Microtechnology for Stem Cell Culture

Stem Cells & Regenerative Medicine

Series EditorKursad [email protected]

For other titles published in this series, go tohttp://www.springer.com/series/7896

Krishnarao Appasani

Raghu K. AppasaniEditors

Stem Cells & Regenerative Medicine

From Molecular Embryology to Tissue Engineering

Foreword bySir John B. Gordon

EditorsKrishnarao AppasaniGeneExpressions Systems, IncWaltham, MA [email protected]

Raghu K. AppasaniStudent ScienceBoston, [email protected]

ISBN 978-1-60761-859-1 e-ISBN 978-1-60761-860-7DOI 10.1007/978-1-60761-860-7Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2010938600

© Springer Science+Business Media, LLC 2011All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

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v

Preface

Embryology is a branch of biology that has an immediate bearing on the problem of “life.” Life cannot be fully accounted for without an understanding of its dynamic nature, which expresses itself in the incessant production of new organisms in the process of ontogenetic development. Therefore, embryology is defined as the sci-ence of the development of an embryo from the fertilization of the ovum to the fetus stage. Teaching of embryology has long been an established feature at universities throughout the world, both for students in biology and students in medical sciences. During the twentieth century most of this science has been overshadowed by exper-imental-based genetics and cell biology, which have turned classical embryology into “developmental biology.” Several universities are now teaching developmental biology instead of embryology as a course in biology programs. Significant contri-butions made in the twenty-first century in the fields of molecular biology, biochem-istry, and genomics, integrated with embryology and developmental biology, provide an understanding of the molecular portrait of a “developmental cell.” This integrated approach to development is incorporated in the present book as “stem cell biology,” a new sister branch of embryology/developmental biology that emphasizes the study of self-renewal, differentiation, pluripotency, and nuclear programming, which are characteristics of stem cells. In a broad sense, stem cell biology is nothing more than an understanding of embryology and development together at the molecular level using engineering, imaging, and cell culture principles. With such a wide scope, this book can only be an introduction to stem cell biology.

Stem Cells and Regenerative Medicine: From Embryology to Tissue Engineering is mainly intended for readers in the biotechnology and molecular medicine fields. Although quite a number of books already exist covering stem cells, this book dif-fers, in that is the first text completely devoted to the basic developmental, cellular, and molecular biologic aspects of stem cells and their clinical applications in tissue engineering and regenerative medicine. We took serious consideration in choosing the chapters and sections in this book to maintain the theme of Molecular Embryology to Tissue Engineering.

This book focuses on the basic biology of embryonic and cancer cells and their key involvement in self-renewal, muscle repair, epigenetic processes, and therapeu-tic applications. Significant contributions, such as nuclear reprogramming–induced pluripotency, and stem cell culture techniques using novel biomaterials, are also

vi Preface

covered. This text consists of 36 chapters, grouped into six parts. Most of the chap-ters are written by experts in the field from academia and industry. The goal is to have this book serve as a reference for graduate students, post-docs, and teachers and as an explanatory analysis for executives and scientists in biotech and pharma-ceutical companies. Our hope is that this volume will provide a prologue to the field for both newcomers and those already active in the field.

The term “stem cell” appeared in the scientific literature as early as 1868 in the work of the eminent German biologist Ernst Haeckel. Haeckel, a supporter of Darwinian evolution, developed a number of phylogenetic trees to represent the evolution of organisms from common ancestors and called these trees Stammbaume (“stem trees”). In this context, he used the term Stammzelle (“stem cell”) to describe the ancestor unicellular organism from which he presumed all multicellular organ-isms evolved. He referred to fertilized egg as the source that gives rise to all cells of the organism. Later, in 1887, Theodor Boveri and Valentin Hacker identified the earliest germ cells in animal embryos. In 1892, Valentin Hacker described stem cells as the cells that later in development produce oocytes in the gonads. Thus, in these early studies, the term stem cell referred to what we call the “germline line-age,” “primordial germ cells,” and “germline stem cells.” In 1896, Edmund Wilson, an embryologist, reviewed the finding of these German scientists in his book The Cell in Development and Inheritance, which was published in English and became an inspirational work for a generation of embryologists and geneticists, especially in United States. Given his wide readership, Wilson is generally credited as having coined the term “stem cell.”

Nuclear programming is the process that instructs specialized adult cells to form early pluripotent stem cells. Pluripotent stem cells posses the capacity to become any type of mature cell in the body and therefore have great potential for experi-mental and therapeutic purposes. Using the concept of “cellular reprogramming,” Briggs and King in 1952 produced normal tadpoles by transplanting nuclei from blastula cells to enucleated eggs in the frog Rana pipens. However, transplanting nuclei from differentiated cells was achieved by John Gurdon in 1962 in the African clawed toad, Xenopus laevis, which is now known as the classic nuclear transfer experiment. It took more than another decade (1975) for Gurdon to succeed in producing healthy and sexually mature fertile frogs with functional muscle, beat-ing hearts, well-differentiated eyes, and all of the other organs. This experiment provided the first clear evidence that cell specialization does not involve irreversible inactivation in the genes required for development of an animal. This conceptual framework led to the start of the field of nuclear reprogramming, and Gurdon became known as the “father” of nuclear reprogramming (cloning). It took almost another 10 years to clone an adult sheep, Dolly (in 1996), by Kevin Campbell and Ian Wilmut of the Roslin Institute in Edinburgh, Scotland. This experiment dra-matically extended Gurdon’s concept from frogs to mammals. The Dolly-related work of somatic cell nuclear transfer was further extended to produce monkeys, cows, dogs, mice, and other animals. These remarkable contributions stimulated other researchers to think about using nuclear transfer to generate pluripotent human embryonic stem cells for cell replacement therapy.

viiPreface

The road to embryonic stem cells and beyond began in the 1960s with the work of Leroy Stevens from the Jackson Labs, Bar Harbor, Maine, who discovered embryonal carcinoma cells while studying testicular carcinomas. Later Stevens and colleagues demonstrated that these embryonal carcinoma cells are indeed pluripo-tent stem cells. In the mid-1970s, Gail Martin’s postdoctoral work with Martin Evans at the University of Cambridge led her to develop new in vitro clonal culture methods of embryoid cells. In the early 1980s, Martin, then at the University of California at San Francisco, and Martin Evans and Matthew Kaufman of the University of Cambridge independently isolated stem cells from mouse embryos and coined the term “embryonic stem cells.” It took almost 10 years for Jamie Thompson of the University of Wisconsin to culture monkey embryonic stem cells and subsequently human embryonic stem cells in 1999. Thompson’s work pro-pelled the activity of stem cell research and cell propagation technologies in general.

There are two routes to producing a living animal: (1) injection of a somatic cell nucleus into an enucleated egg (nuclear reprogramming) and (2) use of an embryo to produce embryonic stem cells. In a quite astonishing discovery, Kazutoshi Takahashi and Shinya Yamanaka of Kyoto University in Japan in 2006 for the first time turned adult mouse skin fibroblast cells into pluripotent cells. This break-through of inducing fribroblasts was achieved by stable transfection of only four transcription factors (Oct4, Sox2, Klf4, and c-Myc), and these are now referred to as induced pluripotent stem (iPS) cells. The discovery of iPS cells turned the field of nuclear reprogramming upside down. This work was extended and further con-firmed by several groups that generated iPS cells from individuals with various neurodegenerative diseases, raising the hope of cell replacement therapy and mak-ing personalized medicine a reality. A section of this book with six chapters details the concepts behind nuclear reprogramming and induced pluripotent stem cells.

In 1868, Ernst Neumann suggested that hematopoiesis occurs in bone marrow. He used the term “stem cell” to refer to the common precursor of the blood system in 1912. The debate about the existence of a common hematopoietic stem cell con-tinued for several decades until definitive evidence was provided in 1961 by two Canadian scientists, James Till and Ernest McCulloch. Blood and the system that forms it, known as the hematopoietic system, consists of many cell types with spe-cialized functions (some of these include red blood cells, platelets, granulocytes, macrophages, B and T lymphocytes, etc). Generally, the hematopoietic system is destroyed by radiation and chemotherapeutic agents that kill dividing cancer cells. In order to quantitatively assess the radiation sensitivity of normal bone marrow cells, a colony-forming unit assay was developed by Till and McCulloch, who coined the term “pluripotent hematopoietic stem cells” (HSCs). Today, we know that the best locations for HSCs are bone marrow, umbilical cord blood, and embry-onic stem cells. In 1959, for the first time, Edward Donnall Thomas of the University of Washington used HSCs for treating leukemia and lymphomas through bone marrow transplantation. The efficient expansion of HSCs in culture remains one of the major research themes of stem cell biology. Combined applications of genomics, proteomics, and gene therapy approaches will further help to widen the

viii Preface

horizon for clinical applications. According to Irving Weissman of Stanford University Medical School, the progeny produced from hematopoietic stem cells exhibits properties that include self-renewal, differentiation, migration, and apopto-sis. A few chapters in the third part of this book highlight of the use of HSCs for bone marrow cell therapy, heart transplantation, and cell replacement therapy for neurologic disorders.

The term “tissue engineering” was first used by Eugene Bell of MIT in 1984, and later was also used extensively by Wolter and Meyer in 1984. Tissue engineer-ing combines cells, engineering, and materials methods with suitable biochemical and physiochemical factors to improve or replace biologic functions. In other words, it deals with the repair or replacement of portions of or whole tissues such as bone, blood vessels, bladder, skin, and artificial organs. According to Robert Langer and Joseph Vacanti, it “applies the principles of engineering and life sci-ences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.” Powerful developments in the multidis-ciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices (“scaffolds”), cells, and biologically active mol-ecules. A section of this book with five chapters highlights recent developments in biomaterials, three-dimensional culture systems, lab-on-a-chip concepts, and microtechnologies used in attempts to understand stem cell biology.

Regenerative medicine is a new branch of medicine that attempts to change the course of chronic disease, in many instances regenerating failing organ systems lost due to age, disease, damage, or congenital defects. The term “regenerative medi-cine” was first referred to in 1992 by Leland Kaiser and then popularly used by William Haselstine of Human Genome Sciences. The term regenerative medicine is often used synonymously with tissue engineering, although those involved in regenerative medicine place more emphasis on the use of stem cells to treat diseases using cell therapies or transplantation methods. This field holds the promise of regenerating damaged tissues and organs in the body by stimulating previously irreparable organs to heal themselves. Regenerative medicine also empowers scien-tists to grow tissues and organs in the laboratory and safely implant them when the body cannot heal itself. A section in this book is entirely devoted to describing the use of stem cells in muscle repair and treating cardiac and urologic diseases.

Gurdon has spent much of his career deciphering the molecules and mechanisms that an egg uses to “rejuvenate” nuclei. We know a lot about nuclear transfer, but the question remains of how to regulate and control the most efficient way to repro-gram the nucleus. Although both Gurdon’s (nuclear reprogramming) and Yamanaka’s (iPS) technologies can generate living animals, we do not know the molecular mechanisms underlying these two strategies. The potential of iPS cell technology in biology and medicine is enormous; however, it is still in its infancy, and there are many challenges to overcome before various applications can be used successfully. We still need to understand the components of oocytes or eggs used to depress

ixPreface

somatic gene expression and discover the direct cell-type switches by over-expressed transcription factors. It is also important to identify the basis for the stability of the differentiated state of cells, which will help us to understand how egg-reprogramming factors operate. Finally, mapping of the “embryome” is a necessity, and is looks as though it will become available soon, which will help us to understand the intricacies and epigenetic imprints of embryos.

Many people have contributed to making our involvement in this project possi-ble. We thank our teachers for their excellent teaching, guidance, and mentorship, which helped us to bring this educational enterprise. We are extremely thankful to all of the contributors to this book, without whose commitment this book would not have been possible. Many people have had a hand in the preparation of this book. Each chapter has been passed back and forth between the authors for criticism and revision; hence each chapter represents a joint composition. We thank our readers, who have made our hours putting together this volume worth it. We are indebted to the staff of Springer Science + Business Media (Humana Press), and in particular Mindy Okura-Marszycki and Vindra Dass for their generosity in giving time and effort throughout the editing of this book. This book is dedicated to memory of my late friend, Prof. Xiangzhong (Jerry) Yang of the University of Connecticut, Storrs, who was the first to clone a cow (Amy, the calf) and a strong proponent of stem cell research here in US and China. This book is also dedicated to memory of my late friend, Prof. C.M. Habibullah of the Deccan College of Medical Sciences, India, who was a strong supporter of stem cell research in India. We especially thank Prof. John Gurdon, a researcher of great distinction, for his kindness and support in writ-ing the Foreword to this book. Last, but not least, we thank Shyamala Appasani for her understanding and cooperation during the development of this book.

This book is the first joint project of father and son. A portion of the royalties will be contributed to the Dr. Appasani Foundation, a nonprofit organization devoted to bringing social change through the education of youth in developing nations.

Waltham, MA Krishnarao AppasaniBoston, MA Raghu K. Appasani

xi

I am very grateful to Krishnarao and Raghu Appasani for preparing this volume on the massively expanding fields of stem cells and regenerative medicine and for inviting me to offer a few introductory comments.

The prospect of being able to rejuvenate cell types of almost any kind from easily accessible cells of an adult makes it realistic to envisage cell replacement. Most important, this possibility would provide a patient with new cells of their own genetic type, thereby avoiding the necessity of immunosuppression, as would be required for any cells derived from any other individual, except an identical twin. The great interest in this field has been enormously stimulated by the recent discov-ery of induced pluripotency stem cells but has depended on several much earlier discoveries, most notably on that of embryonic stem cells.

There has been something of a tidal wave of interest in stem cells and regenera-tive medicine as researchers all over the world become active in it. Almost every day there are new papers published on various aspects of pluripotency, and it would be hard, even for those intimately involved in experimental work of this kind, to keep up to date with every advance. It is therefore very valuable to have a volume of 36 contributions summarizing the current status of progress in the various fields that contribute to regenerative medicine. Krishnarao and Raghu Appasani have assembled the contributing chapters into six main areas, ranging from stem cell biology through tissue engineering and therapeutic possibilities. The component chapters will be valuable not only to those who are experimentally active in an aspect of regenerative medicine, but also to those concerned with potential thera-peutic applications. This volume also contains a valuable historical perspective by the Appasanis explaining key events in the development of this field over the last 150 years.

Although there is great enthusiasm for the eventual therapeutic value of work in this field for human health, scientists are very cautious about the time scale of human benefit. Bone marrow cells have been of great clinical value for a number of years. However, there is a long way to go before the brain and heart, to take two examples, can benefit from laboratory-created stem cells. It is indeed remarkable that beating heart cells or dopamine-secreting brain cells can now be derived from human skin and can be proliferated in the laboratory. However, substantial advances will be needed for it to be possible to integrate these laboratory-grown cells into

Foreword

xii Foreword

organs or tissues of living individuals and to arrange for these new cells to continue their newly acquired activity once transplanted into a patient. It is unlikely that a complex organ, often consisting of many different cell types, will soon be able to be constructed in the laboratory. The number of cells required for human therapy is also of concern, since a human heart or brain consists of more than 1 million mil-lion (1012) cells. On the other hand, some cells make their contribution by secret-ing products or by providing critical neural connections, and even 10,000 cells of one kind could be valuable, as, for example, in the retina of the eye. I believe there is a cautious optimism in this field. It is generally true that once scientists find out how to achieve a desired result to a small extent, it is only a question of time before this advance is made to work enormously more efficiently.

My last comment concerns the reliability and safety of stem cells in regenerative medicine. There is understandable concern that any stem cells used for therapeutic purposes should be completely free of potential cancer cells or potentially harmful viruses. However, I submit that a situation might be reached where, even though one patient may suffer, more than 99.9% of other patients may derive enormous benefit. I hope that the fear of an occasional harmful replacement cell will not dis-courage continuing attempts to derive replacement cells that could be of enormous therapeutic value for a great number of other patients.

Cambridge, UK John. B. Gurdon

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Part I Stem Cell Biology

Introduction to Stem Cells and Regenerative Medicine .............................. 3Krishnarao Appasani and Raghu K. Appasani

Embryonic Stem Cells: Discovery, Development, and Current Trends ..... 19Elias Theodorou and Michael Snyder

Bmi1 in Self-Renewal and Homeostasis of Pancreas ................................... 45Eugenio Sangiorgi and Mario Capecchi

Cancer Stem Cells in Solid Tumors ............................................................... 59Elodie du Potet, Lauren Cameron, Nagy A. Habib, and Natasa Levicar

Adipose-Derived Stem Cells and Skeletal Muscle Repair ........................... 77Claude A. Dechesne, Didier F. Pisani, Sébastien Goudenege, and Christian Dani

Regeneration of Sensory Cells of Adult Mammalian Inner Ear ................ 89Dongguang Wei and Ebenezer N. Yamoah

Stem Cells and Their Use in Skeletal Tissue Repair .................................... 103Laura Baumgartner, Vuk Savkovic, Susanne Trettner, Colette Martin, and Nicole I. zur Nieden

Part II Epigenetic and microRNA Regulation in Stem Cells

Epigenetic Identity in Cancer Stem Cells ..................................................... 127Maria Ouzounova, Hector Hernandez-Vargas, and Zdenko Herceg

Function of MicroRNA-145 in Human Embryonic Stem Cell Pluripotency ................................................................................... 141Na Xu, and Kenneth S. Kosik

Contents

xiv Contents

Mesenchymal Stem Cells for Liver Regeneration ........................................ 155Tom K. Kuo, Yueh-Hsin Ping, and Oscar K. Lee

The Role of Time-Lapse Microscopy in Stem Cell Research and Therapy .................................................................................................... 181Kevin E. Loewke and Renee A. Reijo Pera

Part III Stem Cells for Therapeutic Applications

Therapeutic Applications of Mesenchymal Stem/Multipotent Stromal Cells ................................................................................................... 195Weian Zhao, Debanjan Sarkar, James Ankrum, Sean Hall, Weili Loh, Wei Suong Teo, and Jeffrey M. Karp

Gastrointestinal Stem Cells ............................................................................ 219N. Parveen, Aleem A. Khan, M. Aejaz Habeeb, and C. M. Habibullah

Lung Epithelial Stem Cells ............................................................................. 227Magnus Karl Magnusson, Olafur Baldursson, and Thorarinn Gudjonsson

Placental-Derived Stem Cells: Potential Clinical Applications .................. 243Sean Murphy, Euan Wallace, and Graham Jenkin

Bone Marrow Cell Therapy for Acute Myocardial Infarction: A Clinical Trial Review ................................................................................... 265Franca S. Angeli and Yerem Yeghiazarians

Stem Cell Transplantation to the Heart ........................................................ 279Michael J. Mann

Adult Neural Progenitor Cells and Cell Replacement Therapy for Huntington Disease ................................................................... 299Bronwen Connor

Migration of Transplanted Neural Stem Cells in Experimental Models of Neurodegenerative Diseases ......................................................... 315Nathaniel W. Hartman, Laura B. Grabel, and Janice R. Naegele

Prospects for Neural Stem Cell Therapy of Alzheimer Disease ................. 337Thorsten Gorba, Sarah Harper, and P. Joseph Mee

Part IV Nuclear Reprogramming and Induced Pluripotent Stem Cells

Nuclear Transfer Embryonic Stem Cells as a New Tool for Basic Biology ..................................................................................... 351Sayaka Wakayama, Eiji Mizutani, and Teruhiko Wakayama

xvContents

Pluripotent Stem Cells in Reproductive Medicine: Formation of the Human Germ Line in Vitro............................................... 371Sofia Gkountela, Anne Lindgren, and Amander T. Clark

Prospects for Induced Pluripotent Stem Cell Therapy for Diabetes .......... 387Robert J. Drummond, James A. Ross, and P. Joseph Mee

Keratinocyte-Induced Pluripotent Stem Cells: From Hair to Where? ..................................................................................... 399Trond Aasen and Juan Carlos Izpisúa Belmonte

Generation and Characterization of Induced Pluripotent Stem Cells from Pig......................................................................................... 413Toshihiko Ezashi, Bhanu Prakash V. L. Telugu, and R. Michael Roberts

Induced Pluripotent Stem Cells: On the Road Toward Clinical Applications ..................................................................................................... 427Fanyi Zeng and Qi Zhou

Direct Reprogramming of Human Neural Stem Cells by the Single Transcription Factor OCT4 ........................................................................... 439Jeong Beom Kim, Holm Zaehres, and Hans R. Schöler

Part V Tissue Engineering

Stem Cells and Biomaterials: The Tissue Engineering Approach ............. 451Stefania Antonini, Angelo Vescovi, and Fabrizio Gelain

Microtechnology for Stem Cell Culture ........................................................ 465Elena Serena, Elisa Cimetta, Camilla Luni, and Nicola Elvassore

Using Lab-on-a-Chip Technologies for Stem Cell Biology .......................... 483Kshitiz Gupta, Deok-Ho Kim, David Ellison, Christopher Smith, and Andre Levchenko

The Development of Small Molecules and Growth Supplements to Control the Differentiation of Stem Cells and the Formation of Neural Tissues ............................................................ 499Victoria B. Christie, Daniel J. Maltman, Andy Whiting, Todd B. Marder, and Stefan A. Przyborski

Long-Term Propagation of Neural Stem Cells: Focus on Three-Dimensional Culture Systems and Mitogenic Factors ................ 515Rikke K. Andersen, Jens Zimmer, and Morten Meyer

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Part VI Regenerative Medicine

Stem Cells and Regenerative Medicine in Urology ...................................... 541Anthony Atala

Muscle-Derived Stem Cells: A Model for Stem Cell Therapy in Regenerative Medicine ............................................................................... 565Burhan Gharaibeh, Lauren Drowley, and Johnny Huard

Regenerative Strategies for Cardiac Disease ................................................ 579Xiaojing Huang, James Oh, and Sean M. Wu

Collecting, Processing, Banking, and Using Cord Blood Stem Cells for Regenerative Medicine .......................................................... 595David T. Harris

Index ................................................................................................................. 615

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Contributors

Trond Aasen, PhD Institut de Recerca Hospital Vall d’Hebron, 08035 Barcelona, Spain and Pathology Department Fundació Institut de Recerca Hospital Vall d’ Hebron, 08035 Barcelona, Spain

Rikke K. Andersen Department of Anatomy and Neurobiology Institute of Medical Biology, University of Southern Denmark, Odense C, DK-5000, Denmark

Franca S. Angeli, MDDivision of Cardiology, University of California at San Francisco Medical Center, 505 Parnassus Avenue, San Francisco, CA 94143-0124, USA

James Ankrum Harvard-MIT Division of Health Sciences and Technology, Department of Medicine Brigham and Women’s Hospital, Harvard Medical School, and Harvard Stem Cell Institute, 65 Landsdowne Street PRB325, Cambridge, MA 02139, USA

Stefania Antonini Center for Nanomedicine and Tissue Engineering and Department of Biotechnology and Biosciences, University of Milan-Bicocca, A.O. Ospedale Niguarda Ca’ Granda, Milan 20126, Italy

Krishnarao Appasani, PhD, MBA Gene Expression Systems, Inc., P.O. Box 540170, Waltham, MA 02454, USA

Anthony Atala, MD Wake Forest Institute for Regenerative Medicine and Department of Urology Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA

xviii Contributors

Olafur BaldurssonDepartment of Pulmonary Medicine, Landspitali University Hospital, Reykjavik, Iceland

Laura BaumgartnerFraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, Leipzig, 04103, Germany

Juan Carlos Izpisúa Belmonte, PhDCenter of Regenerative Medicine in Barcelona, Dr. Aiguader 88, Barcelona, 08003, Spain and Gene Expression Laboratory, Salk Institute for Biological Stududies, 10010 North Torrey Pines Rd., La Jolla, CA 92037, USA

Lauren Cameron Department of Surgery, Imperial College of London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK

Mario R. Capecchi, PhDDistinguished Professor of Human Genetics & Biology Investigator, Howard Hughes Medical Institute, University of Utah School of Medicine, 15 North 2030 East, Room 5440, Salt Lake City, UT 84112, USA

Victoria B. ChristieSchool of Biological and Biomedical Sciences, Durham University and Reinnervate Limited, Durham, DH1 3LE, UK

Elisa CimettaDepartment of Chemical Engineering, University of Padua, Via Marzolo 9, Padova, 35131, Italy

Amander T. Clark, PhDDepartment of Molecular Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA

Bronwen Connor, PhDDepartment of Pharmacology and Clinical Pharmacology, Centre for Brain Research, FMHS, University of Auckland, Private Bag 92019, Auckland, New Zealand

Christian Dani, PhDInstitute of Developmental Biology and Cancer, University of Nice Sophia-Antipolis, CNRS, UMR 6543, Nice, France

xixContributors

Claude A. Dechesne Institute of Developmental Biology and Cancer, University of Nice Sophia-Antipolis, CNRS, UMR 6543, Nice, France

Lauren Drowley, PhD Stem Cell Research Center, University of Pittsburgh, Bridgeside Point 2, Suite 206, 450 Technology Drive, Pittsburgh, PA 15219, USA

Robert J. DrummondTissue Injury and Repair Group, School of Clinical Sciences and Community Health, University of Edinburgh Room FU501, Chancellors Building, 49 Little France Crescent, Edinburgh, Scotland, UK

Elodie du PotetDepartment of Surgery , Imperial College of London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK

David EllisonDepartment of Biomedical Engineering, Johns Hopkins University Clark Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA

Nicola Elvassore, PhDDepartment of Chemical Engineering, University of Padua and Venetian Institute of Molecular Medicine, Via Marzolo 9, 35131, Padova, Italy

Toshihiko Ezashi, PhDDivision of Animal Sciences, University of Missouri-Columbia, Christopher S. Bond Life Sciences Center, 1201 E. Rollins Street, Columbia, MO 65211-7310, USA

Fabrizio Gelain, PhDCenter for Nanomedicine and Tissue Engineering, and Department of Biotechnology and Biosciences, University of Milan-Bicocca, A.O. Ospedale Niguarda Ca’ Granda, 20126 Milan, Italy

Burhan Gharaibeh, PhDStem Cell Research Center, University of Pittsburgh Bridgeside Point 2, Suite 206, 450 Technology Drive, Pittsburgh, PA 15219, USA

Sofia Gkountela, PhDDepartment of Molecular Cell and Developmental Biology, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute and Jonsson Comprehensive Cancer Center , College of Letters and Science, University of California Los Angeles, Los Angeles, CA 90095, USA

xx Contributors

Thorsten GorbaStem Cell Sciences UK Ltd., Minerva Building 250, Babraham Research Campus, Cambridge, CB22 3AT, UK

Sébastien GoudenegeInstitute of Developmental Biology and Cancer, University of Nice Sophia-Antipolis, CNRS, UMR6543, Nice, France

Laura B. Grabel, PhD, Lauren B. Dachs Professor of Science in Society, Department of Biology, Hall-Atwater Labs, Wesleyan University, Middletown, CT 06459, USA

Thorarinn Gudjonsson, PhDStem Cell Research Unit, Dapartment of Anatomy, Faculty of Medicine, University of Iceland and Department of Laboratory Hematology, Landspitali – University Hospital, Reykjavik, Iceland

Kshitiz GuptaDepartment of Biomedical Engineering, Johns Hopkins University, Clark Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA

John Gurdon, DPhil, FRSEmeritus Professor, Gurdon Institute, University of Cambridge, Cambridge, UK

M. Aejaz HabeebCentre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, A.P., India

Nagy A. Habib, MBCh, PhD, FRCSDepartment of Surgery, Imperial College of London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK

C. M. Habibullah, MD (deceased)Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, A.P. India

Sean HallDivision of Newborn Medicine and Pulmonary and Critical Care, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA

Sarah HarperStem Cell Sciences UK Ltd., Minerva Building 250, Babraham Research Campus, Cambridge, CB22 3AT, UK

xxiContributors

David T. Harris, PhDDepartment of Immunobiology Organization, University of Arizona, 1656 E. Mabel, MRB 221, Tucson, AZ 85724, USA

Nathaniel W. HartmanDepartment of Biology Wesleyan University, Middletown, CT 06459, USA

Zdenko Herceg, PhDEpigenetics Group Leader, International Agency for Research on Cancer, 150 cours Albert-Thomas Lyon, Cedex 08, 69372, France

Xiaojing HuangDivision of Cardiology, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School CPZN 3224, Simches Building, 185 Cambridge Street, Boston, MA 02114 USA

Johnny Huard, PhDDepartment of Orthopaedic Surgery and Molecular Genetics and Biochemistry, and Stem Cell Research Center, University of Pittsburgh, Pittsburgh, PA 15219, USA

Graham Jenkin, PhDThe Ritchie Center, Monash Institute of Medical Research, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC 3168, Australia

Jeffrey M. Karp, MD, PhDDepartment of Medicine, Harvard-MIT Division of Health Sciences and Technology, Brigham and Women’s Hospital, Harvard Medical School and Harvard Stem Cell Institute, 65 Landsdowne Street, Cambridge, MA 02139, USA

Aleem A. KhanCentre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, A.P. India

Deok-Ho Kim, PhDDepartment of Biomedical Engineering, Johns Hopkins University, 207 Clark Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA

Jeong Beom KimDepartment of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, NRW, Germany

xxii Contributors

Kenneth S. Kosik, MDDepartment of Molecular, Cellular and Developmental Biology, Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106, USA

Tom K. KuoStem Cell Research Center, National Yang-Ming University, Taipei, Taiwan

Oscar K. Lee, MD, PhDDepartment of Medical Research and Education, Taipei Veterans General Hospital, Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan

Andre Levchenko, PhDDepartment of Biomedical Engineering, Johns Hopkins University, 208 Clark Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA

Natasa LevicarDepartment of Surgery, Imperial College of London, Hammersmith Campus, Du Cane Road, London, W12 ONN, UK

Anne Lindgren, PhDDepartment of Molecular Cell and Developmental Biology, College of Letters and Science, University of California at Los Angeles, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Molecular Biology Institute and Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095, USA

Kevin E. LoewkeDepartment of Mechanical Engineering, Stanford University, Auxogyn, Inc., 1490 O’Brien Drive, Menlo Park, CA 94025, USA

Weili LohHarvard-MIT Division of Heath Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School and Harvard Stem Cell Institute, 65 Landsdowne Street PRB325, Cambridge, MA 02139, USA

Camilla LuniDepartment of Chemical Engineering, University of Padua, Via Marzolo 9, Padova, 35131, Italy

Magnus Karl MagnussonStem Cell Research Unit, Biomedical Center, University of Iceland, Department of Laboratory Hematology, Landspitali University Hospital, Reykjavik, Iceland

xxiiiContributors

Daniel J. MaltmanSchool of Biological and Biomedical Sciences, Durham University and Reinnervate Limited, Durham, DH1 3LE, UK

Michael J. Mann, MDDivision of Cardiothoracic Surgery, Director, Cardiothoracic Translational Research Laboratory, University of California at Director, San Francisco, CA, USA

Todd B. MarderDepartment of Chemistry, Durham University, Durham, DH1 3LE, UK

Colette MartinDepartment of Cell Biology and Neuroscience and Stem Cell Center, University of California Riverside, Riverside, CA 92521, USA

P. Joseph Mee, PhD Stem Cell Sciences PLC, Minerva Building 250, Babraham Research Campus, Cambridge, CB22 3AT, UK

Morten Meyer, PhDDepartment of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, J.B. Winslows Vej 21, DK-5000 Odense C, Denmark

Sean Murphy, PhDFaculty of Medicine, Nursing Health Sciences, Monash Immunology and Stem Cell Laboratories, Monash University, Wellington Road, Clayton, Victoria 3800, Australia

Eiji MizutaniLaboratory for Genomic Reprogramming, RIKEN Center for Developmental Biology 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan

Janice R. Naegele, PhDDepartment of Biology, Hall-Atwater Labs, Wesleyan University, Middletown, CT 06459, USA

James OhDivision of Cardiology, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School CPZN 3224, Simches Building, 185 Cambridge Street, Boston, MA 02114, USA

xxiv Contributors

Maria OuzounovaEpigenetics Group, International Agency for Research on Cancer, 150 cours Albert-Thomas, Lyon cedex 08, 69372 France

Yueh-Hsin Ping, PhDStem Cell Research Center and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan

Didier F. PisaniInstitute of Developmental Biology and Cancer, University of Nice Sophia-Antipolis, CNRS, UMR6543, Nice, France

N. ParveenCentre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad, 500058, A.P., India

Stefan A. Przyborski, PhDSchool of Biological and Biomedical Sciences, Durham University, Durham, DH1 3LE, UK and Reinnervate Limited, Durham, DH1 3HP, UK

Renee A. Reijo Pera, PhDProfessor, Center for Human Embryonic Stem Cell Research and Education, Department of Obstetrics and Gynecology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, CA 94304-5542, USA

Michael R. Roberts, PhDEmeritus Professor, Department of Animal Sciences, 240h, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia, 1201 E. Rollins Street, Columbia, MO 65211-7310, USA

James A. RossTissue Injury and Repair Group, School of Clinical Sciences and Community Health, University of Edinburgh, Room FU501, Chancellors Building, 49 Little France Crescent, Edinburgh, Scotland, UK

Eugenio Sangiorgi, MDIstituto di Genetica Medica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168, Roma, Italy

Debanjan SarkarHarvard-MIT Division of Heath Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School and Harvard Stem Cell Institute, 65 Landsdowne Street PRB325, Cambridge, MA 02139, USA

xxvContributors

Vuk SavkovicFraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, Leipzig, 04103, Germany

Hans R. Schöler, PhD Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, NRW, Germany

Elena SerenaDepartment of Chemical Engineering, University of Padua and Venetian Institute of Molecular Medicine, Via Marzolo 9, Padova, 35131, Italy

Christopher SmithDepartment of Biomedical Engineering, Johns Hopkins University, Clark Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA

Michael Snyder, PhDDepartment of Genetics, Stanford University School of Medicine, MC: 5120, 300 Pasteur Dr., M-344, Stanford, CA 94305-2200, USA

Bhanu Prakash Telugu, PhDDepartment of Animal Sciences, 240h, Christopher S. Bond Life Sciences Center, University of Missouri-Columbia, 1201 E. Rollins Street, Columbia, MO 65211-7310, USA

Wei Suong TeoDepartment of Medicine, Harvard-MIT Division of Health Sciences and Technology, Brigham and Women’s Hospital, Harvard Medical School and Harvard Stem Cell Institute, 65 Landsdowne Street PRB325, Cambridge, MA 02139, USA

Elias TheodorouMolecular, Cellular and Developmental Biology Department, Yale University, P.O. Box 208103, New Haven, CT 06511, USA

Susanne TrettnerFraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, Leipzig 04103, Germany

Hector Hernandez VargasEpigenetics Group Leader, International Agency for Research on Cancer, 150 cours Albert-Thomas, Lyon cedex 08, 69372, France

xxvi Contributors

Angelo VescoviCenter for Nanomedicine and Tissue Engineering and Department of Biotechnology and Biosciences, University of Milan-Bicocca A.O. Ospedale Niguarda Ca’ Granda, Milan 20126, Italy

Sayaka WakayamaLaboratory for Genomic Reprogramming, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan

Teruhiko Wakayama, PhDLaboratory for Genomic Reprogramming, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan

Euan Wallace, MDFaculty of Medicine, Nursing and Health Sciences, Department of Obstetrics and Gynaecology, Monash Institute of Medical Research, Monash University, Wellington Road, Clayton, Victoria 3800, Australia

Dongguang Wei, PhDDepartment of Anesthesiology and Pain Medicine, Center for Neuroscience, University of California at Davis, 1544 Newton Court, Davis, CA 95618, USA

Andy WhitingDepartment of Chemistry, Durham University, Durham, DH1 3LE, UK

Sean M. Wu, MD, PhDCardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Harvard Stem Cell Institute, Simches Building, CPZN 3224 185 Cambridge Street, Boston, MA 02114, USA

Na Xu, PhDDepartment of Molecular, Cellular and Developmental Biology, Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106, USA

Ebenezer N. Yamoah, PhDDepartment of Anesthesiology and Pain Medicine, Center for Neuroscience, Program in Communication Science, University of California at Davis, 1544 Newton Court, Davis, CA 95618, USA

Yerem Yeghiazarians, MDDivision of Cardiology, Cardiac Stem Cell Translational Development Program, University of California at San Francisco Medical Center, 505 Parnassus Avenue, San Francisco, CA 94143-0124, USA

xxviiContributors

Holm ZaehresDepartment of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, NRW, Germany

Fanyi Zeng, MD, PhDShanghai Institute of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai, 200040, P.R. China

Weian Zhao, PhDDepartment of Medicine, Harvard-MIT Division of Heath Sciences and Technology, Brigham and Women’s Hospital, Harvard Medical School and Harvard Stem Cell Institute, 65 Landsdowne Street, Cambridge, MA 02139, USA

Qi ZhouShanghai Institute of Medical Genetics, Shanghai Stem Cell Institute, Shanghai Jiao Tong University School of Medicine, 280 S. ChongQing Road, Bldg 5, Room 707, Shanghai 200025, China

Jens ZimmerDepartment of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense C, DK-5000, Denmark

Nicole I. zur Nieden, PhDDepartment of Cell Biology, and Neuroscience and Stem Cell Center, University of California Riverside, 1113 Biological Sciences Building, Riverside, CA 92521, USA and Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany

1

1 Micro-technology for stem cell culture

Elena Serena1,2

, Elisa Cimetta1, Camilla Luni

1, Nicola Elvassore

1,2*

(1) Department of Chemical Engineering, University of Padua, Via Marzolo 9, I-35131 Padova,

Italy

(2) Venetian Institute of Molecular Medicine, Via Orus 2, I-35131 Padova, Italy

(*) correspondence to: Nicola Elvassore, Ph.D.

Department of Chemical Engineering, University of Padua, Via Marzolo 9, I-35131 Padova, Italy

Phone: +390498275469; Fax: +390498275461

Email: [email protected]

Chapter in Book: Stem Cells & Regenerative Medicine

Editor: Dr. Krishna Appasani

2

Abstract

The advances in stem cell research of the last decades have been also sustained by the progresses

made in the development of novel technologies aimed at being coupled with biological systems. In

parallel, mimicking in vitro the stem cell niche is becoming crucial for both basic stem cell research

and for the development of innovative therapies based on stem cells.

Innovative microscale-technologies can contribute to quantitative understanding of how phenomena

at the microscale can determine the stem cell behavior, increasing the fidelity in controlling the

culture conditions and the throughput of the data, while reducing times and costs. In particular,

micro-technologies must be designed and developed in order to capture the complexity of cell-

substrate, cell-cell and cell-soluble environment interactions considering both the characteristic time

and length scales of biological phenomena. While acknowledging the advantages of applying these

technologies to stem cell culture, this chapter is focused on the relevant issues related to the control

and mimicking of the microenvironmental cues of the stem cell niche such as the substrate

properties, the cell topology, the soluble environment and the electro-physiology.

Key words (4-5): stem cells, niche, micro-technology, patterning, microfluidics.

3

1.1 Introduction

The nowadays scientific community is becoming more and more aware of the importance of the

concept of stem cell niche and how to accurately control its temporal evolution in vitro1. Innovative

technology can enhance our capability to gain new insight into stem cell research and, in particular,

to contribute to quantitative understanding of how phenomena at the microscale can determine the

stem cell behavior at higher level of complexity2. To elucidate the potentiality of using novel micro-

technology in stem cell research, fundamental principles of biological and cellular stem cell

functions have also been analyzed with reference to their characteristic time and length scales.

Cells in their native tissues reside in an extremely complex environment constituted of other cell

types, extracellular matrix proteins (ECM), and structural templates guiding their orientation and

maintaining the three-dimensional (3D) architecture. In addition, it must also be considered the

intricate network of short and long range communications between single cells and/or tissues that

occurs through a vast cascade of signaling pathways and regulates their behavior3-7

. Such

complexity of cellular functions and their response to internal events and environmental

perturbations or stimuli, develop in both time and space with characteristic scales spanning over

orders of magnitude8,9

.

Figure 1A shows a simple schematization of the components of the in vitro cell microenvironment

constituting the so-called niche which is mainly given by: i) substrate, ii) soluble environment and

iii) electrical properties. The cells adhere to a specific substrate, which is defined by a set of

chemico-physical properties, such as chemical composition, stiffness or surface topology. The cells

are immersed in a soluble environment composed by a complex ensemble of factors that can be

either produced by the cells themselves, endogenous factors, or by other cells in the surroundings,

exogenous factors. In particular, the temporal evolution of composition of both endogenous and

exogenous factors within the microenvironment surrounding the cell is strictly related to cell local

density and cell topology landscape. Finally, another important component of the stem cell niche is

4

given by the electro-physiological stimuli which are particularly relevant for electro-excitable cells.

Figure 1B shows approximate ranges for both space and time scales of some relevant biological

phenomena. In this sight, it is evident how biological phenomena cannot be simply considered as

the sum of "building blocks" whose final structure recapitulates the function or behavior of a cell or

tissue. Each "level" is tightly interconnected with all the others and phenomena occurring on

smaller scales influence also the large-scale ones. For example, in the case of electrically excitable

cells or tissues, intracellular events triggered by small ion dynamics can affect the whole single-cell

or even cell cluster behavior in terms of contractile properties and/or gene transcription10

.

Conventional stem cell cultures are typically performed in Petri dishes, multiwell plates or T-flasks

in a volume ranges from some microliters to several milliliters. While they are simple and widely-

used devices, the culture conditions in these systems show some intrinsic limitations in resembling

both the temporal evolution and the constitutive characteristics of the stem cell niche. First of all,

medium is stagnant over the cells, and the transport of metabolites, gases and signaling molecules

from the bulk to the cell surface is slowly driven only by diffusion, producing a time-dependent

environment. Thus, the culture system inherent properties can be accurately defined only at the

beginning whereas its temporal evolution pattern is hardly controllable and, often, unpredictable.

Moreover, the batch-wise operation associated with the periodic medium change does not allow

neither stationary culture conditions nor the generation of precise temporal patterns of biochemical

stimulation.

On the other hand, the ability of controlling cellular local environment surrounding it is not

straightforward in conventional systems. For instance, the stiffness of cell culture surface (typically

made of glass or polystyrene) which was found extremely relevant in stem cell defferentation11

,

cannot be properly tuned, even when surface functionalization technique is used.

Cell density and cell topology landscape are key factors in stem cell differentiation and

proliferation, but they are hardly controlled in a Petri dish12

. In fact, after manual seeding, cells are

spread according to a random two-dimensional spatial distribution, which is scarcely repeatable and

5

produces uncontrollable cell-cell interactions; this aspect is particularly relevant when cell co-

culture is required to have a given cell-cell pattern. Finally, electrical stimulation is totally neglected

in conventional cultures.

These observations lead to the impelling necessity to develop novel technologies for studying stem

cell biology under controllable conditions, able to capture the complexity of the native environment

and, at the same time, able to meet the demand for increased throughput13

. To this aim,

miniaturization of the culture systems is an important step towards accurate control of the cultured

stem cells and tissues14-16

. Microscale technologies are of paramount importance in successfully

controlling stem cell cultures, allowing for the precise definition of the local microenvironment

surrounding the cells, and responding to the above-mentioned specific requirements for accurately

mimicking the natural niche encountered in vivo. In addition, a major advantage introduced by

microscale technologies resides in the feasibility of producing parallel high-throughput systems and

simultaneously acquiring vast amounts of information at affordable operative costs17,18

.

In perspective, these technologies have a wide field of application that includes:

- basic science knowledge on normal or pathological stem cell behavior, both at the cellular

and molecular level. Phenomena such as stem cell activation, proliferation, differentiation,

and cell cycle progression can be investigated in a defined microenvironment by systematic

experimental campaigns;

- the precise control over stem cell differentiation, that enables the obtainment of cell

constructs as reliable in vitro models for the development and screening of new therapeutic

strategies and drugs;

- the transfer of biological knowledge for production of clinical grade stem cells on larger

scale. Once the critical operative parameters have been identified, the repeatability of the

experimental conditions and the safety of the process will increase;

- the development of small-scale highly-reliable diagnostic technology.

6

In this perspective, benefits from microscale technologies can derive from an accurate

investigation of the following specific aspects:

1) cell-substrate interactions through micrometric thin films of biomaterials: mechanical

properties of the substrate, surface morphology and surface functionalization with specific

cell binding ligands (peptides, proteins, etc.) have been demonstrated to be important cues

which can substantially affect the cellular response and development;

2) cell-cell interactions: cell pattern and topology are extremely relevant in stem cell culture

and biology, since their control allows the investigation of specific effects of cell

organization and cell-cell cross-talking at single-cell level, in cell clusters and in

multicellular co-culture systems;

3) cell-soluble environment interactions: the miniaturized and specific control of the soluble

environment over the cells allows the investigation of precise concentration profiles,

enabling the simultaneous investigation of several conditions with great reduction of

experimental time and costs;

4) cell-electrical fields coupling: microelectrode-based technology is at the basis of both the

exogenous cell electrical stimulation, and the recording of biochemical and physiological

signals in the sensor field.

In the next paragraphs, an overview will be presented of the principles and applications of micro-

technology, according to the relevant stem cell issues cited above.

1.2 Micro-structured substrate and cell topology

Mammalian cells integrate and respond to a combination of factors in the microscale environment,

such as mechanical and chemical properties of the substrates, substrate surface morphology, and the

overall cell topology that strongly affects cell-cell interactions.

7

Substrate mechanical properties can be properly designed by physical or chemical deposition of

micrometric thin layers of soft biomaterials. Among different biomaterials, hydrogels have recently

captured attention in the field of tissue engineering because of their high water content,

biocompatibility and elastic properties, resembling those of native tissues. It is worth to underline

that the elastic properties can be finely controlled and modulated by changing the amount of

polymer and crosslinker ratio during the production process. The stiffness of the hydrogels affects

the cell behavior at the cytoskeletal level and the transcriptional activity of mesenchimal stem

cells19-21

. Hydrogels, such as poly(ethylene glycol) (PEG) and derivatives, have been widely used as

substrates for cell culture or for encapsulating living cells22,23

. Their non fouling surface hinders cell

adhesion, thus allowing the formation and suspension cultures of a cell type such as embryoid

bodies (EB), cells aggregates derived from embryonic stem cells, which initiate differentiation and

recapitulate embryonic development to a limited extent24

. On the other hand, in order to enhance

and promote cell attachment, a wide variety of copolymers, composed of a synthetic backbone and

grafted biomolecules (or vice versa), such as fibrinogen25

, hyaluronic acid26

, or short peptides

sequences such as RGD27

, were developed. These biomimetic substrates were proposed and tested

to reproduce biological cues required for stem cell differentiation.

Besides cell-substrate interactions, the control of cell topology is of paramount importance for

studying stem cell differentiation, which requires a particular cell template, and for controlling cell-

cell interactions. For example, this latter aspect could be extremely relevant when a specific cell

pattern of two different cell types is required in a co-culture systems. To this aim, two different

methods which are based on different principles, were widely developed and applied: i) “physical”

method based on microstructured surface morphology; ii) “biochemical” method based on

micropatterning of functionalized surface.

In the first case, the topologic control over cell culture can be achieved through the use of substrates

(generally synthetic polymeric membranes) whose surface carries a three-dimensional morphology:

nanopatterned gratings, microgrooves and microtextures28-30

. Cell adhesion is thus guided by the

8

three-dimensional architecture imposed. These substrates are produced by soft-lithography

technique (see paragraph 1.5 for definition and methodologies), and have found several

applications, including cardiomyocyte cultures where topographical and electrical cues on a single

chip were incorporated31

, osteoblast differentiation32

, and liver bioreactor33

. Microwell arrays allow

the culture of single or cluster of stem cells in defined microenvironment and facilitates experiments

designed to dissect the interplay of soluble and insoluble molecules that comprise the stem cell

niche and allow analyses of fate changes of large numbers of single cells in a high-throughput

manner34

.

On the other hand, an alternative strategy to control cell culture topology is the selective deposition

of adhesion proteins (protein patterning) onto cell-repellent surfaces. Many patterning techniques

have been proposed and widely reviewed over the last few years35-39

, a more detailed list of such

techniques will be given in paragraph 1.5. Protein patterning allow the deposition of adhesion

proteins with a resolution of ∼1 μm, the immobilization on the surface occurs through physical or

chemical adsorption and allows proteins to maintain their activity without suffering from major

denaturation phenomena. Consequently, cell adhesion is controlled with micrometric precision

(Figure 2A-B).

The main advantage of this technique resides in the possibility of guiding cell adhesion with such

high fidelity, being the surface used cell repellent, allowing to study the effects of specific

topologies on stem cell behavior. Patterns of proteins have been created on glass or polystyrene

surfaces via micro-contact printing (μCP)40

, and laminin lanes have been patterned using the same

technique on polymeric films for the subsequent adhesion of cardiomyocytes41-43

. The patterning

can be designed to mimic the topology of specific tissues and thus to facilitate stem cell

differentiation towards a specific lineage44-46

. It can also be used in co-cultures in order to control

cell-cell contact and interaction at the microscale by topologically organizing two different cell

types47-49

(Figure 2C).

9

As frequently argued, performing stem cell cultures onto surfaces, 2D systems, do not mimic with

high fidelity the natural 3D architecture of in vivo tissues. In this sight, Albrecht and colleagues

explored the role of microscale organization of tissues in three-dimensions through the integration

of co-culture and topological cell organization in a 3D model50

, and they demonstrated that

chondrocyte biosynthesis specifically modulates microscale organization.

Another application of micropatterning aims at studying, in a highthroughput fashion, the effect of

the extracellular matrix (ECM) on stem cell behavior (Figure 2D). Microarray platforms of ECM

proteins were developed for screening their effects on stem cells in a combinatorial and parallel

fashion. A conventional DNA robotic spotter has been modified for the immobilization of specific

ECM components (collagen I, collagen III, collagen IV, laminin and fibronectin) or combinations of

these molecules on hydrogel surfaces51

. The developed array was used for studying the commitment

of mouse ES cells towards an early hepatic fate, leading to the identification of several

combinations of ECM components that synergistically impact ES cell differentiation and hepatocyte

function. This platform was further developed to analyze the interactions between ECM

components and soluble growth factors on stem cell fate, resulting in a multiwell microarray

platform that allows 1200 simultaneous experiments on 240 unique signaling microenvironments.

These studies were consistent with results previously reported in the literature, confirming the

reliability of the platform. Similarly, a microarray of biomaterials has been developed and used for

the high-throughput and rapid synthesis and screening of combinatorial libraries of

photopolymerizable biomaterials to identify compositions that influence human ES cell attachment,

growth and differentiation52

. Over 1700 human ES cell-material interactions were simultaneously

characterized and a multitude of unexpected materials effects were identified, offering new levels of

control over human embryonic stem cell behavior.

10

1.3 Soluble environment control

Another aspect of stem cell niche involves the soluble local microenvironment. Metabolites, gases,

and growth factors are transported between the bulk of the culture medium and the cell surface in

either direction. In standard culture systems these components are exclusively delivered by a

mechanism of diffusion. When cells uptake or release a specific chemical component at a higher

rate than that of the diffusion transport process, a concentration gradient is produced from the cell

surface to the bulk of the culture medium. In other words, when diffusive mass transport has a

characteristic time larger than that of cell uptake or release, a depletion or accumulation of that

component occurs at the cell surface53

. Note that the two processes occur in series. Furthermore, if

cell density in culture is not uniform, as it is the case in standard culture systems, the concentration

gradients surrounding single cells can intersect and larger-scale regions of accumulation or

depletion of that component are produced. These phenomena affect the homogeneity of the cell

population and the definition of the actual local concentration at the cell surface, complicating an

insight into cell behavior.

In perfused systems, the cell culture is performed under a precise control of the conditions, and

convection occurs as a further mechanism of mass transport from the bulk of the culture medium to

the cell surface and vice versa. Convection takes place in parallel with respect to diffusion, and it is

the dominant mass transport mechanism only when its characteristic time, τC, is smaller than the

diffusion characteristic time, τD, meaning that the ratio τC/τD is less than 1. The prevalence of either

mechanism is dependent on the specific geometry of the culture system and on the operative

conditions.

Left panel of Figure 3A schematizes a typical micro-perfused culture chamber. As an example,

assuming fixed the inlet medium flowrate, Q, it is possible to decide to grow the cells at the bottom

of the well under either diffusive or convective regime. In fact, τC is proportional to the cross

section area of the well, d·h, where d and h are the well width and height respectively, and τD is

11

proportional to the square of well height, h2. Thus, their ratio, τC/τD, is proportional to d/h. This is

the most important geometrical variable that needs to be accurately selected for establishing the

desired mass transport regime (either diffusive or convective) within microfluidic systems53

.

Typically, for Q=1 l/min and d/h=1, h must be in the range of few millimeters or less to obtain a

diffusion-limited regime (right panel of Figure 3A).

Despite its simplicity, the system in Figure 3A shows all the potential of microfluidic devices in

defining the microenvironment in direct contact with the cells. Furthermore, as shown in the

example above, it is possible to regulate the mass transport rates at which different components

reach cell surface by choosing the appropriate mass transport regime. This technical solution allows

stationary culture conditions if medium inlet composition is kept constant, or defined temporal

patterns of biochemical stimulation if an upstream system of micro-valves temporally regulates the

composition of the inflow medium.

Being again interested on the microscale, we focus on the field of stem cell culture integrated with

microfluidics: “the science and technology of systems that process or manipulate small amounts of

fluids, using channels with dimensions of tens to hundreds of micrometres”54

.

At the relevant scale for microflows, meaning inside the microchannels and culture chambers of

microfluidic devices, the dominating forces change and differ from the most known, macroscale

ones. Excellent descriptions can be found in the literature55-59

, extensively analyzing the physics of

fluids at the micro- and nano-scale and illustrating the most relevant applications giving insights

into the operating principles for system configurations of possible interest. Particular attention has

also been focused on the accurate description and characterization of the dominant phenomena

within microfluidic bioreactors, giving the design specifications for important operating parameters

and the principles for their optimization at the microscale level53

.

The feasible control over the operating parameters allows the application of complex and well-

defined patterns of stimulation not only, as already said, in terms of temporal concentration profiles,

but also of spatial dynamic perturbations, such as precise and localized concentration gradients or

12

steep compartments (Figure 3B). Examples include studies of chemotaxis in cell culture systems

utilizing hydrogels of differential compositions60

, short-term perfusion of graded concentrations of

regulatory factors over seeded cells61

, and the assembly of membrane-based diffusion chips62

.

However, one must be well aware that the use of such microfluidic gradient generators is generally

associated with hydrodynamic shear stresses exerted on cultured cells, that arise from the small

characteristic dimensions of microfluidic channels63

.

Examples of applications of microfluidic platforms for lab-on-a-chip applications have been

extensively reviewed64,65

and point at the advantages deriving from the miniaturization, integration

and automation of biochemical assays, while giving more insights also in the fabrication processes

and properties of the materials that are typically used66-68

. Since its first appearance decades ago,

microfluidics has been adapted to a multiplicity of different applications. Recent literature reflects

increased interest in interfacing microfluidic devices with biological systems54,69-72

and in their use

in drug discovery process73-75

, or molecular detection76

. High-tech platforms involving integrated

microdevices such as micro-valves, injectors, pumps or mixers77

are also being considered for use

in live cell experimentation. Microscale technologies were designed for applications ranging from

studies at a single cell level78

to the recreation of more complex 3D structures79-82

and the

development of diagnostics platforms for clinical and medical research83

. Concerning a specific

application for this latter aspect, Toner and Irimia84

have reviewed the use such devices for blood

manipulation at the microscale, highlighting the importance of having small, portable,

highthroughput but yet reliable and ready-to-use instruments for specific blood analysis tasks. In

addition, microfluidics has been adopted for tissue engineering purposes, and examples exist in

applications involving basal lamina, vascular tissue, liver, bone, cartilage and neurons85

. The most

interesting and relevant recent applications, have seen the use of microfluidic devices in studies on

human embryonic stem cells86-88

(Figure 3C-D).

13

Research is being conducted to improve the cell culture soluble environment also at larger scales

(hundreds of milliliters) using suspension bioreactors where stirring is mechanically provided to

improve the transport of metabolites, gases, and growth factors89-91

. These devices have been used

to culture stem cells that can grow in suspension as single cells or as cell clusters, such as

hematopoietic stem cells92-95

, neural stem cells96

, or embryonic stem cells97

. The potential of these

bioreactors has also been extended to other types of cells that are suspended after encapsulation in

gelatin microspheres98

or inside porous polymeric microcarriers99-101

. These large-scale devices

have currently been limited in their use because of the high costs and the large number of cells

needed91

. In perspective, scaling down these systems to stirred microliter bioreactors, taking

advantages from knowledge of microfluidic stirring mechanisms already studied for other

applications, such as buoyancy-driven thermoconvection and electrokinetic flow102,103

, would be

extremely useful.

1.4 Electrical stimulation and recording

Endogenous electric fields play a key role in several biological processes (cell differentiation,

movement and growth). They are present in developing and regenerating animal tissues, both in the

extracellular space or in cell cytoplasm, and have magnitude that ranges from few to several

hundreds of mV/mm104-106

. Electrical stimulation and recording of excitable tissues (brain, muscles,

peripheral nerves or sensory system) are the subjects of interest of electrophysiological research and

clinical functional electrical stimulation107

. Consequently, the integration of electrical stimulation

devices with stem cell cultures could be a powerful tool for studying the effects of an exogenous

electric field on stem cell biology and give important insight into cell physiology.

The exposure of stem cell to neuro-physiological stimulation can be achieved through co-culture

with neuronal tissue108,109

, or the modulation of ion channel expression110

or finally the generation

14

of an electric field between electrodes, the latter being the most efficacious, as it allows the fine

control of crucial parameters such as amplitude, duration and frequency of the imposed pulse111

.

The need for increasing highthroughput characteristics of the experimentations, has led to the

development of miniaturized and arrayed platforms allowing for the generation of highthroughput

data112

. Among these systems, microelectrode arrays (MEA) are the most used and have been

proven to be a valuable tool for detecting and measuring extracellular potentials for excitable cell

types such as cardiomyocytes and neurons31,113-117

(Figure 4A). In general, microelectrodes allow

chemical measurements in spatial and temporal domains that were previously inaccessible118

.

In addition, microelectrodes platforms can be used as biosensors for recording signals released from

the cells themselves, thus allowing to monitor and record changes in defined physiological and

biochemical signals for applications ranging from pharmacology, cell biology, toxicology, and drug

discovery119,120

(Figure 4B). Several examples exist in the literature, illustrating basic rules dictating

their operative performances121

and demonstrating the ease of use in effective signal

recording122,123

. The most advanced systems integrate nanostructured materials, such as carbon

nanotubes, within the sensing elements, thus greatly improving the performance of the

devices124,125

.

1.5 Micro-technology overview

As well described in some of the above cited works55,58,59

, a multiplicity of techniques have been

applied in order to gain micrometric control over culture systems.

One of the first techniques ever applied to fabricate microelectromechanical systems (MEMS) is

silicon or glass micromachining. However, even if this method is still extensively used for

applications involving strong solvents or high temperatures, its use for biologically inspired studies

is limited due to its high costs, difficult realization, and limited compatibility of the materials (i.e.

silicon is not transparent).

15

To date, lithographic techniques, and especially photolithography, are the most commonly used to

fabricate microscale features on glass or silicon substrates to be subsequently used as masters for

obtaining the final devices. Since the early ’90’90, when Whitesides and others initiated the

exploration of its use in the field of biological studies36,126-128

, soft lithography has emerged among

all lithographic techniques and has been exploited for its many advantages such as the ease of

application, low cost, high versatility and properties of the usable materials. Soft lithography is

commonly used to create structures on surfaces for controlling cell-substrate interactions. It

basically covers a group of techniques with the common feature that at some stage of the process an

elastomeric (soft) material is used to create the desired structures. Such elastomeric devices

(typically in poly(dimethyl siloxane), PDMS) can be obtained through replica molding on the above

mentioned microstructured silicon masters. Photolithography and PDMS replica molding are the

leading techniques for the production of microfluidic platforms and microbioreactors.

Similarly micro-contact printing (µCP), first described by Whitesides in 1993129

, is the technique

for micropatterned substrates production, and consists in using a microstructured elastomeric stamp

with relief features to transfer an “inked” material (adhesion proteins in the case of cell culture)

onto a substrate. Similarly to µCP, other techniques such as microtransfer molding, micromolding

in capillaries and solvent-assisted micromolding use microstructured PDMS replica masters to

transfer its features on other materials. Alternatively, microscale features could be created on

supporting materials via selective chemical vapor deposition, wet or dry etching processes, or more

advanced micromachining processes such as bulk and surface micromachining, microstereo

lithography or finally laser mediated ablations. We again refer to specific publications for a more

detailed description of all these techniques59

.

16

Figure captions

Figure 1. Stem cell niche. A. Schematic representation of the stem cell niche, where

matrix/substrate interactions, cell-cell contact and soluble factors direct cell fate. B. Length and

time scales for some relevant biological phenomena.

Figure 2. Micropatterning and microstructured cultures. A. Examples of µCP of laminin and

resulting mouse satellite cell culture in circular dots (A1) of 300µm diameter and parallel lanes (A1)

75µm wide and 125µm spaced .Scale bars, 300µm. B. Human embryonic stem cell colonies

patterned at 200µm, 400µm, and 800µm diameters. Scale bars, 250µm. [PERMISSION]. C. Phase

contrast images of hepatocyte-fibroblast co-cultures. Scale bars, 200µm. [PERMISSION]. D.

Schematic representation of the layout of the extracellular matrix array; bright field of hepatocytes

cultured onto the array; immunofluorescence of albumin (a marker of differentiated hepatocyte

function). Scale bar, 1mm; inset scale bar, 500µm. [PERMISSION]

Figure 3. Soluble environment control. A1. A schematic representation of a typical culture chamber

with the microfluidic channels for fluid perfusion. The characteristic dimensions are its width d and

height h (distance between the bottom of the chamber and the microfluidic channels). A2. Graph

exemplifying the results of computational modeling of the averaged properties of the system and in

particular of the diffusion and convection time constants as a function of medium flow rate and

culture chamber geometry. Results are presented for two chemical species representative of a small

metabolite (higher diffusion coefficient D) and of a larger growth factor (lower D). The existence of

two distinct mass transport regimes is evident, one dominated by molecular diffusion, and the other

17

by convection. B. Two examples of microfluidic platforms capable of recreating defined and

compartmentalized concentration patterns on adherent cell populations within a microchannel. The

microbioreactors designs and images of their validation are presented. C. An image of a

microbioreactor array platform constituted of 3 independent rows, each composed of 4 individual

culture chambers (4mm diameter) [PERMISSION] that was used for the culture of hESC colonies

(image on the right). D. Results of a long-term culture of hESC colonies within a microfluidic

channel [PERMISSION] .

Figure 4. Electrical stimulation and recording. A. ES cell-derived neurons at different time points

after initiating the differentiation. Phase-contrast images of ES cell-derived neurons on

microelectrode arrays (MEAs) at 7 (left) or 28 days (right) after initiating the differentiation.

[PERMISSION]. B. an example of an integrated multi-electrodes biosensor array is reported.

specifically, it allows detections of glucose, lactate, glutamine, and glutamate. [PERMISSION]

18

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Figure 4