Preface Since their isolation over twenty years ago, embryonic stem (ES) cells have come to play a prominent role in many different fields in biomedical research While used extensively for gene targeting studies for the generation of ‘‘knock-out mice,’’ it is their capacity to differentiate into a wide array of lineages in culture that has most recently captured the attention of basic scientists, clinical researchers, and the lay public The capacity of an ES cell to differentiate into almost any cell type in a culture dish offers unprecedented opportunities for studies in lineage commitment and development, gene function, and cancer In addition, the development of human ES cells in 1998 has expanded the potential uses of the in vitro differentiation approach to include the generation of specific cell types for cell replacement therapy and regenerative medicine It has been known for some time that mouse ES cells can differentiate in culture and generate derivatives of the three primary germ cell layers: ectoderm, endoderm, and mesoderm In many of these early studies, however, differentiation was not well controlled and the cultures often consisted of mixtures of tissues Significant advances in the methodologies of ES cell differentiation have been made in recent years, and it is now possible to generate relatively pure populations of cells from a number of different lineages in a reproducible fashion These new approaches have enabled investigators to begin to use the system to probe the molecular events regulating early lineage commitment, as well as to generate populations appropriate for cell replacement therapy in preclinical models With the establishment of human ES cells, the challenge for those in the field of stem cell and developmental biology is to now translate the information from the mouse to the human system In this volume, we have brought together a comprehensive collection of the most up-to-date methods for the differentiation of both mouse and human ES cells into a broad spectrum of lineages Together with the differentiation protocols, we have solicited a select set of protocols that highlight approaches for gene discovery and lineage selection using ES cells The chapters are written by leaders in the field from the international community and provide technical details that will enable the reader to establish and maintain ES cell cultures and induce their differentiation into a large number of different lineages We hope that this volume on Differentiation of Embryonic Stem Cells will find its way into many laboratories and proves to be useful at the bench xv xvi PREFACE for investigators world-wide We are extremely grateful to the many authors for their excellent contributions to this volume and their patience in dealing with publication schedules Finally, we extend our appreciation to Shirley Light at Academic Press who organized the assembly of this and hundreds of other volumes of Methods in Enzymology over several decades PAUL M WASSARMAN GORDON M KELLER Table of Contents CONTRIBUTORS TO VOLUME 365 ix PREFACE xv VOLUMES IN SERIES xvii Section I Differentiation of Mouse Embryonic Stem Cells Early Commitment Steps and Generation of Chimeric Mice Lineage Specific Differentiation of Mouse ES Cells: Formation and Differentiation of Early Primitive Ectoderm-like (EPL) Cells JOY RATHJEN AND PETER D RATHJEN Differentiation of F1 Embryonic Stem Cells into Viable Male and Female Mice by Tetraploid Embryo Complementation KEVIN EGGAN AND RUDOLF JAENISCH 25 Differentiation to Mesoderm Derivatives: Hematopoietic and Vascular Hematopoietic Commitment of ES Cells in Culture MARION KENNEDY AND GORDON M KELLER 39 In Vitro Differentiation of Mouse Embryonic Stem Cells: Hematopoietic and Vascular Cell Types STUART T FRASER, JUN YAMASHITA, L MARTIN JAKT, MITSUHIRO OKADA, MINETARO OGAWA, SATOMI NISHIKAWA, AND SHIN-ICHI NISHIKAWA 59 KENJI KITAJIMA, MAKOTO TANAKA, JIE ZHENG, EIKO SAKAI-OGAWA, TORU NAKANO 72 In Vitro Differentiation of Mouse Embryonic Stem Cells to Hematopoietic Cells on an OP9 Stromal Cell Monolayer AND In Vitro Differentiation of Mouse ES Cells: Hematopoietic and Vascular Development JOSEPH B KEARNEY AND VICTORIA L BAUTCH 83 In Vitro Differentiation of Mouse ES Cells into Hematopoietic, Endothelial, and Osteoblastic Cell Lineages: The Possibility of In Vitro Organogenesis MOTOKAZU TSUNETO, TOSHIYUKI YAMANE, HIROMI OKUYAMA, HIDETOSHI YAMAZAKI, AND SHIN-ICHI HAYASHI 98 v vi TABLE OF CONTENTS Development of Hematopoietic Repopulating Cells from Embryonic Stem Cells The In Vitro Differentiation of Mouse Embryonic Stem Cells into Neutrophils 10 Development and Analysis of Megakaryocytes from Murine Embryonic Stem Cells 11 Development of Lymphoid Lineages Embryonic Stem Cells In Vitro from 12 Probing Dendritic Cell Function by Guiding the Differentiation of Embryonic Stem Cells 13 Gene Targeting Strategies for the Isolation of Hematopoietic and Endothelial Precursors from Differentiated ES Cells 14 Establishment of Multipotent Hematopoietic Progenitor Cell Lines from ES Cells Differentiated In Vitro 15 Vasculogenesis and Angiogenesis from In Vitro Differentiation of Mouse Embryonic Stem Cells MICHAEL KYBA, RITA C R PERLINGEIRO, AND GEORGE Q DALEY 114 JONATHAN G LIEBER, GORDON M KELLER, G SCOTT WORTHEN 129 AND KOJI ETO, ANDREW L LEAVITT, TORU NAKANO, AND SANFORD J SHATTIL 142 SARAH K CHO AND JUAN CARLOS ă ZUNIGA-PFLUCKER 158 PAUL J FAIRCHILD, KATHLEEN F NOLAN, HERMAN WALDMANN AND 169 WEN JIE ZHANG, YUN SHIN CHUNG, BILL EADES, AND KYUNGHEE CHOI 186 LEIF CARLSSON, EWA WANDZIOCH, ´ ´ PERPETUA PINTO DO O, ˚ AND ASA KOLTERUD 202 OLIVIER FERAUD, ´ ` MARIE-HELENE PRANDINI, AND DANIEL VITTET 214 Other Derivatives of Mesoderm 16 ES Cell Differentiation to the Cardiac Lineage KENNETH R BOHELER 228 17 Bone Nodule Formation via In Vitro Differentiation of Murine Embryonic Stem Cells SARAH K BRONSON 241 18 In Vitro Differentiation of Mouse ES Cells: Bone and Cartilage JAN KRAMER, CLAUDIA HEGERT, AND ă JURGEN ROHWEDEL 251 BRIGITTE WDZIEKONSKI, PHI VILLAGEOIS, AND CHRISTIAN DANI 268 19 Development of Adipocytes from Differentiated ES Cells Differentiation to Endoderm Derivatives 20 In Vitro Differentiation of Embryonic Stem Cells into Hepatocytes TAKASHI HAMAZAKI NAOHIRO TERADA AND 277 vii TABLE OF CONTENTS 21 Differentiation of Mouse Embryonic Stem Cells into Pancreatic and Hepatic Cells GABRIELA KANIA, PRZEMYSLAW BLYSZCZUK, JAROSLAW CZYZ, ANNE NAVARRETE-SANTOS, AND ANNA M WOBUS 287 Differentiation to Ectoderm Derivatives 22 Generating CNS Neurons from Embryonic, Fetal, and Adult Stem Cells JONG-HOON KIM, DAVID PANCHISION, RAJA KITTAPPA, AND RON MCKAY 303 23 Defined Conditions for Neural Commitment and Differentiation QI-LONG YING AND AUSTIN G SMITH 327 24 Development of Melanocytes from ES Cells TAKAHIRO KUNISADA, TOSHIYUKI YAMANE, HITOMI AOKI, NAOKO YOSHIMURA, KATSUHIKO ISHIZAKI, AND TSUTOMU MOTOHASHI 341 Section II Differentiation of Mouse Embryonic Germ Cells 25 Isolation and Culture of Embryonic Germ Cells MARIA P DE MIGUEL AND PETER J DONOVAN 353 Section III Gene Discovery by Manipulation of Mouse Embryonic Stem Cells 26 Gene Trap Mutagenesis in Embryonic Stem Cells WEISHENG V CHEN PHILIPPE SORIANO AND 367 27 Gene Trap Vector Screen for Developmental Genes in Differentiating ES Cells HEIDI STUHLMANN 386 28 Gene-Based Chemical Mutagenesis in Mouse Embryonic Stem Cells YIJING CHEN, JAY L VIVIAN, AND TERRY MAGNUSON 406 Section IV Differentiation of Monkey and Human Embryonic Stem Cells 29 Growth and Differentiation Monkey ES Cells of Cynomolgus 30 Isolation, Characterization, and Differentiation of Human Embryonic Stem Cells HIROFUMI SUEMORI NORIO NAKATSUJI AND MARTIN F PERA, ADAM A FILIPCZYK, SUSAN M HAWES, AND ANDREW L LASLETT 419 429 viii TABLE OF CONTENTS 31 Factors Controlling Human Embryonic Stem Cell Differentiation MAYA SCHULDINER NISSIM BENVENISTY 32 Development of Cardiomyocytes from Human ES Cells IZHAK KEHAT, MICHAL AMIT, AMIRA GEPSTEIN, IRIT HUBER, JOSEPH ITSKOVITZ-ELDOR, AND LIOR GEPSTEIN AND 446 461 AUTHOR INDEX 475 SUBJECT INDEX 501 Contributors to Volume 365 Article numbers are in parentheses following the names of contributors Affiliations listed are current MICHAL AMIT (32), Department of Physiology and Biophysics, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, POB 9649, Haifa 31096, Israel Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, A2-025, P.O Box 19024, Seattle, Washington 98109-1024 YIJING CHEN (28), Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 HITOMI AOKI (24), Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, 5008705, Japan SARAH K CHO (11), Division of Biological Sciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093 VICTORIA L BAUTCH (6), Department of Biology and Program in Genetics, University of North Carolina at Chapel Hill, CB#3280, Chapel Hill, North Carolina 27599 KYUNGHEE CHOI (13), Department of Pathology & Immunology, Washington University School of Medicine, 660 S Euclid Avenue, Box 8118, St Louis, Missouri 63110 NISSIM BENVENISTY (31), Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel YUN SHIN CHUNG (13), Department of Pathology & Immunology, Washington University School of Medicine, 660 S Euclid Avenue, Box 8118, St Louis, Missouri 63110 PRZEMYSLAW BLYSZCZUK (21), In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, Corrensstr 3, Gatersleben, D-06466, Germany JAROSLAW CZYZ (21), In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research Corrensstr 3, Gatersleben, D-06466, Germany KENNETH R BOHELER (16), Molecular Cardiology Unit, Laboratory of Cardiovascular Science, National Institute on Aging, NIH 5600 Nathan Shock Drive, Baltimore, Maryland 21224 GEORGE Q DALEY (8), Whitehead Institute for Biomedical Research, Harvard Medical School, Cambridge, Massachusetts 021421479 SARAH K BRONSON (17), Department of Cellular & Molecular Physiology, Penn State College of Medicine, The Milton S Hershey Medical Center, H166, 500, University Drive, Hershey, Pennsylvania 17033-0850 CHRISTIAN DANI (19), Centre de Biochimie, Institut de Recherches Signalisation, Biologie du Developpement et Cancer, Universite´ de Nice-Sophia Antipolis, UMR 6543 CNRS, Parc Valrose, Nice cedex 2, 06108 France ˚ LEIF CARLSSON (14), Umea Centre for Mole˚ cular Medicine, Umea University, 901 87 ˚, Umea Sweden MARIA P DE MIGUEL (25), Department of Microbiology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 WEISHENG V CHEN (26), Program in Developmental Biology, Division of Basic ix x CONTRIBUTORS PETER J DONOVAN (25), Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 BILL EADES (13), Department of Pathology & Immunology, Washington University School of Medicine, 660 S Euclid Avenue, Box 8118, St Louis, Missouri 63110 KEVIN EGGAN (2), Whitehead Institute for Biomedical Research, Cambridge Center, Cambridge, Massachusetts 02142 KOJI ETO (10), Division of Vascular Biology, Department of Cell Biology, Scripps Research Institute, 10550 North Torrey Pines Road, VB-5, La Jolla, California 92037 PAUL J FAIRCHILD (12), Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK OLIVIER FERAUD (15), Laboratoire Developpement et Vieillissement de L’endothelium, EMI INSERM 0219, DRDC/DVE, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble cedex 09, France ADAM A FILIPCZYK (30), Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria, 3168, Australia STUART T FRASER (4), Laboratory of Molecular Mouse Genetics, Institute for Toxicology, Johannes Gutenberg-University, Obere Zahlbacher Strasse 67, Mainz 55131, Germany LIOR GEPSTEIN (32), Cardiovascular Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, POB 9649, Haifa 31096, Israel Medicine, P.O Box 100275, Gainesville, Florida 32610 0275 SUSAN M HAWES (30) Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria, 3168, Australia SHIN-ICHI HAYASHI (7), Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 NishiMachi, Yonago, Tottori 683-8503, Japan CLAUDIA HEGERT (18), Department of Medical Molecular Biology, Medical University of Lu ăbeck, Ratzeburger Allee 160, Luăbeck, D-23538, Germany IRIT HUBER (32), Cardiovascular Research Laboratory, Department of Biophysics and Physiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, POB 9649, Haifa 31096, Israel KATSUHIKO ISHIZAKI (24), Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu 500-8705, Japan JOSEPH ITSKOVITZ-ELDOR (32), Department of Physiology and Biophysics, Bruce Rappaport Faculty of Medicine, TechnionIsrael Institute of Technology, Efron Street, POB 9649, Haifa 31096, Israel RUDOLF JAENISCH (2), Whitehead Institute for Biomedical Research, Cambridge Center, Cambridge, Massachusetts 02142 L MARTIN JAKT (4), RIKEN Centre for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, Japan 650-0047 AMIRA GEPSTEIN (32), Cardiovascular Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, POB 9649, Haifa 31096, Israel GABRIELA KANIA (21), In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, Corrensstr 3, Gatersleben, D-06466, Germany TAKASHI HAMAZAKI (20), Department of Pathology University of Florida College of JOSEPH B KEARNEY (6), Program in Genetics and Molecular Biology, University of North CONTRIBUTORS xi Carolina at Chapel Hill CB#3280, Chapel Hill, North Carolina 27599 University, 246 Clayton Road, Clayton, Victoria, 3168, Australia IZHAK KEHAT (32), Cardiovascular Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, POB 9649 Haifa 31096, Israel ANDREW L LEAVITT (10), Departments of Laboratory Medicine and Internal Medicine, University of California at San Francisco, San Francisco, California 94143 GORDON M KELLER (3,9), Carl C Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1496, New York, New York 10029-6574 MARION KENNEDY (3), Carl C Icahn Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1496, New York, New York 10029-6574 JONG-HOON KIM (22), Laboratory of Molecular Biology, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4092 KENJI KITAJIMA (5), Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita Osaka, Osaka 565-0871, Japan RAJA KITTAPPA (22), Laboratory of Molecular Biology, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4092 ˚ ˚ ASA KOLTERUD (14), Umea Centre for Mole˚ cular Medicine, Umea University, 901 87 ˚, Umea Sweden JAN KRAMER (18), Department of Internal Medicine I, University of Luăbeck, Lu ăbeck, D-23538, Germany TAKAHIRO KUNISADA (24), Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu 500-8705, Japan MICHAEL KYBA (8), Center for Developmental Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9133 ANDREW L LASLETT (30), Monash Institute of Reproduction and Development, Monash JONATHAN G LIEBER (9), Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206 TERRY MAGNUSON (28), Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 RON MCKAY (22), Laboratory of Molecular Biology, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4092 TSUTOMU MOTOHASHI (24), Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu 500-8705, Japan TORU NAKANO (5,10), Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita Osaka, Osaka 565-0871, Japan NORIO NAKATSUJI (29), Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan ANNE NAVARRETE-SANTOS (21), Department of Anatomy and Cell Biology, Martin Luther University, Halle-Wittenberg, Halle (Saale), D-06108, Germany SATOMI NISHIKAWA (4), RIKEN Centre for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe 650-0047, Japan SHIN-ICHI NISHIKAWA (4), RIKEN Centre for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, Japan 650-0047 xii CONTRIBUTORS KATHLEEN F NOLAN (12), Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK University of Luăbeck, Ratzeburger Allee 160, Luăbeck, D-23538, Germany MINETARO OGAWA (4), Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan EIKO SAKAI-OGAWA (5), Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita Osaka, Osaka 565-0871, Japan MITSUHIRO OKADA (4), Department of Cell Differentitation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan 650-0047 MAYA SCHULDINER (31), Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel HIROMI OKUYAMA (7), Biotechnology Research Laboratories, Takara Bio Inc., Otsu, Siga 520-2193, Japan SANFORD J SHATTIL (10), Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, VB-5, La Jolla, California 92037 DAVID PANCHISION (22), Laboratory of Molecular Biology, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4092 MARTIN F PERA (30), Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria 3168, Australia RITA C R PERLINGEIRO (8), ViaCell Inc, 26 Landsdowne St, Cambridge, MA 2139 ´ ´ ˚ PERPETUA PINTO DO O (14), Umea Centre ˚ for Molecular Medicine, Umea University, ˚, 901 87 Umea Sweden ´ ` MARIE-HELENE PRANDINI (15), Laboratoire Developpement et Vieillissement de L’endothelium, EMI INSERM 0219, DRDC/ DVE, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble cedex 09, France JOY RATHJEN (1), Department of Molecular Biosciences and ARC Special Research Centre for the Molecular Genetics of Development, Adelaide University, Molecular Life Sciences/335, Adelaide, 5005, Australia AUSTIN G SMITH (23), Institute for Stem Cell Research, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, Scotland EH9 3JQ, UK PHILIPPE SORIANO (26), Program in Developmental Biology, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, A2-025, P.O Box 19024, Seattle, Washington 98109-1024 HEIDI STUHLMANN (27), Department of Cell Biology, Division of Vascular Biology, Scripps Research Institute, Mail CVN-26, 10550 North Torrey Pines Road, La Jolla, California 92037 HIROFUMI SUEMORI (29), Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan MAKOTO TANAKA (5), Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita Osaka, Osaka 565-0871 Japan PETER D RATHJEN (1), Department of Molecular Biosciences and ARC Special Research Centre for the Molecular Genetics of Development, Adelaide University, Molecular Life Sciences/335, Adelaide, 5005, Australia NAOHIRO TERADA (20), Department of Pathology, University of Florida College of Medicine, P.O Box 100275, Gainesville, Florida 32610-0275 ă JURGEN ROHWEDEL (18) Department of Medical Molecular Biology, Medical MOTOKAZU TSUNETO (7), Division of Immunology, Department of Molecular and 495 AUTHOR INDEX Stein, H., 223 Stein, M., 290 Stein, P., 90 Steinbeisser, H., 17 Steinkasserer, A., 182 Steinman, R M., 169, 170 Stern, M D., 181, 236 Stern, P., 435 Stevens, L C., 353 Stevenson, L., 283, 406 Stewart, A F., 367 Stewart, C L., 186, 231, 233, 251 Stewart, T J., 187, 190(26), 252, 328 St Jacques, B., 287 Stock, J L., 242, 244(8) Stocker, E., 283 Stoppaciaro, A., 220 Stott, D., 358 Stracey, C., 447 Strand, A., 385 Strauss, J., 29 Strebhardt, K., 381 Strong, V., 170, 175(8), 177, 177(8), 178(8) Strouboulis, J., 116 Strubing, C., 187, 190(24), 387 ă Studer, L., 303, 304(4), 315, 322(14,16), 327, 335(4) Stuhlmann, H., 78, 84, 85, 143, 147(16), 149(16), 151(16), 155(16), 156(16), 381, 386, 387, 389, 398(30,31), 402(31), 404(30,31), 405, 405(30) Sturm, V., 252 Su, G H., 93 Suda, T., 65 Sudo, T., 73, 98, 131, 147, 159 Suemori, H., 419, 421, 425 Sullivan, A., 60 Sullivan, M., 262 Sun, W., 277 Sun, Y., 315, 322(19), 323(19) Surani, M A., 368 Suratt, B T., 130, 131(1) Sussel, L., 288 Suzuki, T., 170 Swat, W., 127 Swiergiel, J J., 185, 186, 277, 419, 429, 435(1), 446, 461 T Tabar, V., 315, 322(14,16) Tada, T., 421 Taga, T., 280 Tagaya, H., 98 Takagi, T., 79, 80(16), 143, 147(13), 149(13) Takahashi, K., 286, 342 Takahashi, M., 425 Takahashi, T., 78, 79, 80(16), 143, 147(13), 149(13) Takakura, N., 64 Takase, K., 281, 284(33), 289 Takayanagi, K., 372 Takeda, N., 372 Takeichi, M., 65 Takenami, T., 290 Takiguchi, M., 279, 281(21) Tal, D., 471 Tamura, K., 74 Tanaka, M., 72 Tanaka, T., 65 Tanaka, T S., 385 Tanaka, Y., 281, 284(33), 289 Taniguchi, S., 368, 372(16) Tanio, Y., 98 Tapscott, S J., 385 Tarabykin, V., 380 Tarasov, K V., 181, 236 Teepe, M., 119 Teichmann, G., 95 Teillet, M A., 316 Temple, S., 315 Tenen, D G., 93 Terada, N., 277, 281, 284(33), 286, 286(32), 289 Teramoto, K., 281, 284(33), 289 Teraoka, H., 281, 284(33), 289 Tertoolen, L., 449(20), 453, 459(20) Tessier-Lavigne, M., 381 Theill, L E., 98 Theodosiou, N G., 15 Thomas, K R., 367 Thomas, M K., 289, 290(31) Thomas, T., 382 Thompson, S., 180(17), 181, 367 Thompson-Snipes, L., 159 Thomson, J A., 143, 185, 186, 277, 419, 420, 429, 434, 435, 435(1), 446, 448(35), 449(19), 453, 457(19), 460, 461, 463 496 AUTHOR INDEX Thorsteinsdottir, U., 124 Threadgill, D W., 409 Timmermans, J P., 454 Timmons, P M., 14, 15(10), 17(10), 21(10) Ting, Y T., 315, 322(16) Tintrup, H., 386 Titeux, M., 119 Toda, K., 65 Tokunaga, T., 372 Tokusashi, Y., 277 Toman, D., 262 Tone, M., 170, 175(8), 177, 177(8), 178(8) Tone, Y., 170, 175(8), 177, 177(8), 178(8) Tong, J Z., 290 Torii, R., 421 Torres, M., 288 Tosh, D., 284, 289 Trask, T., 219 Trent, J M., 385 Trinder, P K E., 182 Tropepe, V., 328, 329(14) Trounson, A., 419, 429, 435, 446, 448(2), 449(2), 450(2), 454(2), 461, 465(4) Troy, A E., 269 Tsai, B C., 289 Tsai, R Y., 315, 322(18) Tsoulfas, P., 315, 322(17) Tsuchida, K., 64 Tsuji, K., 74, 280 Tsuji, T., 290 Tsujimoue, H., 286 Tsuneto, M., 98, 100 Tsunoda, J., 65 Tsunoda, Y., 284, 289 Tucker, K L., 330, 334(26), 393, 459 Turetsky, D., 252 Turetsky, T., 448(25), 454, 460(25) Tweedie, D., 181, 230, 236 Tzukerman,M.,289,420,449(13),450,459(13) U Uchida, N., 166 Uehara, Y., 280 Uemura, Y., 387 Ullrich, A., 40, 52(13), 60 Unkeless, J C., 197 Urano, F., 425 Urbach, A., 460 Urbanek, P., 26, 31(4) Ure, J M., 283 Usman, N., 380 Uzan, G., 61, 62(9), 90, 143, 187, 190(16), 193(16), 215, 219(4), 221(4), 223(4), 450 V Vaeyens, F., 290 Vainchenker, W., 119, 143 Vallageois, P., 269 Vallejo, M., 289, 290(31) Vallier, L., 381 Vallis, K A., 82, 85, 406 van den Brink, C E., 449(20), 453, 459(20) Vandenhoeck, A., 218 van der Heyden, M., 449(20), 453, 459(20) van der Hoeven, F., 367 van der Kooy, D., 328, 329(14) van der Zee, R., 60 Van Eyken, P., 283 van Putten, W., 435 Vasa, S R., 289 Vecchi, A., 220 Veiby, O P., 159, 160(7) Velasco, I., 278, 289, 290(22) Velculescu, V E., 179 Verma, I M., 457 Vernallis, A., 269 Vernochet, C., 250, 269 Vescovi, A L., 314 Vicario-Abejon, C., 315, 322(17) Vigano, A., 14, 15(10), 17(10), 21(10) Vijaya, S., 369 Vilasante, A., 262 Villacorta, R., 118 Villageois, P., 187, 190(28), 268, 269, 328 Villeval, J L., 143 Visvader, J E., 127, 195 Vitrat, N., 143 Vittet, D., 61, 62(9), 90, 187, 190(16), 193(16), 214, 215, 219(4), 221(4–6), 223(4), 224(5), 450 Vivian, J L., 406, 407, 408(2) Vogelstein, B., 179 von Borstel, N J., 195 von Keitz, A., 435 von Melchner, H., 368, 371(15), 381, 388 von Ruden, T., 61, 62(12) Voss, A K., 382 Vuorio, E., 262 AUTHOR INDEX W Wagner, E F., 26, 31(4), 61, 62(12), 186, 288 Wagner, E G., 233 Wagner, M., 289, 290(33), 291(33), 292(33), 299(33), 300(33) Wainwright, B J., 377 Wakasugi, S., 368, 372(16) Waknitz, M A., 185, 186, 277, 419, 429, 434, 435(1), 446, 461, 463 Walcz, E., 262 Walderich, B., 408, 414(6) Waldmann, H., 169, 170, 175(8), 177, 177(8), 178(8), 180(17), 181, 182(9) Wall, C., 40, 41(5), 82, 190, 193, 193(35), 198(35), 199(35) Wallukat, G., 89, 229 Walluket, G., 464 Walther, B T., 287 Wandzioch, E., 202, 203, 209(2), 210(2) Wang, C.-U., 242 Wang, D., 262 Wang, L C., 127 Wang, P J., 29 Wang, R., 84, 89(6), 187, 190(15), 214, 387 Wang, S., 277 Wang, X., 385 Wang, Y., 393, 459 Wang, Z Q., 26, 31(4), 385 Ward, D., 449(20), 453, 459(20) Wartenberg, M., 216, 239, 462 Washington, J M., 4, 6(3), 8, 8(3), 12(3,7), 16(3), 22(3) Wasserman, R., 167, 205 Wassif, C., 203, 214(3) Watanabe, K., 425 Watanabe, M., 281, 284(33), 289 Watt, A., 283, 406 Watt, F M., 187, 190(27) Watt, S M., 40 Wdziekonski, B., 268, 269 Webb, G C., 15 Webb, S., 130, 131(1), 135, 277 Webber, T D., 78, 160, 166(13), 168(13) Weber, J S., 368, 408 Weidle, U H., 223 Weiher, H., 369 Weiss, S., 314 Weissman, I L., 166 Wellner, M C., 229, 239(6), 263, 295 497 Wendel, M., 262 Wendling, F., 119 Werb, Z., 15 Wernig, M., 448(35), 460 Westphal, H., 203, 214(3) White, G., 143, 154(10), 155(10), 156(10), 157(10) Whitney, M., 381 Whyatt, L M., 251 Wichterle, H., 327, 328(5), 329(5) Widlund, H R., 385 Wiedemann, B., 187, 190(24), 387 Wienholds, E., 408, 414(6) Wiestler, O D., 252 Wilcox, D., 143, 154(10), 155(10), 156(10), 157(10) Wiles, M V., 41, 61, 62(11,13), 73, 84, 115, 130, 134, 136(5), 143, 187, 190(10,11), 193(11), 203, 217, 218(14), 221(14), 328, 329(15), 387 Willbold, E., 290, 294(44) Willhoite, A R., 326 Williams, R L., 15, 186, 233, 251 Willson, T A., 186, 233, 251 Wilson, D B., 223, 288 Wilson, D I., 283, 284, 289 Winkler, T H., 167 Wintrobe, M., 130 Winzeler, E A., 385 Witte, O N., 80, 81(19), 158 Witzemann, V., 267 Witzenbichler, B., 60 Wizigmann-Voos, S., 40 Wobus, A M., 89, 175, 181, 187, 190, 190(18,19,21,22,24,27,30), 229, 230, 231, 236, 239(6), 240, 240(7,9), 251, 252, 254, 259(15,16), 260(16), 263, 264(16), 265(16), 267, 267(15), 268, 268(16), 278, 283, 287, 289, 290(20,33), 291, 291(20,33), 292, 292(33), 295, 295(20), 299(33), 300(33), 327, 387, 452, 464, 466 Wong, G G., 186, 233, 251 Wong, H., 263 Wong, S., 80, 158 Wood, C R., 40 Wood, H., 187, 190(29) Wood, K G., 385 Worthen, G S., 129, 130, 131(1) Woychik, R P., 368, 407, 409(1) Wright, W E., 275 498 AUTHOR INDEX Wu, F J., 290 Wu, X., 262 Wu, X F., 41, 195 Wurst, W., 145, 171, 283, 406 Wutz, A., 120 Wylie, C C., 358 X Xiao, W., 411 Xiong, J.-W., 84, 85, 381, 389, 398(30,31), 402(31), 404(30,31), 405, 405(30) Xu, C., 420, 428, 458, 462 Xu, M., 74 Xu, M J., 280 Xu, Y., 159, 203, 214(3) Xynos, J D., 187, 190(29) Y Yagi, T., 372 Yamada, G., 263, 268(38) Yamada, T., 284, 289 Yamaguchi, T P., 40, 41, 60, 195 Yamaguchi, Y., 65 Yamamoto, M., 61, 62(16) Yamamura, F., 342 Yamamura, I., 279, 281(21) Yamamura, K., 368, 372(16), 386, 387 Yamane, T., 78, 98, 99, 112(9), 341, 342, 343(4) Yamashita, J., 59, 60, 62(6), 187, 190(17), 193(17), 220, 278 Yamato, E., 278 Yamazaki, H., 78, 98, 99, 112(9), 342, 343(4) Yamazaki, N., 170 Yan, H.-C., 219 Yanagisawa, M., 342 Yang, F., 29 Yang, H.-T., 230, 289, 290(20), 291(20), 295(20) Yang, L., 289, 290(29) Yanuka, O., 185, 428, 446, 447, 447(3), 448(3,4,24), 449(3,4), 450(3,4), 453(4), 454, 454(3), 455, 455(4), 457(29), 459(3), 462 Yao, M., 187, 190(23), 328, 387 Ybarrondo, B., 143, 154(10), 155(10), 156(10), 157(10) Ye, W., 335 Yee, D., 368, 407, 408(2), 409, 409(1) Yeom, Y I., 14, 15(12), 17(12), 21(12) Yin, Y., 285 Ying, Q.-L., 327, 329, 330, 333(21), 334(21), 335(21), 336(21), 341(21) Yoder, M C., 60 Yokomizo, T., 61, 62(16) Yonemura, S., 278 Yonemura, Y., 119 Yoshida, H., 61, 62(16), 73, 98, 131, 147, 159 Yoshida, K., 280 Yoshida, M., 372 Yoshida, Y., 277 Yoshikawa, K., 425 Yoshikawa, M., 284, 286, 289 Yoshimura, N., 341 Yoshimuta, J., 386 Yoshinobu, K., 386 Yoshitomi, H., 280 Young, L S., 182 Young, P E., 40 Young, S K., 130, 131(1) Yu, R T., 60 Yu, X., 143 Yu, X.-J., 205 Yu, Y., 85 Yujiri, T., 277, 286 Yurugi, T., 60, 62(6), 187, 190(17), 193(17), 220, 278 Z Zachgo, J., 388, 389(20) Zahn, D., 381 Zambrowicz, B P., 121, 372 Zandstra, P W., 125, 216 Zaret, K S., 280, 287, 288, 288(3), 289 Zehnbauer, B A., 116 Zeigler, F C., 119 Zelenika, D., 180(17), 181 Zeng, H., 119 Zerwes, H G., 61, 62(8), 83, 187, 190(14), 214 Zevnik, B., 329, 330(19), 386 Zhang, L., 179 Zhang, P., 367 Zhang, R., 262 Zhang, S C., 448(35), 460 Zhang, W J., 186, 195 Zheng, B., 368 AUTHOR INDEX Zheng, M., 280, 288, 289 Zheng, Q Y., 409, 414(9) Zheng, Z., 118 Zhou, B., 90 Zhu, L., 205 Zimmerman, L B., 290, 304 Ziomek, C A., 15 Zipori, D., 127 Zlokarnik, G., 381 499 Zon, L I., 281, 286(32), 289 Zorn, A M., 454 Zschiesche, W., 280, 288 Zsebo, K M., 342, 353, 354(4), 362(4), 363(4) Zulewski, H., 289, 290(31) Zuniga-Pflucker, J C., 78, 158, 160, 166, 166(13), 167, 167(17), 168(13) Zuschratter, W., 267, 268 Subject Index A C Adipocyte, formation from mouse embryonic stem cells adipogenesis phases, 269–271 embryoid body differentiation culture, 272–274 embryonic stem cell maintenance, 271–272 gene expression analysis, 275–276 materials and media, 274–275 overview, 268–271 Angiogenesis, see Vasculogenesis Calcium flux, imaging in differentiated cardiomyocytes, 469–470 Cardiomyocyte cynomolgus monkey embryonic stem cell differentiation, 427 human embryonic stem cell formation application potential, 461, 473 calcium flux imaging, 469–470 embryoid body formation, 464–465 embryonic stem cell maintenance, 463–465 immunofluorescence microscopy, 465–467 mechanical activity assay, 472 mouse embryonic fibroblast feeder layer preparation, 462–463 multi-electrode array recording, 470–472 pharmacological studies, 472 proliferation and cell cycle regulation assay, 467–468 transmission electron microscopy, 469 mouse embryonic stem cell formation applications, 229–230 cell lines, 231 contamination prevention, 231–232 embryoid body culture hanging drop, 238–239 mass culture, 239–240 embryonic fibroblast feeder cell preparation, 233–236 embryonic stem cell maintenance, 230–231, 236–237 isolation of cardiomyocytes, 240–241 media and sera, 231–233 Chondrocyte, formation from mouse embryonic stem cells chondrogenic cell isolation from embryoid bodies, 264–265 efficiency optimization, 266–268 embryoid body differentiation hanging drop culture, 258 plating, 258–259 B B cell, OP9 stromal cell monolayer culture of mouse embryonic stem cells for differentiation cytokines, 159–160 differentiation culture, 78–79, 164–166 embryonic stem cell maintenance, 163 flow cytometry analysis, 166–168 kinetics of differentiation, 168 materials, 160–163 OP9 cell maintenance, 163–164, 168 overview, 158–159 Beta cell embryogenesis, 287–289 formation from mouse embryonic stem cells application prospects, 288–289 differentiation culture, 291–292 embryonic stem cell maintenance, 291 histotypic differentiation into spheroids, 294 immunofluorescence analysis, 295–298 immunohistochemistry of spheroids, 298 insulin enzyme-linked immunosorbent assay, 298–300 nestin expression by progenitors and selection, 289–290, 292, 299–300 reverse transcription—polymerase chain reaction analysis, 294–296, 300 Blast colony-forming cell, see Hematopoiesis Bone nodule, see Osteoprogenitor 501 502 SUBJECT INDEX Chondrocyte, formation from mouse embryonic stem cells (Cont.) suspension culture, 258 embryonic fibroblast feeder preparation, 254–255 embryonic stem cell maintenance, 255, 258 gene expression analysis with reverse transcription–polymerase chain reaction, 259–263 histochemical staining, 265–266 immunostaining analysis, 262–263 materials, 252–254 media and additives, 253 whole-mount fluorescence in situ hybridization, 263–264 Confocal laser scanning microscopy megakaryocytes, 156–157 vasculogenesis studies using green fluorescent protein-expressing cells, 85–86, 94–97 D DC, see Dendritic cell Dendritic cell formation from mouse embryonic stem cells applications, 185–186 embryoid body generation, 173–174 embryonic stem cell maintenance, 171–173 genetic modification, 181–184 induction and expansion from embryoid bodies, 174–177 maturation characteristics, 177–178 rationale, 170, 184–185 serial analysis of gene expression, 178–181 functions, 169–170 E EB, see Embryoid body EC cell, see Embryonic carcinoma cell Ectoderm-like cells applications, 4, 24 culture from mouse embryonic stem cells adherent culture, 12–13 embryonic stem cell maintenance and morphology analysis, 10–11 materials consumables, fetal calf serum and assays, 5–8 gelatin, 8–10 media, MEDII media preparation and quality control, 11–12, 23–24 suspension culture, 13–14 ectoderm formation culture conditions, 19–20 marker analysis, 20–21 markers, 14–16 mesoderm formation culture conditions, 16–17 marker analysis, 17–19 trypsinization, 16–18 morphology, 14 reversion to embryonic stem cells, 22–23 EG cell, see Embryonic germ cell ELISA, see Enzyme-linked immunosorbent assay Embryoid body adipocyte formation, see Adipocyte cardiomyocyte formation, see Cardiomyocyte chondrocyte formation, see Chondrocyte cynomolgus monkey cell formation, 426–427 dendritic cell formation, see Dendritic cell development, 387–388 differentiation, 3–4 hanging drop culture, 89, 124–125, 238–239, 258 hematopoietic commitment in culture, see Hematopoiesis hepatocyte formation, see Hepatocyte human embryonic stem cell formation, 445, 450–452 mesoderm formation, see Ectoderm-like cell neuron formation, see Neuron osteoprogenitor formation, see Osteoprogenitor vasculogenesis studies, see Mesoderm; Vasculogenesis Embryonic carcinoma cell aneuploidy, 353 origins, 353 pluripotency, 353 SUBJECT INDEX Embryonic germ cell isolation and culture from mouse derivation from primordial germ cells, 359–360 freezing and thawing, 361–362 materials feeder cells, 362–363 fixatives, 354–355 media, 354 serum, 362 solutions, 354 passaging, 360–361 primordial germ cell isolation and culture culture, 358 embryo dissection, 355–357, 362 identification, 358 proliferation assays, 358–359 trypsinization and homogenization, 357 origins, 353 pluripotency, 353 Embryonic stem cell adipocyte formation, see Adipocyte beta cell formation, see Beta cell cardiomyocyte formation, see Cardiomyocyte chondrocyte formation, see Chondrocyte cynomolgus monkey cells applications, 420, 428–429 colony dissociation, 421–422 differentiation induction, 424–427 embyroid body formation, 426–427 markers, 419, 421 medium, 422 mouse embryonic fibroblast feeder preparation, 422–423 subculture, 422–424 teratoma formation in severe combined immunodeficient mice, 427–428 dendritic cell formation, see Dendritic cell differentiation markers, overview, 277–278 ectoderm-like cell formation, see Ectoderm-like cell endothelial cell formation, see Endothelial cell gene targeting approaches, 85, 251 hematopoietic commitment in culture, see Hematopoiesis hepatocyte formation, see Hepatocyte 503 history of study, 229 human cells comparison with other mammalian cell lines, 429–430 differentiation cardiomyocyte formation, see Cardiomyocyte culture, 444–446 embryoid body formation, 445, 450–452 functional assays, 459 growth factors, 452–455 morphology analysis, 456 potential, 446–450 ethics, 430 flow cytometry, 437–438, 456–457 immunofluorescence microscopy, 436–437 immunohistochemistry, 434–436 immunomagnetic isolation of viable cells, 438, 440 inner cell mass isolation, 431 karyotyping, 434 maintenance, 434 mouse embryonic fibroblast feeder preparation, 431–432 passaging, 434 preimplantation blastocysts as source, 429–430 reverse transcription–polymerase chain reaction analysis of gene expression amplification reactions, 442 controls, 441 primers, 439 reverse transcription, 442 RNA isolation, 441–442 subculture, 432, 434 teratoma formation in severe combined immunodeficient mice, 443–444 tissue integration assessment, 459–460 transfection, 457–459 melanocyte formation, see Melanocyte mesoderm differentiation, see Mesoderm mutagenesis, see also N-Ethyl-Nnitrosourea mutagenesis; Gene trapping approaches, 367, 407 rationale, 367, 406 neuron formation, see Neuron 504 SUBJECT INDEX Embryonic stem cell (Cont.) osteoprogenitor formation, see Osteoprogenitor pluripotency, 98, 130, 186, 251, 303, 353 totipotency, 84, 242 transgenic mouse production, see Tetraploid embryo complementation vasculogenesis studies, see Mesoderm; Vasculogenesis Endothelial cell, see also Vasculogenesis D4T endothelial cell-conditioned medium preparation, 44–45 flow cytometry of differentiated cells CD4 marker analysis, 194–198 cell sorting caveats, 200–201 Flk-1 marker analysis, 194–198 principles, 193–194 replating analysis, 198–200 formation from mouse embryonic stem cells approaches, 215–216 differentiation media, 191–193 embryoid body formation, 190–191, 216 embryonic stem cell maintenance, 188–190, 217 methylcellulose differentiation culture advantages, 216 gene expression analysis, 219–221 growth factors, 218–219 immunohistochemistry, 223 overview, 217–218 vascular morphogenesis in embryoid bodies, 221, 223 overview, 186–187 immunohistochemical staining, 200–201 ENU mutagenesis, see N-Ethyl-N-nitrosourea mutagenesis Enzyme-linked immunosorbent assay albumin assay of hepatocytes, 299, 301 insulin assay of beta cells, 298–300 EPL cells, see Ectoderm-like cells ES cell, see Embryonic stem cell N-Ethyl-N-nitrosourea mutagenesis embryonic stem cell mutagenesis applications, 414–415 clone picking and cryopreservation, 409–410 heteroduplex analysis for screening, 410–411 incubation conditions, 409 mouse mutant generation, 412–414 mutation frequencies, 407–408 preparation of mutagen, 408 principles, 367, 407 transforming growth factor- signaling studies, 407 F Fetal calf serum, ectoderm-like cell culture and quality assays, 5–8 Flow cytometry B cell differentiation analysis, 166–168 fibrinogen binding to megakaryocytes, 155–156, 155–156 hematopoietic and endothelial precursor analysis CD4 marker analysis, 194–198 cell sorting caveats, 200–201 Flk-1 marker analysis, 194–198 principles, 193–194 replating analysis, 198–200 human embryonic stem cells, 437–438, 456–457 neural conversion quantification of embryonic stem cell differentiation, 331–333 neural precursor purification from monolayer culture, 336–337 G Gelatin coating of culture dishes, 45, 104 ectoderm-like cell culture and quality assays, 8–10 Gene trapping cloning of sequences flanking gene trap insertion sites anchoring polymerase chain reaction, 380–381 long and accurate polymerase chain reaction system, 377 rapid amplification of complementary DNA ends, 377–380, 388 consortiums, 388 embryoid body gene expression analysis alkaline phosphatase reporter assay, 400–402 applications, 405–406 SUBJECT INDEX colocalization of alkaline phosphatase reporter and developmental marker expression, 402, 404–405 culture, 400–402 differential expression screening, 398, 400 markers, 399 RNA in situ hybridization, 404 embryonic stem cells clone manipulation, 375–376 culture, 372–374, 392–395 electroporation, 374–375 embryonic fibroblast feeder cell preparation, 393–394 retrovirus infection, 374, 396–397 titering, 395–396 selection of clones, 374, 397–398 mouse strain production and analysis, 382–384 mutagenesis screens, 384–386 principles, 368–369, 386–387 reporter assays of trapped gene expression, 381–382, 388, 400 retroviral vectors bifunctional vectors, 389–392 reverse-orientation-splice-acceptor vector, 369–370 vector design, 387–388 H Hemangioblast, see Hematopoiesis Hematopoiesis blast colony-forming cell characteristics, 41 definitive hematopoiesis, 40 embryogenesis, 39–40, 84 embryonic stem cell differentiation culture definitive hematopoietic colony culture analysis erythroid colonies, 56–57 macrophage colonies, 57 megakaryocyte colonies, 56–57 multilineage colonies, 57–58 embryoid bodies generation, 51–52, 190–193 harvesting, 52 hemangioblast stage analysis of hematopoietic potential, 53–55 505 hematopoietic stage analysis of hematopoietic potential, 55–56 hematopoietic replating analysis, 198–200 maintenance of embryonic stem cells, 49–50, 188–190 materials ascorbic acid stock solution preparation, 45 cytokines, 43 D4T endothelial cell-conditioned medium preparation, 44–45 dishes, 43 gelatinized flasks and dishes, 45 Matrigel-coated well preparation, 46 media recipes, 46–48, 191–192 methylcellulose stock solution and mixtures, 43–44, 47–48 monothioglcerol, 46 mouse embryonic fibroblast cell preparation, 45 serum, 58 primitive erythroid colony culture and analysis, 56 fluorescence analysis flow cytometry CD4 marker analysis, 194–198 cell sorting caveats, 200–201 Flk-1 marker analysis, 194–198 principles, 193–194 replating analysis, 198–200 macrophage differentiation analysis, 94 primitive erythrocyte differentiation analysis, 93–94 hemangioblast as progenitor, 40 lateral plate mesoderm production of hematopoietic cells definitive hematopoietic cultures, 69 erythroid cultures, 68 materials, 65 overview, 62 lymphocyte formation, see B cell; Natural killer cell neutrophil formation, see Neutrophil OP9 stromal cell monolayer culture of mouse embryonic stem cells for differentiation B cell formation, 78–79, 164–166 comparison with embryoid body sytem, 71, 81–83 506 SUBJECT INDEX Hematopoiesis (Cont.) conditional gene expression using tetracycline-regulated expression, 80–89 erythrocyte formation, 78 gene expression analysis, 79–80 induction of differentiation, 75–77 maintenance embryonic stem cells, 75 OP9 cells, 74–75, 107–109, 113, 147–148 megakaryocyte formation, 78, 147–151 natural killer cell formation, 164–166 OP9 cell characteristics, 73–74 osteoclast formation, 78, 99 primitive hematopoiesis, 39–40 progenitor cell line formation from Lhx2expressing mouse embryonic stem cells clonal assays of embryoid body cells, 207–208 embryoid body formation, 206–207 embryonic stem cell maintenance, 204 materials, 211–213 overview, 203 progenitor cell line generation and maintenance, 208–211 progenitor chaaracteristics, 210–211 rationale, 202–203, 214 retroviral transduction infection and selection, 206 retrovirus production, 205 vectors, 204–205 repopulating hematopoietic stem cell derivation from mouse embryonic stem cells hanging drop embryoid body culture, 124–125 HoxB4 induction of hematopoietic cultures, 125–128 leukemic engraftment using Bcr/Abl induction of hematopoietic cultures, 116–119 Lox-in to derive inducible embryonic stem cell lines from Ainv15 targeting cells, 122, 124 non-oncogenic engraftment, 119–123 overview, 114–116 therapeutic repopulation, 128–129 Hepatocyte embryogenesis, 280, 287–288 formation from mouse embryonic stem cells albumin enzyme-linked immunosorbent assay, 299, 301 application prospects, 286–288 differentiation culture, 291–293 embryoid body-independent culture, 284–286 embryonic stem cell maintenance, 278–279, 291 -galactosidase as reporter, 283 green fluorescent protein as reporter, 281, 283, 285 growth factors, 280 histotypic differentiation into spheroids, 294 immunofluorescence analysis, 295–298, 301–302 immunohistochemistry of spheroids, 298 indocyanine green uptake, 284 markers, 279–281, 283 nestin expression by progenitors and selection, 289–290, 292, 299, 301 reverse transcription—polymerase chain reaction analysis, 294–296 urea synthesis in embryoid bodies, 284 I ICM, see Inner cell mass Immunofluorescence microscopy beta cells, 295–298 cardiomyocytes, 465–467 hepatocytes, 295–298, 301–302 human embryonic stem cell differentiation, 436–437 Immunohistochemistry beta cell spheroids, 298 endothelial cells, 200–201, 223 hepatocyte spheroids, 298 human embryonic stem cell differentiation, 434–436 Inner cell mass ectoderm differentiation, isolation from human embryos, 431 L Leukemic engraftment, see Hematopoiesis M Macrophage, see Hematopoiesis MEA, see Multi-electrode array 507 SUBJECT INDEX Megakaryocyte, see also Hematopoiesis formation from mouse embryonic stem cells analysis cell preparation, 153–154 confocal microscopy, 156–157 flow cytometry, 155–156 immunocytochemistry, 154–155 applications, 143 differentiation culture, 56–57, 148–151 embryonic stem cells freezing, 147 maintenance, 144 passaging, 146–147 thawing, 146 flask preparation with feeder cells, 146 irradiated embryonic fibroblast cell preparation, 145–146 OP9 cell maintenance and passaging, 147–148 OP9 stromal cell monolayer culture of mouse embryonic stem cells, 78 prospects for study, 158 retrovirus-mediated gene transduction infection, 153 materials, 151–152 retrovirus preparation and titering, 152–153 Melanocyte development, 341–342 formation from mouse embryonic stem cells applications, 343 cell counting, 348 cell lines, 348 differentiation culture, 346–347 embryonic fibroblast feeder cell preparation, 344–345 embryonic stem cell maintenance, 345–346 materials, 343–344–344 overview, 342–343 plating density, 349 serum, 348–349 ST2 stromal cell preparation, 346 Mesoderm formation from ectoderm-like cells, see Ectoderm-like cells lateral plate mesoderm formation from mouse embryonic stem cells cell lines, 63 culture induction and purification, 66, 71 isolation of differentiated cells, 64, 66–67 medium, 63–64 overview, 61 gene expression analysis data analysis, 70–72 DNA microarrays, 65–66, 70 materials, 65–66 overview, 62–63 RNA isolation and reverse transcription, 65, 69–70 hematopoietic cell generation definitive hematopoietic cultures, 69 erythroid cultures, 68 materials, 65 overview, 62 markers, 60 vascular progenitor cell generation overview, 62 smooth muscle cells, 64, 67, 71 vascular tubes, 64, 67–68 vasculogenesis, 84 Multi-electrode array, electrophysiology recording of differentiated cardiomyocytes, 470–472 N Natural killer cell, OP9 stromal cell monolayer culture of mouse embryonic stem cells for differentiation cytokines, 159–160 embryonic stem cell maintenance, 163 kinetics of differentiation, 168 materials, 160–163 OP9 cell maintenance, 163–164, 168 overview, 158–159 Neuroepithelial stem cell, see Neuron Neuron commitment and differentiation culture from mouse embryonic stem cells cell lines, 329–330, 338 flow cytometry quantification of neural conversion, 331–333 medium, 330–331 monolayer neural differentiation and monitoring, 331, 333–335 overview, 327–329 508 SUBJECT INDEX Neuron (Cont.) precursor purification from monolayer culture flow cytometry, 336–337 OS25 cells, 337–338 puromycin selection, 337 troubleshooting of monolayer cultures cell death, 339 cell lines, 338 coated substrates, 338 medium components, 339–341 plating density, 338–339 serum, 339 tyrosine hydroxylase-positive cell generation from monolayer culture, 335–336 cynomolgus monkey embryonic stem cell differentiation, 425, 427 dopaminergic neuron derivation from mouse embryonic stem cells application potential, 304 differentiation to midbrain dopaminergic neurons, 313–314 embryoid body formation, 310–311 embryonic stem cell maintenance, 308–309 expansion of midbrain precursor cells, 312 materials coated culture dish preparation, 308 equipment, 305 growth factors and supplements, 307–308 media, 305–307 solutions, 307 nestin expression and selection, 311–312 overview, 304–305 transfection with Nurr1 construct, 309–310 neuroepithelial stem cell culture from rats adult rat cell culture, 324–327 coated culture dish preparation, 318 critical parameters, 322–323 fetal brain dissection, 318–319 materials, 316–318 modification for mice, 323–324 neurosphere culture, 324–325 overview, 314–316 passaging, 321–322 plating and culture, 320–321 Neutrophil, formation from mouse embryonic stem cells using OP9 stromal cell feeder layer applications, 141–142 embryonic stem cell maintenance, 136–137 harvesting of neutrophils, 139–140 materials cytokines, 135 medium, 132–134 plasticware, 135–136 reagents, 133 reducing agents, 136 sera, 133–135 OP9 cell culture, 137 overview, 129–131 primary differentiation culture for embryoid body formation, 137 regional areas of neutrophil production, 140–141 secondary differentiation culture, 137–138 tertiary differentiation culture, 138–139 yield of neutrophils, 139–140 NK cell, see Natural killer cell O OP9 stromal cell, see B cell; Hematopoiesis; Natural killer cell; Neutrophil; Osteoclast Osteoclast formation from mouse embryonic stem cells alkaline phosphatase staining, 110–111 CD31 staining, 111 differentiation without stromal cell lines, 109–110, 113 embryonic fibroblast preparation, 105–106 gelatin coating of culture dishes, 104 maintenance of embryonic stem cells, 106–107, 112 materials and media, 100–104 OP9 stromal cell monolayer culture, 78, 99, 107–109, 113 prospects, 112 single-step culture, 99, 107, 109, 112–113 ST2 stromal cell maintenance, 107, 112 ... TABLE OF CONTENTS Development of Hematopoietic Repopulating Cells from Embryonic Stem Cells The In Vitro Differentiation of Mouse Embryonic Stem Cells into Neutrophils 10 Development and Analysis of. .. from Murine Embryonic Stem Cells 11 Development of Lymphoid Lineages Embryonic Stem Cells In Vitro from 12 Probing Dendritic Cell Function by Guiding the Differentiation of Embryonic Stem Cells 13... Section I Differentiation of Mouse Embryonic Stem Cells [1] Lineage Specific Differentiation of Mouse ES Cells: Formation and Differentiation of Early Primitive Ectoderm-like (EPL) Cells By JOY