Methods in Bioengineering: Stem Cell Bioengineering (The Artech House Methods in Bioengineering Series)

223 Pages·2009·7.22 MB·English

by  Biju Parekkadan

Methods in Bioengineering
Stem Cell Bioengineering
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Methods in Bioengineering
Stem Cell Bioengineering
Biju Parekkadan
Massachusetts General Hospital/Harvard Medical School
Martin Yarmush
Massachusetts General Hospital/Harvard Medical School

Editors
artechhouse.com
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Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the U. S. Library of Congress.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library.
ISBN-13: 978-1-59693-402-3
Text design by Darrell Judd

Cover design by Igor Valdman
© 2009 Artech House. All rights reserved.
Printed and bound in the United States of America. No part of this book may be reproduced or
utilized in any form or by any means, electronic or mechanical, including photocopying, record-
ing, or by any information storage and retrieval system, without permission in writing from the
publisher.
All terms mentioned in this book that are known to be trademarks or service marks have
been appropriately capitalized. Artech House cannot attest to the accuracy of this information.
Use of a term in this book should not be regarded as affecting the validity of any trademark or
service mark.
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Contents
Preface xiii
CHAPTER 1
Somatic Cell Nuclear Transfer and Derivation of Embryonic Stem Cells 1
1.1 Introduction 2
1.2 Materials for Nuclear Transfer 2
1.2.1 Equipment for mouse nuclear transfer 2
1.2.2 Reagents for mouse nuclear transfer 2
1.3 Methods for Nuclear Transfer 4
1.3.1 Preparation of enucleation and nuclear transfer pipettes 4
1.3.2 Medium preparation 4
1.3.3 Animal preparation 5
1.3.4 Nuclear transfer 5
1.3.5 Enucleation 5
1.3.6 Preparation of donor cells 6
1.3.7 Nuclear transfer 7
1.3.8 Activation 11
1.3.9 Embryo culture and embryo transfer 12
1.4 Derivation of Mouse ntES Cells 13
1.5 Materials for ES Cell Derivation 13
1.6 Methods for ES Cell Derivation 14
1.6.1 Derivation of ntES cells 14
1.6.2 In vitro characterization of ntES cells 15
1.6.3 In vivo characterization of ntES cells 16
1.7 Discussion and Commentary 19
Troubleshooting Table 19
1.8 Summary Points 21
Acknowledgments 21
References 21
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Contents
CHAPTER 2
Derivation of Mouse Parthenogenetic Embryonic Stem Cells 23
2.1 Introduction 24
2.2 Materials 24
2.2.1 Reagents 24
2.2.2 Equipment 26
2.2.3 Media recipe 27
2.3 Methods 29
2.3.1 Generation of p(MI) embryos 30
2.3.2 Generation of p(MII) embryos 30
2.3.3 Generation of p(hap) embryos 31
2.3.4 Derivation of p(MI), p(MII), and p(hap) ES cells 32
2.3.5 ES cell characterization 34
2.3.6 Teratoma induction 34
2.4 Data Acquisition, Anticipated Results, and Interpretation 35
2.5 Discussion and Commentary 35
Troubleshooting Table 36
2.6 Summary Points 36
Acknowledgments 37
References 37
CHAPTER 3
Generation of Mice from Embryonic Stem Cells Using Tetraploid Embryos as Hosts 39
3.1 Introduction 40
3.2 Experimental Design 40
3.3 Materials 41
3.3.1 Preparation of 4n embryos: Mice and reagents 41
3.3.2 Preparation of 4n embryos: Equipment 41
3.3.3 ES cells and culture conditions 41
3.3.4 Micromanipulation system 42
3.4 Methods 42
3.4.1 Preparation of host embryos 42
3.4.2 Electrofusion of two-cell stage embryos 43
3.4.3 Removal of the zona pellucida 43
3.4.4 Generation of ES ´ multiple 4n embryos by aggregation 43
3.4.5 Generation of ES ´ 4n embryos by blastocyst injection 44
3.4.6 Data acquisition 45
3.5 Anticipated Results 45
3.6 Discussion and Commentary 45
Troubleshooting Table 46
3.7 Application Notes 47
3.8 Summary Points 47
Acknowledgments 48
References 48
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Contents
CHAPTER 4
Bioreactor Design and Implementation 49
4.1 Introduction 50
4.2 Experimental Methods and Materials 51
4.2.1 General system description 51
4.2.2 Closed system 52
4.2.3 Hollow-fiber bioreactor 53
4.2.4 CES fluid circuit 54
4.2.5 Oxygenator design 55
4.2.6 Monitoring 56
4.2.7 User interface 56
4.3 Anticipated Results 56
4.3.1 MSC source 1: Loading whole BM marrow into the CES 58
4.3.2 MSC source 2 or 3: Loading preselected MSC into the CES 59
4.4 Discussion and Commentary 60
Troubleshooting Table 61
4.5 Application Notes 61
4.5.1 Therapeutic dose of MSC grown from a whole BM sample 61
4.5.2 Nonadherent cell culture: Kg1a cells grown in suspension 61
4.6 Summary Points 62
CHAPTER 5
Extracellular Matrix Microarrays and Stem Cell Fate 63
5.1 Introduction 64
5.2 Experimental Design 65
5.3 Materials 65
5.4 Methods 66
5.4.1 Preparation of substrates for arrays 66
5.4.2 Fabrication of ECM arrays 67
5.4.3 Cell culture on ECM arrays 68
5.4.4 Staining, imaging, and data acquisition 68
5.4.5 Data analysis 69
5.5 Anticipated Results 69
5.6 Discussion and Commentary 70
Troubleshooting Table 71
5.7 Application Notes 72
5.8 Summary Points 72
Acknowledgments 73
References 73
CHAPTER 6
Microfluidic Culture Platform for Investigating the Proliferation and
Differentiation of Stem Cells 75
6.1 Introduction 76
6.2 Experimental Design 76
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Contents
6.2.1 Microfluidic platform for differentiation of human neural
progenitor cells 76
6.2.2 A hybrid microfluidic platform for stem cell biology 77
6.2.3 ESC response under dynamically controlled gradient condition 79
6.3 Materials and Methods 80
6.3.1 Fabrication of the microfluidic device 80
6.3.2 Fabrication of a hybrid microfluidic platform 81
6.3.3 Human neural stem cells 81
6.3.4 Mouse neural stem cells 82
6.3.5 Culturing cells inside the microfluidic chamber and time-lapse
microscopy 82
6.3.6 Immunocytochemistry 83
6.4 Data Acquisition, Anticipated Results, and Interpretation 83
6.4.1 hNSC proliferation in the gradient chamber 83
6.4.2 Differentiation of hNSCs into astrocytes in the gradient chamber 84
6.4.3 ESC response to BMP signaling 85
6.5 Discussion and Commentary 86
Troubleshooting Table 86
6.6 Application Notes 87
6.7 Summary Points 87
Acknowledgments 87
References 87
CHAPTER 7
Analysis of Mouse Hematopoietic Stem and Progenitor Cells 89
7.1 Introduction 90
7.2 Experimental Design of Lineage Depletion of Whole Bone Marrow Cells 90
7.3 Materials for Lineage Depletion of Whole Bone Marrow Cells 91
7.3.1 Tools and plasticware 91
7.3.2 Reagents 91
7.3.3 Additional equipment and reagents required for method 1
(MACS beads method) 91
7.3.4 Additional equipment and reagents required for method 2
(Dynabeads method) 91
7.3.5 Common protocols for both methods 91
7.3.6 Discussion and commentary 93
Troubleshooting Table for Lineage Depletion Method 94
7.4 Methylcellulose-Based in Vitro Colony-Forming Assay 95
7.4.1 Materials 95
7.4.2 Methods 95
7.4.3 Data acquisition, anticipated results, and interpretation 96
7.4.4 Discussion and commentary 96
Troubleshooting Table for CFU Assay 97
7.5 Radiation of Mice for In Vivo Assays 97
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Contents
Troubleshooting Table for Bone Marrow Transplantation 97
7.6 Colony-Forming Unit-Spleen Assay 98
7.6.1 Materials 98
7.6.2 Methods 99
7.6.3 Data acquisition, anticipated results, and interpretation 99
7.6.4 Discussion and commentary 100
Troubleshooting Table for CFU-S Assay 100
7.7 Quantification of HSCs Using the Limiting Dilution Assay 100
7.7.1 Buffers and materials 101
7.7.2 Methods 101
7.7.3 Data acquisition, anticipated results, and interpretation 102
7.7.4 Discussion and commentary 103
Troubleshooting Table for Limited Dilution Assay 104
7.8 Summary Points 104
References 105
CHAPTER 8
Skeletal Stem Cells and the Hematopoietic Microenvironment: Biology and Assays 107
8.1 Introduction 108
8.2 Experimental Design 108
8.3 Materials 109
8.3.1 Stromal cell isolation and culture 109
8.3.2 Isolation of CD45-CD146+ cells 109
8.3.3 In vivo transplantation 109
8.4 Method 109
8.4.1 Bone marrow single-cell suspensions 109
8.4.2 Isolation of MCAM/CD146-expressing bone marrow
osteoprogenitors 110
8.4.3 In vivo transplantation 111
8.4.4 Analysis of heterotopic ossicles 112
8.5 Anticipated Results 112
8.6 Discussion and Commentary 113
Troubleshooting Table 114
8.7 Application Notes 114
8.8 Summary Points 115
Acknowledgments 116
References 116
CHAPTER 9
Targeting the Stem Cell Niche In Vivo 117
9.1 Introduction 118
9.2 Experimental Design 119
9.3 Materials 120
9.3.1 PTH treatment 120
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