Human Embryonic Stem Cells Edited by Arlene Y Chiu and Mahendra S Rao Human Embryonic Stem Cells Human Embryonic Stem Cells Edited by Arlene Y Chiu National Institute of Neurological Disorders and Stroke National Institutes of Health Bethesda, MD and Mahendra S Rao National Institute on Aging National Institutes of Health Baltimore, MD Humana Press Totowa, New Jersey © 2003 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and not necessarily reflect the views of the publisher This publication is printed on acid-free paper ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials Production Editor: Wendy S Kopf Cover design by Patricia F Cleary Cover illustration: Cover photo shows neural cells that have differentiated from human embryonic stem cells These cells have been immunostained to reveal phenotypic features: red (rhodamine) for neurofilament 200 and green (fluorescein) for GFAP Nuclei (blue) are visualized by Hoechst staining Cover photo provided by Dr Su-Chun Zhang For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-2561699; Fax: 973-256-8314; E-mail: humana@humanapr.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $20.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc The fee code for users of the Transactional Reporting Service is: [1-58829-311-4/03 $20.00] Printed in the United States of America 10 Library of Congress Cataloging in Publication Data Human embryonic stem cells / edited by Arlene Chiu and Mahendra S Rao p cm Includes bibliographical references and index ISBN 1-58829-311-4 (alk paper); e-ISBN 1-59259-423-9 Human embryo Embryonic stem cells I Chiu, Arlene II Rao, Mahendra S QP277.H87 2003 612.6'46 dc21 2003041669 Preface Human embryonic stem cells are derived from the earliest stages of blastocyst development after the union of human gametes Prior to fertilization, the oocyte first requires timed completion of meiosis This vital step does not occur throughout a woman’s life; rather, oocytes are arrested at the first meiotic division until puberty when small numbers mature competitively during the reproductive years Maturation is complete at the one-day event of ovulation that occurs in a regular, approximately monthly cycle In humans, oocytes can be successfully fertilized only during a short period after ovulation; oocytes that are not fertilized are not retained Sperm cells mature from spermatic stem cells through a sequential process that has been well characterized Spermatic stem cells are in turn generated from primordial germ cells set aside during early embryonic development Generally, tens of millions of sperm are present in an ejaculate of which only one will successfully fertilize the mature oocyte Sperm that fail to fertilize are discarded Fertilization initiates the process of cell differentiation Because embryonic transcription is not initiated until later, the earliest developmental events are regulated primarily by maternally inherited mRNA Once the sperm enters the egg, its DNA-associated proteins are replaced by oocyte histones The two pronuclei become enveloped with oocytederived membranes, which fuse and begin the zygote’s mitotic cell cycle Embryonic development starts with a series of cleavages to produce eight undetermined and essentially equivalent blastomeres The pattern of cleavage is well coordinated by cytoplasmic factors and in mammalian eggs is holoblastic and rotational Though genomic DNA is inherited from both parents, mitochondrial DNA is inherited from only the mother Paternal mitochondria transferred at the time of sperm entry are discarded by a little-understood process Human eggs, with a diameter of 100 µm, are generally smaller than eggs of other species They are normally fertilized within the fallopian tubes and undergo cellular division in a defined milieu as they migrate toward the uterus Over the first few days, cellular division follows a predictable 12–18-h cycle resulting in 2- to 16-cell pre-embryos The v vi Preface sperm centrosome controls the first mitotic divisions until day 4, when genomic activation occurs within the morula stage The individual blastomeres are initially totipotent until the morula begins compaction and the cells initiate polarization During compaction, cell boundaries become tightly opposed and cells are no longer equivalent Cells in the inner cell mass (ICM) contribute to the embryo proper, whereas cells on the outside contribute to the trophoectoderm The blastocyst forms approx 24 h after the morula stage by the development of an inner fluid-filled cavity, the blastocell Implantation is the process through which the compact zonula is thinned and the blastocyst released to implant The blastocyst must first hatch from the thinning zona pellucida by alternating expansion and contraction; this process of hatching is critical to further development Implantation of the hatched blastocyst requires several steps, including apposition, attachment, penetration, and trophoblast invasion, and cannot occur until the first cell specification into trophoectoderm has occurred As the trophoblast is developing to form the fetal component of the placenta, the endometrial lining of the maternal uterus is undergoing a decidual reaction to generate the maternal component of the placenta Simultaneously the inner cell mass undergoes gastrulation, defined as a process of complex, orchestrated cell movements that vary widely among species, but include the same basic movements These include epiboly, invagination, involution, ingression, and delamination Thus, several major developmental events have taken place as the fertilized egg migrates from the site of fertilization (fallopian tubes) to the body of the uterus over a period of d At all stages the egg is shielded from the external environment, initially by the zonula and subsequently by the trophoectoderm, but is accessible to ex utero manipulation (see Fig 1) It is important to emphasize that these critical developmental events have occurred prior to implantation and well before blood vessel growth and heart development The early stage fertilized egg that has not yet been implanted has been termed a pre-embryo to distinguish it from the implanted embryo Manipulation of the preimplanted embryo has been feasible for the past three decades, and detailed rules governing the use of blastocysts have been developed After implantation, the ICM proliferates and undergoes differentiation Several results suggest that lineage-specific genes are operating in a totipotent blastocyst cell prior to lineage commitment, and strongly support the concept that stem cells express a multilineage transcriptosome Most genes (including tissue-specific genes) are Preface vii Fig Many techniques have been devised to manipulate the process of fertilization and maturation prior to implantation (summarized at right) The recent development of techniques to generate embryonic stem cell lines and perform somatic nuclear transfer has increased our ability to understand the process of development and intervene therapeutically Note that the fertilized egg and early stage zygote are accessible to manipulation prior to implantation maintained in an open state with low but detectable levels of transcription with higher levels of specific transcription seen in appropriate cell types Maintenance of an open transcriptosome in multipotent cells likely requires both the presence of positive factors as well as the absence of negative regulators Factors that maintain an open transcriptosome include as yet unidentified agents such as demethylases, reprogramming molecules present in blastocyst cytoplasm, and regulators of heterochromatin modeling Global activators, global repressors, and master regulatory genes play important regulatory roles in switching on or off cassettes of genes, whereas methylation and perhaps small interfering RNA (siRNA) maintain a stable phenotype by specifically regulating the overall transcriptional status of a cell Allelic inactivation and genome shuffling further sculpt the overall genome profile to generate sex, organ, and cell-type specification viii Preface Few genes have been identified that are required for the maintenance of the epiblast population Oct4-/- embryos die before the egg cylinder stage and embryonic stem (ES) cells cannot be established from Oct4-/- cells Levels of Oct expression are critical to the fate of the cells Low cell levels lead to differentiation into trophoblast giant cells, whereas high levels cause differentiation into primitive endoderm and mesoderm FGF4 is required for formation of the egg cylinder and FGF 4-/- embryos fail to develop after implantation and ICM cells not proliferate in vitro Foxd3/Genesis is another transcription factor that may be required for early embryonic development TGF-β/ SMADs, Wnts, and FGFs are thought to play an important role in the process of gastrulation BMP4 is essential for the formation of extraembryonic mesoderm and the formation of primordial germ cells Nodal expression is required for mesoderm expansion, maintenance of the primitive streak, and setting up the anterior–posterior and proximo–distal axis FGF4 is secreted by epiblast cells and is required for the maintenance of the trophoblast As our understanding of early developmental events has increased, our ability to safely manipulate the reproductive process has also increased In vitro fertilization is now a relatively commonplace procedure that has been performed for more than 20 yr Today, there are over a thousand established clinics worldwide Even such technically complex procedures as intracytoplasmic sperm injection (ICSI), ooplasm transfer, assisted hatching, intrauterine genomic analysis, intrauterine surgery, and organ transplants are becoming more commonplace More then 70 human embryonic cell lines have been established and their ability to differentiate into ectoderm, endoderm, and mesoderm repeatedly demonstrated Nuclear transfer has become feasible, and the potential of combining ES cell technology with somatic nuclear transfer to clone individuals has caught the attention of people worldwide At each stage of technological sophistication, profound ethical issues have been raised and publicly debated Perhaps the most recent technological breakthroughs are the ones that have created the most controversy, primarily because of their potential to be used on a large scale The ability to generate human ES (hES) cells and the ability to perform somatic nuclear transfer and successfully clone mammalian species raise fears often fueled by limited information An additional potential paradigm shift has been the suggestion that pluripotent ES-like cells may exist and indeed may persist into adulthood (see Fig 2) These cells, while differing subtly from ES cells, may be functionally equivalent for therapeutic applications The possible Preface ix existence of such adult cells with ES cell properties has fanned the debate and fueled a drive to assess the properties of all classes of pluripotent cells and to understand their underlying differences In Human Embryonic Stem Cells we invited leaders in the field to present their work in an unbiased way so that readers can assess the potential of stem cells and the current state of the science The first section covers issues that regulate the use of human pluripotent cells Chapters 1–3 begin with a summary of the ethical debate surrounding the derivation of human stem cells, and the current policies governing their use in the United States and abroad The presidential announcement of August 2001 heralded a change in policy enabling federal support of research with hES cells that meet specific criteria In Chapters and 3, representatives from the National Institutes of Health (NIH) discuss the rules and conditions regulating federal funding, and issues of intellectual property regarding the use of hES cells Chapter delves into what constitutes “allowable” research and provides a guide to researchers interested in acquiring funding from US federal agencies such as the NIH for studies in this field Part II describes the types of human pluripotent cells that are currently being studied, their sources, methods of derivation, and maintenance Many tissues are constantly renewed by the activities of resident, multipotent precursor or progenitor cells that have the ability to produce several different mature phenotypes In the well-characterized hematopoietic system, T and B lymphocytes are derived from the lymphoid stem cell, whereas the myeloid stem cell can generate a host of red and white blood cells, including monocytes, eosinophils, platelets, and erythrocytes However, both the myeloid and lymphoid stem cells are committed precursors, unable to differentiate along other pathways There are only a few examples of truly pluripotent stem cells with the developmental capacity to generate cells representing all three germ layers (see Fig 2) Four types of such pluripotent stem cells are discussed in this section In Chapter 4, Draper, Moore, and Andrews review the tumorigenic origins of embryonal carcinoma (EC) cells and their developmental counterparts, embryonic germ (EG) cells, present in the germinal ridges of young fetuses Although there are many claims that pluripotent and highly plastic stem cells reside in adult tissues, the best characterized are those present in bone marrow Cardozo and Verfaille summarize studies demonstrating their pluripotency in Chapter The high degree of interest in hES cells arise from two properties: their ability to self-renew essentially indefinitely and to be maintained 448 Appendices 448 Appendix 449 449 450 Appendices 450 Index 451 Index A B Abortion, stem cell sources, Aging, see Senescence Allogeneic cell therapy, advantages of human embryonic stem cell therapy, 254, 255, 257 challenges, human embryonic stem cell therapy, 252, 253, 254 somatic stem cell therapy, 251, 252, 253 histocompatibility, 251 prospects, 258 ALS, see Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), hNT studies, 365 Angiogenesis, functions, 201, 211 hematopoietic stem cell role, 91 inhibition model using human embryonic stem cell differentiation, 211, 212 overview, 201 Antisense, gene repression in human embryonic stem cells, 276 Autologous cell therapy, advantages, 242, 246 challenges, conventional therapy, 246–248 therapeutic cloning, 246, 249, 250 transdifferentiation, 246, 248, 249 BAC, see Bioethics Advisory Committee Bayh-Dole Act, 46, 52 Bioethics Advisory Committee (BAC), Singapore, 22, 23 Blood transfusion, human embryonic stem cell-derived cells, 230, 231 BMPs, see Bone morphogenetic proteins Bone marrow, stem cells, see Hematopoietic stem cell; Mesenchymal stem cell; Multipotent adult progenitor cell; Side population cell transplantation applications, 219 Bone morphogenetic proteins (BMPs), cardiogenesis role, 183, 184 C Cardiac myocyte, stem cell derivation, clinical applications and prospects, 181, 195 hematopoietic stem cell differentiation, 91, 93 human embryonic stem cell differentiation, beating frequency, 192 electrophysiology, 192 451 452 embryoid body induction, 187, 189 enrichment of derived cardiomyocytes, 194, 195 markers, 189, 190, 192 pharmacologic responses, 192, 193 prolonged cell culture, 187 mouse embryonic stem cell culture, 186 Cardiogenesis, bone morphogenetic proteins, 183, 184 conservation in vertebrates, 182 endoderm signaling, 183 fibroblast growth factors, 183, 184 overview, 181, 182 transcription factors, 182 transforming growth factor-β role, 182, 183 Wnt role, 184 CBER, see Center for Biologics Evaluation and Research, Cell replacement therapy, see Transplant therapy, embryonic stem cells Center for Biologics Evaluation and Research (CBER), human embryonic stem cellderived therapy safety assurance, culture process control, bovine serum in medium, 331 cell isolation from inner cell mass, 330 nonhuman feeder layers and transplantation, 331–333 overview, 329, 330 stability monitoring of cell lines, 334 Index differentiated cell line characterization, 334–337 donor eligibility, 328 donor medical history and molecular genetic testing, 329 flexibility, 341 goals, 326, 327 histocompatibility, 336, 337 overview, 324–326 pilot clinical study initiation, 326 preclinical animal testing, proof-of-concept investigations, 340, 341 toxicology, 337–341 mission, 323, 324 Web site resources, 396, 397 Culture, human embryonic stem cells, animal-free culture conditions, human foreskin as feeder layer, 138 human serum use, 138 matrices using serum-free medium, 138, 140 rationale, 136, 138 cell line availability, 297 cryopreservation of stem cells, 124, 125 derivation, inner cell mass isolation by immunosurgery, 118, 119, 128 I series cell lines, 128, 129 J series cell lines, 129–132 plating and expansion, 119 preimplantation genetic diagnosis specimens with genetic defects, 132, 133 differentiation culture, see specific tissues Index embryoid body formation, 125, 126, 129 federal regulation for therapeutics, bovine serum in medium, 331 cell isolation from inner cell mass, 330 nonhuman feeder layers and transplantation, 331–333 overview, 329, 330 stability monitoring of cell lines, 334 fibroblast feeder layer culture, 119, 120 incubators, 116 Matrigel culture, conditioned medium preparation, 121 daily maintenance, 123 difficulty, 139 guidelines, 123, 124 Matrigel-coated plate preparation, 121 passage of cells, 121–122 media, 115 mouse embryonic fibroblast feeder preparation, cryopreservation, 116, 117 harvesting, 116 irradiation and plating, 117, 118 thawing and maintenance, 117 scalability for therapeutic use, 255, 258 stock solutions, 114, 115 subclones, 133–136 surface markers, 113 thawing of stem cells, 125 tissue culture plates and flasks, 115 Cystic fibrosis, gene therapy, 132, 133 human embryonic stem cell culture, 133 453 D Diabetes, pancreatic tissue transplantation, 161, 162, 239 prevalence, 161 prospects for study, 174 stem cell therapy, 161, 174 Dolly, public response, Donaldson Report, 19, 20 E Early primitive ectodermlike (EPL) cells, characteristics, 130–132 derivation, 131 EB, see Embryoid body EC cell, see Embryonal carcinoma cell EG cell, see Embryonic germ cell Embryoid body (EB), differentiation, see specific cells and tissues formation from cultured embryonic stem cells, 125, 126, 129, 265 vascular potential, 202 Embryonal carcinoma (EC) cell, see also TERA2; Teratoma, antigens, 69, 70 clinical prospects, 77 definition, 67, 68 differentiation, 70, 71 imprinted genes, 75 murine teratocarcinoma cell lines, 67, 68 stage-specific embryonic antigens in humans versus mice, 69, 70 Embryonic germ (EG) cell, generation, 74 surface markers, 74 Embryonic stem (ES) cell, basic research applications, 316–318 454 commercial sources of cell lines, 391 criteria, 127, 128 culture, see Culture, human embryonic stem cells drug discovery, 314–316 federal regulation of safety, see Center for Biologics Evaluation and Research gene expression profiling and discovery, 313, 314 genetic manipulation, see Genetically-modified human embryonic stem cells heterogeneity of differentiation, 163, 164 neural stem cell fusion, 103 nuclear transfer, see Therapeutic cloning signaling and differentiation in mice, 68 transplant therapy, see Transplant therapy, embryonic stem cells EPL cells, see Early primitive ectodermlike cells ES cell, see Embryonic stem cell Ethics, arguments against stem cell derivation from human embryos, Catholic Church, 5–8 Church of Scotland, United Methodist Church, arguments supporting stem cell derivation from human embryos, biomedical research advocates, 9–11 Islam, Jewish law, 8, Presbyterian Church, embryos as human beings, Index human egg donors for therapeutic cloning, 288 national policies on pluripotent stem cells, Australia, 21, 22 Germany, 21 Singapore, 22, 23 United Kingdom, 18–20 United States, 11–17 Evangelum Vitae, 5, F FDA, see Food and Drug Administration FGFs, see Fibroblast growth factors Fibroblast growth factors (FGFs), cardiogenesis role, 183, 184 FGF-2, neuroepithelia differentiation from embryonic stem cells, 146, 147, 151–153 Food and Drug Administration (FDA), see Center for Biologics Evaluation and Research G GCT, see Germ cell tumor Genetically-modified human embryonic stem cells, clinical applications, major histocompatibility complex gene deletion, 278, 312 nuclear transplantation therapy, 278, 280, 281 overview, 277, 279 Parkinson’s disease, 277, 278 differentiation induction, 272, 273 homologous recombination, 309, 310 marker gene expression, lineage selection, 270, 272 rationale, 269, 270 Index mouse embryonic stem cell manipulation of endogenous genes, antisense, 276 gene targeting, 274–276 overview, 274 RNA inhibition, 276, 277 nuclear transfer, see Therapeutic cloning overview, 265, 266 peptide delivery, 306–310 promoter usage, 273, 274 prospects, 281, 297, 298 retroviral vectors, 267, 268, 308, 309 transfection, electroporation, 266, 267 lipofection, 267 polymer-DNA complexes, 267 transient versus stable, 268, 269 universal donors, 310, 312 Germ cell tumor (GCT), chromosomal rearrangements, 65 extragonadal tumors in neonates and adults, 76 histology, 64 humans versus mice, 69 origins, 65 ploidy, 65 testicular seminomas versus nonseminomas, 64, 65 GFP, see Green fluorescent protein Graft-versus-host disease (GVHD), human embryonic stem cell applications, 228 Grants, see National Institutes of Health Green fluorescent protein (GFP), lineage selection of embryonic stem cells, 270, 272 GVHD, see Graft-versus-host disease 455 H HD, see Huntington’s disease Hematopoietic stem cell (HSC), adult cell potency, 91, 93–96, 99, 100 angiogenesis role, 91 assays, 90, 91 cardiac myocyte differentiation, 91, 93 embryonic stem cell differentiation, engraftment, 223, 227–229 human embryonic stem cells, CFC hematopoietic assay, 225, 226 culture, 225, 226 gene expression analysis, 224 genetic transduction, 224 markers, 225 mouse cell differences, 223, 224 prospects for clinical use, 228–231 xenogeneic transplantation models, 227, 228 mouse embryonic stem cell culture and hematopoiesis, 222, 223 rationale, 220, 221, 2288 engraftment, 99, 100 expansion, 229 graft tolerance induction, 229 hepatocyte differentiation, 93–95 mobilization and collection for transplantation, 219, 220 neuron differentiation, 95 senescence of mouse versus human cells in culture, 244 surface markers, 219 vasculogenesis role, 91 Hepatocyte, hematopoietic stem cell differentiation, 93–96 HFEA, see Human Fertilisation and Embryology Authority 456 hNT, amyotrophic lateral sclerosis studies, 365 derivation from NT-2, 348 differentiation potential, 348, 349, 372, 373 electrophysiology, 350 gene expression profiling, 349 graft characteristics in central nervous system, maturation, 351, 352 neurite outgrowth and synaptogenesis, 354, 355 neurofilament expression, 352 neurotransmitters, 354 pathogenicity potential, 353, 354 survival, 350, 352, 353 Huntington’s disease studies, 363, 365 neuronal character, 348–350 neurotransmitter receptors, 349, 350 Parkinson’s disease studies, 363 prospects for study, 371–375 spinal cord injury studies, clinical trial prospects, 375 connectivity with host tissue, 371, 372 differentiation of grafts, 368 growth and histology, 366–370, 374 motor-evoked potentials, 370, 371 overview, 365, 366 rat spinal cord injury model, 367–371 survival of graft, 367 uninjured spinal cord grafts in immunocompromised nude mice, 366, 367 stroke studies, animal model studies, 355–359 Index mechanisms of benefits, 358, 359 Phase I/II clinical trials, brain imaging, 361, 362, 373, 374 chromosomal polyploidy, 362, 363 concerns, 359, 360 graft preparation, 358 immunosuppression, 360 implantation, 360 postmortem evaluation, 362, 363 safety, 361, 373, 374 study design, 360 survival of graft, 359 survival in culture, 348 ultrastructure, 349, 350 Hogan patent, see Patents HSC, see Hematopoietic stem cell Human Fertilisation and Embryology Authority (HFEA), 18, 20 Huntington’s disease (HD), hNT studies, 363, 365 I ICM, see Inner cell mass Inner cell mass (ICM), isolation by immunosurgery, 118, 119, 128 Institutional review board (IRB), requirements for human embryonic cell research, 34–36 Intellectual property, see Patents Internet resources, Center for biologics Evaluation and Research, 396, 397 National Institutes of Health policy, 395, 396 patents, 396 In utero transplantation, see Transplant therapy, embryonic stem cells IRB, see Institutional review board Index K Karyotype stability, human embryonic stem cells, 133, 134 L Leukemia inhibitory factor (LIF), murine embryonic stem cell culture, 113, 146 LIF, see Leukemia inhibitory factor M Major histocompatibility complex (MHC), federal regulations for differentiated stem cell histocompatibility testing, 336, 337 gene deletion in human embryonic stem cells, 278, 310, 312 immune rejection role, 310, 312 universal cell donors, 310, 312 MAPC, see Multipotent adult progenitor cell Material Transfer Agreement (MTA), 40, 52, 54, 404–409 Matrigel culture, see Culture, human embryonic stem cells, MDSC, see Muscle-derived stem cell Memorandum of Understanding (MOU), 401–411 Mesenchymal stem cell (MSC), adult cell potency, 96, 97 senescence of mouse versus human cells in culture, 244, 245 surface markers, 96 MHC, see Major histocompatibility complex MOU, see Memorandum of Understanding Mouse embryonic fibroblast feeder cells, see Culture, human embryonic stem cells MSC, see Mesenchymal stem cell 457 MTA, see Material Transfer Agreement Multipotent adult progenitor cell (MAPC), angiogenesis role, 102, 103 culture conditions for pluripotency, 76 differentiation induction, 100, 102 engraftment, 102 proliferation, 100 senescence in culture, 241 surface markers, 100 tissue distribution, 100 Muscle-derived stem cell (MDSC), adult cell potency, 98 N National Bioethics Advisory Commission (NBAC), 8, 9, 12–17 National Institutes of Health (NIH), embryonic stem cell line registry, 127, 391, 392 federal funding opportunities for stem cell research, 36 federal requirements for review and approval by institutional review board, 34–36 fund application requirements, 31 history of human embryo research funding, 12, 14, 17, 27–29, 39 human embryonic germ cells in research, 30–33 human embryonic stem cells in research, 30, 391 patent implications of federal funding, 46 prohibited research activities, 33, 34 prospects for stem cell research funding, 37 research agreements, see Research agreements 458 stem cell access facilitation, agreements with stem cell providers, 52–54, 401–411 Research Tools Principles and Guidelines, 52 Web site resources, 395, 396 National policies, pluripotent stem cells, Australia, 21, 22 Germany, 21 Singapore, 22, 23 United Kingdom, 18–20 United States, 11–17 NBAC, see National Bioethics Advisory Commission Neural stem cell (NSC), adult cell potency, 97–99 embryonic stem cell fusion, 103 senescence of mouse versus human cells in culture, 245 Neuroepithelia, differentiation from embryonic stem cells, clinical prospects, 145, 146 human embryonic stem cells, aggregation and differentiation, 150, 151 culture, 148, 149 fibroblast growth factor-2 induction, 151–153 neural rosettes, 151–153 properties of derived cells, 153–155 retinoic acid induction, 149, 153 mouse embryonic stem cells, conditioned media from mesoderm-derived cell lines, 147 culture, 146 fibroblast growth factor-2 induction, 146, 147 innate tendencies of cells, 147, 148 isolation and markers of derived neural cells, 148 Index retinoic acid induction, 146 signaling, 148 neural induction, 145, 149, 150 prospects for research, 156 transplantation studies, 155 Neuron, embryonal carcinoma line derivatives, see hNT; NT-2; NTERA2; TERA2 fetal grafts, 346, 347 hematopoietic stem cell differentiation, 95 mesenchymal stem cell differentiation, 96, 97 progenitor cells, 345, 346 NIH, see National Institutes of Health NSC, see Neural stem cell NT-2, derivation from NTERA2, 348 therapeutic application of derived neurons, see hNT, NTERA2, derivation from TERA2, 348 differentiation, gene expression, 72, 73 induction, 73 features, 71–73 NT-2 subclone, see NT-2 teratoma formation, 71 Nuclear transfer, see Therapeutic cloning Nuffield Council on Bioethics, 18, 19 O Organ replacement, see Transplant therapy, embryonic stem cells P Pancreas development, anatomy, 164 experimental models, 165 mechanisms, 165, 166 neurogenesis similarities, 166, 168 signaling, 166 Index stem cells, 164, 165 transcription factors, 166 Pancreatic differentiation, pluripotent stem cells, culture of embryonic stem cells, 171–174 diabetes management, 161, 174 gene expression of embryoid bodies, 169, 170 growth factors, 169, 170 insulin production and secretion, 170 selection of insulin-secreting cells from differentiated cultures, 170, 171 Parkinson’s disease (PD), fetal cell transplantation, 346, 347 genetically-modified human embryonic stem cell therapy, 277, 278 hNT studies, 363 Patents, claim types, 41, 42 Edinburgh patent, 49 European patent laws and policies, 49, 50 examples, Hogan patent for non-murine pluripotential cells, 415–430 Thomson patent for primate embryonic stem cells, 415, 416, 431–450 federal funding implications, 46 functions, 39, 40 human pluripotent embryonic germ cells, 48 human pluripotent embryonic stem cells, 44–46 National Institutes of Health role in stem cell access facilitation, agreements with stem cell providers, 52–54 459 Research Tools Principles and Guidelines, 52 patent pending technologies, 50, 51 patentability of inventions, 40, 41 primate pluripotent embryonic stem cells, 42–44 prospects for stem cell line access, 54, 55 rights, 40 Thomson patent licensing, 46–48 Web site resources, 396 PD, see Parkinson’s disease PGC, see Primordial germ cell Plasticity, stem cells, see also specific cells and tissues, criteria, 89, 90, 103 fusion between stem cells, 103, 104 mechanisms in adult stem cells, 103–105 prospects for study, 105, 106 Pluripotency, definition, 27, 28 Primordial germ cell (PGC), fate-determining genes, 75 migration, 75 teratocarcinoma development, 66, 67, 74 Promoter, genetically-modified human embryonic stem cell usage, 273, 274 R RA, see Retinoic acid Regenerative medicine, inadequacy of small-molecule drugs, 239, 240 Research agreements, Material Transfer Agreement, 40, 52, 54, 404–409 Memorandum of Understanding, 401–411 Web site resources, 396, 401 Retinoic acid (RA), neuroepithelia differentiation from embryonic stem cells, 146, 149, 153 460 RNA inhibition, gene repression in human embryonic stem cells, 276, 277 S SCI, see Spinal cord injury Senescence, nuclear transfer advantages, 292 rodent versus human stem cells in culture, 242–245 Side population (Sp) cell, adult cell potency, 97 Skin progenitor (SKP) cell, adult cell potency, 98, 99 senescence of mouse versus human cells in culture, 244 SKP cell, see Skin progenitor cell Smooth muscle cell, see Vasculogenesis Sp cell, see Side population cell Spinal cord injury (SCI), fetal cell transplantation, 346, 373 hNT studies, clinical trial prospects, 374, 375 connectivity with host tissue, 370, 371 differentiation of grafts, 368 growth and histology, 366– 370, 374 motor-evoked potentials, 370, 371 overview, 365, 366 rat spinal cord injury model, 367–371 survival of graft, 367 uninjured spinal cord grafts in immunocompromised nude mice, 366, 367 Stem cell, see also specific cells, classification, 89, 162 definition, 89, 162 plasticity criteria, 89, 90, 103 Index Stroke, hNT studies, animal model studies, 355–359 mechanisms of benefits, 358, 359 Phase I/II clinical trials, brain imaging, 361, 362, 372, 373 chromosomal polyploidy, 362, 363 concerns, 359, 360 graft preparation, 358 immunosuppression, 360 implantation, 360 postmortem evaluation, 362, 363 safety, 361, 373, 374 study design, 360 survival of graft, 359 Subcloning, human embryonic stem cells, 133–136 T Telomerase, deficiency and aging, 243 induction, 243 telomere maintenance, 242, 243 Telomere, shortening in cell aging, 242, 243 TERA2, derivation, 71 subclones, see NT-2; NTERA2 teratoma formation, 71 Teratoma, see also Germ cell tumor, histology, 63 history of study, 63 mice, cell lines, 67, 68 induction, 66 primordial germ cell origins, 66, 67, 74 spontaneous formation, 65, 66 tissue distribution, 63, 64 TGF-β, see Transforming growth factor-β Index Therapeutic cloning, advantages, 291, 292 autologous cell therapy challenges, 246, 249, 250 clinical prospects, 278, 280, 281 cross-species nuclear transfer, 291 difficulty in humans, 288 efficiency, 291, 312, 313 embryonic stem cells, 241, 312 historical perspective, 285, 287 human egg requirements, 288, 291 non-egg donor cells, 291 principles, 278, 280, 281, 287, 288 prospects, 292, 293, 297, 298, 312 technical variables influencing outcomes in humans, 290, 291 Thomson patent, see Patents Transdifferentiation, see Plasticity, stem cells; specific cells and tissues Transfection, see Geneticallymodified human embryonic stem cells Transforming growth factor-β (TGF-β), cardiogenesis role, 182, 183 Transplant therapy, embryonic stem cells, cell replacement therapy, advantages, 300 clinical prospects, 300, 301 limitations, 300 principles, 300 drug delivery, 306 genetic engineering, see Genetically-modified human embryonic stem cells inductive therapy, 306, 307 in utero transplantation, delivery techniques, 301 461 indications, 302 rationale, 301 surgical intervention, 302 organ generation in vivo, 305, 306 organ replacement, cell types, 303, 305 principles, 303 tissue candidates, 304 overview, 299, 300 V Vasculogenesis, embryoid body potential, 202 hematopoietic stem cell role, 91 human embryonic stem cell differentiation, applications, angiogenesis inhibition model, 211, 212 cell transplantation and tissue engineering, 212, 213 culture, 202, 203 prospects for study, 213 rationale, 207, 208, 213 smooth muscle cell markers, 203 vascular progenitor cell differentiation culture, 208, 209 vasculogenic features, 209, 211 overview, 201 vascular progenitor cells, candidate markers, 205, 207 emergence, 204, 205 smooth muscle cell formation, 205 types, 204 W Web sites, see Internet resources Wnt, cardiogenesis role, 184 Human Embryonic Stem Cells Edited by Arlene Y Chiu Mahendra S Rao National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD National Institute on Aging, National Institutes of Health, Baltimore, MD Although there is great interest in the potential for using stem cells as cell replacements and other treatments for diseases that currently have no cure, research on the biology of human embryonic stem cells is still in its infancy In Human Embryonic Stem Cells, pioneers, leaders, and experts in this emerging field join forces to address all the key issues in the use of human pluripotent stem cells for treating degenerative diseases or for replacing tissues lost from trauma On the practical side, these topics range from the problems of deriving human embryonic stem cells and driving their differentiation along specific lineages, regulating their development into mature cells, and bringing stem cell therapy to clinical trials The authors cover the criteria used by investigators in different fields to recognize mature phenotypes of specific tissues Regulatory issues are addressed in discussions of the ethical debate surrounding the derivation of human embryonic stem cells and the current policies governing their use in the United States and abroad, including the rules and conditions regulating federal funding and questions of intellectual property Reviewing the most pressing issues involved in human embryonic stem cell research, Human Embryonic Stem Cells, provides an invaluable sourcebook for researchers seeking a review of their basic biology and an unbiased assessment of their potential for new therapies Features • Unbiased presentation of the potential of embryonic stem cells and the current state of the science • Discussion of the potential uses of human embryonic stem cells in a variety of applications • Summary of the ethical debate surrounding stem cells and the rules governing their use • Review of the sources, derivation, and maintenance of the major human pluripotent stem cells • Appendices detailing websites, stem cell patents, and material transfer agreements for the sharing of cells Contents Part I: Policy Ethical Issues Associated with Pluripotent Stem Cells A Researcher’s Guide to Federally Funded Human Embryonic Stem Cell Research in the United States Intellectual Property of Human Pluripotent Stem Cells Part II: Types of Pluripotent Cells Embryonal Carcinoma Cells: The Malignant Counterparts of ES and EG Cells Human Pluripotent Cells from Bone Marrow Protocols for the Isolation and Maintenance of Human Embryonic Stem Cells Subcloning and Alternative Methods for the Derivation and Culture of Human Embryonic Stem Cells Part III: Differentiation Differentiation of Neuroepithelia from Human Embryonic Stem Cells Pancreatic Differentiation of Pluripotent Stem Cells Human Embryonic Stem Cell-Derived Cardiomyocytes: Derivation and Characterization Vascular Lineage Differentiation from Human Embryonic Stem Cells Hematopoietic Progenitors Derived from Human Embryonic Stem Cells Part IV: Therapeutics Human Embryonic vs Adult Stem Cells for Transplantation Therapies Genetic Manipulation of Human Embryonic Stem Cells Human Therapeutic Cloning Therapeutic Uses of Embryonic Stem Cells Human Embryonic Stem Cells and the Food and Drug Administration: Assuring the Safety of Novel Cellular Therapies Studies of a Human Neuron-Like Cell Line in Stroke and Spinal Cord Injury: Preclinical and Clinical Perspectives Appendix I: Cell Lines and Companies Involved with Human Embryonic Stem Cell Research Appendix II: Useful Websites Appendix III: Research Agreements and Material Transfer Agreements Between Investigator and Stem Cell Provider Appendix IV: Stem Cell Patents Index 90000 HUMAN EMBRYONIC STEM CELLS ISBN: 1-58829-311-4 E-ISBN: 1-59259-423-9 humanapress.com 781588 293114 ... Data Human embryonic stem cells / edited by Arlene Chiu and Mahendra S Rao p cm Includes bibliographical references and index ISBN 1-5 882 9-3 1 1-4 (alk paper); e-ISBN 1-5 925 9-4 2 3-9 Human embryo Embryonic. .. Lineage Differentiation from Human Embryonic Stem Cells Sharon Gerecht-Nir and Joseph Itskovitz-Eldor 12 • Hematopoietic Progenitors Derived from Human Embryonic Stem Cells Dan S Kaufman... attention to stem cells Formal inquiries into the ethics of human pluripotent From: Human Embryonic Stem Cells Edited by: A Chiu and M S Rao © Humana Press Inc., Totowa, NJ Usdin stem cell research