolds and ocular surface reconstruction. Stem Cell Rev 2013;9(5):609e19. 96. Nadri S, Kazemi B, Eslaminejad MB, Yazdani S, Soleimani M. High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds. Mol Biol Rep 2013;40(6):3883e90. 97. Chen FM, Wu LA, Zhang M, Zhang R, Sun HH. Homing of endogenous stem/progenitor cells for in situ tissue regeneration: promises, strategies, and translational perspectives. Biomaterials 2011; 32(12):3189e209. 98. Vanden Berg-Foels WS. In situ tissue regeneration: chemoattractants for endogenous stem cell recruitment. Tissue Eng Part B Rev 2014;20(1):28e39.

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27 Employing Microfluidic Devices to Induce Concentration Gradients Nathalie Brandenberg, Matthias P. Lutolf Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland

O U T L I N E 1. Introduction 1.1 The Importance of Concentration Gradients in Biology 1.2 Molecular Mass Transport Through Tissues: Theoretical Considerations 1.3 Learning From Developing Organisms 2. Engineering Concentration Gradients Using Microtechnology 2.1 Introduction 2.2 Fluid Mechanics at the Microscale 2.3 Micromanipulating Flows to Generate Concentration Gradients 2.4 Hydrogels as a Barrier to Generate Concentration Gradients

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1. INTRODUCTION 1.1 The Importance of Concentration Gradients in Biology Gradients were first proposed to be a critical component of biological processes more than a century ago. Embryologists observed recurrent patterns forming during the development of sea urchins, insects, and amphibians, suggesting the presence of graded signals within developing embryos.1 Alan Turing coined the term morphogens to describe the organized signals. In addition, he defined them precisely as diffusible molecules that induce different cellular responses in a concentration-dependent manner.2 Initially, the hypothesis was undermined by the common belief that the

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00027-5

3. Generating Micro-Engineered Gradients to Control Stem Cell Fate 3.1 Conventional Microfluidics to Spatially Control Stem Cell Fate 3.2 Combining Hydrogel and Microfluidic Technology to Manipulate Stem Cell Fate in Near-Physiological Settings

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Glossary

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Acknowledgment

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rapid development of such systems could not be dictated by diffusion speed, which was known to be slow. However, Crick later provided a mathematical demonstration showing that, in this context, diffusion could in fact correlate with the observed biological phenomena,3 catalyzing widespread study of concentration gradients in the organization of cells. More recently, the importance of morphogens and even gas gradients was described in diverse adult tissues. A notable example is the initiation of the formation of blood vessels, where, after activation of the endothelium, selected endothelial cells sprout and sense their elongation direction by following a gradient of vascular endothelial growth factor secreted by the surrounding tissue.4e6 Interestingly, graded signals have also been shown to be an important component of several

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adult stem cell niches in the body. Remarkably, the hematopoietic stem cell (HSC) niche, located in the bone marrow, displays graded signals from the sinusoids to the endosteal surface that are responsible for maintaining long-term HSCs (LT-HSCs).7 In particular, low oxygen tension in the bone marrow niche seems to be a key regulator of quiescence because of the related low oxidative stress that permits LT-HSCs to maintain their slow proliferation rate.8 Other niches, such as the intestinal stem cell (ISC) niche, maintain their homeostasis with a tightly regulated balance between bone morphogenetic proteins (BMPs) and Wnt pathway proteins. BMPs are secreted by the villi mesenchyme, whereas Wnts are secreted in the intervilli crypts. These proteins seem to form an inverse pair of concentration gradients from the bottom of the crypts, where ISCs reside, to the top of the villi, where the fully differentiated cells are located. This maintains the fidelity of the organ structure by keeping the various cellular compartments of the organ in specific locations.9,10

1.2 Molecular Mass Transport Through Tissues: Theoretical Considerations To engineer appropriate gradients in vitro, engineers must first understand their dynamics and the processes by which tissues establish them. Typically, morphogenesis induced by concentration gradients occurs by the secretion of morphogens from a source followed by the spread of morphogens into the target tissue to then be degraded. Although the gradient is being established or remains dynamic, the concentration of effectors will vary in space and time. At equilibrium, the gradient reaches a steady state. Concentration gradients of both morphogens and chemoattractants have been characterized in various manners in the literature.11 Among them, two main hypotheses have been presented. First, the system can be described as discrete entities that individually aid in the transport of a specific effector molecule. Although this description gives a high-resolution picture of all the cellular processes and the contribution of each entity in forming the gradient, this type of model is highly specific to the tissues and molecules of interest. Moreover, although the dynamic of a whole tissue is conserved over individuals and species, it is very unlikely that cellular processes between individuals are identical. These complexities render the approach difficult to generalize and, thus, to engineer. An obvious alternative to these discrete models is to describe the dynamic of a tissue as a continuum. Here, the effective molecules vary continuously in time and space, whereas individual entities of the tissue are not distinguished. In this case, the sum of every entity in the tissue can be recapitulated in specific parameters such as the effective

diffusion coefficient of the molecule of interest or its effective degradation rate. It is of high interest to understand and formalize such gradients mathematically to engineer devices that accurately generate concentration gradients in vitro. In 2009, Gonza´les-Gaita´n and colleagues described different diffusion models inspired by embryonic development from a theoretical perspective and mathematically formalized them.12 Because most of the gradients studied in vivo display symmetry, the mathematical descriptions are simplified to one spatial dimension. Four different types of gradients are described. The first case study is gradients formed solely by diffusion (Fig. 27.1A). In the ideal situation, the molecules produced by the source spread in a manner proportional to their effective diffusion coefficient (D). Having production but not depletion, the concentration in the target tissue increases constantly with time, which leads to the formation of Gaussian gradients that never reach steady state. Even so, transient states of these gradients can instruct cells, as is suggested for the anterioreposterior patterning of Drosophila embryos, which we will describe later in more detail. To achieve steady state, effector molecules must be degraded. The simplest model considers diffusion coupled with linear degradation (Fig. 27.1B). In this case, the molecule is being degraded at a single rate (represented by a constant, k), independent of location. This model is characterized by an analytical solution, which provides the concentration profile at steady state (solid line). The amplitude of the gradient is proportional to the source concentration, and the decay length is proportional to the effective diffusion coefficient (D) and inversely proportional to the degradation rate (k). During Drosophila wing development, it has been demonstrated using fluorescence recovery after photobleaching that the gradient of Decapentaplegic (Dpp) behaves similarly to the model of diffusion with linear degradation; the time needed to establish the Dpp gradient is on the same time scale as the time to reach steady state. In addition, measurements of its degradation rate were observed as constant.13 However, morphogen degradation rates can also vary nonlinearly. Diffusion with nonlinear degradation (Fig. 27.1C) is typically observed when feedback loop mechanisms depend upon the morphogen concentration. In many cases, such as Hedgehog (Hh) proteins, an intracellular negative feedback loop causes high concentrations of protein to result in higher degradation rates. Although linear degradation translates into exponential gradients, nonlinear degradation gives rise to power-law gradients. Gonza´les-Gaita´n and colleagues postulate that robustness is the key characteristic that distinguishes exponential from power-law gradients. In the exponential case, there is no limiting factor as

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FIGURE 27.1 (A) Free diffusion model. The molecules diffuse in the cellular compartment without any interactions with the surrounding tissue. This model does not give rise to a steady state. (B) Diffusion and linear degradation model. The molecules diffuse in the cellular compartment and are being degraded by the cells at an equivalent rate throughout the tissue. This model allows for a steady-state gradient with an exponential shape. (C) Diffusion and nonlinear degradation model. The molecules diffuse in the cellular compartment and are being degraded at unequal rates by the different cells composing the tissue. This model allows for a steady-state gradient with a power law shape. (D) Cell lineage transport model. The molecules are not secreted in the extracellular space, which forms a gradient through cell growth and division. This model allows for a steady-state gradient following a power law as well. (E) Fluorescent images of a Drosophila embryo at the blastoderm stage of development. Bicoid and Hunchback proteins are fluorescently tagged using immunofluorescence in red and blue, respectively. The protein concentrations are represented as normalized in arbitrary units. (F) Scanning electron micrograph of the closed neural tube. Counter gradients of BMPs and Sonic Hedgehog contribute to the dorsaleventral patterning of the cells along the developing neural tube. Adapted from Wartlick O, Kicheva A, Gonzalez-Gaitan M. Morphogen gradient formation. Cold Spring Harb Perspect Biol 2009;1:a001255; Reinitz J. Developmental biology: a ten per cent solution. Nature 2007;448:420e421; and Briscoe J, Novitch BG. Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube. Philos Trans R Soc Lond B Biol Sci 2008;363:57e70.

the gradient is only proportional to the flux of molecules coming from the source, leading to a general increase of concentration over the whole spatial dimension. In contrast, in the power-law case, a higher production leads to a higher concentration, which in turn increases the degradation rate. Thus, the effective increase in concentration is smaller than that in exponential gradients. The latter model shows a self-regulatory component that has the potential to mimic proteins displaying

feedback loop mechanisms. Lastly, gradients can be generated without secretion or diffusion in the target tissues. Cell growth and division itself can propagate effectors and thus establish graded intracellular signals. This cell lineage transport model (Fig. 27.1D) takes single cells as “volume elements” of the tissue. Therefore, concentration is inversely proportional to the volume, which implies that if an element grows exponentially, the concentration will decrease at the same rate; the molecules

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get diluted because of growth. The corresponding mathematical model gives a steady-state solution that follows a power law, which is similar to the model of diffusion with nonlinear degradation. On a final note, it is important to specify that cellular proliferation during development most likely couples cell lineage transport with gradients established through diffusion, regardless of degradation characteristics. However, if the growth of the tissue is much slower than diffusion turnover, which is often the case, the dilution effect of cell division becomes negligible. Overall, the models described above are valuable tools because they can recapitulate the fine modulation of gradients that occurs through complex interactions between receptors, extracellular matrix (ECM) components, and inhibitors12 by including these interactions in the model as different variables.

1.3 Learning From Developing Organisms As a perspective, we will detail two extensively studied examples to illustrate how these models can be relevant for understanding in vivo morphogenesis. In Drosophila embryos, anterioreposterior patterning is initiated by a gradient of the transcription factor Bicoid (Bcd) established from maternal Bcd mRNA deposited in the embryonic syncytium. Even though detailed mechanistic studies are still pursued to understand precisely how the gradient develops, Bcd is widely described as a simple diffusion model, where the Bcd protein is produced in the anterior part of the embryo and diffuses and decays throughout the tissue. Although it is still debated whether this system reaches a steady state before instructing the cells, its “instructive state” can be represented by an exponential profile14 (Fig. 27.1E). Experimentally, the Bcd profile was found to deviate slightly from the exponential model at the anterior region because the synthesis of Bcd protein is not localized at a single point source. However, the fidelity of the model is sufficient to recapitulate tissue dynamics such as pattern scaling.15 Vertebrate development is also orchestrated by tightly regulated morphogen gradients. In this context, neural tube patterning has been the gold standard for studying gradient-mediated dorsaleventral patterning. In developing neural tissue, the source of the ventralizing morphogen, Sonic Hedgehog (SHH), is located outside of the developing tissue and induces the most ventral cells of the forming neural tube to differentiate16 (Fig. 27.1F). Interestingly, cell patterning in the neural tube is proportional to SHH concentration and the duration of SHH exposure. Thus, the SHH gradient acts in a concentration-dependent manner and is increasing in amplitude in the neural tube, establishing then an exponential gradient, which resembles to diffusion with linear reaction. However, cells seem to be responsive

to SHH only during specific time windows, suggesting mechanisms exist that modify the signaling activity of differentiating cells and desensitize them to the Hh signaling.17 These intriguing mechanisms fortify the patterning process of developing tissues, such as the neural tube.18

2. ENGINEERING CONCENTRATION GRADIENTS USING MICROTECHNOLOGY 2.1 Introduction Microfluidic technology has unique features that allow the manipulation of incredibly small amounts (109e1018 L) of fluids at the micrometer scale. Operating fluids at such a small scale provides two major advantages for biological applications: First, fluid behavior at this scale is mostly laminar, which permits the flow to be manipulated with fine control. In a laminar regimen, flows do not mix convectively; each particle follows its specific streamline. Thus, mixing can only occur by diffusion within the fluid or across the interface of different fluids. Furthermore, by reducing or eliminating the presence of chaotic turbulent flow, microfluidics and its laminar nature allow users to precisely predict critical geometrical and fluidic parameters19 to engineer relevant devices. Second, the order of magnitude of microfluidic devices, which ranges from tens of nanometers to hundreds of micrometers, fits particularly well with the scale at which cellular processes occur.20 Moreover, the standardized fabrication process allows an exceptionally high degree of freedom in planar designs and even in multilayered devices, although the fabrication of more intricate features along the z-axis still remains a challenge. So far, microfluidics has been applied primarily for analytical biology or fabricating microparticles from emulsions. Despite notable elegant demonstrations using robust systems, such as Drosophila embryos,21 microorganisms,22 or cell culture,23,24 coupling living organisms with microfluidics is still a relatively early application of the technology. Today, facilities supporting development of microfluidics technology is common in many institutes around the world where biologists, physicists, chemists, and microengineers develop cutting-edge, application-based microfluidic systems. First, we will give a basic overview of the fluid mechanics knowledge required to build successful microfluidics systems, and then we will study how gradients can be generated using such systems.

2.2 Fluid Mechanics at the Microscale When studying flows at the microscale, several considerations must be taken into account. Specific, intrinsic

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parameters define fluids, whereas the microfluidic channels themselves are subject to physical constraints. Every fluid is characterized by two important parameters; its density, r, which measures the ratio of mass to volume, and its viscosity, m, which represents the flow response of the material. Even though all materials are compressible to some degree, while looking at fluids in motion, especially biological fluids that can be considered analogous to water, their densities remain constant. Thus, they are taken as incompressible, which drastically simplifies the mathematical characterization of the system. Microfluidic channels display interesting characteristics to consider when calculating flows. First, actuation of flow solely in the horizontal direction and the relatively inconsequential gravitational effects at this dimension render vertical fluctuations negligible. In addition, most of these devices are fabricated using planar lithography techniques, which gives the channels rectangular cross-sections. Thus, they are characterized by the channel width, w, and height, h. Finally, these flows are mostly pressure driven, and it has been demonstrated extensively that fluids at the surface of the microchannels obey the no-slip boundary condition (i.e. there is no flow at the fluidechannel interface), as they wet the surface.25 The best qualitative measure of a fluid in motion is the Reynolds number, a dimensionless number that assesses the ratio of convective forces to viscous forces. It is described as follows: Re ¼

rvDh m

(27.1)

where Dh is the characteristic length, and v is the average fluid velocity in the channel. Typically, the characteristic length for a rectangular channel can be calculated as follows: Dh ¼

2wh ðwþ hÞ

(27.2)

We usually define flow as laminar when Re is lower than 2300. All microfluidic devices possess an Re below this threshold, showing that viscous forces are dominant in this environment. Lastly, flows can be calculated from the Naviere Stokes equations, whose mathematical development is beyond the scope of this book. However, after simplification, multiple parameters can be calculated using Eqs. (27.3) and (27.4). Flow rates, Q, are typically formalized as a function of the pressure drop, Dp, over a channel length, L, and can be written as follows for rectangular cross-sections: Q ¼ vA ¼ vwh ¼

wh3 Dp 12mL

(27.3)

It is noteworthy to specify that the pressure drop can be written as a relation between the hydrodynamic resistance in the channel, RH, and the flow,26 mirroring voltage drops in electrical current. From Eqn (27.3), this relation can be written as follow: Dp ¼ QRH ¼ Q

12mL wh3

(27.4)

Hydrodynamic resistance, which is cumulative like in electrical circuits, is a critical parameter to consider when designing microfluidic devices.

2.3 Micromanipulating Flows to Generate Concentration Gradients As described above, flows are laminar in microfluidic devices. Thus, the simplest way to generate a gradient is to bring two streams composed of different molecular species together using a T-shaped or Y-shaped junction.27e29 However, despite their simplicity, these types of gradient generators are limited to certain profiles, such as sigmoidal shapes. Mixing mediated by microfluidics has been extensively characterized in the literature and has created a foundation for more elaborate gradient generators. Using a premixing microchannel network, Jeon et al.30 developed the first device capable of generating many distinct profiles. This designdsubsequently improved by Dertinger et al.31 and commonly known in the field as the “Christmas tree gradient generator”dharbors two main regions (Fig. 27.2A). The upper part of the device has a series of bifurcated microchannels that repetitively split and recombine. As the horizontal channel length was designed to be three orders of magnitude smaller than the vertical channel (V) length, the resistance of the horizontal channel can be neglected, allowing only the serpentine resistance to be considered when modeling the flow being split. In addition, each serpentine channel is congruent, and each branched path has an equal amount of branching layers (B). Thus, the resistance and the flux across each serpentine are equal. Finally, because of its vertical symmetry, the splitting ratios are symmetric at each branching point. Together, these characteristics allow the dilution of the initial concentrations to be precisely calculated across the whole mixing region.31 The lower part of the device is made of a larger channel, in which all the mixed branches join to create a quasi-continuous gradient over its width, whose cross-section, L, represents the analyzed section of the resulting gradient in Fig. 27.2A. As a result, many profiles can be generated using this device. Two variables can be changed to modify the profiles: the flow rates of the entering fluids and the contribution of each in the whole system. When the flow rates are all equal and a fluorophore is incorporated only in

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FIGURE 27.2 (A) Scheme of the Christmas tree microfluidic design. B represents the number of branching points, V, the number of vertical channels. The resulting gradient is analyzed in L. (BeG) For every generated gradient, a wide-field fluorescent micrograph is shown with a dashed line indicating where the gradient was analyzed. The concentrations were normalized for comparison purposes. (BeD) Gaussian-shaped gradient, generated using equal flows in all inlets of 1, 10, and 100 mm/s, respectively. (E) Linear, (F) modified belleshaped, and (G) parabolic gradients are generated using different contributions of the inlets. Adapted from Jeon NL, et al. Generation of solution and surface gradients using microfluidic systems. Langmuir 2000;16:8311e8316; and Kim S, Kim HJ, Jeon NL. Biological applications of microfluidic gradient devices. Integr Biol 2010;2:584e603.

the middle input channel, bell-shaped profiles are produced (Fig. 27.2B). In addition, an equal increase from 1 to 100 mm/s in all input flow rates sharpens the profile peak (Fig. 27.2C and D). To produce more intricate profiles, the contribution of each input flow can be varied. For example, linear gradients can be generated while stopping the left flow, and keeping the middle flow at 50% of the right flow rate (Fig. 27.2E). Any combination can be used to create varying parabolic profiles (Fig. 27.2F and G). Based on this concept, adding parallel mixing regions gives a higher resolution in the output channel and permits the generation of more complex shapes, such as triangular or bimodal profiles.32 Although these active approaches to engineer gradients are interesting in terms of control, they possess two main disadvantages. First, the produced gradients are not purely continuous, but could be considered more accurately as “digital,” because each contributing branch has a single, specific concentration. Second, high shear stresses induced by high flow rates tend to become deleterious for cells.

An alternative to using active flows is the use of free diffusion from a source to a sink compartment. To avoid convective flow between the two compartments and isolate the fluid of the gradient-forming region, microchannels with high fluidic resistance,33e35 porous semipermeable membranes,36e38 and hydrogels, typically composed of collagen or agarose,39,40 have been used to form gradients. Here, the concentration gradient evolves over time as the molecules are transported through the convection barrier until a steady-state is reached, when the diffusive influx and outflux are balanced. To describe in details these convection-free cases, we will focus on hydrogel barriers, which are more relevant for live cell or organism culture.

2.4 Hydrogels as a Barrier to Generate Concentration Gradients Hydrogels are porous natural or synthetic polymer networks with high water content. Their porosity allows molecules sized up to a few hundred kilodaltons to freely

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FIGURE 27.3

(A) Graphical representation of hydrogel barriereinduced gradients. The left panel shows the insertion of the hydrogel from the side channels. The right panel shows the insertion of the hydrogel from a specific hydrogel loading channel. In both cases, the number of microgrooves on the sink and the source side will define the gradient shape. (B) Different gradient shapes. Left, wide-field fluorescent micrographs of the forming gradients. Right, expected theoretical gradients are shown with a gray solid line, and experimental analysis are shown with a black solid line. From Kim S, Kim HJ, Jeon NL. Biological applications of microfluidic gradient devices. Integr Biol 2010;2:584e603.

diffuse through the network while preventing convective flows. This characteristic renders hydrogels the ideal candidates for forming convection-free gradients. Saadi et al. first demonstrated hydrogel-mediated gradient generators. They developed a simple ladder-shaped device, called the “Ladder Chamber,” which could generate stable gradients across two-dimensional (2D) cell culturing microchannels or three-dimensional (3D) hydrogels. The device is composed of two parallel but separated channels, the source channel and the sink channel, connected with small, perpendicular, rectangular microchannels containing the cells or the hydrogel, which may or may not harbor cells.33 Because only diffusion is involved and the device geometry is symmetric, this design is limited in the variety of gradients that can be generated. Mosadegh et al. achieved greater versatility of the ladder chamber by modifying the geometry of the flow-free chambers (Fig. 27.3). Indeed, varying the area of the interface between the source or sink and the hydrogel modifies the influx and outflux of molecules within the gel. This results in local accumulations or depletions of molecule and thus modifies the concentration gradient profile.41 For example, a triangular-shaped chamber with its tip at the sink side allows the formation of a square rooteshaped gradient (Fig. 27.3A). Differentially varying the number of microgrooves touching the hydrogel on the source and the sink side generates different types of profiles, including

linear and sigmoidal shapes (Fig. 27.3B). Variations of this concept can give rise to a broad range of 2D and 3D gradient generators suited for different applications, from conventional cell culture to complex 3D cellular constructs. Finally, it is noteworthy to point out here that using hydrogels in gradient-generating devices is of high interest as it has been shown extensively over the past decade that, in many cases, cells display completely different phenotypes when cultured in 3D as opposed to 2D. In particular, certain cell types, such as cancer cells,42 seem to lose their aberrant characteristics in 2D, which may pose fundamental issues for drug screening accuracy, whereas they maintain the tumorigenic phenotype in 3D.43 Thus, assessing the behavior of cells in 3D, notably under a specific molecular gradient, is critical to observe reactions closest to in vivo.

3. GENERATING MICRO-ENGINEERED GRADIENTS TO CONTROL STEM CELL FATE 3.1 Conventional Microfluidics to Spatially Control Stem Cell Fate T/Y-shaped gradient generators have been primarily used to locally control stem cell differentiation. Their ease of use and their high predictability permit sensitive

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FIGURE 27.4 (A) Schematic representation of a mammalian blastocyst. The Nanog-positive and Nanog-negative cells segregate into distinct regions of the inner cell mass. (B) Microfluidic model of this natural organization. (C) Control condition. Constant leukemia inhibitory factor (LIF) exposure and constant Nanog expression (Dox inducible) leads to a homogenous population of Nanog-positive cells. (D) Counter gradients of LIF þ Dox and RA allow the generation of distinct and neighboring populations of Nanog-positive and Nanog-negative cells. From Zhang YS, Sevilla A, Wan LQ, Lemischka IR, Vunjak-Novakovic G. Patterning pluripotency in embryonic stem cells. Stem Cells 2013;31:1806e1815.

cells such as pluripotent stem cells and mesenchymal stem cells (MSCs) to be cultured in microfluidic devices for limited time periods. At first, phenotypic changes were induced using growth factor or morphogen gradients in such devices. For example, Park et al. could locally manipulate the number of neuronal cell bodies and the density of the neurite bundle networks upon exposure of counter-gradients of SHH and FGF8 and SHH and BMP4.44 Similarly, multiple studies show gradient-induced migration or differentiation of MSCs into the three main lineages (i.e., adipogenesis, ostegenesis, chondrogenesis).45,46 Moreover, it was recently demonstrated that the spatial control of early differentiation of mouse embryonic stem cells (mESCs) through gradient generators can mimic in vivo processes to a certain extent.47 It is well known that during the early organization of the inner cell mass, Nanog-positive and Nanog-negative cells

segregate into, respectively, the epiblast that will later differentiate into all the embryonic tissues, the extraembryonic mesoderm, and the primitive endoderm that will differentiate into extraembryonic tissues, namely the visceral and parietal endoderm48 (Fig. 27.4A). Furthermore, it has been shown that the heterogeneity of Nanog is also found in ESC cultures.49 Zhang and colleagues developed a microfluidic model (Fig. 27.4B) of this early segregation by locally differentiating mESCs into Nanog-negative cells while keeping the neighboring cells Nanog positive. Using doxycycline (Dox)inducible Nanog-expressing cells, they demonstrate that upon exposure of Dox plus leukemia inhibitory factor (LIF) on one side and retinoic acid (RA) minus LIF on the other side, the cells located on the right half of the culture chamber stay pluripotent, whereas the cells on the left half of the chamber lose their pluripotency and, therefore, expression of Nanog (Fig. 27.4C and D).

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This study highlights the ability of microfluidic approaches to recapitulate, in vitro, important events of early development. Similarly, microfluidic bioreactors for the gradient-induced differentiation of human ESCe based embryoid bodies (EBs) have been developed. These systems typically possess two side channels, which act as source/sink for each other, and the EB culture chamber in the middle50 (Fig. 27.5). Even though such system could be interesting for screening applications, in this configuration, within the same connecting channel, the aggregates are not only exposed to the molecule of interest but also to the signaling activity of the neighboring aggregates. This has been suggested to increase the variability of the response of aggregates and, thus, of the results. Overall, the deviations from nature in these systems are still profound as the cells reside on hard, glass, or poly(dimethylsiloxane) (PDMS) surfaces, which is far from their in vivo microenvironment. Because of these differences, cell behavior can be dramatically altered in multiple ways. First, it is suggested that differentiation of mESCs is influenced by the stiffness of their substrate.51 Therefore, glass, plastic, or PDMS surfaces have limited biologically relevant adaptations as they cover a restricted range of stiffness. Second, conventional microfluidic devices are primarily made of PDMS. Despite its sufficient biocompatibility, ease-of-use, optical transparency, and gas permeability, the cross-linking agent of this silicon-based polymer can display some toxicity. The uncross-linked oligomers can diffuse into the culture chambers, particularly when the surface to volume ratio is high. In addition, technical issues can arise because of the adsorption of molecules at the surface of PDMS, which causes local changes in molecular concentration and osmolarity changes that can result from the evaporation of water.52e54

3.2 Combining Hydrogel and Microfluidic Technology to Manipulate Stem Cell Fate in Near-Physiological Settings In addition to their excellent mass transport properties, which allows molecular gradients to form, both naturally derived hydrogels, such as Matrigel or collagen, and synthetic hydrogels can be engineered to elicit physiologically highly relevant multicellular phenomena.55 In particular, synthetic hydrogels such as those composed of poly(ethylene glycol) (PEG) are attractive because of their highly controllable physicochemical and biological properties.56e58 With these biomaterials, many parameters can be tested on cells or cellular constructs in two and three dimensions. As stated before, the stiffness of the microenvironment may play an important role in cellular processes,59e62

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and it can be tuned extremely precisely63 in synthetic hydrogels to probe cellular behavior at a higher resolution than conventionally used biomaterials. Second, synthetic hydrogels can employ “cell-friendly” and highly selective chemistry to attach biochemical cues that direct both the behavior and fate of cells.64,65 Finally, the polymer backbone can also be modified to allow cellmediated remodeling of the substrate, notably through the addition of matrix metalloproteinaseesensitive sites,56e58 which permits cells to remodel their environment and deposit their own matrix. The combination of these parameters in cellular assays can lead to a complex analysis scheme,64,66 which can be addressed either by formulating educated guesses from in vivo studies to find the accurate microenvironments for the cell types of interest67 or by developing 3D ECM screening systems using robotic liquid dispensers to elucidate functionally relevant conditions.66 Despite the high versatility of these emerging biomaterials, cells in these culture systems are still exposed to homogenous mixtures of signaling cues, which does not mimic the dynamic nature of developmental and adult regeneration processes. To begin to trigger the organization of cells into more relevant tissue models, dynamic conditions must be interfaced with these model matrices. Thus, coupling microfluidic technologies with synthetic ECMs may be an attractive approach to probe yet unexplored, more complex biological processes in vitro.68 As one of the first proof-of-concepts, Lutolf and coworkers developed a method that allows the attachment of biomolecular cues in a graded manner onto synthetic PEG hydrogel surfaces.69,70 By incorporating protein Aand/or NeutrAvidine-maleimide molecules in PEGbased hydrogels, the surface of these natively inert substrates can be functionalized with Fc-tagged and/ or biotinylated biomolecules. They could, for example, interface microfabricated Y-shaped gradient generators (Fig. 27.6A) and biofunctionalized surfaces to flow fluorescently labeled Fc-tagged bovine serum albumin (BSA) at the surface of the hydrogel (Fig. 27.6B), generating a variety of gradient shapes, including linear, exponential, or Gaussian.69 Perhaps more interestingly, they could show that, upon the formation of a graded, tethered LIF signal, the pluripotency of Rex1:GFP reporter mESCs could be spatially influenced after the gradient of the molecular effector. Cells forming colonies on the highest tethered area were maintaining Rex1 expression, whereas colonies on the other end of the gradient were exiting pluripotency (Fig. 27.6C). This system may also be explored to screen concentrations of biomolecules on adherent stem cells. Yet, thus far these innovative microchip-based hydrogel culture platforms have been explored exclusively in 2D setting, whereas the culture of cells in three

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FIGURE 27.5 (A) Schematic representation of the microbioreactor including representative results of the computational modeling of mass transport within the bioreactor using a 70-kDa fluorescent molecule. The right panel shows the theoretical concentration profile across the microwells. (B) Photograph showing the entire microbioreactor seeded with EBs. From Cimetta E, et al. Microfluidic bioreactor for dynamic regulation of early mesodermal commitment in human pluripotent stem cells. Lab Chip 2013;13:355e364.

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FIGURE 27.6 (A) Schematic representation of the gradient-generating microfluidic design. (B) Left, device holding the microfluidic chip onto the hydrogel surface, allowing the formation of the gradient and the attachment of the molecules onto the hydrogel. Right, chemistry used to attach the flowed molecules to the noncrosslinked polymer arms. (C) Bright-field and fluorescence micrographs showing Rex1:GFP reporter cells responding to the tethered LIF gradient. The GFP intensity of the colonies decreases along the gradient and the colony area increases inversely to the gradient, indicating differentiation. (D) Conceptual scheme of the combination of aggregate culture with microfluidic-induced gradients. (E) Profile of the gradient forming over the first hour, right, and over the following 6 h, left, upon active perfusion. (F) Bright-field and fluorescent micrographs showing sox1:GFP reporter ESCs derived EBs as a response to graded RA signaling. Expression of sox1 and the size of the colony increase with higher concentrations of RA. From Cosson S, Allazetta S, Lutolf, MP. Patterning of cell-instructive hydrogels by hydrodynamic flow focusing. Lab Chip 2013;13:2099e2105; and Cosson S, Lutolf MP. Hydrogel microfluidics for the patterning of pluripotent stem cells. Sci Rep 2014;4:4462.

dimensions is arguably more physiological.71e73 What is more, existing 3D culture systems are still mostly static, enabling morphogens delivered in a rather unspecific manner. Thus, it is of high interest to merge microfluidic technology with 3D biomatrices affording precise delivery of specific biomolecules to cells in space and in time. Perhaps one of the first demonstration for such a combination of hydrogel and microfluidic technology with 3D cell culture was described by Cosson and Lutolf.74 Using patterns made with soft lithography and plastic supports,

they molded agarose gels with pyramidal-shaped microwells on the upper part of device and microchannels in the lower part of the device to allow perfusion of molecules of interest (Fig. 27.6D). They show that green and red fluorescent BSA molecules diffuse across the aggregate culture upon active perfusion (Fig. 27.6E). The left panel shows a higher resolution of the gradient behavior in the initial phase, and the right panel indicates that the gradient stabilizes after 3 h. To demonstrate the advantage of the system to analyze the interaction of molecules

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of interest with the aggregates, RA was delivered to mESCs in a graded manner. With the green fluorescent reporter indicating SOX1 expression and, therefore, cell fate commitment into the neural lineage, they could observe that below a certain threshold of concentration, SOX1 was not expressed anymore, implying the cells were not committed toward the neural lineage (Fig. 27.6F). Exposing cells to graded signals provides the elegant ability to analyze their behavior under continuous concentration changes in which the effective concentration can be discerned immediately. Finally, gradients in hydrogels in three dimensions have been recently shown in an elegant study by Ehrbar and coworkers to influence MSCs morphogenesis.75 These authors aimed at mimicking native gradients of PDGF-BB that may exist in the perivascular niche to probe MSC behavior. They could observe that cells residing closer to the source were more elongated and displayed a higher migratory activity compared with the cells residing further away. These early studies highlight the exciting potential in combining engineering of microfluidics and extracellular matrices to impact cell biology and tissue engineering.

4. FUTURE DIRECTIONS In recent years, adult and pluripotent stem cells have shown the remarkable ability to self-organize and differentiate into multicellular constructs termed organoids. These organoids can be defined as “a collection of organ-specific cell types that develops from stem cells or organ progenitors and self-organizes through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo.76” This nascent field holds tremendous promise to create relevant human in vitro organ models for drug screening and tissue engineering for cell therapy and organ replacement.76e78 Furthermore, deriving these structures from pluripotent stem cells gives developmental biology researchers a unique tool for studying inaccessible cellular processes while working with animal models or for better understanding events specific to humans.79,80 Yet, organoid systems can thus far only be obtained in rather ill-defined and clinically irrelevant native ECMderived microenvironments (e.g., Matrigel) in which they are homogeneously flooded with biomolecules that induce in vitro development. Unsurprisingly, in vitro self-organization is very difficult to control and also mechanistically poorly understood. Therefore, a major challenge will be to replace current 3D culture systems with chemically defined biomaterials. Moreover, to render in vitro organoid development more robust and ultimately predictable, we expect that the abovedescribed microtechnology-based approaches may be

crucial for delivering developmental signals in a spatiotemporally controlled manner in vitro. To address this formidable engineering and biological challenge, one has to take into consideration the final size of the developing tissues which can be several millimeters (e.g., 1e5 mm for neural organoids and up to 500 mm for epithelial organoids) and the often very long time scales of organogenesis which may be up to several months. This implies that common microfabrication techniques will have to be adaptable over this extended culture time to accommodate the size and also allow sufficient access to nutrients for defined culture windows.

ABBREVIATIONS AND ACRONYMS Bcd Bicoid BMP Bone morphogenetic protein BSA Bovine serum albumin 2D Two-dimensional 3D Three-dimensional D Diffusion coefficient Dh Characteristic length Dox Doxycycline Dpp Decapentaplegic EB Embryoid body ECM Extracellular matrix FGF Fibroblasts growth factor GFP Green fluorescent protein h Microchannel height Hh Hedgehog HSC Hematopoietic stem cell ISC Intestinal stem cell k Degradation rate L Microchannel length LIF Leukemia-induced factor LT-HSC Long-term hematopoietic stem cell mESC Mouse embryonic stem cell MSC Mesenchymal stem cell PDGF Platelet-derived growth factor PDMS Poly(dimethylsiloxane) PEG Poly(ethylene glycol) PSC Pluripotent stem cell Q Flow rate RA Retinoic acid Re Reynolds number RH Hydrodynamic resistance SHH Sonic sedgehog w Microchannel width Dp Pressure drop m Viscosity r Density v Average fluid velocity

Glossary Concentration gradient The gradual difference in concentration of a molecule, soluble or tethered, between a region of high density and one of lower density. Convection The transport of material within a moving fluid or between a boundary surface and a moving fluid.

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REFERENCES

Fluid mechanics The study of fluid properties (liquids and gases) such as velocity, pressure, density and temperature, as functions of space and time. Hydrogel A porous, natural or synthetic, polymer networks with high water content. Microfluidics The science of systems that process or manipulate small (nano to atto-liters) volumes of fluids, using micrometer-sized channels. Molecular diffusion The macroscopic transport of mass, independent of any convection. Organoid A collection of organ-specific cell types that develop from stem cells or organ progenitors and self-organizes through cell sorting and spatiotemporally restricted lineage commitment in a manner similar to in vivo.

Acknowledgment We thank Sylke Hoehnel for significant contributions to the figure design.

References 1. Locke M. The cuticular pattern in an insect e the intersegmental membranes. J Exp Biol 1960;37:398e406. 2. Turing AM. The chemical basis of morphogenesis. 1953. Bull Math Biol 1990;52:153e97. discussion 119e152. 3. Crick F. Diffusion in embryogenesis. Nature 1970;225:420e2. 4. Gerhardt H. VEGF and endothelial guidance in angiogenic sprouting. Organogenesis 2008;4:241e6. 5. Davis GE, Stratman AN, Sacharidou A, Koh W. Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. Int Rev Cell Mol Biol 2011;288:101e65. 6. Geudens I, Gerhardt H. Coordinating cell behaviour during blood vessel formation. Development 2011;138:4569e83. 7. Hanoun M, Frenette PS. This niche is a maze; an amazing niche. Cell Stem Cell 2013;12:391e2. 8. Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 2010;7:150e61. 9. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 2006;7:349e59. 10. Davis H, et al. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat Med 2015;21:62e70. 11. Reeves GT, Muratov CB, Schupbach T, Shvartsman SY. Quantitative models of developmental pattern formation. Dev Cell 2006; 11:289e300. 12. Wartlick O, Kicheva A, Gonzalez-Gaitan M. Morphogen gradient formation. Cold Spring Harb Perspect Biol 2009;1:a001255. 13. Kicheva A, et al. Kinetics of morphogen gradient formation. Science 2007;315:521e5. 14. Reinitz J. Developmental biology: a ten per cent solution. Nature 2007;448:420e1. 15. Cheung D, Miles C, Kreitman M, Ma J. Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos. Development 2014;141:124e35. 16. Briscoe J, Novitch BG. Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube. Philos Trans R Soc Lond B Biol Sci 2008;363:57e70. 17. Cohen M, et al. Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms. Nat Commun 2015;6:6709. 18. Kicheva A, Briscoe J. Developmental pattern formation in phases. Trends Cell Biol 2015;25:579e91.

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19. Sia SK, Whitesides GM. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 2003;24: 3563e76. 20. Whitesides GM. The origins and the future of microfluidics. Nature 2006;442:368e73. 21. Chung K, et al. A microfluidic array for large-scale ordering and orientation of embryos. Nat Methods 2011;8:171e6. 22. Dai J, Suh SJ, Hamon M, Hong JW. Determination of antibiotic EC using a zero-flow microfluidic chip based growth phenotype assay. Biotechnol J 2015;10(11):1783e91. 23. Hung PJ, Lee PJ, Sabounchi P, Lin R, Lee LP. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol Bioeng 2005;89:1e8. 24. Du G, Fang Q, den Toonder JM. Microfluidics for cell-based high throughput screening platforms-A review. Anal Chim Acta 2016; 903:36e50. 25. Stone HA, Stroock AD, Ajdari A. Engineering flows in small devices: microfluidics toward a lab-on-a-chip. Annu Rev Fluid Mech 2004;36:381e411. 26. Stone HA. CMOS biotechnology. (US): Springer; 2007. 27. Hatch A, et al. A rapid diffusion immunoassay in a T-sensor. Nat Biotechnol 2001;19:461e5. 28. Kamholz AE, Yager P. Theoretical analysis of molecular diffusion in pressure-driven laminar flow in microfluidic channels. Biophys J 2001;80:155e60. 29. Ismagilov RF, Stroock AD, Kenis PJA, Whitesides G, Stone HA. Experimental and theoretical scaling laws for transverse diffusive broadening in two-phase laminar flows in microchannels. Appl Phys Lett 2000;76:2376e8. 30. Jeon NL, et al. Generation of solution and surface gradients using microfluidic systems. Langmuir 2000;16:8311e6. 31. Dertinger SKW, Chiu DT, Jeon NL, Whitesides GM. Generation of gradients having complex shapes using microfluidic networks. Anal Chem 2001;73:1240e6. 32. Kim S, Kim HJ, Jeon NL. Biological applications of microfluidic gradient devices. Integr Biol 2010;2:584e603. 33. Saadi W, et al. Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber. Biomed Microdevices 2007;9:627e35. 34. Atencia J, Morrow J, Locascio LE. The microfluidic palette: a diffusive gradient generator with spatio-temporal control. Lab Chip 2009;9:2707e14. 35. Shamloo A, Ma N, Poo MM, Sohn LL, Heilshorn SC. Endothelial cell polarization and chemotaxis in a microfluidic device. Lab Chip 2008;8:1292e9. 36. Diao J, et al. A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis. Lab Chip 2006;6:381e8. 37. Abhyankar VV, Lokuta MA, Huttenlocher A, Beebe DJ. Characterization of a membrane-based gradient generator for use in cellsignaling studies. Lab Chip 2006;6:389e93. 38. Kim D, Lokuta MA, Huttenlocher A, Beebe DJ. Selective and tunable gradient device for cell culture and chemotaxis study. Lab Chip 2009;9:1797e800. 39. Cheng SY, et al. A hydrogel-based microfluidic device for the studies of directed cell migration. Lab Chip 2007;7:763e9. 40. Haessler U, Kalinin Y, Swartz MA, Wu M. An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies. Biomed Microdevices 2009;11:827e35. 41. Mosadegh B, et al. Generation of stable complex gradients across two-dimensional surfaces and three-dimensional gels. Langmuir 2007;23:10910e2. 42. Kenny PA, et al. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol 2007;1:84e96.

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43. Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 2014;12:207e18. 44. Park JY, et al. Differentiation of neural progenitor cells in a microfluidic chip-generated cytokine gradient. Stem Cells 2009;27: 2646e54. 45. Lai N, Sims JK, Jeon NL, Lee K. Adipocyte induction of preadipocyte differentiation in a gradient chamber. Tissue Eng Part C Methods 2012;18:958e67. 46. Zhang Y, Gazit Z, Pelled G, Gazit D, Vunjak-Novakovic G. Patterning osteogenesis by inducible gene expression in microfluidic culture systems. Integr Biol 2011;3:39e47. 47. Zhang YS, Sevilla A, Wan LQ, Lemischka IR, Vunjak-Novakovic G. Patterning pluripotency in embryonic stem cells. Stem Cells 2013; 31:1806e15. 48. Mitsui K, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003; 113:631e42. 49. Singh AM, Hamazaki T, Hankowski KE, Terada N. A heterogeneous expression pattern for nanog in embryonic stem cells. Stem Cells 2007;25:2534e42. 50. Cimetta E, et al. Microfluidic bioreactor for dynamic regulation of early mesodermal commitment in human pluripotent stem cells. Lab Chip 2013;13:355e64. 51. Poh YC, et al. Generation of organized germ layers from a single mouse embryonic stem cell. Nat Commun 2014;5:4000. 52. Regehr KJ, et al. Biological implications of polydimethylsiloxanebased microfluidic cell culture. Lab Chip 2009;9:2132e9. 53. Berthier E, Young EW, Beebe D. Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip 2012;12:1224e37. 54. Halldorsson S, Lucumi E, Gomez-Sjoberg R, Fleming RM. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens Bioelectron 2015;63:218e31. 55. Gjorevski N, Ranga A, Lutolf MP. Bioengineering approaches to guide stem cell-based organogenesis. Development 2014;141: 1794e804. 56. Ehrbar M, et al. Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 2007;28:3856e66. 57. Ehrbar M, et al. Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions. Biomacromolecules 2007;8:3000e7. 58. Ehrbar M, et al. Elucidating the role of matrix stiffness in 3D cell migration and remodeling. Biophys J 2011;100:284e93. 59. Charras G, Sahai E. Physical influences of the extracellular environment on cell migration. Nat Rev Mol Cell Biol 2014;15:813e24. 60. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677e89. 61. Gilbert PM, et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 2010;329:1078e81.

62. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 2005;23:47e55. 63. Lutolf MP, Hubbell JA. Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition. Biomacromolecules 2003;4:713e22. 64. Gobaa S, et al. Artificial niche microarrays for probing single stem cell fate in high throughput. Nat Methods 2011;8:949e55. 65. Mosiewicz KA, et al. In situ cell manipulation through enzymatic hydrogel photopatterning. Nat Mater 2013;12:1072e8. 66. Ranga A, et al. 3D niche microarrays for systems-level analyses of cell fate. Nat Commun 2014;5:4324. 67. Gobaa S, Hoehnel S, Lutolf MP. Substrate elasticity modulates the responsiveness of mesenchymal stem cells to commitment cues. Integr Biol 2015;7:1135e42. 68. Kobel S, Lutolf MP. Biomaterials meet microfluidics: building the next generation of artificial niches. Curr Opin Biotechnol 2011;22: 690e7. 69. Allazetta S, Cosson S, Lutolf MP. Programmable microfluidic patterning of protein gradients on hydrogels. Chem Commun 2011;47:191e3. 70. Cosson S, Allazetta S, Lutolf MP. Patterning of cell-instructive hydrogels by hydrodynamic flow focusing. Lab Chip 2013;13: 2099e105. 71. Sato T, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262e5. 72. Eiraku M, et al. Self-organizing optic-cup morphogenesis in threedimensional culture. Nature 2011;472:51e6. 73. Lancaster MA, et al. Cerebral organoids model human brain development and microcephaly. Nature 2013;501:373e9. 74. Cosson S, Lutolf MP. Hydrogel microfluidics for the patterning of pluripotent stem cells. Sci Rep 2014;4:4462. 75. Lienemann PS, et al. Locally controlling mesenchymal stem cell morphogenesis by 3D PDGF-BB gradients towards the establishment of an in vitro perivascular niche. Integr Biol 2015;7:101e11. 76. Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 2014;345:1247125. 77. Woodford C, Zandstra PW. Tissue engineering 2.0: guiding selforganization during pluripotent stem cell differentiation. Curr Opin Biotechnol 2012;23:810e9. 78. Sato T, Clevers H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 2013;340: 1190e4. 79. Huch M, Koo BK. Modeling mouse and human development using organoid cultures. Development 2015;142:3113e25. 80. Meinhardt A, et al. 3D reconstitution of the patterned neural tube from embryonic stem cells. Stem Cell Rep 2014;3:987e99.

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C H A P T E R

28 Engineering Niches for Embryonic and Induced Pluripotent Stem Cells Hongli Mao1, Yoshihiro Ito2 1

RIKEN, Saitama, Japan; 2RIKEN Brain Science Institute, Saitama, Japan

O U T L I N E 1. Introduction

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2. Niches for Embryonic Stem/Induced Pluripotent Stem Cell Maintenance 2.1 Live Feeder Cells 2.2 Cell-Derived Substrates 2.2.1 Chemically Fixed Cells 2.2.2 Decellularized Extracellular Matrix 2.3 Artificial Reconstruction 2.3.1 Matrigel 2.3.2 Extracellular Matrix Components 2.3.3 Cell Adhesion Molecules 2.3.4 Cytokines 2.4 Synthetic Substrates

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1. INTRODUCTION Stem cells hold great potential for applications in regenerative medicine and as powerful model systems for basic biological and biomedical studies.1,2 Two hallmark properties of stem cells are self-renewal and differentiation potency. Self-renewal is the ability of stem cells to proliferate indefinitely while maintaining their cellular identity, whereas differentiation potency refers to the capacity of stem cells to differentiate into specialized cell types. These features of stem cells are determined by a complex series of extrinsic cues in collaboration with intrinsic genetic programs.3,4 Therefore, understanding how to precisely control the behavior of these cells has become a challenging

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00028-7

3.1 Live Feeder Cells 3.2 Cell-Derived Substrates 3.3 Artificial Reconstruction 3.3.1 Matrigel 3.3.2 Extracellular Matrix Components 3.3.3 Cell Adhesion Molecules 3.3.4 Cytokines (Growth Factors) 3.4 Synthetic Substrates

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issue in stem cell-based therapeutic and scientific applications. Stem cells reside in specific environments that not only physically anchor them via either cell membraneassociated proteins or localized extracellular matrix (ECM) components, but also dynamically affect the fate of stem cells, including maintenance of a constant number of slowly dividing stem cells by balancing the proportion of quiescent and activated cells.4 Consequently, the effects of such extrinsic stimuli as determinants of cell fate have been studied in parallel with the role of intrinsic regulation. In 1978, the concept of the stem cell niche was first proposed by Schofield, who stated that “the cellular environment which retains the stem cell I shall call a stem cell ‘niche’.5”

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Copyright © 2017 Elsevier Inc. All rights reserved.

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As shown in Fig. 28.1, the stem cell niche consists of soluble cytokines, ECM interactions, and neighboring cell interactions, which play important roles in stem cell maintenance. Since the initial proposal, studies on the extrinsic cues and their downstream intracellular signaling pathways have given rise to significant progress in understanding how to control the expansion and differentiation of stem cells. Furthermore, engineered artificial niches are being increasingly developed to control stem cell fate by mimicking the intrinsic regulatory factors of native stem cell niches. Among the various types of stem cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have shown great promise for use in stem cell-based applications. In this chapter, we focus on the studies describing engineering niches for maintenance of the undifferentiated state and regulating the differentiation of ES and iPS cells. ES Cells: ES cells are pluripotent, self-renewing stem cells derived from the inner cell mass (ICM) of blastocyst-stage early mammalian embryos. Mouse ES cells were first isolated and described in 1981 by Martin6 and Evans et al.,7 whereas the first viable human ES cells were derived from the ICM of human blastocysts and successfully subcultured on irradiated mouse embryonic fibroblasts (MEFs) as a cell line by Thompson et al.8 Since the establishment of human ES cells, stem cell research expanded rapidly because of their importance in regenerative medicine. These cells possess the ability to propagate and differentiate into any cell type in the body. In vitro, ES cells can self-renew indefinitely without genetic transformation, and can be expanded clonally without losing their pluripotency, which is the ability to differentiate into all adult cell types including germ cells. iPS Cells: The pluripotent state of ES cells is largely governed by core transcription factors octamer-binding

transcription factor ¾ (Oct3/4), Sox2, and Nanog.9,10 Several other genes that are frequently upregulated in tumors, such as signal transducer and activator of transcription 3 (STAT3), c-Myc, and Klf4, have been shown to function in long-term maintenance of the ES cell phenotype and their rapid proliferation.11e13 In 2006, by retrovirus-mediated introduction of four transcription factors, Oct3/4, Sox2, c-Myc, and Kruppel-like factor 4 (Klf4), iPS cells were derived from mouse embryonic and adult fibroblasts.14 These mouse iPS cells were similar to ES cells in terms of their morphology, proliferation, and teratoma formation. Subsequently, in 2007, human iPS cells were established using the same four factors15 and a slightly different combination.16 Human iPS cells are also similar to human ES cells in many aspects. The discovery of iPS cells has changed the perception of cellular reprogramming, revealing that the plasticity of somatic cells is much greater than had been previously thought. Moreover, the derivation of human iPS cells offers an appealing solution to the likely immune rejection of ES cell-derived cells upon their transplantation into an unmatched patient, thus providing potential avenues for patient-specific cell therapy. Importantly, iPS cells avoid the ethical issues that have been raised by the derivation of human ES cells, which is a highly controversial topic in many countries.

2. NICHES FOR EMBRYONIC STEM/ INDUCED PLURIPOTENT STEM CELL MAINTENANCE Culture of ES/iPS cells in an undifferentiated state without losing their pluripotency is important for ES/ iPS cell-based applications. To support the growth of ES/iPS cells in an undifferentiated state, one attractive strategy is engineering niches by mimicking in vivo cellular microenvironments that consist of cells,

FIGURE 28.1 Schematic of a stem cell niche. The niche essentially consists of soluble and extracellular matrix (ECM)-bound factors, ECM component molecules and membraneassociated ligands and receptors including cell adhesion molecules.

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2. NICHES FOR EMBRYONIC STEM/INDUCED PLURIPOTENT STEM CELL MAINTENANCE

cytokines, and the ECM as shown in Fig. 28.1. To date, various engineered niches have been developed for stem cell culture.

2.1 Live Feeder Cells To culture ES/iPS cells in an undifferentiated state, it is a common practice to use live feeder cells that provide direct cellecell interactions, produce a bioactive matrix for celleECM interactions, and release nutrients and other complex molecules into the culture medium to support cell growth, prevent spontaneous differentiation, and maintain the pluripotency of ES/iPS cells during cultivation8,14 (Fig. 28.2A). As feeders, the proliferation of these cells must be inhibited completely, so they can be used as nonproliferating viable support cells.17 Mitomycin-C (MMC) and irradiation treatments are commonly used to prepare nonproliferating and metabolically active feeder cells. Mouse-derived cells, such as MEFs and SNL 76/7 cells, are the most frequently used feeders to maintain the pluripotency of ES/iPS cells. However, the use of mouse feeder cells for human ES/iPS cell culture poses the risk of cross-species contamination of the human cells with animal pathogens that may be present in feeder cells, attachment matrices, or conditioned medium, thereby greatly compromising the future clinical applications of human ES/iPS cells. Therefore, feeder layers derived from human fetal muscle, fetal skin, human adult fallopian tubal epithelial layers, and other isogenic parental autologous feeder cells have been used to support the undifferentiated growth of human

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ES/iPS cells.17e19 For example, human adult bone marrow mesenchymal stem cells (MSCs) have been tested as a potent feeder system. The results showed that MSCs support prolonged in vitro proliferation of human iPS cells along with maintenance of their pluripotency. However, human iPS cells cultured on human MSC feeders are slightly thinner and flatter than those cultured on other systems.20

2.2 Cell-Derived Substrates Although live feeder cells are generally used as niches in the culture of ES/iPS cells, many disadvantages also exist in this culture system, such as the laborious procedure21 (cells must be cultured and treated with MMC or X-ray irradiation for growth inactivation prior to use as feeders) and the high risk of contamination during passaging (feeder cells may detach from the culture surface when ES/iPS cells are digested for passaging, which may affect the differentiation efficiency of stem cells and cause severe problems in clinical applications). To avoid these disadvantages, chemically fixed feeder cells and decellularized ECMs have been developed and used as culture substrates for ES/iPS cell culture. 2.2.1 Chemically Fixed Cells Chemically fixed cells are used in fundamental cell biology to reveal the mechanisms by which membraneassociated factors or proteins affect cells. It has been shown that membrane proteins retain their mobility and biological activity even after chemical fixation.22,23 Furthermore, direct cellecell interactions among

FIGURE 28.2 Schematic of engineered niches using (A) mitotically inactivated live cells, (B) chemically fixed feeder cells, and (C) decellularized extracellular matrix.

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stem cells and feeder cells, which play important roles in maintaining the undifferentiated state of stem cells, are provided by this culture system. Therefore, chemically fixed feeder cells can be considered as niches that support the undifferentiated growth of ES/iPS cells (Fig. 28.2B). Ito’s research group have applied chemically fixed human stromal cells to support ex vivo expansion of human cord blood hematopoietic progenitor cells.22,24 In addition, they demonstrated the utility of chemically fixed nurse cells as feeders to maintain ES cells in an undifferentiated state.25 MEFs fixed with formaldehyde (FA) or glutaraldehyde (GA) have also been used to maintain the pluripotency of mouse iPS cells.26 The pluripotency-maintaining ability of fixed MEFs can be explained by the comparable mobility of the membrane proteins of fixed feeder cells with those of MMC-treated live MEFs.27 Currently, an optimized chemical fixation protocol using GA and FA has been developed to prepare a niche matrix from autologous feeder cells for human iPS cell culture28 (Fig. 28.3). Human iPS cells cultured on chemically fixed feeders exhibit both in vitro and in vivo pluripotency.

cells. There is a review article and report that summarized the preparation methods for a decellularized ECM and highlighted the dependence of ECM components and structures on such methods.29,30 Klimanskaya et al. prepared a decellularized ECM from MEFs monolayers by treatment with sodium deoxycholate to maintain human ES cells in serum-free medium.31 The human ES cells cultured on the decellularized ECM maintained a normal karyotype and expression of stem cell markers such as Oct-4, stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumor-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase (ALP). Lim et al. used sodium dodecyl sulfate (SDS) to remove cellular components including DNA without a detectable change in the ECM architecture and integrity.32 The human ES/iPS cells cultured on this decellularized matrix maintained gene expression of pluripotency markers and had a differentiation potency for all three germ layers.

2.2.2 Decellularized Extracellular Matrix Similar to chemically fixed feeder cells, cell-formed ECM as a cell-derived substrate has been used as a niche for stem cell culture after decellularization treatment (Fig. 28.2C). Such decellularized ECMs retain the complex ECM composition to present essential factors to the cells and have been reported to play important roles in maintenance of the undifferentiated state of stem

2.3 Artificial Reconstruction To replace feeder cells, researchers have performed a molecular level of artificial reconstitution or reconstruction. The most commonly used substrate is Matrigel. However, considering that Matrigel is a mouse-derived product with a mixture of biomacromolecules, more defined and homogeneous substrates are desired for basic science and further applications. Therefore, studies

FIGURE 28.3

Phase contrast images of the morphology and stage-specific antigen (SSEA)-4 expression of human iPS cells cultured on (A and B) MMC-treated SNL cells, (C and D) MMC-treated human dermal fibroblasts (HDFs), and chemically fixed HDFs treated with either (E and F) glutaraldehyde (GA) or (G and H) formaldehyde (FA). Very few SSEA-4-expressing human iPS colonies can be detected on (I and J) laminin-5 and are completely absent on (K and L) gelatin. Green indicates positive SSEA-4 expression. Scale bar, 500 mm. From Joddar B, Nishioka C, Takahashi E, Ito Y. Chemically fixed autologous feeder cell-derived niche for human induced pluripotent stem cell culture. J Mater Chem B 2015;3:2301e07.

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FIGURE 28.4 Schematic of engineered niches using (A) immobilized extracellular matrix components, (B) cell adhesion molecules (CAMs) and (C) cytokines.

have also investigated immobilization of ECM components, cell adhesion molecules (CAMs), and cytokines. 2.3.1 Matrigel Matrigel, also known as Engelbreth-Holm-Swarm (EHS) extract, reconstituted basement membrane (BM), and Cultrex, is an assortment of gelatinous proteins extracted from EHS tumors in mice. Matrigel forms a three-dimensional (3D) gel at 37 C and is enriched with ECM components such as collagen IV, laminin, and fibronectin, which support cell morphogenesis, proliferation, and differentiation. Matrigel provides good adhesion for ES cells, possibly via integrin-mediated binding.33 ES cells have been cultured under feederfree conditions on Matrigel with MEF-conditioned medium, which can be used for applications such as genetic modification of ES cells without feeder cell contamination.34 A combination of Matrigel and growth factors has also been developed for long-term maintenance of mouse ES cells in an undifferentiated state.35 Takahashi et al. generated human iPS cells from adult human dermal fibroblasts (HDFs) by introduction of four defined factors (Klf4, Oct4, Sox2, and c-Myc) and the generated iPS cells maintained an undifferentiated state on Matrigel-coated plates in MEF-conditioned primate ES cell medium.15 Totonchi et al. cultured human iPS cells under serum- and feeder-independent conditions and they found that the number of ALP-positive iPS cell colonies was increased significantly in both conditioned and feeder-free media in the presence of Matrigel.36 Human iPS cells have been also generated from adult human adipose stem cells by culture on Matrigel-coated surfaces that also supported the culture of human iPS cells for at least 20 days.37 In another study, a culture technique using the Rho kinase inhibitor Y-27632 on Matrigel-coated dishes was developed for human ES and iPS cell culture. Under these conditions, the cells retain their typical morphology and a stable karyotype, express pluripotency markers, and have the potential to differentiate into derivatives of all three germ layers after long-term culture.38

2.3.2 Extracellular Matrix Components The ECM is a complex structural entity surrounded by supporting cells which include three major classes of biomolecules: structural proteins (e.g., collagen and elastin), specialized proteins (e.g., laminin, fibronectin, and fibrillin), and proteoglycans that consist of long chains of repeating disaccharide units termed glycosaminoglycans attached to a protein core. Single component substrates of ECM proteins such as laminin,39 fibronectin,40 vitronectin,41,42 and collagen I,43 as well as ECM polysaccharides such as hyaluronic acid (HA),44 have been used for ES/iPS cell culture (Fig. 28.4A). For example, to understand the molecular mechanisms of iPS cell adhesion and proliferation, Rowland et al. coated vitronectin onto culture dishes as a defined culture substrate for human iPS cell culture.42 They found that integrin avb5 was required for initial attachment on vitronectin-coated dishes, whereas inhibition of both integrins avb5 and b1 resulted in a significant decrease in iPS cell proliferation. Furthermore, iPS cells cultured on vitronectin for up to nine passages retained a normal karyotype, pluripotency marker expression, and the capacity to differentiate in vitro. HA has been used for expansion of mouse ES cells, and the effects of the molecular weight (MW) of HA on the maintenance of the pluripotency and proliferation of mouse ES cells were investigated in vitro.44 The results showed that high MW (1000 kDa) HA interacted with mouse ES cells via CD44, whereas low MW HAs (4e8 kDa) interacted with these cells mostly via CD168. ES cells cultured on both high and low MW HAs appeared to be undifferentiated after 3 days. However, low MW HA substrates were effective to maintain mouse ES cells in a viable and undifferentiated state, which favors their use in the propagation of ES cells for tissue engineering. In a previous study, various human ES cell lines after long-term culture were maintained on fibronectin using MEF-conditioned medium and passaged with ethylenediaminetetraacetic acid (EDTA)-free trypsin.45 These cultures exhibited normal karyotypes and the expected

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morphology of monolayer growth and prominent nucleoli of human ES cells cultured under these conditions. In combination with a completely xeno-free medium, StemFit, recombinant laminin-511 E8 fragments are a useful niche for maintenance of human ES and iPS cells.46 Using this culture system, stem cells can be easily and stably passaged for long periods without any karyotypic abnormalities by dissociating the cells into single cells. 2.3.3 Cell Adhesion Molecules CAMs are proteins located on the cell surface, which contribute to cellecell or celleECM adhesion. An increasing number of reports has revealed that CAMmediated cohesive interactions among ES/iPS cells and between the cells and their neighboring instructive cues, including niche cells and ECM, contribute significantly to the pluripotency of ES/iPS cells47 (Fig. 28.4B). For example, E-cadherin is an important member of all the main classes of CAMs and interacts with key components of the naive stemness pathway. Ablation of E-cadherin prevents stem cells from forming welldifferentiated teratomas and contributing to chimeric animals.48 Loss of E-cadherin in mouse ES cells results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways.49 In the absence of the transcription factor Oct-3/4, murine ES cells can be reprogrammed to their pluripotent state simply by addition of viral E-cadherin.50 Therefore, E-cadherin is essential to permit cellular cross talk and the maintenance of pluripotency in mouse ES cells.51 By culturing cells on CAM-modified substrates, cellecell interactions can be effectively alternated to cellesubstrate interactions, and the pluripotency of ES cells can be maintained successfully. For example, murine ES cells cultured on a plate coated with a fusion protein of E-cadherin and the IgG Fc domain (E-cad-Fc) retain all ES cell features including pluripotency to differentiate into cell types of all three germ layers, as well as germline transmission after extended culture without cellecell contacts or colony formation.52 Furthermore, ES cells cultured on E-cad-Fc-coated surfaces show a higher proliferative ability, lower dependency on leukemia inhibitory factor (LIF), and higher transfection efficiency than in colonyforming conditions. Human ES and iPS cells have also been maintained on plates coated with a fusion protein consisting of human E-cadherin and the IgG Fc domain in the absence of feeder cells.53 Cells grown under these conditions maintain a similar morphology and growth rate to those grown on Matrigel and retain all pluripotent features including the ability to differentiate into multiple cell lineages in teratoma assays.

2.3.4 Cytokines When a specific receptor located on the cell surface or inside the cell is activated by a signaling molecule such as a growth factor, a biochemical chain of events will be triggered inside the cell, leading to a cellular response. Therefore, many types of signaling proteins have been immobilized and shown to activate cellular transduction pathways54 (Fig. 28.4C). LIF was initially identified as a cytokine capable of inducing the differentiation of M1 myeloid leukemia cells, and was later shown to have a strong differentiation-inhibiting effect on pluripotent cells. LIF combined with photoreactive gelatin has been immobilized on tissue culture plastic by photoirradiation and found to maintain mouse ES cells in a pluripotent state for at least 6 days without further addition of soluble LIF at each medium change.55 Immobilized LIF stimulated activation of STAT3 signaling. In another study, LIF and stem cell factors were immobilized on maleic anhydride copolymer thin-film coatings at defined concentrations, which supported mouse ES cell pluripotency for at least 2 weeks in the absence of soluble LIF.56 The immobilized LIF activated STAT3 and mitogen-activated protein kinase signaling pathways in a dose-dependent manner.

2.4 Synthetic Substrates A compositionally defined niche that supports ES/ iPS cell expansion is important to determine factors that regulate the fate of stem cells, expand the use of ES/iPS cells in biotechnologies, and enable potential clinical applications. In this sense, synthetic substrates are the most desired for ES/iPS cell culture. Thus far, various synthetic substrates have been developed as niches for ES/iPS cell culture, and their effects on the fate of ES/iPS cells have been investigated. For example, mouse ES cells have been cultured on conventional polystyrene cell-culture dishes photoimmobilized with four types of polymers, poly(acrylic acid) (PAAc), polyallylamine, gelatin, and poly(2-methacryloyloxyethyl phosphorylcholine-co-methacrylic acid) (PMAc50), all of which were coupled with azidophenyl groups.57 The results showed that the morphology and growth rate of mouse ES cells were significantly affected by the polymer surface properties. ES cells attached to gelatin and polyallylamine surfaces, but colonies formed only on the former. In addition, significant enhancement of growth was observed on the gelatin surface. In contrast, ES cells aggregated to form embryoid bodies (EBs) on the photoimmobilized PAAc and PMAc50 surfaces, although cell growth was reduced. Significant enhancement of ES cell aggregation on the PMAc50 surface was observed in morphology and gene expression.

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In a hallmark study investigating 1700 human ES cellematerial interactions by microarray technology, a new range of synthetic polymers was identified for ES cell culture.58 The effects of various acrylate, diacrylate, dimethylacrylate, and triacrylate monomers on ES cells were analyzed in vitro. The results from this study showed that the majority of polymers that allowed cell attachment and spreading also induced differentiation into a cytokeratin-positive epithelial cell type. In contrast, although some polymers initially inhibited cell adhesion, they supported cell proliferation by diffusion of soluble growth factors from the surface. Villa-Diaz et al. coated six polymers by surfaceinitiated graft polymerization and tested their ability to promote the attachment and proliferation of undifferentiated human ES cells.59 They found that poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] sustained long-term human ES cell growth in several culture media including commercially available defined serum-free media. Polysulfone membranes coated with polymerized 3,4dihydroxy-L-phenylalanine have also been used to support long-term self-renewal of undifferentiated human ES/iPS cells under defined conditions.60 The pluripotency of the cells could be maintained for at least 10 passages even without using serum or coating with proteins. Human ES cells cultured on a completely synthetic ECM consisting of a semiinterpenetrating polymer network (a polymer hydrogel) remained viable, maintained cell morphology, and expressed markers of undifferentiated ES cells.61 Human iPS cells have been maintained in a pluripotent state on porous polyethylene terephthalate membranes with proliferative human adipose-derived stromal cells seeded on the bottom surface of the membranes.62 The presence of the membrane physically separated the two types of cells, but allowed diffusion of soluble factors through the pores. Surface topography and elasticity of engineered niches are also important for maintenance of pluripotency in stem cells. For example, fibronectin-coated poly(dimethyl siloxane) substrates with line grating (600 nm ridges with 600 nm spacing and 600  150 nm feature height) induce alignment, mediate organization of cytoskeletal components including actin, vimentin, and a-tubulin, and reduce the proliferation of cultured human ES cells. These findings suggest that the interplay between cytoskeleton and substrate interactions is a key modulator of morphological and proliferative responses in stem cells cultured on a specific surface topography.63

3. NICHES FOR REGULATION OF DIFFERENTIATION An important defining property of stem cells is the ability to differentiate into specialized cell types.

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Therefore, great efforts have been made to develop suitable niches for regulation of differentiation in ES/iPS cells.

3.1 Live Feeder Cells Conventionally, the formation of EBs is the principal step in differentiation of colony-forming stem cells. For this reason, EB formation has been widely used as a trigger of in vitro differentiation in colony-forming stem cells.64 Ou et al. found that the combined effect of 3D clusters and coculture with fibroblasts mimicked the in vivo physiological environment and influenced EB formation, which in turn improved cardiomyocyte differentiation from ES cells.65 By direct culture on a monolayer of M15 mesodermderived supporting cells under selective culture conditions, ES cells were induced sequentially into regional-specific gut endoderm lineages such as pancreatic, hepatic, and other cell lineages.66,67 A detailed chronological analysis revealed that activin, fibroblast growth factor, and bone morphogenetic protein (BMP) signals were critical at various steps, and that additional short-range signals were required for differentiation into pancreatic and duodenal homeobox 1 (Pdx1)-expressing cells. Moreover, analysis of the hepatic cells derived from ES cells showed that these cells resembled mature hepatocytes by storing glycogen and expressing various molecular markers such as albumin, bile acid transporters, and cytochrome P450 metabolic enzymes. Many other cell types also affect stem cell differentiation. For example, PA6 cell spots induce neuronal differentiation of ES cells through microarray methodology.68 Human embryonic fibroblasts (HEFs) have been used as feeder cells to support undifferentiated growth of human ES cells and induce differentiation of definitive endoderm.69 The differentiation of human ES cells under the influence of periodontal ligament cells in vitro has been investigated by coculture of human ES cells with human periodontal ligament fibroblastic cells. The results indicated the feasibility to direct the differentiation of human ES cells toward periodontal ligament fibroblastic progenitors for periodontal tissue engineering applications.30 By coculture with progenitor cells from limb buds of the developing embryo, ES cells were induced to differentiate into chondrocytes (cartilage-producing cells).70 Almost 60e80% of cells exhibited phenotypic characteristics of mature chondrocytes and expressed genes such as Sox9, collagen II, and proteoglycans, which were accompanied by a decrease in expression of the ES cell-specific transcription factor Oct-4. Collagen II, which is expressed as two forms during chondrogenic differentiation because of alternate mRNA splicing,

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was also regulated properly. These results suggest that the signals produced by progenitor cells in the developing embryo induce lineage-specific differentiation.

notochordal cell-like cells expressed typical notochordal marker genes including brachyury, cytokeratin-8, and cytokeratin-18 and had the ability to generate nucleus pulposus-like tissue in vitro, which was enriched with aggrecan and collagen II.

3.2 Cell-Derived Substrates Although chemically fixed feeder cells have not been used for regulation of differentiation, decellularized ECM has been used in such applications because the ECM is regarded as a key factor that regulates the fate of stem cells, including cell differentiation.71 Artificial BM with a highly integrated structure including ECM molecules has been prepared and applied to differentiate ES and iPS cells into pancreatic b cells without the need for a feeder layer.72 The major components of most BMs are collagen IV, laminins, entactin (nidogen), and heparin sulfate proteoglycans (HSPGs). Together with secreted cytokines, these molecules are integrated into the structure of the BM and assemble to form an optimal extracellular environment for regulation of cell fate. The BM substratum was prepared from the HEK293 cell line that stably expresses laminin-511. The HEK293 cells were cultured for deposition of BM components, and then the cells were removed from the substrate to prepare the artificial BM. Subsequently, mouse ES or iPS cells were seeded on the BM and sequentially differentiated into definitive endoderm, pancreatic progenitor cells, and then insulin-secreting pancreatic b cells. It has been considered that the differentiation signals from BM are transduced, in part, through laminineintegrin interactions and growth factor receptors, which function in the presence of HSPGs in the BM. A synthesized BM has been demonstrated to support highly efficient regional-specific differentiation of mouse and human ES cells into hepatic endoderm, and integrin-b1 played an important role in mediation of hepatic differentiation.73 The derived cells were able to differentiate further into hepatic cells expressing mature hepatocyte markers and secreting albumin. Notochordal cells are a promising cell source for cellbased therapy to treat certain types of intervertebral disc degeneration. However, a major limitation of this cellbased application is the lack of available notochordal cells as a potential therapeutic cell source. Native nucleus pulposus tissue harbors a number of notochordal cells, and it is believed that the nucleus pulposus matrix alone may contain sufficient regulatory factors that can generate notochordal cells from human pluripotent stem cells. Recently, a method employing porcine nucleus pulposus matrix to direct notochordal differentiation of human iPS cells was reported by Liu et al.74,75 They found that human iPS cells were able to differentiate into notochordal cell-like cells under the influence of devitalized porcine nucleus pulposus matrix. The

3.3 Artificial Reconstruction 3.3.1 Matrigel Matrigel is commonly used to regulate the differentiation of ES/iPS cells. Following adipogenic induction in vitro, differentiated human iPS and ES cells have been seeded in Matrigel and transplanted into the subcutaneous tissue of nude mice.76 After 1e4 weeks, cells with adipocyte-like features were observed in the transplanted construct by histological analysis, and gene expression of adipocyte markers was found in the transplanted cells. These results suggested that the iPS/ES cells were differentiated into adipocytes in the transplanted construct employing Matrigel. Kohen et al. found that human ES cells respond differently to Matrigel depending on the properties of the underlying substrates.77 Matrigel forms a globular network on polystyrene and fibrillar networks on hydrophilic surfaces (glass and oxygen plasma-treated polystyrene). Protein networks adsorbed on glass are denser than those on oxygen plasma-treated polystyrene, and stem cell differentiation is affected by the density and structure of the Matrigel network. Massumi et al. coated poly(lactic-co-glycolic acid) nanofibrous scaffolds with Matrigel and revealed a synergistic effect on differentiation of mesoderm-derived cells and germ cells from ES cells, but inhibition of endodermal cell lineages.78 Mouse iPS cells cultured as monolayers on Matrigel maintain an undifferentiated state and can differentiate into cells with a definitive endoderm phenotype in differentiation medium containing activin A.79 Implantation of human iPS cells encapsulated within Matrigel into an in vivo-vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. The differentiated cells were identified by teratoma formation with all three germ lineages evident within 4 weeks.80 Pompe-iPS cells have been successfully generated from a mouse model of Pompe disease, which differentiate into skeletal muscle cells in Matrigel-coated plates.81 The spindle-shaped skeletal muscle cells derived from Pompe-iPS cells may be useful for the treatment of skeletal muscle in Pompe disease. 3.3.2 Extracellular Matrix Components The role of the composition and structure of ECM components has been investigated in the promotion of stem cell differentiation. For example, by culturing

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mouse ES cell-derived EBs within various semiinterpenetrating polymer networks consisting of collagen, fibronectin, and laminin, Battista et al. found that high collagen concentrations inhibit EB cavitation and subsequent differentiation by inhibiting apoptosis.82 The presence of fibronectin in 3D collagen constructs strongly stimulates endothelial cell differentiation and vascularization. Conversely, laminin increases the ability of ES cells to differentiate into beating cardiomyocytes. Another study explored the role of the ECM in mouse ES cell differentiation using a multiwell microarray platform.83 Mixtures of ECM proteins (fibronectin, laminin, collagen I, collagen III, and collagen IV) were arrayed to culture mouse ES cells in the presence of combinations of soluble factors (Wnt3a, activin A, BMP-4, and fibroblast growth factor-4). Interestingly, addition of growth factors such as Wnt3a inhibited ES cell differentiation. In contrast, activin A and BMP-4 stimulation promoted ES cell cardiogenesis, whereas inhibition of Wnt signaling promoted cardiac differentiation. The ECM-immobilized arrays allowed cross talk such that the individual presence of collagen I or III inhibited ES cell differentiation, and their simultaneous presence stimulated ES cell differentiation. Conversely, collagen I and fibronectin exhibited an antagonistic interaction effect. Among the ECMgrowth factor interactions, fibronectin exhibited antagonistic interactions with Wnt3a and activin A. Collagen I and BMP-4 also exerted antagonistic effects. Based on culturing cells on thin, fibrillar, collagen I coatings that mimic the structure of physiological collagen, MSC-like cells have been derived from human ES and iPS cells.84 Although fewer cells attached to the collagen surface initially than standard tissue culture plastic, resilient colonies of homogenous spindleshaped MSC-like cells were finally obtained. These cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146, and CD166, and were negative for hematopoietic markers CD34 and CD45. Moreover, the cells were successfully differentiated into osteogenic, chondrogenic, and adipogenic lineages in vitro. 3.3.3 Cell Adhesion Molecules Although EB-derived differentiation protocols are comparatively easy and require relatively short periods of time, the variability of the EB size and asynchronous distribution of morphogens that reach the innermost layers of the EBs affect the yield of differentiated cells. Furthermore, as aggregates of many cells, EBs present difficulties in monitoring cell morphology during differentiation. To overcome this problem, Haque et al. prepared two recombinant CAM (namely E-cadherin-Fc and N-cadherin-Fc)-coated surfaces for ES and iPS cell culture and differentiation.85 As a result, completely homogeneous populations of mouse ES and iPS cells could be maintained on E-cadherin-based substrata under feeder- and serum-free culture conditions to

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induce neural differentiation. Using defined monolayer differentiation conditions on the E-cadherin and N-cadherin (E-/N-cad-Fc) hybrid substratum, a highly homogeneous population of primitive ectoderm and neural progenitor cells were routinely obtained. Using such an adherent culture system, ES/iPS cells can be differentiated as a monolayer without the need for cellular aggregates, resulting in uniform exposure to morphogens. 3.3.4 Cytokines (Growth Factors) Because of the complexity of differentiation protocols that require large amounts of expensive growth factors, many research groups have used immobilized growth factors instead of soluble factors in culture medium.86 Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and BMPs have been mixed with fibronectin and collagen I, and then printed as microspots onto silane-modified glass slides. Mouse ES cells cultured on HGF spots exhibit features of hepatic differentiation and upregulation of early hepatic markers such as albumin and a-fetoprotein. The hepatic differentiation can be enhanced further by addition of hepatic stellate cells to surfaces that already contain mouse ES cells on growth factor-printed spots. The same group also investigated hepatic differentiation of human ES cells cultured on glass substrates that were imprinted with a mixture of ECM molecules and growth factors.87 The stem cells cultured on these growth factorcontaining surfaces were positive for endoderm markers Sox17 and Foxa2, as well as early liver markers such as a-fetoprotein and albumin. These results suggested that the human ES cells received differentiation-inducing signals from the growth factor-containing surfaces, which directed differentiation along the hepatic lineage. Minato et al. reported cardiomyocyte differentiation of ES cells using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4).88 IGFBP4 was stably immobilized to polystyrene dishes through fusion with elastin-like polypeptides. By culturing mouse ES cells on the engineered niches, they found that cardiomyocyte differentiation of ES cells was effectively promoted by strong and continuous inhibition of Wnt/bcatenin signaling with IGFBP4-immobilized substrates. To guide the differentiation of iPS cells toward neurons, neuron growth factor has been grafted with alginate-chitosan-gelatin and poly(ε-caprolactone)-poly(b-hydroxybutyrate) (PCL-PHB) scaffolds.89,90 The differentiation of iPS cells into neurons was improved in both of these scaffolds. In addition, the differentiation of iPS cells into the neuronal lineage was affected by the composition of alginate-chitosan-gelatin scaffolds. An inhibiting effect on differentiation into lineages other than neurons was also detected in iPS cells cultured in neuron growth factor-grafted PCL-PHB scaffolds.

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Compared with traditional approaches, the advantages of growth factor immobilization include a reduction in the cost of experiments, stronger and longer stimulation, and the possibility of screening growth factorestem cell interactions in a multiplexed manner.

3.4 Synthetic Substrates The application of synthetic substrates as niches has been reported for the regulation of ES/iPS cell differentiation. Culturing mouse ES cells on electrospun poly(3-caprolactone) fiber scaffolds not only enhances differentiation into neural lineages but also promotes and guides neurite outgrowth.91 Levenberg et al. generated complex structures with the features of various committed embryonic tissues in vitro by inducing the differentiation of ES cells in supportive 3D biodegradable synthetic polymer [such as poly(lactic-co-glycolic acid)/poly(L-lactic acid)] scaffolds.92 The differentiation and organization of ES cells were influenced by the scaffold. In addition, the cells were induced to differentiate into 3D structures with the characteristics of various developing tissues, such as neural, cartilage, or liver, using different growth factors. An in vivo bone induction approach has been demonstrated by culturing human iPS cells in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique and transplanting such cell-scaffold constructs into subcutaneous sites of male athymic mice.93 Ardeshirylajimi et al. evaluated osteogenic differentiation of human iPS cells cultured on polyethersulfone nanofibrous scaffold.94 Significantly higher expression of common osteogenesisrelated genes such as Runx2, collagen I, osteocalcin, and osteonectin was observed in the iPS cells seeded on the polyethersulfone nanofibrous scaffold compared with cells cultured on tissue culture polystyrene. Alizarin red staining and an ALP activity assay of the differentiated iPS cells suggested a significant osteoblastic differentiation potential. Thus, the nanofiberbased scaffold promoted the differentiation capacity of iPS cells into osteoblast-like cells. Substrate properties and mechanical stimulation can also affect the differentiation of stem cells. For example, Li et al. investigated the effects of scaffold properties on tenogenic differentiation of human iPS cell-derived MSCs by developing braided submicron fibrous scaffolds and seeding the derived stem cells on the prepared scaffolds.95 Scaffolds with different mechanical properties were produced by changing the fiber chemistry and/or braiding angle. The fiber chemistry dictated cell adhesion, while the braiding angle dictated the tissue-specific lineage commitment of the stem cells. Scaffolds braided with large angles better supported tenogenic differentiation as evidenced by the production

of T/L-associated markers, downregulation of osteogenic markers, and a fibroblast-like, spindle cell morphology compared with scaffolds braided with small angles. Currently, nanotechnology platforms are being developed to regulate stem cell behaviors. The native ECM in vivo is enriched with nanostructured components, and cells are inherently sensitive to local nanoscale structures.96,97 Artificial niches with nanoscale surfaces or structures have been used to investigate the contribution of nanoscale cues to maintain the undifferentiated state or direct differentiation of stem cells. For example, a commercially available 3D nanofibrillar organization (Ultra-Web) formed by electrospinning greatly enhances the proliferation and self-renewal of mouse ES cells.98 As a unique alternative to organic polymeric nanomaterials, carbon-based nanomaterials have been implemented in stem cell research. Hu et al. reported that graphene and graphene oxide support mouse iPS cell culture and allow spontaneous differentiation.99 Mouse iPS cells cultured on both graphene and graphene oxide surfaces differentiate spontaneously into ectodermal and mesodermal lineages without significant disparity. On the other hand, graphene suppresses iPS cell differentiation toward the endodermal lineage, whereas graphene oxide augments endodermal differentiation compared with uncoated glass surfaces.

4. CONCLUSION AND OUTLOOK Many challenges including precise regulation of stem cell functions outside of their native environments still remain. Various interactions of soluble and immobilized factors, heterogeneous cell types, and ECM molecules collectively comprise stem cell niches. These components vary in a spatiotemporal fashion. Identification and mechanistic understanding of the contributions of the components of stem cell niches are important to control the presentation of multiple signals to cells. For successful medical applications of ES and iPS cells, the importance of large-scale culture systems is increasing. Recently, a suspension culture bioreactorbased method was reported to sufficiently expand and induce cardiac differentiation of human iPS cells.100 After bioreactor culture of approximately 2  107 human iPS cells for 14 days, w8  107 EB cells were able to differentiate further into cardiomyocytes, leading to about 80% cardiac troponin T-positive cells. Looking to the future, flexible consideration is also required for efficient cell culture with or without artificial niches. Stem cell culture engineering, including feeder- and xeno-free conditions, and minimum supplementary factor-requiring systems, should be considered from multiple standpoints for regenerative medicine and other clinical benefits.

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REFERENCES

ABBREVIATIONS AND ACRONYMS 3D Three-dimensional ALP Alkaline phosphatase bFGF Basic fibroblast growth factor BMP Bone morphogenetic protein CAMs Cell adhesion molecules CD Cluster of differentiation EBs Embryoid bodies ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid ES cells Embryonic stem cells FA Formaldehyde GA Glutaraldehyde HA Hyaluronic acid HDFs Human dermal fibroblasts HEFs Human embryonic fibroblasts HGF Hepatocyte growth factor HSPGs Heparin sulfate proteoglycans ICM Inner cell mass IGFBP4 Insulin-like growth factor binding protein 4 iPS cells Induced pluripotent stem cells Klf4 Kruppel-like factor 4 LIF Leukemia inhibitory factor MEFs Mouse embryonic fibroblasts MMC Mitomycin-C MSCs Mesenchymal stem cells MW Molecular weight Oct3/4 Octamer-binding transcription factor 3/4 PAAc Poly(acrylic acid) PCL Poly(caprolactone) PCL-PHB Poly(ε-caprolactone)-poly(b-hydroxybutyrate) Pdx1 Pancreatic and duodenal homeobox 1 Runx2 Runt-related transcription factor 2 SDS Sodium dodecyl sulfate SSEA-3 Stage-specific embryonic antigen 3 SSEA-4 Stage-specific embryonic antigen 4 STAT3 Signal transducer and activator of transcription 3 TRA Tumor-rejection antigen

Glossary c-Myc A regulator gene that codes for a transcription factor E-cad-Fc A fusion protein of E-cadherin and the IgG Fc domain. Foxa2 Forkhead box protein A2, also known as hepatocyte nuclear factor 3-beta (HNF-3b) HEK293 A specific cell line originally derived from human embryonic kidney cells grown in tissue culture. M15 A mesoderm-derived supportive cell line Matrigel The trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced and marketed by Corning Life Sciences. Nanog A transcription factor critically involved with self-renewal of undifferentiated stem cells. N-cad-Fc A fusion protein of N-cadherin and the IgG Fc domain. PA6 A stromal cell line derived from newborn mouse calvaria. SNL 76/7 cells Clonally derived from a mouse fibroblast STO cell line transformed with neomycin resistance and murine LIF genes. SOX 2 A transcription factor that is essential for maintaining selfrenewal of undifferentiated embryonic stem cells, also known as SRY (sex determining region Y)-box 2. Y-27632 A cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).

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References 1. Gepstein L. Derivation and potential applications of human embryonic stem cells. Circ Res 2002;91:866e76. 2. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105e11. 3. Blank U, Karlsson G, Karlsson S. Signaling pathways governing stem-cell fate. Blood 2008;111:492e503. 4. Watt FM, Huck WTS. Role of the extracellular matrix in regulating stem cell fate. Nat Rev Mol Cell Biol 2013;14:467e73. 5. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7e25. 6. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981;78:7634e8. 7. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154e6. 8. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282: 1145e7. 9. Silva J, Smith A. Capturing pluripotency. Cell 2008;132:532e6. 10. Niwa H. How is pluripotency determined and maintained? Development 2007;134:635e46. 11. Li Y, Mcclintick J, Zhong L, Edenberg HJ, Yoder MC, Chan RJ. Brief report Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Evaluation 2005;105:635e7. 12. Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, Dalton S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 2005;132:885e96. 13. Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048e60. 14. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663e76. 15. Takahashi K, Tanabe K, Ohnuki M, et al. induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861e72. 16. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917e20. 17. Richards M, Fong C-Y, Chan W-K, Wong P-C, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20: 933e6. 18. Fu X, Toh WS, Liu H, et al. Autologous feeder cells from embryoid body outgrowth support the long-term growth of human embryonic stem cells more effectively than those from direct differentiation. Tissue Eng C Methods 2010;16:719e33. 19. Takahashi K, Narita M, Yokura M, Ichisaka T, Yamanaka S. Human induced pluripotent stem cells on autologous feeders. PLoS One 2009;4:e8067. 20. Havasi P, Nabioni M, Soleimani M, Bakhshandeh B, Parivar K. Mesenchymal stem cells as an appropriate feeder layer for prolonged in vitro culture of human induced pluripotent stem cells. Mol Biol Rep 2013;40:3023e31. 21. Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19: 971e4. 22. Ito Y, Hasauda H, Kitajima T, Kiyono T. Ex vivo expansion of human cord blood hematopoietic progenitor cells using glutaraldehyde-fixed human bone marrow stromal cells. J Biosci Bioeng 2006;102:467e9. 23. Tanaka KAK, Suzuki KGN, Shirai YM, et al. Membrane molecules mobile even after chemical fixation. Nat Methods 2010;7:865e6.

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24. Joddar B, Ito Y. Artificial niche substrates for embryonic and induced pluripotent stem cell cultures. J Biotechnol 2013;168: 218e28. 25. Ito Y, Kawamorita M, Yamabe T, Kiyono T, Miyamoto K. Chemically fixed nurse cells for culturing murine or primate embryonic stem cells. J Biosci Bioeng 2007;103:113e21. 26. Yue X-S, Fujishiro M, Nishioka C, et al. Feeder cells support the culture of induced pluripotent stem cells even after chemical fixation. PLoS One 2012;7:e32707. 27. Zhou Y, Mao H, Joddar B, et al. The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness. Sci Rep 2015;5:11386. 28. Joddar B, Nishioka C, Takahashi E, Ito Y. Chemically fixed autologous feeder cell-derived niche for human induced pluripotent stem cell culture. J Mater Chem B 2015;3:2301e7. 29. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials 2006;27:3675e83. 30. Lu H, Hoshiba T, Kawazoe N, Chen G. Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture. J Biomed Mater Res A 2012;100:2507e16. 31. Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet 2005;365:1636e41. 32. Lim ML, Jungebluth P, Sjo¨qvist S, et al. Decellularized feeders: an optimized method for culturing pluripotent cells. Stem Cells Transl Med 2013;2:975e82. 33. Siti-Ismail N, Bishop AE, Polak JM, Mantalaris A. The benefit of human embryonic stem cell encapsulation for prolonged feederfree maintenance. Biomaterials 2008;29:3946e52. 34. McElroy SL, Reijo Pera RA. Culturing human embryonic stem cells in feeder-free conditions. Cold Spring Harb Protoc 2008;3:9e11. 35. Greenlee AR, Kronenwetter-Koepel TA, Kaiser SJ, Ellis TM, Liu K. Combined effects of Matrigel (TM) and growth factors on maintaining undifferentiated murine embryonic stem cells for embryotoxicity testing. Toxicol Vitr 2004;18:543e53. 36. Totonchi M, Taei A, Seifinejad A, et al. Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells. Int J Dev Biol 2010;54:877e86. 37. Sun N, Panetta NJ, Gupta DM, et al. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci USA 2009;106:15720e5. 38. Pakzad M, Totonchi M, Taei A, Seifinejad A, Hassani SN, Baharvand H. Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev Rep 2010;6:96e107. 39. Beattie GM, Lopez AD, Bucay N, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 2005;23:489e95. 40. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layerand serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837e45. 41. Braam SR, Zeinstra L, Litjens S, et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells 2008; 26:2257e65. 42. Rowland TJ, Miller LM, Blaschke AJ, et al. Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin. Stem Cells Dev 2010;19:1231e40. 43. Furue MK, Na J, Jackson JP, et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci USA 2008;105:13409e14. 44. Joddar B, Kitajima T, Ito Y. The effects of covalently immobilized hyaluronic acid substrates on the adhesion, expansion, and differentiation of embryonic stem cells for in vitro tissue engineering. Biomaterials 2011;32:8404e15.

45. Brimble SN, Zeng X, et al. Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev 2004;13:585e97. 46. Nakagawa M, Taniguchi Y, Senda S, et al. A novel efficient feederfree culture system for the derivation of human induced pluripotent stem cells. Sci Rep 2014;4:3594. 47. Li L, Bennett SAL, Wang L. Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells. Cell Adh Migr 2012;6:59e70. 48. Pieters T, van Roy F. Role of cell-cell adhesion complexes in embryonic stem cell biology. J Cell Sci 2014;127:2603e13. 49. Soncin F, Mohamet L, Ritson S, et al. E-cadherin acts as a regulator of transcripts associated with a wide range of cellular processes in mouse embryonic stem cells. PLoS One 2011;6:e21463. 50. Redmer T, Diecke S, Grigoryan T, Quiroga-Negreira A, Birchmeier W, Besser D. E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO Rep 2011;12:720e6. 51. Fok EYL, Zandstra PW. Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 2005;23:1333e42. 52. Nagaoka M, Koshimizu U, Yuasa S, et al. E-cadherin-coated plates maintain pluripotent ES cells without colony formation. PLoS One 2006;1:e15. 53. Nagaoka M, Si-Tayeb K, Akaike T, Duncan SA. Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. BMC Dev Biol 2010;10:1e12. 54. Joddar B, Ito Y. Biological modifications of materials surfaces with proteins for regenerative medicine. J Mater Chem 2011;21: 13737e55. 55. Makino H, Hasuda H, Ito Y. Immobilization of leukemia inhibitory factor (LIF) to culture murine embryonic stem cells. J Biosci Bioeng 2004;98:374e9. 56. Alberti K, Davey RE, Onishi K, et al. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods 2008;5:645e50. 57. Konno T, Kawazoe N, Chen G, Ito Y. Culture of mouse embryonic stem cells on photoimmobilized polymers. J Biosci Bioeng 2006; 102:304e10. 58. Anderson DG, Levenberg S, Langer R. Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nat Biotechnol 2004;22:863e6. 59. Villa-Diaz LG, Nandivada H, Ding J, et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat Biotechnol 2010;28:581e3. 60. Kandasamy K, Narayanan K, Ni M, Du C, Wan ACA, Zink D. Polysulfone membranes coated with polymerized 3, 4-dihydroxy-l-phenylalanine are a versatile and cost-effective synthetic substrate for defined long-term cultures of human pluripotent stem cells. Biomacromolecules 2014;15:2067e78. 61. Li YJ, Chung EH, Rodriguez RT, Firpo MT, Healy KE. Hydrogels as artificial matrices for human embryonic stem cell self-renewal. J Biomed Mater Res A 2006;79:1e5. 62. Hwang S-T, Kang S-W, Lee S-J, et al. The expansion of human ES and iPS cells on porous membranes and proliferating human adipose-derived feeder cells. Biomaterials 2010;31:8012e21. 63. Gerecht S, Bettinger CJ, Zhang Z, Borenstein JT, VunjakNovakovic G, Langer R. The effect of actin disrupting agents on contact guidance of human embryonic stem cells. Biomaterials 2007;28:4068e77. 64. Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 2007;103:389e98. 65. Ou DB, He Y, Chen R, et al. Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes. J Cell Biochem 2011;112:3555e62.

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REFERENCES

66. Shiraki N, Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm. Stem Cells 2008;26:874e85. 67. Shiraki N, Umeda K, Sakashita N, Takeya M, Kume K, Kume S. Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells 2008;13(7):731e46. 68. Yamazoe H, Iwata H. Cell microarray for screening feeder cells for differentiation of embryonic stem cells. J Biosci Bioeng 2005;100: 292e6. 69. Zhou J, Ou-Yang Q, Li J, Zhou X-Y, Lin G, Lu G-X. Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells. Stem Cells Dev 2008;17: 737e49. 70. Sui Y, Clarke T, Khillan JS. Limb bud progenitor cells induce differentiation of pluripotent embryonic stem cells into chondrogenic lineage. Differentiation 2003;71:578e85. 71. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science 2009;324:1673e7. 72. Higuchi Y, Shiraki N, Yamane K, et al. Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages. J Cell Sci 2010;123:2733e42. 73. Shiraki N, Yamazoe T, Qin Z, et al. Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum. PLoS One 2011;6:e24228. 74. Liu Y, Fu S, Rahaman MN, Mao JJ, Bal BS. Native nucleus pulposus tissue matrix promotes notochordal differentiation of human induced pluripotent stem cells with potential for treating intervertebral disc degeneration. J Biomed Mater Res A 2015;103:1053e9. 75. Liu Y, Rahaman MN, Bal BS. Modulating notochordal differentiation of human induced pluripotent stem cells using natural nucleus pulposus tissue matrix. PLoS One 2014;9:e100885. 76. Noguchi M, Hosoda K, Nakane M, et al. In vitro characterization and engraftment of adipocytes derived from human induced pluripotent stem cells and embryonic stem cells. Stem Cells Dev 2013;22:2895e905. 77. Kohen NT, Little LE, Healy KE. Characterization of Matrigel interfaces during defined human embryonic stem cell culture. Biointerphases 2009;4:69e79. 78. Massumi M, Abasi M, Babaloo H, et al. The effect of topography on differentiation fates of Matrigel-coated mouse embryonic stem cells cultured on PLGA nanofibrous scaffolds. Tissue Eng A 2012; 18:609e20. 79. Si-Tayeb K, Noto FK, Nagaoka M, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010;51:297e305. 80. Lim SY, Lee DG, Sivakumaran P, et al. In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation. Biochem Biophys Res Commun 2012;422: 75e9. 81. Kawagoe S, Higuchi T, Meng X-L, et al. Generation of induced pluripotent stem (iPS) cells derived from a murine model of Pompe disease and differentiation of Pompe-iPS cells into skeletal muscle cells. Mol Genet Metab 2011;104:123e8. 82. Battista S, Guarnieri D, Borselli C, et al. The effect of matrix composition of 3D constructs on embryonic stem cell differentiation. Biomaterials 2005;26:6194e207. 83. Flaim CJ, Teng D, Chien S, Bhatia SN. Combinatorial signaling microenvironments for studying stem cell fate. Stem Cells Dev 2008; 17:29e40.

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84. Liu Y, Goldberg AJ, Dennis JE, Gronowicz GA, Kuhn LT. One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One 2012;7:e33225. 85. Haque A, Yue X-S, Motazedian A, Tagawa Y, Akaike T. Characterization and neural differentiation of mouse embryonic and induced pluripotent stem cells on cadherin-based substrata. Biomaterials 2012;33:5094e106. 86. Tuleuova N, Lee JY, Lee J, Ramanculov E, Zern MA, Revzin A. Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells. Biomaterials 2010;31:9221e31. 87. Ghaedi M, Duan Y, Zern MA, Revzin A. Hepatic differentiation of human embryonic stem cells on growth factor-containing surfaces. J Tissue Eng Regen Med 2014;8:886e95. 88. Minato A, Ise H, Goto M, Akaike T. Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides. Biomaterials 2012;33:515e23. 89. Kuo Y-C, Wang C-C. Guided differentiation of induced pluripotent stem cells into neuronal lineage in alginateechitosanegelatin hydrogels with surface neuron growth factor. Colloids Surf B Biointerfaces 2013;104:194e9. 90. Kuo YC, Huang MJ. Material-driven differentiation of induced pluripotent stem cells in neuron growth factor-grafted poly(ε-caprolactone)-poly(b-hydroxybutyrate) scaffolds. Biomaterials 2012; 33:5672e82. 91. Xie J, Willerth SM, Li X, et al. The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages. Biomaterials 2009;30:354e62. 92. Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc Natl Acad Sci USA 2003;100:12741e6. 93. Jin G-Z, Kim T-H, Kim J-H, et al. Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold. J Biomed Mater Res A 2013;101:1283e91. 94. Ardeshirylajimi A, Hosseinkhani S, Parivar K, Yaghmaie P, Soleimani M. Nanofiber-based polyethersulfone scaffold and efficient differentiation of human induced pluripotent stem cells into osteoblastic lineage. Mol Biol Rep 2013;40:4287e94. 95. Czaplewski SK, Tsai TL, Duenwald-Kuehl SE, Vanderby R, Li WJ. Tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds. Biomaterials 2014;35: 6907e17. 96. Stevens MM, George JH. Exploring and engineering the cell surface interface. Science 2005;310:1135e8. 97. Aumailley MGB. Structure and biological activity of the extracellular matrix. J Mol Med 1998;76:253e65. 98. Nur-E-Kamal A, Ahmed I, Kamal J, Schindler M, Meiners S. Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells. Stem Cells 2006;24:426e33. 99. Chen GY, Pang DWP, Hwang SM, Tuan HY, Hu YC. A graphenebased platform for induced pluripotent stem cells culture and differentiation. Biomaterials 2012;33:418e27. 100. Matsuura K, Wada M, Shimizu T, et al. Creation of human cardiac cell sheets using pluripotent stem cells. Biochem Biophys Res Commun 2012;425:321e7.

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C H A P T E R

29 Engineering Niches for Cardiovascular Tissue Regeneration Kay Maeda, Erik J. Suuronen, Marc Ruel University of Ottawa Heart Institute, Ottawa, ON, Canada

O U T L I N E 1. Introduction

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2. The Cardiac Microenvironment 2.1 Cardiac Cells and Major Components of the Cardiac Extracellular Matrix 2.2 Growth Factors and Other Signaling Molecules 2.3 The Role of Extracellular Matrix in Cardiac Damage and Repair

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3. Cell-Based Cardiac Regenerative Therapy

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4. Biomaterial Engineering for Cardiac Regeneration 4.1 Biomaterials as Stem Cell Niches 4.2 Biomaterial Properties in Cardiovascular Regenerative Therapy 4.3 Scaffolds for Codelivery With Growth Factors 5. Types of Biomaterials and Their Applications

460 461 461

463 463

467 467 470 471 471 471 472 472

6. Biomaterial-Based Cardiovascular Devices

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7. Future Direction for Cardiac Regeneration

473

Abbreviations and Acronyms 466 467

474

Glossary

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467

References

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1. INTRODUCTION The World Health Organization estimates that around 30% of all global deaths are caused by cardiovascular diseases.1 The financial burden of managing cardiovascular diseases continues to increase. There is an enormous social demand for new cardiac regenerative therapy. Despite tremendous advances in current surgical approaches (e.g., ventricular assist devices and heart transplants) for patients with end-stage heart failure, the limited availability of these strategies is a major impediment to wide usage. Given these limitations, much

Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00029-9

5.1 Natural Biomaterials 5.1.1 Collagen 5.1.2 Fibrin 5.1.3 Hyaluronic Acid 5.1.4 Natural Polysaccharide 5.1.5 Cardiogel 5.1.6 Self-Assembling Peptides 5.2 Synthetic Biomaterials

attention has been directed toward cardiac regenerative therapy as a potentially revolutionary therapeutic modality. However, the adult mammalian heart, formed from terminally differentiated and highly organized tissue, lacks the ability to regenerate tissue once damaged. Because of this inadequate endogenous regenerative potential, stem/progenitor cell transplantation has received focus as a promising strategy to gain an intrinsic restorative response within the diseased heart. Cell-based therapy has been demonstrated success over the past few decades, with numerous promising outcomes and ongoing clinical trials; however, there

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Copyright © 2017 Elsevier Inc. All rights reserved.

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has not yet been any major achievement in improving cardiac function. In the myocardium, the cardiac extracellular matrix (ECM) provides important biophysical, biochemical, and topographical cues that help regulate cell behavior in cardiac development, homeostasis, and pathological progression. Ideally, a supportive microenvironment is required at the time of cell transplantation to assist cell delivery and provide essential cues to support survival, and to stimulate cell differentiation and the therapeutic potential of cells.2,3 Biomaterials can provide not only mechanical support but also essential signals, which enhance the potential capacity of endogenous and exogenous therapeutic elements, including cells and bioactive molecules. Over the last decade, many biomaterials have been developed and investigated. This chapter provides an overview of the biomaterials most commonly used for cardiac tissue engineering and discusses their roles in cellular support and cardiac repair.

2. THE CARDIAC MICROENVIRONMENT 2.1 Cardiac Cells and Major Components of the Cardiac Extracellular Matrix In a mammalian heart, although the exact cellular population can vary from species to species, the major cell types in the myocardium are cardiomyocytes (CMs), the main constituent of cardiac muscle, as well as fibroblasts, endothelial cells (ECs), and smooth muscle cells (SMCs).4 Cardiac fibroblasts play a prominent role in defining cardiac structure and in maintaining homeostasis.5 They also play numerous roles in cardiac remodeling through their proliferation, migration, and myofibroblast differentiation.5 ECs and SMCs, as the main components of the cardiac vasculature, play a critical role in transporting nutrients and oxygen, and secrete a wide range of important soluble proteins, various growth factors (GFs), and cytokines that regulate myocardial conditions and support the heart’s contractile function. Native cardiac cells are surrounded by a structurally and biochemically supportive protein matrix called the cardiac ECM. The types of molecules found in the cardiac ECM include collagen fibers, elastic fibers, glycosaminoglycans, proteoglycans, polysaccharides, and glycoproteins.6 Collagens, as the principal components of the cardiac ECM, provide structural scaffolding and mechanical integrity and are the most studied ECM proteins for use in natural biomaterials for cardiac tissue engineering.7e10 The collagen network in the myocardium is composed mainly of type I and III collagen, which together account for 90% of the total collagen in the healthy heart, with minor amounts of others, such

as types IV, V, VI, and VIII.6 Collagen type I fibers are the main cardiac ECM component of the heart and provide structural support, maintaining ventricular stiffness and shape. Collagen type III fibers form a fine meshwork, known as reticular fibers, which provide flexibility and act as a supporting mesh between the contractile elements of CMs. These two types of collagens jointly support beating CMs and help translate the contraction of the CMs into ventricular pumping for maintaining their alignment, shape, and function. The ECM supports intercellular signaling and regulates cell adhesion, migration, differentiation, proliferation, and apoptosis.6 It also plays a role in cytokine activity and intracellular signaling. GFs and signaling molecules can be stored within the ECM to protect them against degradation or they may attach to the surface of the ECM to present themselves more efficiently to the cell receptors.11e14 The exact ECM composition of the heart varies with the stage of development and with the health or disease state. Differential cardiac expression of ECM proteins, including collagen, fibronectin, and laminin, is observed in dilated, ischemic, and valvular cardiomyopathies,15 suggesting that different ECM protein expression may result in different cardiac pathologies. The balance between ECM synthesis and ECM degradation is physiologically regulated by a multitude of proteases. Of these, matrix metalloproteinases (MMPs) are important for ECM remodeling in the heart; these are a family of matrix proteases that contribute to the metabolism of ECM proteins.16 Tissue inhibitors of MMPs (TIMPs) also regulate ECM turnover through the blocking of MMP activity. The activity of MMPs and TIMPs can promote/inhibit cell growth, regulate cell survival or death, and affect angiogenesis.6,16 To understand how the ECM influences cell behavior and tissue function, it is important to understand the celleECM and ECMeECM interactions. Cells attach to and receive signals from the ECM through the binding of adhesion molecules, primarily integrins, which are adhesive transmembrane receptor proteins. Integrins facilitate bidirectional signaling between the cells and the ECM, resulting in a variety of cell responses, such as cell survival, cell differentiation, tissue formation, and sequential tissue repair and regeneration. For instance, the attachment and migration of ECs depends on the interaction between collagens and integrins; ECs adhere to collagen through specific integrins that subsequently activate intracellular pathways to promote cell migration and neovascularization capacity in the ischemic tissue.17 Much attention has been paid to integrin-mediated celleECM interactions because such interactions also affect the homing of stem/progenitor cells to an injury site and subsequently activate intracellular signaling to control the fate of the cells.

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2. THE CARDIAC MICROENVIRONMENT

2.2 Growth Factors and Other Signaling Molecules Cardiac cells and the ECM maintain the electrical, chemical, and biomechanical responsive nature of the heart. Moreover, these store and secrete GFs and signaling molecules to preserve the three-dimensional (3D) structure and contribute to myocardial health via autocrine/paracrine action, as well as via direct cellecell and celleECM interactions.11,14 A brief overview of several essential factors for a healthy cardiac environment is provided here. ECs and SMCs are the most abundant source of instructive signals for cells in the perivascular niche. These cells secrete a wide range of bioactive molecules, such as vascular endothelial growth factor (VEGF), stromal cell-derived factor 1 (SDF-1), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF-b1), and insulin growth factor (IGF), which have been implicated in the reparative function of cells.13,18,19 VEGF is crucial for EC proliferation and migration during cardiac development and promotes both angiogenesis and vasculogenesis during tissue remodeling following injury.13 VEGF therapy is used for therapeutic revascularization in patients with myocardial ischemia and peripheral vascular diseases. TGF-b signaling plays a role in epithelial-tomesenchymal transformation that contributes to the coronary vasculature and the cardiac fibroblast population in epicardial cells during cardiac development.11 IGF-1 can promote antiapoptotic and antiinflammatory effects in the vascular wall by inhibiting endothelial dysfunction, atherosclerotic plaque development, and myocardial infarction (MI) damage.12 IGF-1 also stimulates the expression of tropoelastin mRNA and elastic matrix deposition by poorly elastogenic adult vascular SMCs,20 which may result in successful attempts to regenerate elastin matrix structures. As congenital disruption of GF signaling leads to morphogenetic heart defects, further understanding of how GFs can affect the cardiac environment may be important to improving our ability to design strategies to improve it in heart disease patients. For cardiovascular regeneration, such GFs are commonly used to stimulate angiogenesis, support survival of cardiomyocytes, and regulate ECM composition and quality to recover matrix homeostasis (Fig. 29.1).

2.3 The Role of Extracellular Matrix in Cardiac Damage and Repair The cardiac ECM plays an important role not only in heart development and homeostasis but also in several cardiac disorder and repair processes. This section will

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highlight the role of the ECM in several abnormal heart conditions. Atrial fibrillation (AF) is the most commonly acquired arrhythmia, increasing the risk of stroke and premature death. The initiation and maintenance of AF are strongly associated with arterial fibrosis, which is characterized by disrupted MMP/TIMP activity and subsequent severe alteration of collagen type I and III synthesis and degradation.21 The expression of other noncollagenous ECM proteins, such as fibrillin-1, fibronectin-1, and fibromodulin, is also upregulated in porcine AF atria compared with those in a sinus rhythm heart.22 The role of these ECM elements and the downstream signaling pathways associated with atrial fibrosis has been a recent focus of targeted drug research. MI is irreversible myocardial cell damage that results from persistent blockage of blood flow. ECM remodeling in MI and its progression to heart failure have been well characterized. Myocardial cell death triggers the upregulation of various integrins, selectins, and components of their complementary signaling cascades.6 These signals regulate the production and secretion of GFs and cytokines, such as tumor necrosis factor (TNF)-a, monocyte chemoattractant protein-1, and interleukin-6 and interleukin-8; and this sequentially recruits platelets, neutrophils, and mononuclear cells to the ischemic site. The resultant inflammatory reaction amplifies the upregulation of cytokines, chemokines, and GFs. Cardiac fibroblasts then break down the existing ECM and synthesize a new matrix composed mainly of collagen type I and III that forms the post-MI scar tissue in the infarcted area.5,6 Experimental observations have shown that collagen type I content decreased from 80% to 40% in the infarct area.15 The balance between collagen synthesis and degradation regulates each stage of infarct progression. MMP-1 and MMP-8 degrade collagen into fragments, which are further degraded into smaller peptides by MMP-2, MMP-3, and MMP-9. This early proteolytic activity is responsible for collagen degradation at the infarct tissue site. In the subsequent weeks post MI, increasing TIMP synthesis at the infarct area suppresses the activity of MMPs and promotes progressive collagen synthesis. The balance in the synthesis and degradation of not only collagen type I and III but also types IV, V, and VI (the nonfibrillar collagens) has a role to play in cardiac repair.23 ECM properties in MI command attention since the ECM proteins provide crucial signals for both endogenous and exogenous cell responses, such as recruitment, activation, and maturation. The health of heart valves is also dependent on the state of the ECM. For example, calcific aortic valve stenosis, a common type of adult valve disease, is characterized by fibrotic valve leaflets that ultimately obstruct blood flow and aggravate cardiac function. Dramatic changes in the ECM, including collagen, hyaluronan,

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29. ENGINEERING NICHES FOR CARDIOVASCULAR TISSUE REGENERATION

FIGURE 29.1 Codelivery of growth factors within a scaffold for cardiovascular regeneration. Cells bind to the extracellular matrix scaffold through integrins and membrane-bound proteoglycans. Codelivered growth factors activate cell signaling pathways, promote cell homing, differentiation, and proliferation, and enhance angiogenesis and vascularization in the damaged tissue.

and elastin, occur in calcific aortic valve stenosis and affect its progression.24 The disorganized valve ECM which results from deregulated ECM synthesis/degradation, leads to the loss of its trilayer structure and also its mechanical and biological properties.24 Overall, the ECM provides matricellular cues that contribute to disease progression and remodeling, as well as to healthy heart homeostasis.

3. CELL-BASED CARDIAC REGENERATIVE THERAPY Different populations of resident cardiac stem/progenitor cells have been identified in the adult myocardium.25 In native cardiac repair, for example, bone marrowderived c-kitþ cells play a cardioprotective role in endogenous cardiac repair. MI activates the mobilization of these cells to the infarcted heart, where they act to promote angiogenesis and tissue repair. A limited number of new CMs are generated in the peri-infarcted

area, which is explained by the proximity of surviving capillaries and preserved perfusion. Hsieh et al. showed that the percentages of new CMs after MI were 34.6%  2.8% in MI border areas and 28.1%  2.4% in MI remote areas, compared with 14.5%  2.0% in control mice.26 However, these cells are insufficient in number and/or regenerative capacity to endogenously repair the damaged heart. Cell transplantation is thought to be a promising therapeutic strategy to enhance the inadequate restorative response within the diseased heart, with multiple different stem cell sources being tested, including pluripotent stem cells (PSCs) and various somatic stem cells such as mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and skeletal myoblasts.27 PSCs, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the prominent potential to differentiate into all three germ layers.27 PSCs possess great advantages for cardiovascular regeneration because of the following points: (1) high proliferative capacity for cardiovascular cell differentiation and high survival rate; (2) unlimited amounts of

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4. BIOMATERIAL ENGINEERING FOR CARDIAC REGENERATION

autologous cells (iPSCs); and (3) lesser ethical and immunogenic concerns (iPSCs). However, further careful investigation about in vivo safety and functionality of these cells will be needed to apply this in the clinical setting, due to their potential risk of teratoma formation and poor efficiency in the successful induction of their pluripotency to fully mature adult cells.27 MSCs are a stem cell population that can differentiate into osteoblasts, adipocytes, and also small numbers of CMs (under controlled conditions). The advantage of MSCs is that they are initially less immunogenic than other stem cell populations. In the POSEIDON clinical trial, MSC injection in patients with left ventricular (LV) dysfunction improved functional capacity, ventricular morphology, and quality of life index without evidence of a directed allogeneic MSC donor-specific immune response at 1 year follow-up.28 The disadvantage for clinical application is the broad differentiation capacity. Some studies have shown that, after transplantation, MSCs differentiated into osteoblasts in ventricular tissue.29 EPCs play a key role in vascular homeostasis and angiogenesis. They accumulate at vascular injury sites after being mobilized from the bone marrow or more local tissue sites and have the potential to proliferate and differentiate into ECs.18,30 However, it is primarily through the secretion of various proangiogenic cytokines and GFs that EPCs can promote vascular repair through the proliferation of ECs and migration to the injury site.18 Preclinical studies of EPC transplantation showed improved cardiac function in regions of neovascularized ischemic myocardium.30 For cell therapy, a supportive microenvironment is needed to direct tissue repair, which is vital for determining transplanted cell fate. The damaged myocardial environment results in an early loss of transplanted cells and the lack of cell persistence in the host tissue reduces the expected effects of cell therapy.3 A viable myocardium has essential signals for cell survival and development, transmitted through cellecell and celleECM adhesion. Ideally, these signals would be present at the time of cell transplantation into the damaged myocardium to support transplanted cells and their maximal regenerative capability. Controlling external conditions, thereby influencing internal cellular responses, can promote cell migration, engraftment, proliferation, differentiation, and function.15,31 Biomaterials could provide such a microenvironment with the signals to enhance the potential of transplanted cells, augment their angiogenic/cardiomyogenic responses, and support the electrical and mechanical integration of newly formed CMs.3 Another goal of regenerative therapy (cells þ/ biomaterials) is to promote the body’s intrinsic selfhealing ability by providing cells with a suitable microenvironment incorporated directly into the reparative process of the tissue.

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4. BIOMATERIAL ENGINEERING FOR CARDIAC REGENERATION Biomaterials can be used as a cell-seeded tissue scaffold and/or as a carrier for the delivery of cellular material and/or signaling molecules in the heart. Numerous studies have tested delivering stem cells with biomaterials as a solution to improve cell engraftment, survival, and proliferation after transplantation.3 There are two main sources of the biomaterial: natural and synthetic.32 Natural biomaterials used for scaffolds consist of ECM components, such as collagen, fibrin, and even the whole decellularized natural ECM. These have the benefits of being bioactive and biocompatible, with mechanical properties potentially more closely matched to those of the native tissue. The primary advantage of natural biomaterials is their ability to interact with cells through adhesion molecules, thus providing native signaling necessary for proper cell function. Synthetic materials consist of classically distinct materials, including metals, polymers, and ceramics. Their mechanical properties can be made to be superior to natural materials. Therefore, the advantages of synthetic materials are their strength, durability, and availability. These natural and synthetic biomaterials can be used with cells and/or various signaling molecules to augment the intrinsic repair process and provide a suitable environment for the desired cells (host or transplanted).3,32 At present, there are three main approaches to tissue regeneration: (1) in situ injection of cells with or without a supporting matrix, into the damaged tissues33,34; (2) cell implantation within a preformed 3D scaffold generated by bioreactor systems35; and (3) the scaffoldbased delivery of signaling molecules, low-molecularweight drugs, and oligonucleotides that support endogenous cell recruitment, migration, growth, and differentiation.36e39 In the following sections, we provide an overview of biomaterials as stem cell niches and their application in cardiac regeneration. Fig. 29.2 provides a simplified view of biomaterial-based strategies for introducing regenerative cells and approaches of administrating tissue engineered constructs.

4.1 Biomaterials as Stem Cell Niches Stem cells are stored in niches throughout the body. Within the heart, the niches control the physiological turnover of cardiac cells and the migration and proliferation of cardiac stem cells to replace damaged cells in the myocardium.25 The cardiac ECM can promote stem cell differentiation toward the CM lineage.25 Thus ECMbased biomaterials, derived from human or animal tissue, may serve as an appropriate scaffold for the generation of newly developed tissues. The most direct

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FIGURE 29.2 Cardiac tissue engineering strategies. Cells, scaffolds, and signaling molecules can be introduced alone or in combination at the injury site. Scaffolds provide biophysical, topographical, and biochemical microenvironments to the transplanted and host cells. Mechanical stiffness of biomaterials can guide proper stem cell differentiation. Stretch is a typical function of the cardiovascular system and has been shown to guide differentiation of stem cells toward cardiomyocytes or smooth muscle cells. Nanotopography of the biomaterial can affect stem cell phenotype, cellular alignment, and electrophysical properties.

approach to mimic the native environment is to use the myocardial ECM itself. Decellularization is a process whereby living cells and nuclear material are removed from tissues without affecting the structural integrity and desired composition of the ECM.40,41 Due to the high conservation of ECM elements, the decellularized ECM scaffolds can be integrated or incorporated into the body and provide cell- or tissue-specific support. Ott et al. have demonstrated whole heart engineering by decellularizing hearts using a detergent extraction method, retaining the underlying ECM and vascular architecture with intact chambers.40 When cardiac-derived cells and ECs were reseeded into the decellularized heart, these cells could self-assemble, reorganize, and produce contractile responses when electrically

stimulated (Fig. 29.3). Whole heart engineering still requires optimization for clinical applications, but it has the potential to transplant new functional autologous hearts in part or as an entire donor organ for patients who need a transplant. Another approach is to use decellularized cardiac ECM to generate an injectable cardiac ECM hydrogel. Singelyn et al. have developed decellularized porcine myocardial ECM as an injectable scaffold that can retain components of the natural cardiac ECM.41 In both in vitro and in vivo experiments, this decellularized myocardial matrix increased neovascularization and the recruitment of endogenous ECs and SMCs into the infarct area, resulting in preserved cardiac function. Such a cardiac ECM has been shown to be safe and effective in a clinically relevant porcine MI model.42

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FIGURE 29.3 Decellularized whole adult rat hearts could contain instructive signals for cardiac function. (A) Macroscopic view of coronary corrosion casts of cadaveric and decellularized rat hearts shows that decellularization could retain underlying extracellular matrix and vascular architecture with intact chambers. (B) Recellularized heart with a mixed population cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells, takes on functional properties in a matter of days when electrically stimulated. (Left) Representative functional assessment tracing of decellularized whole heart construct paced in a working heart bioreactor preparation at day 0. Real-time tracings of ECG, aortic pressure (afterload), and left ventricular pressure (LVP) are shown. (Center) Images of recellularized hearts on culture day 4 with pump turned off. Realtime tracings of a region of movement are shown below each image in blue, green, and red. (Right) Tracing of ECG, aortic pressure (afterload), and LVP of the paced construct are shown on 8 days after recellularization and on day 8 after stimulation with physiological (50e100 M) doses of phenylephrine. From Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14(2):213e21.

Biomaterials cannot only provide a naturally occurring extracellular environment but can also control the biochemical microenvironment of transplanted stem cells and enhance stem cell function. For example, an ECM scaffold derived from porcine small intestinal submucosa (SIS-ECM) has been one of the most extensively characterized decellularized ECM materials. SISECM scaffold is often cited as a prototypical ECM scaffold, and it could provide an ideal extracellular environment for cardiac cells because of its high content of cardiac ECM elements, such as collagen types I, III, IV, V, and VII, fibronectin, elastin, glycosaminoglycans, glycoproteins, and various GFs.

Injectable decellularized SIS-ECM was shown to promote cellular infiltration (c-kitþ cells, myofibroblasts, and macrophages) and improve cardiac function in an MI rat model.43 It has been demonstrated that a SISECM material seeded with stem cells is successful in treating MI. In a rabbit MI model, MSC-seeded SISECM patches significantly improved LV contractile function and dimensions and the capillary density of the infarcted area.44 MSCs migrated into the infarcted area and differentiated to CMs and SMCs in the SISECM treated groups. SIS-ECM treatment may have enhanced local cardiomyogenesis and limited the extent of adverse LV remodeling. The damaged tissue

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FIGURE 29.4 Engineering of electrically conductive scaffolds. (A) Gold nanowires act as conductive bridges when embedded in macroporous alginate hydrogels to allow better electrical signal propagation and contractile behavior of cardiomyocytes. (B) Nanoelectronics integrated into cardiac tissue allows spatiotemporal electrical signal propagation. (C) Methacrylated gelatin hydrogel sheets containing carbon nanotubes influence cardiomyocyte cell alignment and mechanical properties. (A) From Dvir T, Timko BP, Brigham MD, Naik SR, Karajanagi SS, Levy O, et al. Nanowired three-dimensional cardiac patches. Nat Nanotechnol 2011;6(11):720e25. (B) From Tian B, Liu J, Dvir T, Jin L, Tsui JH, Qing Q, et al. Macroporous nanowire nanoelectronic scaffolds for synthetic tissues. Nat Mater 2012;11(11):986e94. (C) From Shin SR, Jung SM, Zalabany M, Kim K, Zorlutuna P, Kim S, et al. Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators. ACS Nano 2013;7(3):2369e80.

forms a barrier that disrupts the electrical conduction system of the heart, resulting in an arrhythmia, including ventricular tachycardia and AF. In a retrospective clinical study on patients undergoing primary isolated coronary artery bypass grafting, pericardial reconstruction with SIS-ECM for pericardial closure contributed to a statistically significant decrease in the rate of postoperative AF,45 showing enhanced electrical signal transmission between cells via the material. Overall, these studies demonstrate that a naturally derived decellularized ECM has the potential in the near future for clinical use as a scaffold therapy with or without stem cells.

4.2 Biomaterial Properties in Cardiovascular Regenerative Therapy An ideal biomaterial should meet various required properties for application in humans. One indispensable

property is biocompatibility, such that it is biodegradable with degradation products that are nontoxic and nonimmunogenic. The following properties are also essential for clinical use: biocompatible mechanical properties (e.g., supporting cell construct, resistant to stress/strain), ability to be sterilized, and biomechanical characteristics similar to those of the tissue it is replacing.46,47 During the last 5 years, understanding in the field of electrically conductive scaffolds for heart tissue regeneration has brought promising attempts to produce more functional cardiac patches. The native myocardium has an organized conduction system facilitated by fastsigning bundles and Purkinje fibers.48,49 Most scaffolds used in cardiac tissue engineering are electrically insulating. Novel biomaterials have recently been developed to improve cardiac electrical signal propagation and cell alignment (Fig. 29.4).48,50 You et al. have developed microporous synthetic polymeric scaffolds with immobilized gold nanoparticles, resulting in an increment in

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the expression of connexin43, known to be gap-junction proteins, in embryonic rat CMs.48 Dvir et al. have created alginate hydrogels with incorporation of gold nanowires in the macroporous walls.50 Gold nanowires act as conductive bridges, resulting in improved CM electrophysiological and contractile behavior. A similar concept was later employed for the development of macroporous nanowire nanoelectronic scaffolds for sensing various microenvironmental conditions.51 Cells integrated with these nanohybrid scaffolds could allow spatiotemporal electrical signal propagation, and exhibited a morphology resembling those found in a natural heart tissue. Gelatin hydrogel containing carbon nanotubes improved the adherence of CMs, their actinic and troponin expression, and their mechanical properties.49 Mooney et al. have reported that MSCs could be stimulated and induced to differentiate into CMs on a polylactic acid scaffold embedded with carbon nanotubes.52 The addition of carbon nanotubes creates a nanofibrous structure that closely mimics the size scale of intrinsic ECM.49 Topological properties act together with environmental induction signals under different conditions to regulate cell behavior. Heidi et al. have developed a microfabricated system incorporating biphasic electrical pulses and topographical cues on cell culture chips.53 The chips were hot embossed into polystyrene surrounded by gold electrodes to create microgrooves and microridges of exactly defined depth, width, and ridge. Topography had a greater influence on CMs’ phenotype and cellular alignment. The cultivation of CMs on nanogrooves patterned on poly(ethylene glycol) (PEG) hydrogels resulted in a significantly functional increase in cell alignment, Cx43 expression, and conduction velocity.54 Chiu et al. have demonstrated that CMs cultured on photocrosslinkable collagen-chitosan hydrogels with microgrooves significantly improved electrophysical properties compared with smooth hydrogels, with the smaller groove producing the best results for cell elongation and orientation.55 The topographical roughness of scaffold surfaces has proved to enhance the cell attachment and proliferation.

4.3 Scaffolds for Codelivery With Growth Factors One of the limitations to the scaffold-based approach for the delivery of cells is the potential deficiency of oxygen, nutrition, and signals supplied to the cells within the scaffold matrix. However, biomaterials can also be used to deliver proteins, genes, or small RNAs together with therapeutic cells, and such a codelivery strategy may overcome these inadequacies.32,39 In one of the first investigations of the codelivery of cells and GFs within a scaffold for myocardial tissue

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repair, IGF-1 was tethered to self-assembling peptide nanofibers (NF-IGF-1), leading to prolonged IGF-1 release into the myocardium and improved cardiac function of neonatal CMs in a rat MI model compared with that of cell-seeded NF without GFs and GFs alone.56 In another study, NF-IGF-1 local injection along with cardiac progenitor cells (CPCs) was shown to enhance the differentiation of resident and delivered CPCs into mature CMs, resulting in improved cardiac function in a rat MI model, compared with CPCs and to NF-IGF-1 alone.57 Self-assembling peptide nanofibers with SDF-1, known to be a chemotactic protein for EPCs, led to enhanced EPC homing, increased capillary density and improved cardiac function.19 Codelivery of stem cells with GFs has been shown to enhance angiogenesis and vasculogenesis in vitro and in vivo.13 Silva et al. have shown that an injection of vasculogenic progenitor cells, delivered from macroporous alginate scaffolds that release VEGF, improved engraftment of delivered cells in ischemic murine hind limb musculature, increased blood vessel densities, and further improved limb perfusion compared with stand-alone delivery.31 Introducing EPCs within scaffolds and GF-recruited circulating EPCs increased the local EPCs, which contributed to enhanced vascularization.58 Our group has developed encapsulated SDF-1 release system in ischemic hind limb mice models.59 Injectable collagen matrix integrated with SDF-1encapsulating alginate microspheres stimulated endogenous stem cell-mediated regenerative responses and neovascularization in the ischemic hind limb of mice. Codelivery of cells and GFs within scaffolds could maximize their effectiveness for functional tissue regeneration. Ultimately, many approaches will likely require organization of molecularly designed biomaterials with stem cells to develop stable tissue regeneration.

5. TYPES OF BIOMATERIALS AND THEIR APPLICATIONS This section introduces various natural/synthetic biomaterials used for cardiovascular tissue engineering and their respective roles in cellular support (exogenous and endogenous) and cardiac repair. The selected combination of biomaterials, stem/progenitor cells, and signaling factors for the treatment of MI models is shown at the end of this chapter (Table 29.1).

5.1 Natural Biomaterials 5.1.1 Collagen Collagen is the most widely investigated protein for natural ECM-like biomaterials because it is the most abundant protein in the body, and in the cardiac ECM. Collagen-based constructs have been developed for

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Natural/Synthetic Biomaterials and Cell Combinations for the Treatment for MI (Selected) Cells/Factors

Approach

MI Model

Functional Effects

References

MSCs

Cardiac patch

Rat

LV function[, wall thickness[, cell engraftment[

107

MSCs

Intramyocardial injection

Rat

Reduced relocation

108

BMCs

Cardiac patch

Human

Wall thickness[, Ventricular remodelingY

8

EPCs SDF-1

Cell sheets

Rat

Cardiac function[, neovascularization

60

CACs

Intramyocardial injection

Mouse

Cardiac function[, vascular density[, viability[, perfusion[, wall thickness[

63

Skeletal myoblasts

Epicardial sponge

Rat

LV function[, angiogenesis

109

ADSCs

Intramyocardial injection

Rat

LV function[, retention

110

Cardiomyoblasts

Intramyocardial injection

Rat

LV function[, cell survival[

111

VEGF

Cardiac patch

Rat

Cell recruitment, survival, proliferation Angiogenesis

46

MSCs

Cardiac patch

Pig

Wall thickness[, neovascularization

67

BMCs

Intramyocardial injection

Rat

LV function[, cell retention[

33

MCSCs

Intramyocardial injection

Rat

Cardiac function[, vascular density[, infarct sizeY, cell survival, proliferation, differentiation

70

Skeletal myoblasts

Intramyocardial injection

Rat

Cardiac function[, wall thickness[

68

ADSCs

Intramyocardial injection

Rat

LV perimeterY, capillary density[

110

BMNCs/ HGF

Intramyocardial injection

Mouse

Cardiac function[, cell engraftment

74

BMNCs/ TGF-b1

Intramyocardial injection

Rat

Cardiac function[, cell differentiation[

73

bFGF

Transmyocardial channels

Dog

Cardiac function[, angiogenesis

72

MSCs

Cardiac patch

Rat

Scar fibrosisY, vascularization

78

BMNCs

Intramyocardial injection

Rat

Cardiac function[, scar sizeY, inflammatory cell infiltrationY, cell retention, survival, differentiation, angiogenesis

77

BMNCs

Cardiac patch

Rat

Wall thickness[, cell retention, survival, vascularization

112

SDF-1

Intramyocardial injection

Mouse

Circulating BMCs homing

113

MSCs

Hydrogel patch

Rat

Cardiac function[, cell viability, retention

81

VEGF-A/ PDGF-BB

Intramyocardial injection

Rat

Cardiac function[, vessel density[

38

IGF-1/HGF

Intramyocardial injection

Rat

Scar sizeY, fibrosisY, cell survival, angiogenesis

37

NATURAL BIOMATERIALS Collagen

þ

Fibrin

HA

Alginate

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TABLE 29.1

Natural/Synthetic Biomaterials and Cell Combinations for the Treatment for MI (Selected)dcont’d

Materials

Cells/Factors

Approach

MI Model

Functional Effects

References

Chitosan

ESCs

Intramyocardial injection

Rat

Cardiac function[, wall thickness[, vessel density[, cell retention

83

ADSCs

Intramyocardial injection

Rat

Cell engraftment, survival, homing

82

bFGF

Intramyocardial injection

Rat

Cardiac function[, arteriogenesis

114

MSCs

Intramyocardial injection

Rat

Cardiac function[, scar sizeY, cell survival, differentiation

115

BMNCs

Intramyocardial injection

Pig

Cardiac function[, vessel density[, cell retention

90

Neonatal CM

Intramyocardial injection

Mouse

Cell survival, vascular cell recruitment

89

IGF-1

Intramyocardial injection

Rat

Sustained IGF-1 delivery, activate signaling pathway

56

PDGF-BB/ FGF-2

Intramyocardial injection

Rat

Cardiac function[, vessel formation[, scar sizeY, cell survival

36

Self-assembling peptide

SYNTHETIC BIOMATERIALS (BIODEGRADABLE) PGA

ESCs

Cardiac patch

Mouse

Cardiac function[, survival rate[

93

PGCL

BMNCs

Cardiac patch

Rat

Lessening LV remodeling, cell migration, differentiation, neovascularization

94

PLCL

MSCs

Cardiac patch

Rat

Cardiac function[, scar sizeY, cell survival, differentiation

116

Polyurethane

Skeletal myoblasts

Cardiac patch

Rat

Cell survival, proliferation

117

Skeletal myoblasts þSDF-1

Cardiac patch

Rat

Cardiac function[, scar sizeY

118

Skeletal myoblasts þAkt1

Cardiac patch

Rat

Cardiac function[, scar sizeY, neovascularization

119

BMNCs

Intramyocardial injection

Rabbit

Cardiac function[, scar sizeY, cell engraftment, neovascularization

120

bFGF

Intramyocardial injection

Rat

Cardiac function[, angiogenesis

121

NIPAAm

SYNTHETIC BIOMATERIALS (NONBIODEGRADABLE) PEG-based

BMCs

Intramyocardial injection

Rat

Cardiac function[, scar sizeY, vessel density[, cell retention

122

VEGF

Intramyocardial injection

Rat

Cardiac function[, vessel density[

123

ADSCs, adipose-derived stem cells; bFGF, basic fibroblast growth factor; BMCs, bone marrow cells; BMNCs, bone marrow mononuclear cells; CACs, circulating angiogenic cells; CMs, cardiomyocytes; EPCs, endothelial progenitor cells; ESCs, embryonic stem cells; HA, hyaluronic acid; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; LV, left ventricular; MCSC, marrow-derived cardiac stem cells; MI, myocardial infarction; MSCs, mesenchymal stem cells; NIPAAm, poly(N-isopropylacrylamide); PDGF, platelet-derived growth factor; PEG, poly(ethylene glycol); PGA, poly(glycolic acid); PGCL: PLCL, poly(lactide-co- ε -caprolactone); SDF, stromal cell-derived factor; SMCs, smooth muscle cells; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

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multiple cardiac tissue engineering applications, such as for ventricular regeneration, vascular grafts, and heart valves. As described in Section 2.3, the content of the cardiac ECM is dramatically changed in damaged hearts, including a significant decrease in the proportion of collagen type I relative to other ECM proteins. Collagen type I matrix has been reported to control collagen synthesis in SMCs and fibroblasts, demonstrating its potential to control ECM remodeling. Collagen-based matrices have also been used to deliver stem/progenitor cells and enhance the viability of these therapeutic cells, resulting in reduced scar formation, increased neovascularization, and improved cardiac function after MI. For example, an EPC-seeded collagen matrix has been shown to induce neovasculogenesis and preserve cardiac function in MI rat hearts.60 Our group has evaluated various injectable collagen-based matrices to facilitate the delivery of therapeutic cells into ischemic tissue and enhance their retention and angiogenic potential.7,9,34,61 The collagen type I matrix can be effectively injected into the beating heart where it is retained and conforms to the shape of the ischemic myocardium upon gelation.62 Notably, it was shown that the interaction of the biomaterial with the delivered cells through specific integrin receptors was required for the cell-matrix therapy to synergize and improve the perfusion, viability, and function of the infarcted myocardium.63 In a phase I clinical trial, a collagen matrix seeded with autologous bone marrow cells (BMCs) increased the thickness of the infarct scar and helped to reduce cardiac wall stress in the injured regions, thus limiting ventricular remodeling and improving diastolic function, to a greater degree than cell treatment alone [the MAGNUM (Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium) trial].8 Biomaterials alone (acellular approach) have also demonstrated success in regenerating tissues, overcoming biological and possibly ethical issues related to cell transplantation.2,10,39,64 The biomechanical properties of a collagen-based scaffold can be controlled to generate a distinctive tissue microenvironment, similar to the cardiac ECM that supports cell infiltration and growth. In MI mouse hearts treated with our collagen matrix, we found that the matrix interacts with the host tissue to alter the myocardial cytokine profile, promotes angiogenesis, mediates inflammation, and reduces fibrosis and cell death, leading to improved cardiac function.65 Collagen can also be a template for “smart” biomaterials. To attract reparative cells, we developed a collagen-based matrix containing sialyl LewisX (sLeX), a ligand for the adhesion receptor L-selectin of circulated progenitor cells. The sLeX-collagen matrix increased the recruitment of CD133þCD34þcells and enhanced

angiogenic/chemotactic cytokine production in vitro. In hind limbs treated with a sLeX-collagen matrix, the recruitment and engraftment of endogenous progenitor cells were observed, with greater arteriole density and increased perfusion.64 Angiogenic GFs and cytokines can also be delivered within the collagen matrix to enhance angiogenesis and vascularization. Miyagi et al. showed that a porous collagen patch with covalently immobilized VEGF enhanced cell recruitment and proliferation in vitro and in vivo.46 In a right ventricular free wall defect rat model, the VEGF-treated collagen patch led to increased vessel density, and greater wall thickness correlated with the level of vascularization. 5.1.2 Fibrin Fibrin is a natural component that can be prepared from plasma, and has been widely used for various bioengineering applications due to its biocompatibility, ease of polymerization, and rich bioactivity.66 Fibrin as a biomaterial is a commercially available product approved by the Food and Drug Administration (FDA), such as the fibrin glue used as a surgical wound sealant. A fibrin network can provide physical support for cells and important cues for cell behavior in tissue repair through its binding sites for integrins, other ECM components, and GFs.66 Degradation products of fibrin are also known as activators of tissue repair. Fibrin-based biomaterials can serve as cell instructive scaffolds, guide stem cell phenotype and differentiation, and induce angiogenesis. There are three primary strategies for delivering stem cells using fibrin: (1) a fibrin patch for epicardial application67; (2) an injectable scaffold68; and (3) fibrin glue for epicardial applications.69 Epicardial transplantation of a fibrin patch with MSCs increased cell differentiation and neovascularization in a swine MI model.67 The use of injectable fibrin glue with skeletal myoblasts in an MI rat model was first demonstrated by Christman et al.68 Injectable fibrin glue significantly improved cell survival, increased arteriolar density, and enhanced blood flow to the ischemic myocardium, which was associated with infarct size reduction. Various stem cell types, such as BMCs, marrow-derived cardiac stem cells, and adipose-derived stem cells, have been delivered in fibrin to treat ischemic heart tissue.33,70 Epicardial fibrin glue transplantation was used to improve the retention of previously injected stem cells69; when fibrin glue was applied epicardially directly over the injection site subsequent to stem cell injection, the fibrin provided a seal and prevented backwash of the transplanted cells. In addition, fibrin has been used to deliver proteins, plasmids, and viral vectors in many different tissue engineering applications and extends the duration of the release of factors for up to 2 weeks.71 Fibrin can also be incorporated with several biological molecules to increase the local concentrations of GFs and mimic the natural ECM that

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surrounds the cells. The administration of GFs, such as basic fibroblast growth factor (bFGF), HGF, and TGFb1 within fibrin have been investigated to treat MI.72e74 5.1.3 Hyaluronic Acid HA is abundant in the heart and plays a role in cardiac development during embryogenesis, cell stabilization, morphogenesis, and matrix organization. In addition, HA is significantly involved in cardiovascular disorders, such as atherosclerosis and MI, and in tissue repair. Some advantages to the use of HA-based biomaterials in tissue engineering, especially as a vehicle for stem cells, include their simplicity, ease of applicability, and regenerating potential through stimulating angiogenesis, suppressing inflammation, and supporting cells in their reparative functions. In a rat MI model, an injectable HA-based hydrogel increased wall thickness, decreased scar formation, enhanced neovascularization, and significantly facilitated functional recovery.75 Injectable HA-based hydrogels have been used in combination with stem cells. Administration of cardiosphere-derived cells in HA hydrogels was optimized for cell retention and longterm cell engraftment, which promoted neovascularization and reduced adverse remodeling in a mouse MI model.76 Injection of HA and BMCs in a rat MI model significantly reduced the inflammatory response and CM apoptosis, and improved cell retention, angiogenesis, and arteriogenesis, and ultimately, cardiac function, compared with treatment groups using HA or BMC alone.77 HA-based patches seeded with MSCs increased vascularization and attenuated the fibrotic process in a rat MI model.78 In vitro, EPCs extensively adhered to and showed viability on an HA-based patch, showing active protein synthesis and endothelial differentiation.79 5.1.4 Natural Polysaccharide Polysaccharide biomaterials exhibit excellent biocompatibility, biodegradability, and low toxicity. Alginate, widely used in tissue engineering, is a natural seaweedderived anionic polysaccharide that can be applied in native or modified forms, such as hydrogels, fibers, microspheres, and microcapsules. Alginate has an analogous macromolecular structure to native ECM and has affinity-binding sites to support cell adhesion and GF response. Leor et al. have performed preclinical work in a swine MI model, showing that intracoronary injection of alginate hydrogel prevented adverse remodeling and increased wall thickness through replacement by myofibroblasts and collagen.80 Alginate scaffolds can serve as a unique platform for stem cells and the combination strategy of alginate scaffolds, stem/progenitor cells, and GFs has achieved success in cardiac regeneration. Alginate scaffolds were able to enhance cell survival and retention of transplanted MSCs compared with treatment with cells alone, resulting in improved cardiac function

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in vivo.81 Intramyocardial administration of alginate hydrogels with VEGF-A/PDGF-BB induced mature a-actin positive vessels and improved cardiac function in MI rats compared with administration of the biomaterial alone or gels with single factors.38 Ruvinov et al. have also shown that the dual delivery of IGF/HGF within an alginate biomaterial attenuated scar fibrosis and cell apoptosis and increased angiogenesis in MI rats.37 Our group showed that SDF-1-containing alginate microspheres, which were further integrated into an injectable collagen matrix, enhanced the endogenous stem cell response and restored perfusion via neovascularization in hind limb ischemic mice.59 Chitosan is a natural polysaccharide derived from crustacean shells. Chitosan hydrogels have been developed as biomaterials for myocardial tissue engineering as delivery vehicles for stem cells and as stand-alone therapies. For example, adipose-derived MSCs82 and embryonic stem (ES) cells83 were delivered within chitosan hydrogels and the cell-chitosan treatment enhanced cell engraftment and survival, and improved cardiac function. In our study, chitosan improved the physical properties of the collagen matrix and enhanced the maturation of significantly more ECs toward a vessellike structure, inducing greater angiogenesis than collagen-only matrix.61 In vivo, the collagenechitosan matrix stimulated vascular growth and recruited von Willebrand factorþ and CXCR4þ endothelial/angiogenic cells to a greater degree than the collagen-only matrix when implanted subcutaneously. When applied as treatment in MI mice, the collagenechitosan matrix altered the fibrotic process and improved cardiac remodeling.84 Of note is that Matrigel, isolated from the natural basement membrane of Engelbreth-Holm-Swarm mouse tumor cells, contains many important ECM components such as laminin, collagen, heparin sulfate proteoglycan, and several GFs, including bFGF, epidermal GF, IGF-1, PDGF, and TGF-b1. Given these properties, Matrigel is a natural ECM mimic that considerably influences cell survival, engraftment, differentiation, and proliferation. Intracardiac Matrigel injection resulted in endogenous stem cell recruitment or transplanted cell retention and survival within the ischemic myocardium and subsequently improved cardiac function following MI.85 However, Matrigel is still not applicable for clinical use, as it has the potential for tumorigenesis in vivo because of its derivation from mouse tumors. 5.1.5 Cardiogel Cardiogel is a natural, heterogeneous ECM product derived from in vitro cultured cardiac fibroblasts, consisting of laminin, fibronectin, collagen, proteoglycans, and GFs.86 The ECM components in cardiogel can provide a substrate environment for CM growth and

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differentiation, and influence the development of spontaneous contractile activity and phenotype.86 BMCs cultured on cardiogel showed enhanced cardiomyogenic differentiation and improved survival under oxidative stress conditions. Cardiogel also enhanced cell proliferation, adhesion, and angiogenesis in the absence of any additional inducers.87 Cardiogel possesses potential biochemical stimuli and can act as a 3D scaffold for stem cells with positive therapeutic effects for cardiac regeneration and repair. 5.1.6 Self-Assembling Peptides Self-assembling peptides are short sequences, typically of 8e16 amino acids with alternating hydrophobic and hydrophilic residues, exhibiting biocompatibility, biodegradability, ease of manufacture with standard chemical peptide synthesis, and easy handling. These peptide chains can rapidly form nanofibers and create 3D microenvironments at a physiological pH and osmolarity when introduced into the myocardium.88 The microenvironments can support endogenous/exogenous cell attachment, differentiation, and survival and promote vascularization. Furthermore, self-assembling peptides can potentially be modified to add GFs and specific cell recognition sites to influence cell signaling. When peptide nanofibers were injected into an infarcted myocardium, the scar size was decreased, vascular density was increased, and cardiac function was improved.36,89,90 Self-assembling peptide nanofibers have been employed to deliver cells, such as neonatal CMs89 and BMCs,90 resulting in enhanced retention, viability, growth, and differentiation of these transplanted cells, and reduced infarct size and significantly improved cardiac function. Codelivery of GFs with cells in peptide nanofibers have also been reported in vivo. For example, tethering of IGF-1 to peptide nanofibers and their coadministration with neonatal CMs improved systolic function compared with untethered GFs in MI rats.56 In other work, self-assembling peptides utilized for the dual delivery of PDGF and FGF-2 in MI rats resulted in reduced infarct size, and increased capillary and arteriole density.36 The RGD (arginine-glycine-aspartic acid) sequence is an essential cell attachment site and it is recognized by numerous cell adhesion molecules, including integrins. RGD peptides in biomaterials are highly effective at enhancing cell/biomaterial interaction. The incorporation of the RGD motif in self-assembling peptides has been investigated to enhance the efficacy of stem cell transplantation for repairing the myocardium. RGDcontaining peptide scaffolds showed greater MSC attachment and increased cell viability, and led to improved cardiac function in MI rat hearts compared with those without the RGD motif.91 The addition of cell-specific sequences is a promising area for the further

development and customization of self-assembling peptides to meet specific therapeutic requirements.

5.2 Synthetic Biomaterials The use of synthetic biomaterials in cardiac tissue engineering is being developed at a rapid pace, and they have many attractive properties compared with natural biomaterials: off-the-shelf availability, consistency in the manufacturing process, and controlled physical properties, such as stiffness, porosity, hydrophilic/hydrophobic ratio, and gelation time. Among their physical properties, degradability is an important issue because, ideally, the implanted material should be replaced by cardiac ECM during the tissue remodeling process. The molecular weight, copolymerization ratio, and polydispersity of the polymers are properties that can be exploited to control the degradation rate. For example, polylactic-co-glycolic acid (PLGA) is a copolymer used for biodegradable sutures and is approved by the FDA for drug delivery systems. The degradation products of this polymer are nontoxic lactic and glycolic acid moieties, and the degradation rate can be modified by controlling the ratio of lactic to glycolic acid.92 Among synthetic polymers, hydrolytically degradable polymers [polyglycolic acid (PGA), polylactic acid (PLA), and PLGA] are widely tested as 3D scaffold materials. Ke et al. have developed an embryonic stem cell (ESC)-seeded biodegradable PGA patch, resulting in enhanced ESC survival and improved cardiac function when transplanted in MI mice.93 Another biodegradable material, a poly-(glycolide-co-caprolactone) patch seeded with BMCs transplanted into MI rat hearts, exhibited enhanced cell migration and differentiation into CMs and induced neovascularization and preserved LV function.94 An injectable MMP-responsive, bioactive PEG hydrogel loaded with human ES cell-derived vascular cells prevented adverse remodeling and decreased the infarct size in MI rat hearts.95 The gel also promoted structural organization of native ECs and the delivered cells formed de novo capillaries. Several other applications of PEG-based gels to create 3D microenvironments and provide physical support as delivery vehicles for cells, GFs, and drugs have been reported (as reviewed in Ref. 96). A clinically relevant synthetic matrix for the delivery of cells would prove to be an advantageous strategy for cardiovascular regeneration. Despite these attractive properties for tissue engineering, there are still concerns regarding the application of synthetic biomaterials: the potential toxicity of the degradation products that could be absorbed in host tissues and the low bioactivity (biomimicry) of synthetic biomaterials and their insufficient interaction with cells or other signaling molecules essential for cell behavior

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7. FUTURE DIRECTION FOR CARDIAC REGENERATION

and tissue formation. The combination strategy of synthetic and natural materials to create a hybrid material allows biomaterial development to take advantage of the attractive properties of both synthetic and natural materials. For example, benzotriazole carbonate based on PEG modifies fibrinogen and generates a PEGylated fibrin patch, conferring superior differentiation in MSCs consistent with ECs within the infarcted myocardium.97 The introduction of collagen, fibronectin, fibrinogen, or laminin may also modify the biocompatibility of synthetic materials. For example, injectable hybrid hydrogels based on thiolated collagen and multiple acrylate containing oligo(acryloyl carbonate)-b-poly(ethylene glycol)-b-oligo(acryloyl carbonate) copolymers were developed to combine the intrinsic bioactivity of collagen, along with the controlled mechanical properties and enhanced stability of the synthetic copolymers.98 The hybrid hydrogel was used to deliver bone marrow MSCs to the MI rat heart, resulting in enhanced persistence of the transplanted cells, reduced scar size, and improved cardiac function.

6. BIOMATERIAL-BASED CARDIOVASCULAR DEVICES In valve replacement surgery, tissue valves remain a favorable option compared with the available mechanical valves due to their antibacterial properties and the fact that they do not require the lifetime anticoagulant treatment that accompanies the use of mechanical valves. For these reasons, tissue engineered heart valves have been a research focus for the past three decades. The ultimate goal of the tissue engineered heart valves is to develop valves that are capable of natural biological reactions to chemical and physical stimuli in their microenvironment, while maintaining durability and biocompatibility. For the tissue engineered heart valves, there are two well-defined approaches: (1) natural or synthetic biomaterials such as decellularized xenograft or homograft tissues with or without cells and (2) bioresorbable scaffolds that recruit reparative cells from the blood.99 Rippel et al. suggested the use of nanomaterial synthetic scaffolds, such as a biodegradable PGA and PLA blend scaffold, which is capable of remodeling and recruits circulating progenitor cells or can act as a cell delivery system.100 These valves in combination with bone marrow derived-MSCs were found to be clinically viable for >4 months at the pulmonary position.101 Still, size remodeling and thickening of leaflets were observed to negatively impact the long-term functionality of these valves.101 Alavi and Kheradvar developed a hybrid tissue engineered heart valve, composed of three different cell types arranged in a similar fashion to a native valve around the Nitinol mesh leaflets; aortic SMCs as the first

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layer, fibroblast/myofibroblast cells as the second layer with an endothelialized third layer.102 In vivo, the TNF-a response and inflammatory responses were reduced within the tissue construct.102 However, the exact mechanism of the immune response and the cell recruitment and tissue formation remain to be fully elucidated prior to being ready for human use. Another area of intense investigation is in the development of more advanced stents and vascular grafts. The ultimate goal of the use of stents and vascular grafts is to mimic native blood vessels and restore natural vessel function. For two decades, various materials, including metallic, decellularized tissues, biodegradable polymers, and cell sheets have been tested to prevent restenosis, thrombotic effects, and immunogenic responses. Such materials are required to provide scaffolds for vessel healing. Shirota et al. have shown that a microporous polyurethane film coated with a gelatin layer was capable of providing a scaffold for endogenous EPCs to promote reendothelialization and reform the vessel surface.103 Takeuchi et al. have shown that a partially degradable PET/ PGA stent-graft can be remodeled by recruited host cells and integrated with native aortic wall.104 The use of stem cell seeding on the surfaces is one of the promising ways to mimic natural endothelial function, thereby inhibiting intimal hyperplasia and thrombotic events. Noishiki et al. demonstrated that BMC-seeded vascular grafts formed an endothelial monolayer and that autocrine signals released by the BMCs contribute to endothelialization and vasculogenesis.105 Matsumura et al. have shown that in an adult beagle model, a poly(chitosan-g-lactic acid)/PGA matrix seeded with BMCs enriched for the mononuclear fraction promoted organized neotissue formation without any thrombotic events, stenosis, or aneurysm formation.106

7. FUTURE DIRECTION FOR CARDIAC REGENERATION In the years 2006e16, there has been impressive progress in the development of new biomaterials for cardiac regenerative therapy with many promising results in preclinical studies. The functions of transplanted stem cells are influenced by the surrounding microenvironment, including an array of instructive cues. Biomaterials as biological scaffolds can resemble myocardial architecture using nano- and microscale technologies, supporting the delivery of therapeutic cells and providing biochemical and biological cues essential for proper cell behavior and function. However, a number of challenges still remain to be overcome for their application in clinical settings. The selection of sources and populations of cells and the types of biomaterials most suitable for therapy is a

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challenge. Greater understanding of how both endogenous and exogenous cells interact with and respond to any given biomaterial is needed. Furthermore, an understanding of ECM elasticity, topography, and the mechanisms of ECM in wound healing will be needed to guide the development of clinically successful biomimetic scaffolds to achieve better functional outcomes. Biomaterials-based approaches have shown positive outcomes in large animal models and in small-scale clinical trials (e.g., the MAGNUM trial) but functional improvements can be described as modest.8,34,80 Ideally, for cardiac biomaterials to be clinically relevant, they must be electromechanically comparable to the host myocardium, or allow the remodeling of the ECM to achieve this, in order to reduce the formation of arrhythmias. Furthermore, the patches should possess an intrinsic contractile capacity to assist in decreasing wall tension in damaged hearts. A catheter-based transendocardial injection system may be favored compared with more invasive procedures to minimize surgical risk and to potentially allow multiple applications of therapy if needed. Presently, the PRESERVATION I clinical trial of the Bioabsorbable Cardiac Matrix based on alginate is being conducted using such a catheter delivery system approach (IK-5001 for the prevention of remodeling of the ventricle and congestive heart failure after acute MI. Study NCT01226563). Prior to their widespread use in humans, more large-scale and long-term clinical studies are needed to ensure the safety and efficacy of biomaterial treatments. Another area of interest for the translation of biomaterial therapy into clinical trials is the optimal timing for biomaterial administration. Our group showed greater preservation of cardiac function in MI mice when the matrix was applied early compared with when it was applied at later time-points post MI.65 The myocardial environment dramatically changes post MI and this affects the therapeutic potential of cells and the behaviors of biomaterials that are activated through their interactions in the myocardium. Careful attention should be paid to understanding the complicated interaction between cells, the cardiac ECM, biomaterials, and bioactive molecules during the cardiac repair process. Continued success in the field will provide great hope for the lives of millions; yet, this still requires a considerably greater understanding of the repair process over the coming years in order to reach this potential.

ABBREVIATIONS AND ACRONYMS AF Atrial fibrillation bFGF Basic fibroblast growth factor BMCs Bone marrow cells CMs Cardiomyocytes CPCs Cardiac progenitor cells ECM Extracellular matrix

ECs Endothelial cells EPCs Endothelial progenitor cells GFs Growth factors HA Hyaluronic acid hESCs Human embryonic stem cells HGF Hepatocyte growth factor IGF Insulin growth factor iPSCs Induced pluripotent stem cells LV Left ventricular MI Myocardial infarction MMPs Matrix metalloproteinases MSCs Mesenchymal stem cells PDGF Platelet-derived growth factor PEG Poly(ethylene glycol) PGA Polyglycolic Acid PLA Polylactic acid PLGA Polylactic-co-glycolic Acid PSCs Pluripotent stem cells RGD Arginine-glycine-aspartic acid SDF Stromal cell-derived factor SIS Small intestinal submucosa sLeX Sialyl LewisX SMCs Smooth muscle cells TGF-b Transforming growth factor-beta TIMPs Tissue inhibitors of MMPs TNF Tumor necrosis factor VEGF Vascular endothelial growth factor

Glossary Angiogenesis Remodeling or expansion of preexisting blood vessel network. Biomaterial A substance that interacts with components of living systems. It has been engineered to take a form as a whole or a part of a living structure or a biomedical device, which replaces a natural function. Cardiac extracellular matrix Structurally and biochemically supportive protein matrix that surrounds native cardiac cells. Integrins Adhesive transmembrane receptor proteins that facilitate attachment and bidirectional signaling between the cells and the extracellular matrix. Tissue remodeling Reorganization of existing tissue, resulting in tissue morphogenesis in development, homeostasis, and wound healing. Vasculogenesis De novo formation of blood vessels from mesodermal-derived hemangioblast.

References 1. Organization WH. Cardiovascular diseases (CVDs). Fact sheet 317. WHO Media Centre; March 2013. Available from: http://www. who.int/mediacentre/factsheets/fs317/en/. 2. Kuraitis D, Giordano C, Ruel M, Musaro A, Suuronen EJ. Exploiting extracellular matrix-stem cell interactions: a review of natural materials for therapeutic muscle regeneration. Biomaterials 2012; 33(2):428e43. 3. Segers VFM, Lee RT. Biomaterials to enhance stem cell function in the heart. Circ Res 2011;109(8):910e22. 4. Banerjee I, Fuseler JW, Price RL, Borg TK, Baudino TA. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse. Am J Physiol Heart Circ Physiol 2007;293(3):H1883e91. 5. Souders CA, Bowers SL, Baudino TA. Cardiac fibroblast: the renaissance cell. Circ Res 2009;105(12):1164e76. 6. Jourdan-Lesaux C, Zhang J, Lindsey ML. Extracellular matrix roles during cardiac repair. Life Sci 2010;87(13e14):391e400.

IV. BIOENGINEERING STRATEGIES TO MODEL SYNTHETIC STEM CELL NICHES

REFERENCES

7. Suuronen EJ, Veinot JP, Wong S, Kapila V, Price J, Griffith M, et al. Tissue-engineered injectable collagen-based matrices for improved cell delivery and vascularization of ischemic tissue using CD133þ progenitors expanded from the peripheral blood. Circulation 2006; 114(Suppl. 1):I138e44. 8. Chachques JC, Trainini JC, Lago N, Cortes-Morichetti M, Schussler O, Carpentier A. Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM trial): clinical feasibility study. Ann Thorac Surg 2008;85(3):901e8. 9. Kuraitis D, Hou C, Zhang Y, Vulesevic B, Sofrenovic T, McKee D, et al. Ex vivo generation of a highly potent population of circulating angiogenic cells using a collagen matrix. J Mol Cell Cardiol 2011;51(2):187e97. 10. Kuraitis D, Ebadi D, Zhang P, Rizzuto E, Vulesevic B, Padavan DT, et al. Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury. Eur Cell Mater 2012;24:175e95. 11. Azhar M, Schultz Jel J, Grupp I, Dorn 2nd GW, Meneton P, Molin DG, et al. Transforming growth factor beta in cardiovascular development and function. Cytokine Growth Factor Rev 2003; 14(5):391e407. 12. Conti E, Carrozza C, Capoluongo E, Volpe M, Crea F, Zuppi C, et al. Insulin-like growth factor-1 as a vascular protective factor. Circulation 2004;110(15):2260e5. 13. Hoeben Ann LB, Highley Martin S, Wildiers H, Van Oosterom Allan T, De Bruijn Ernst A. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 2004;56:549e80. 14. Doyle B, Sorajja P, Hynes B, Kumar AHS, Araoz PA, Stalboerger PG, et al. Progenitor cell therapy in a porcine acute myocardial infarction model induces cardiac hypertrophy, mediated by paracrine secretion of cardiotrophic factors including TGFbeta1. Stem Cells Dev 2008;17(5):941e51. 15. Herpel E, Pritsch M, Koch A, Dengler TJ, Schirmacher P, Schnabel PA. Interstitial fibrosis in the heart: differences in extracellular matrix proteins and matrix metalloproteinases in end-stage dilated, ischaemic and valvular cardiomyopathy. Histopathology 2006;48(6):736e47. 16. Singh RB, Dandekar SP, Elimban V, Gupta SK, Dhalla NS. Role of proteases in the pathophysiology of cardiac disease. Mol Cell Biochem 2004;263(1e2):241e56. 17. Senger DR, Perruzzi CA, Streit M, Koteliansky VE, de Fougerolles AR, Detmar M. The alpha(1)beta(1) and alpha(2) beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. Am J Pathol 2002;160(1):195e204. 18. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, et al. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol 2005;39(5):733e42. 19. Segers VF, Tokunou T, Higgins LJ, MacGillivray C, Gannon J, Lee RT. Local delivery of protease-resistant stromal cell derived factor-1 for stem cell recruitment after myocardial infarction. Circulation 2007;116(15):1683e92. 20. Kothapalli CR, Ramamurthi A. Benefits of concurrent delivery of hyaluronan and IGF-1 cues to regeneration of crosslinked elastin matrices by adult rat vascular cells. J Tissue Eng Regen Med 2008; 2(2e3):106e16. 21. Polyakova V, Miyagawa S, Szalay Z, Risteli J, Kostin S. Atrial extracellular matrix remodelling in patients with atrial fibrillation. J Cell Mol Med 2008;12(1):189e208. 22. Lin CS, Lai LP, Lin JL, Sun YL, Hsu CW, Chen CL, et al. Increased expression of extracellular matrix proteins in rapid atrial pacinginduced atrial fibrillation. Heart Rhythm 2007;4(7):938e49. 23. Shamhart PE, Meszaros JG. Non-fibrillar collagens: key mediators of post-infarction cardiac remodeling? J Mol Cell Cardiol 2010; 48(3):530e7.

475

24. Chen JH, Simmons CA. Cell-matrix interactions in the pathobiology of calcific aortic valve disease: critical roles for matricellular, matricrine, and matrix mechanics cues. Circ Res 2011;108(12): 1510e24. 25. Bearzi CRM, Hosoda T, Tillmanns J, Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins RW, Lecapitaine N, Cascapera S, Beltrami AP, D’Alessandro DA, Zias E, Quaini F, Urbanek K, Michler RE, Bolli R, Kajstura J, Leri A, Anversa P. Human cardiac stem cells. Proc Natl Acad Sci USA 2007;104:14068e73. 26. Hsieh PCH, Segers VFM, Davis ME, MacGillivray C, Gannon J, Molkentin JD, et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 2007;13(8):970e4. 27. Dixit P, Katare R. Challenges in identifying the best source of stem cells for cardiac regeneration therapy. Stem Cell Res Ther 2015;6(1): 26. 28. Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, et al. Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 2012; 308(22):2369e79. 29. Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation 2004;109(25): 3154e7. 30. Asahara T, Kawamoto A, Masuda H. Concise review: circulating endothelial progenitor cells for vascular medicine. Stem Cells 2011; 29(11):1650e5. 31. Silva EA, Kim ES, Kong HJ, Mooney DJ. Material-based deployment enhances efficacy of endothelial progenitor cells. Proc Natl Acad Sci USA 2008;105(38):14347e52. 32. Lam MT, Wu JC. Biomaterial applications in cardiovascular tissue repair and regeneration. Expert Rev Cardiovasc Ther 2012;10(8): 1039e49. 33. Nakamuta JS, Danoviz ME, Marques FL, Leonardo DS, Becker C, Gonc¸alves GA, et al. Cell therapy attenuates cardiac dysfunction post myocardial infarction: effect of timing, routes of injection and a fibrin scaffold. PLoS One 2009;4(6):e6005. 34. Giordano C, Thorn SL, Renaud JM, Al-Atassi T, Boodhwani M, Klein R, et al. Preclinical evaluation of biopolymer-delivered circulating angiogenic cells in a swine model of hibernating myocardium. Circ Cardiovasc Imaging 2013;6(6):982e91. 35. Schussler O, Chachques JC, Mesana TG, Suuronen EJ, Lecarpentier Y, Ruel M. 3-dimensional structures to enhance cell therapy and engineer contractile tissue. Asian Cardiovasc Thorac Ann 2010;18(2):188e98. 36. Kim JH, Jung Y, Kim SH, Sun K, Choi J, Kim HC, et al. The enhancement of mature vessel formation and cardiac function in infarcted hearts using dual growth factor delivery with selfassembling peptides. Biomaterials 2011;32(26):6080e8. 37. Ruvinov E, Leor J, Cohen S. The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction. Biomaterials 2011;32(2):565e78. 38. Hao X, Silva EA, Mansson-Broberg A, Grinnemo KH, Siddiqui AJ, Dellgren G, et al. Angiogenic effects of sequential release of VEGF-A165 and PDGF-BB with alginate hydrogels after myocardial infarction. Cardiovasc Res 2007;75(1):178e85. 39. Johnson TD, Christman KL. Injectable hydrogel therapies and their delivery strategies for treating myocardial infarction. Expert Opin Drug Deliv 2013;10(1):59e72. 40. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14(2):213e21.

IV. BIOENGINEERING STRATEGIES TO MODEL SYNTHETIC STEM CELL NICHES

476

29. ENGINEERING NICHES FOR CARDIOVASCULAR TISSUE REGENERATION

41. Singelyn JM, DeQuach JA, Seif-Naraghi SB, Littlefield RB, SchupMagoffin PJ, Christman KL. Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. Biomaterials 2009;30(29):5409e16. 42. Seif-Naraghi SB, Singelyn JM, Salvatore MA, Osborn KG, Wang JJ, Sampat U, et al. Safety and efficacy of an injectable extracellular matrix hydrogel for treating myocardial infarction. Sci Transl Med 2013;5(173):173ra25. 43. Zhao ZQ, Puskas JD, Xu D, Wang NP, Mosunjac M, Guyton RA, et al. Improvement in cardiac function with small intestine extracellular matrix is associated with recruitment of C-kit cells, myofibroblasts, and macrophages after myocardial infarction. J Am Coll Cardiol 2010;55(12):1250e61. 44. Tan MY, Zhi W, Wei RQ, Huang YC, Zhou KP, Tan B, et al. Repair of infarcted myocardium using mesenchymal stem cell seeded small intestinal submucosa in rabbits. Biomaterials 2009; 30(19):3234e40. 45. Boyd WD, Johnson WE, Sultan PK, Deering TF, Matheny RG. Pericardial reconstruction using an extracellular matrix implant correlates with reduced risk of postoperative atrial fibrillation in coronary artery bypass surgery patients. Heart Surg Forum 2010; 13(5):311e6. 46. Miyagi Y, Chiu LLY, Cimini M, Weisel RD, Radisic M, Li R-K. Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair. Biomaterials 2011;32(5):1280e90. 47. Tiwari-Pandey R, Toeg H, Sellke FW, Ruel M. Clinically relevant extracellular-matrix scaffolds for cell transplantation and vascular repair. Curr Vasc Pharmacol 2012;10(3):322e30. 48. You JO, Rafat M, Ye GJC, Auguste DT. Nanoengineering the heart: conductive scaffolds enhance connexin 43 expression. Nano Lett 2011;11(9):3643e8. 49. Shin SR, Jung SM, Zalabany M, Kim K, Zorlutuna P, Kim S, et al. Carbon-nanotube-embedded hydrogel sheets for engineering cardiac constructs and bioactuators. ACS Nano 2013;7(3):2369e80. 50. Dvir T, Timko BP, Brigham MD, Naik SR, Karajanagi SS, Levy O, et al. Nanowired three-dimensional cardiac patches. Nat Nanotechnol 2011;6(11):720e5. 51. Tian B, Liu J, Dvir T, Jin L, Tsui JH, Qing Q, et al. Macroporous nanowire nanoelectronic scaffolds for synthetic tissues. Nat Mater 2012;11(11):986e94. 52. Mooney E, Mackle JN, Blond DJ, O’Cearbhaill E, Shaw G, Blau WJ, et al. The electrical stimulation of carbon nanotubes to provide a cardiomimetic cue to MSCs. Biomaterials 2012;33(26):6132e9. 53. Heidi Au HT, Cui B, Chu ZE, Veres T, Radisic M. Cell culture chips for simultaneous application of topographical and electrical cues enhance phenotype of cardiomyocytes. Lab Chip 2009;9(4):564e75. 54. Kim DH, Lipke EA, Kim P, Cheong R, Thompson S, Delannoy M, et al. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proc Natl Acad Sci USA 2010; 107(2):565e70. 55. Chiu LL, Janic K, Radisic M. Engineering of oriented myocardium on three-dimensional micropatterned collagen-chitosan hydrogel. Int J Artif Organs 2012;35(4):237e50. 56. Davis ME, Hsieh PCH, Takahashi T, Song Q, Zhang S, Kamm RD, et al. Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci USA 2006;103(21):8155e60. 57. Padin-Iruegas ME, Misao Y, Davis ME, Segers VF, Esposito G, Tokunou T, et al. Cardiac progenitor cells and biotinylated insulin-like growth factor-1 nanofibers improve endogenous and exogenous myocardial regeneration after infarction. Circulation 2009;120(10):876e87. 58. Saif J, Schwarz TM, Chau DY, Henstock J, Sami P, Leicht SF, et al. Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization. Arterioscler Thromb Vasc Biol 2010;30(10): 1897e904.

59. Kuraitis D, Zhang P, Zhang Y, Padavan DT, McEwan K, Sofrenovic T, et al. A stromal cell-derived factor-1 releasing matrix enhances the progenitor cell response and blood vessel growth in ischaemic skeletal muscle. Eur Cell Mater 2011;22:109e23. 60. Frederick JR, Fitzpatrick 3rd JR, McCormick RC, Harris DA, Kim AY, Muenzer JR, et al. Stromal cell-derived factor-1alpha activation of tissue-engineered endothelial progenitor cell matrix enhances ventricular function after myocardial infarction by inducing neovasculogenesis. Circulation 2010;122(Suppl. 11): S107e17. 61. Deng C, Zhang P, Vulesevic B, Kuraitis D, Li F, Yang AF, et al. A collagen-chitosan hydrogel for endothelial differentiation and angiogenesis. Tissue Eng A 2010;16(10):3099e109. 62. Ahmadi A, Thorn SL, Alarcon EI, Kordos M, Padavan DT, Hadizad T, et al. PET imaging of a collagen matrix reveals its effective injection and targeted retention in a mouse model of myocardial infarction. Biomaterials 2015;49:18e26. 63. Ahmadi A, McNeill B, Vulesevic B, Kordos M, Mesana L, Thorn SL, et al. The role of integrin a2 in cell and matrix therapy that improves perfusion, viability and function of infarcted myocardium. Biomaterials 2014;35(17):4749e58. 64. Suuronen EJ, Zhang P, Kuraitis D, Cao X, Melhuish A, McKee D, et al. An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model. FASEB J 2009;23(5):1447e58. 65. Blackburn NJ, Sofrenovic T, Kuraitis D, Ahmadi A, McNeill B, Deng C, et al. Timing underpins the benefits associated with injectable collagen biomaterial therapy for the treatment of myocardial infarction. Biomaterials 2015;39:182e92. 66. Brown AC, Barker TH. Fibrin-based biomaterials: modulation of macroscopic properties through rational design at the molecular level. Acta Biomater 2014;10(4):1502e14. 67. Liu J, Hu Q, Wang Z, Xu C, Wang X, Gong G, et al. Autologous stem cell transplantation for myocardial repair. Am J Physiol Heart Circ Physiol 2004;287(2):H501e11. 68. Christman KL, Fok HH, Sievers RE, Fang Q, Lee RJ. Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction. Tissue Eng 2004;10(3e4): 403e9. 69. Terrovitis J, Lautamaki R, Bonios M, Fox J, Engles JM, Yu J, et al. Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery. J Am Coll Cardiol 2009; 54(17):1619e26. 70. Guo HD, Wang HJ, Tan YZ, Wu JH. Transplantation of marrowderived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction. Tissue Eng A 2011; 17(1e2):45e58. 71. Breen A, O’Brien T, Pandit A. Fibrin as a delivery system for therapeutic drugs and biomolecules. Tissue Eng B Rev 2009;15(2):201e14. 72. Nie SP, Wang X, Qiao SB, Zeng QT, Jiang JQ, Liu XQ, et al. Improved myocardial perfusion and cardiac function by controlled-release basic fibroblast growth factor using fibrin glue in a canine infarct model. J Zhejiang Univ Sci B 2010;11(12): 895e904. 73. Yang HS, Bhang SH, Kim IK, Lee TJ, Kang JM, Lee DH, et al. In situ cardiomyogenic differentiation of implanted bone marrow mononuclear cells by local delivery of transforming growth factor-b1. Cell Transplant 2012;21(1):299e312. 74. Zhang G, Hu Q, Braunlin EA, Suggs LJ, Zhang J. Enhancing efficacy of stem cell transplantation to the heart with a PEGylated fibrin biomatrix. Tissue Eng A 2008;14(6):1025e36. 75. Abdalla S, Makhoul G, Duong M, Chiu RC, Cecere R. Hyaluronic acid-based hydrogel induces neovascularization and improves cardiac function in a rat model of myocardial infarction. Interact Cardiovasc Thorac Surg 2013;17(5):767e72.

IV. BIOENGINEERING STRATEGIES TO MODEL SYNTHETIC STEM CELL NICHES

REFERENCES

76. Smith RR, Marban E, Marban L. Enhancing retention and efficacy of cardiosphere-derived cells administered after myocardial infarction using a hyaluronan-gelatin hydrogel. Biomatter 2013; 3(1):e24490. 77. Chen CH, Wang SS, Wei EI, Chu TY, Hsieh PC. Hyaluronan enhances bone marrow cell therapy for myocardial repair after infarction. Mol Ther 2013;21(3):670e9. 78. Fiumana E, Pasquinelli G, Foroni L, Carboni M, Bonafe F, Orrico C, et al. Localization of mesenchymal stem cells grafted with a hyaluronan-based scaffold in the infarcted heart. J Surg Res 2013;179(1):e21e9. 79. Pasquinelli G, Vinci MC, Gamberini C, Orrico C, Foroni L, Guarnieri C, et al. Architectural organization and functional features of early endothelial progenitor cells cultured in a hyaluronan-based polymer scaffold. Tissue Eng A 2009;15(9):2751e62. 80. Leor J, Tuvia S, Guetta V, Manczur F, Castel D, Willenz U, et al. Intracoronary injection of in situ forming alginate hydrogel reverses left ventricular remodeling after myocardial infarction in Swine. J Am Coll Cardiol 2009;54(11):1014e23. 81. Ceccaldi C, Fullana SG, Alfarano C, Lairez O, Calise D, Cussac D, et al. Alginate scaffolds for mesenchymal stem cell cardiac therapy: influence of alginate composition. Cell Transplant 2012;21(9): 1969e84. 82. Liu Z, Wang H, Wang Y, Lin Q, Yao A, Cao F, et al. The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment. Biomaterials 2012;33(11):3093e106. 83. Lu WN, Lu SH, Wang HB, Li DX, Duan CM, Liu ZQ, et al. Functional improvement of infarcted heart by co-injection of embryonic stem cells with temperature-responsive chitosan hydrogel. Tissue Eng A 2009;15(6):1437e47. 84. Ahmadi A, Vulesevic B, Blackburn NJ, Joseph J, Ruel M, Suuronen EJ. A collagen-chitosan injectable hydrogel improves cardiac remodeling in a mouse model of myocardial infarction. J Biomater Tissue Eng 2014;4(4):886e94. 85. Ou L, Li W, Zhang Y, Wang W, Liu J, Sorg H, et al. Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model. J Cell Mol Med 2011;15(6):1310e8. 86. VanWinkle WBSM, Buja LM. Cardiogel: a biosynthetic extracellular matrix for cardiomyocyte culture. In Vitro Cell Dev Biol Anim 1996;32(8):478e85. 87. Sreejit P, Verma RS. Enhanced cardiomyogenic lineage differentiation of adult bone-marrow-derived stem cells grown on cardiogel. Cell Tissue Res 2013;353(3):443e56. 88. Segers VF, Lee RT. Local delivery of proteins and the use of selfassembling peptides. Drug Discov Today 2007;12(13e14):561e8. 89. Davis ME, Motion JPM, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, et al. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation 2005;111(4):442e50. 90. Lin Y-D, Yeh M-L, Yang Y-J, Tsai D-C, Chu T-Y, Shih Y-Y, et al. Intramyocardial peptide nanofiber injection improves postinfarction ventricular remodeling and efficacy of bone marrow cell therapy in pigs. Circulation 2010;122(Suppl. 11):132e41. 91. Guo HD, Cui GH, Wang HJ, Tan YZ. Transplantation of marrowderived cardiac stem cells carried in designer self-assembling peptide nanofibers improves cardiac function after myocardial infarction. Biochem Biophys Res Commun 2010;399(1):42e8. 92. Lu JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 2009;9(4): 325e41. 93. Ke Q, Yang Y, Rana JS, Chen Y, Morgan JP, Xiao Y-F. Embryonic stem cells cultured in biodegradable scaffold repair infarcted myocardium in mice. Sheng Li Xue Bao 2005;57(6):673e81.

477

94. Piao H, Kwon JS, Piao S, Sohn JH, Lee YS, Bae JW, et al. Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model. Biomaterials 2007;28(4):641e9. 95. Kraehenbuehl TP, Ferreira LS, Hayward AM, Nahrendorf M, van der Vlies AJ, Vasile E, et al. Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction. Biomaterials 2011;32(4):1102e9. 96. Arnal-Pastor M, Chachques JC, Monleo´n Pradas M, Valle´sLluch A. Biomaterials for cardiac tissue engineering. InTech; 2013. 97. Zhang G, Wang X, Wang Z, Zhang J, Suggs L. A PEGylated fibrin patch for mesenchymal stem cell delivery. Tissue Eng 2006;12(1): 9e19. 98. Xu G, Wang X, Deng C, Teng X, Suuronen EJ, Shen Z, et al. Injectable biodegradable hybrid hydrogels based on thiolated collagen and oligo(acryloyl carbonate)-poly(ethylene glycol)-oligo(acryloyl carbonate) copolymer for functional cardiac regeneration. Acta Biomater 2015;15:55e64. 99. Vesely I. Heart valve tissue engineering. Circulation Res 2005;97(8): 743e55. 100. Rippel RA, Ghanbari H, Seifalian AM. Tissue-engineered heart valve: future of cardiac surgery. World J Surg 2012;36(7): 1581e91. 101. Sutherland FW, Perry TE, Yu Y, Sherwood MC, Rabkin E, Masuda Y, et al. From stem cells to viable autologous semilunar heart valve. Circulation 2005;111(21):2783e91. 102. Alavi SH, Liu WF, Kheradvar A. Inflammatory response assessment of a hybrid tissue-engineered heart valve leaflet. Ann Biomed Eng 2013;41(2):316e26. 103. Shirota T, He H, Yasui H, Matsuda T. Human endothelial progenitor cell-seeded hybrid graft: proliferative and antithrombogenic potentials in vitro and fabrication processing. Tissue Eng 2003;9(1):127e36. 104. Takeuchi M, Kuratani T, Miyagawa S, Shirakawa Y, Shimamura K, Kin K, et al. Tissue-engineered stent-graft integrates with aortic wall by recruiting host tissue into graft scaffold. J Thorac Cardiovasc Surg 2014;148(4):1719e25. 105. Noishiki Y, Tomizawa Y, Yamane Y, Matsumoto A. Autocrine angiogenic vascular prosthesis with bone marrow transplantation. Nat Med 1996;2(1):90e3. 106. Matsumura G, Miyagawa-Tomita S, Shin’oka T, Ikada Y, Kurosawa H. First evidence that bone marrow cells contribute to the construction of tissue-engineered vascular autografts in vivo. Circulation 2003;108(14):1729e34. 107. Simpson D, Liu H, Fan T-HM, Nerem R, Dudley SC. A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling. Stem Cells 2007;25(9):2350e7. 108. Dai W, Hale SL, Kay GL, Jyrala AJ, Kloner RA. Delivering stem cells to the heart in a collagen matrix reduces relocation of cells to other organs as assessed by nanoparticle technology. Regen Med 2009;4(3):387e95. 109. Hamdi H, Furuta A, Bellamy V, Bel A, Puymirat E, Peyrard S, et al. Cell delivery: intramyocardial injections or epicardial deposition? A head-to-head comparison. Ann Thorac Surg 2009; 87(4):1196e203. 110. Danoviz ME, Nakamuta JS, Marques FLN, dos Santos L, Alvarenga EC, dos Santos AA, et al. Rat adipose tissue-derived stem cells transplantation attenuates cardiac dysfunction post infarction and biopolymers enhance cell retention. PLoS One 2010;5(8):e12077. 111. Kutschka I, Chen IY, Kofidis T, Arai T, von Degenfeld G, Sheikh AY, et al. Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts. Circulation 2006;114(Suppl. 1): 167e73.

IV. BIOENGINEERING STRATEGIES TO MODEL SYNTHETIC STEM CELL NICHES

478

29. ENGINEERING NICHES FOR CARDIOVASCULAR TISSUE REGENERATION

112. Chi NH, Yang MC, Chung TW, Chen JY, Chou NK, Wang SS. Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials 2012;33(22):5541e51. 113. Purcell BP, Elser JA, Mu A, Margulies KB, Burdick JA. Synergistic effects of SDF-1alpha chemokine and hyaluronic acid release from degradable hydrogels on directing bone marrow derived cell homing to the myocardium. Biomaterials 2012;33(31): 7849e57. 114. Wang H, Zhang X, Li Y, Ma Y, Zhang Y, Liu Z, et al. Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive chitosan hydrogel. J Heart Lung Transplant 2010;29(8):881e7. 115. Cui XJ, Xie H, Wang HJ, Guo HD, Zhang JK, Wang C, et al. Transplantation of mesenchymal stem cells with self-assembling polypeptide scaffolds is conducive to treating myocardial infarction in rats. Tohoku J Exp Med 2010;222(4):281e9. 116. Jin J, Jeong SI, Shin YM, Lim KS, Shin H, Lee YM, et al. Transplantation of mesenchymal stem cells within a poly(lactideco-epsilon-caprolactone) scaffold improves cardiac function in a rat myocardial infarction model. Eur J Heart Fail 2009;11(2): 147e53. 117. Siepe M, Giraud MN, Liljensten E, Nydegger U, Menasche P, Carrel T, et al. Construction of skeletal myoblast-based polyurethane scaffolds for myocardial repair. Artif Organs 2007; 31(6):425e33.

118. Blumenthal B, Poppe A, Golsong P, Blanke P, Rylski B, Beyersdorf F, et al. Functional regeneration of ischemic myocardium by transplanted cells overexpressing stromal cell-derived factor-1 (SDF-1): intramyocardial injection versus scaffold-based application. Eur J Cardiothorac Surg 2011;40(4):e135e41. 119. Siepe M, Golsong P, Poppe A, Blumenthal B, von Wattenwyl R, Heilmann C, et al. Scaffold-based transplantation of akt1overexpressing skeletal myoblasts: functional regeneration is associated with angiogenesis and reduced infarction size. Tissue Eng A 2011;17(1e2):205e12. 120. Li XY, Wang T, Jiang XJ, Lin T, Wu DQ, Zhang XZ, et al. Injectable hydrogel helps bone marrow-derived mononuclear cells restore infarcted myocardium. Cardiology 2010;115(3):194e9. 121. Garbern JC, Minami E, Stayton PS, Murry CE. Delivery of basic fibroblast growth factor with a pH-responsive, injectable hydrogel to improve angiogenesis in infarcted myocardium. Biomaterials 2011;32(9):2407e16. 122. Wang T, Jiang XJ, Tang QZ, Li XY, Lin T, Wu DQ, et al. Bone marrow stem cells implantation with alpha-cyclodextrin/ MPEG-PCL-MPEG hydrogel improves cardiac function after myocardial infarction. Acta Biomater 2009;5(8):2939e44. 123. Wu J, Zeng F, Huang XP, Chung JC, Konecny F, Weisel RD, et al. Infarct stabilization and cardiac repair with a VEGF-conjugated, injectable hydrogel. Biomaterials 2011;32(2):579e86.

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30 Engineering Niches for Blood Vessel Regeneration Quinton Smith, Michael Blatchley, Sharon Gerecht Johns Hopkins University, Baltimore, MD, United States

O U T L I N E 1. Introduction 1.1 Bench-to-Beside: The Role of Vascular Tissue Engineering in Translational Medicine 1.2 Learning From Nature: In Vivo Vascularization 1.2.1 Mature Blood Vessels 1.2.2 Vasculogenesis and Angiogenesis 1.2.3 Oxygen 1.2.4 Shear Stress 2. Elements for Recapitulating the Vascular Regeneration Niche In Vitro 2.1 Cell Sources 2.1.1 Pluripotent Stem Cells 2.1.2 Mesenchymal Stem Cells 2.1.3 Endothelial Progenitor Cells 3. Material Design Parameters for Controlling the Vascular Niche 3.1 Hydrogels 3.2 Growth Factor Release 3.3 Substrate Stiffness

479 479 480 480 480 481 481 483 483 483 483 483 485 485 486 486

3.4 Substrate Adhesion 3.5 Degradation 3.6 Hypoxia

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4. Strategies in Engineering Artificial Niches for Vascular Regeneration 4.1 Decellularized Constructs 4.2 3D Printing 4.3 Micropatterned Substrates 4.4 Electrospinning

488 488 489 490 492

5. Inducing Vascularization Through Biomaterials 492 5.1 Subcutaneous Injection 493 5.2 Wound Healing 493 6. Conclusion and Future Directions

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Glossary

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References

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1. INTRODUCTION 1.1 Bench-to-Beside: The Role of Vascular Tissue Engineering in Translational Medicine The circulatory system is the first organ to develop post gastrulation, marking its importance as a sustaining network, facilitating the removal of waste, exchange of oxygen, and delivery of nutrients to the maturing embryo. De novo blood vessel formation, also known as Biology and Engineering of Stem Cell Niches http://dx.doi.org/10.1016/B978-0-12-802734-9.00030-5

vasculogenesis, and the subsequent expansion of the nascent vascular network via angiogenesis, constitute a complex niche essential to embryonic development, female reproduction, wound healing, and disease progression. In addition to contributing to cancer metastasis, postnatal angiogenesis complicates many diseases, causing vascular complications that lead to poor prognoses in patients with inflammatory rheumatoid arthritis, diabetic retinopathy, and renal kidney diseases just to name a few. In fact, ailments affecting the cardiovascular

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system have contributed to an alarming epidemiological transition, in which cardiovascular diseases (CVD) have emerged as the number one cause of death, surpassing perinatal, infectious, and nutritional deficiency diseases globally.1 In the United States alone, over 700,000 people succumb to these diseases annually. These ailments, negatively impacting the homeostasis of the heart and vascular system, are typically initiated by atherosclerosis, a process of thickening, narrowing, and hardening of blood vessels. As the vascular structure changes in this diseased phenotype, the ability to elastically deform in response to hemodynamic forces generated by heart contraction is lost. Ischemic disorders, such as peripheral artery disease, result from vessel narrowing by plaque accumulation, thereby obstructing blood flow, and causing surrounding tissue to undergo necrosis due to a lack of oxygen and nutrients. Although CVD is mostly preventable, strategies for its treatment and management have been undermined by complex interactions between wellunderstood risk factors such as hypertension, obesity, smoking, and diabetes with gender, race, and socioeconomic status.2 Vascular tissue engineering has arisen as a promising multidisciplinary approach to combat vascular diseases. By combining knowledge in the fields of stem cell biology, biomaterials, and biomedical engineering, novel therapeutic interventions have emerged to not only augment, but to also replace diseased vasculature.

1.2 Learning From Nature: In Vivo Vascularization 1.2.1 Mature Blood Vessels Mature blood vessels consist of a luminal monolayer of quiescent endothelial cells (ECs) that serve as a semipermeable barrier, mediating the transfer of small molecules to underlying tissue. In addition to serving as a barrier, ECs respond to shear stress by elongating and aligning parallel to the applied hemodynamic pressure and secreting a variety of vasodilators including nitric oxide (NO). Beyond the thin laminin-rich basement membrane in which ECs adhere, a hydrated threedimensional extracellular matrix (ECM), comprised of a fibrous structural meshwork of sequestered proteins and molecules, harbors a population of supporting mural cells including pericytes and vascular smooth muscle cells (vSMCs). Pericytes found in smaller diameter vessels including capillaries, venules, and arterioles, intimately associate with the endothelium, sharing the basement membrane and often forming discontinuous layers around the vessels. In larger arteries and veins, vSMCs regulate blood pressure through their synthetic phenotype, where elastic ECM is deposited, or their contractile phenotype, relaxing or constricting in response to the pulsatile nature of blood perfusion by the heart. In

addition to this immediate control of vessel diameter, smooth muscle cells (SMCs) provide dynamic regulation of ECM composition amenable to structural remodeling.3 1.2.2 Vasculogenesis and Angiogenesis Targeted gene deletion and image analysis allowed for the identification of vascular endothelial growth factor (VEDF) ligands (VEGF-A, VEGF-B)4 and their coinciding tyrosine kinase receptors VEGFR1 (Fms-like tyrosine like kinase 1/Flt-1) and VEGFR2 (kinase insert domain receptor protein/KDR, fetal liver kinase 1/Flk-1), expressed by ECs as potent regulators of vasculogenesis. During development, the endoderm serves as a reservoir of VEGF, coaxing splanchnic angioblast differentiation from the mesoderm.5 Vasculogenesis begins with aggregation of angioblast endothelial precursor cells that elongate into cord-like structures as they coalesce, eventually forming luminal endothelialized capillary-like plexus networks, which stay as nascent endothelial tubes prior to stabilization by stromal cells (Fig. 30.1A). VEGFs are structurally related to the platelet-derived growth factor (PDGF) family, and are biologically active in the form of heterodimers or homodimers. Four polypeptides make the family of PDGF ligands, which bind and signal through tyrosine kinase surface receptors PDGFRa and PDGFR-b expressed on vSMCs or pericytes. Late gestational death results when mice are null for PDGF-B and PDGFR-b. The phenotypes of null mice can be characterized by a lack of pericyte progenitors/vSMCs at vessel walls, capillary micro-aneurysms, and dramatic vessel dilation.6 VEGF alone induces vasculogenesis, but results in leaky, underdeveloped vasculature characterized by an immature endothelium. When mice are genetically ablated of VEGFR-2, very few ECs develop; however, in the absence of VEGFR-1 an overgrowth of blood vessels ensues, leading to early embryonic lethality due to an abnormal vascular plexus. These studies suggest that VEGFR-1 modulates VEGFR-2 signaling by acting as a decoy receptor, competitively sequestering VEGF.7 Blood vessel maturity continues with the expansion of the primitive vascular plexus into the somatic mesoderm via angiogenesis. This process is coaxed by a cocktail of growth factors (GFs) including VEGF, fibroblast growth factor (FGF), placental growth factor (PGF), insulin-like growth factor (IGF), and cytokine transforming growth factors b1 and b2 (TGF-b1, TGF-b2) that act to regulate anti- and proangiogenic ECM production (Fig. 30.1B). In the remodeling process, proteolytic enzymes, such as matrix metalloproteinases (MMPs), act to remodel the ECM to support sprout propagation during angiogenesis. In a specific spatiotemporal manner, ECs release PDGF-BB, which binds to heparin sulfate proteoglycans native to the ECM or PDGFR-b expressing mural cells, thereby recruiting these cells for vascular stabilization. Expression of sphingosine-1-

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1. INTRODUCTION

(A)

(B)

Vasculogenesis

Angiogenesis

Oxygen Exchange Capillaries

Blood Islands Angiogenic Sprouts Fusion

Smooth Muscle Cells Pericytes

Artery

Vein

Endothelial Cells

Flow

Flow

Primary Endothelial Plexus

FIGURE 30.1 Schematic representation of vasculogenesis and angiogenesis. (A) Vasculogenesis, the de novo formation of blood vessels, initiates with mesodermal differentiation of endothelial progenitor cells, which subsequently form blood islands that fuse and coalesce into a nascent primary capillary plexus. (B) This primitive plexus expands via angiogenic sprouting and remodeling events, and becomes stabilized by supportive smooth muscle cells and pericytes, mediated by an array of growth factors including but not limited to vascular endothelial growth factor, angiopoietin 1, and platelet-derived growth factor.

phosphate-1 (S1P1) by both ECs and mural cells results in signaling to provide additional mural cell recruitment and supplement vessel stabilization. Additional chemokines and proteins such as stromal-cell-derived factor-1 (SDF-1) and angiopoietins play a role in postnatal angiogenesis as well. Angiopoietin ligands (Ang-1, -2, -3, and -4) bind to tyrosine kinase receptor Tie2 present on ECs. A primitive vascular network can be formed in mouse embryos genetically modified to lack Tie-2 and Ang-1, but cannot undergo further vascular remodeling. This inhibition of vascular remodeling is consequential to the lack of interaction between Ang-1 secreting mural cells and the endothelium. Vessel circumference is dramatically enhanced and vessel number is increased with overexpression of Ang-1. Embryonic fatality due to defects in angiogenic remodeling potential results when ephrin B-2, expressed in primordial arterial vessels, and its Eph receptor tyrosine kinase EphB4, expressed in on primordial venous vessels, are knocked out.8 1.2.3 Oxygen Varying oxygen tension throughout embryonic development not only influences vasculogenesis and angiogenesis, but also stem cell fate.9 The hypoxia-inducible factor 1-alpha (HIF-1a) encodes a ubiquitous transcription factor that mediates cellular behavior in conditions where tissue oxygen tension is