The derivation, propagation, storage and gene expression of human embryonic stem cells on human feeders

GENERAL INTRODUCTION Several types of stem cells have been discovered from germ cells, the embryo, fetus and adult Each of these has promised to revolutionize the future of regenerative medicine through the provision of cell replacement therapies to treat a variety of debilitating diseases The tremendous versatility of embryonic stem cells versus the unprecedented reports describing adult stem cell plasticity have ignited debates as to the choice of one cell type over the other for future applications However, the biology of these mysterious cells have yet to be understood through a lot more basic research before new therapies using stem cell differentiated derivatives can be applied Everyday, we read and listen to news reports about how stem cells promise to revolutionize medicine and change our lives with panaceas for every imaginable disease including rhetoric that stem cell therapy will some day delay the process of ageing Embroiled in the hype and media frenzy are also political agendas, numerous religious and genuine ethical concerns To further fuel the debate, embryonic stem cell research is often unjustly associated with reproductive cloning Stem cell research is politically charged, receives considerable media coverage, raises many ethical and religious debates and generates a great deal of public interest Stem cell research also opens the new field of ‘cell based therapies’ and as such several safety measures have also to be evaluated The hope that someday many debilitating human diseases may be treated with stem cell therapy is inspired by remarkable examples of whole organ and limb regeneration in animals as well as the historical success of bone marrow transplants that have improved the lives of many patients suffering from leukemia, immunological and other blood disorders Clearly, stem cell research leading to prospective therapies in reparative medicine has the potential to affect the lives of millions of people around the world for the better and there is good reason to be optimistic However, the road towards the development of an effective cell-based therapy for widespread use is long and involves overcoming numerous technical, legislative, ethical and safety issues Embryos of most mammals are comprised of a special group of cells that have the potential to give rise to all the tissues and organs of the fetus and future adult This group of cells called the inner cell mass (ICM) cells evolves into embryonic stem cells (ESCs) in vitro Unlike other cell types that can only divide a maximum of 50 times or so in tissue culture dishes (Hayflick & Moorhead 1961), ESCs can divide indefinitely without losing their ability to form different cell types Human embryonic stem cells (hESCs) derived from isolation and serial sub-culture of ICMs from 5-day old human blastocysts hold the promise of revolutionizing the future of medicine by the creation of early developmental models for a multitude of human genetic diseases and through the development of cell and tissue replacement therapies Immense commercial interest as well as ethical controversy surrounds hESC research Several improvements in blastocyst culture techniques were a prerequisite for culturing and harvesting good quality blastocysts with large ICMs These breakthroughs not only led to increased pregnancy rates with blastocyst transfer in patients undergoing in vitro fertlilization (IVF) cycles but also enabled scientists to derive hESC cell lines from human blastocysts hESCs cells were first isolated in 1994 (Bongso et al 1994) while the first continuous immortal hESC lines were established only in 1998 (Thomson et al 1998) hESCs are colony forming social cells that are unspecialized This means that if they are coaxed properly, ESCs have in theory the ability to turn into any of the cell types in the human body In contrast, adult stem cells, which are found in adult tissues and organs, have the ability to transform into only a limited variety of cell types Adult stem cells are also difficult to isolate and very challenging to grow in culture This coupled with their restricted developmental potential are the main reasons why many scientists believe that embryonic stem cells are more promising and better alternatives for developing a wider range of cell based therapies In order for hESCs to retain their ability to form different cell types, they need to be grown on feeder cell supports The feeder layer produces growth factors and extracellular matrix components that may help to keep the hESCs from differentiating into other specialized cells Without the support of a feeder layer, hESCs spontaneously and uncontrollably differentiate into a milieu of mixed cell types This is often undesirable for the researcher as specific cell types are often difficult or near impossible to isolate from this mixed milieu Embryonic stem cells are a unique class of cell type for various reasons Most significantly, they can undergo self-renewal for extended periods of in vitro cultivation, have the ability to form teratomas when injected into severely combined immunodeficient (SCID) mice and can differentiate into a variety of cell types from all primitive germ layers in vitro and in vivo, thus distinguishing them from other adult stem cells hESCs differ in many ways from mouse embryonic stem cells (mESCs) Several lines of evidence suggest that hESCs and mESCs not represent equivalent embryonic cell types In vitro differentiation of hESCs leads to the expression of AFP and HCG, which are typically produced by trophoblast cells in the developing human embryo, while mESCs are generally believed not to differentiate along this lineage In addition, hESCs express SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 surface antigens prior to differentiation but only SSEA-1 upon differentiation, while mESCs only express SSEA1 prior to differentiation (Thomson et al 1998, Reubinoff et al 2000, Henderson et al 2002) The cytokine leukemia inhibitory factor (LIF) has an established facultative role in keeping mESCs undifferentiated and an exogenous supply of LIF in the culture medium is sufficient to keep mESCs undifferentiated for prolonged culture periods (Williams et al 1988, Smith et al 1988) hESCs on the other hand not appear to have a perceivable LIF response (Thomson et al 1998, Reubinoff et al 2000) The molecule or group of molecules involved in autocrine or paracrine signaling in keeping hESCs undifferentiated has also not been identified making the culture of undifferentiated hESCs heavily reliant on feeder layer support It has been over five years since the first hESC lines were established but our understanding of hESC biology is still limited for it to be exploited for clinical application This hopefully would change once hESC lines become widely and routinely available to all researchers Nevertheless, hESC research needs to be pursued aggressively if we are to quickly realize the full therapeutic potential of reparative cell therapy and several areas in particular warrant immediate attention Specifically, advances must be made to improve hESC culture techniques Purer and safer populations of functionally normal undifferentiated hESCs and differentiated hESC progenitor cell types need to be derived All current 78 NIH listed hESC lines approved for US government federal research funding have been derived and propagated on mouse embryonic fibroblast (MEFs) and in the presence of culture medium containing animal based ingredients The use of a feeder layer of animal origin and animal components in the culture media substantially elevates the risk of the crosstransfer of viruses and other pathogens to the hESCs Many studies have focused on the differentiation of MEF supported hESCs into a range of clinically useful cell types, while this is important, the development and refinement of a xeno-free culture system that decreases the risk of hESC contamination with adventitious agents while maintaining pure undifferentiated hESC populations amenable to expansion of cell numbers is critical before any clinical exploitation of hESC technology can occur New hESC lines need to be derived and bulk-cultured in current good manufacture practice (cGMP) conditions according to a xeno-free gold standard The establishment of new hESC lines in cGMP conditions necessitates the development of an effective cryopreservation protocol that minimizes or restricts the possibility of early passage hESC seed stock contamination with adventitious agents such as viruses and other pathogens during long-term liquid nitrogen storage Interestingly, hESC lines have heterogeneous genetic backgrounds unlike mESC lines that are from inbred mouse strains and appear to behave differently in culture For example, not all hESC lines are amenable to bulk and feeder-free culture protocols, doubling times differ considerably between different lines and the degree of spontaneous differentiation in vitro also appears to show much variation (Vogel 2002) Functional genomics data need to be gathered for a better understanding of the genetic pathways that regulate pluripotency, self-renewal and differentiation Identifying “master regulators” and as yet undiscovered genes that control hESC self-renewal and immortality will shed light on cancer genetics as well as have implications in ageing research In an effort to better understand the molecular cascades controlling the pluripotent phenotype in hESCs, transcriptional profiling using, microarray, serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) technology are being undertaken on undifferentiated and differentiated hESCs (Sato et al 2003, Sperger et al 2003, Brandenberger et al 2004, Ginis et al 2004, Rao 2004a) Comparative analysis of the transcriptome profiles using these techniques will reveal several interesting candidate genes that are potentially important to the embryonic stem cell phenotype In principle, hESCs are capable of differentiating into all cell types in the adult human, therefore they have the potential to provide a source of tissues for replacement in diseases in which native cell types are inactivated or destroyed However, the wideranging diverse nature of human diseases and technical shortcomings will in most cases limit the promise of hESC replacement therapy to a few common human diseases In contrast, the value of hESCs as a developmental model in helping us gain insight to virtually all human diseases with a genetic basis appears limitless hESCs also hold promise in screening and toxicity testing assays in the pharmaceutical industry Established hESC lines provide a convenient tool for investigating cell differentiation in a way that is pertinent to human embryonic development, providing insights into the causes of birth defects and diseases such as cancer that involve aberrant cell proliferation and differentiation Perhaps even more powerful than generating healthy tissues from existing hESC lines for cell replacement therapy, would be the ability to generate diseased hESC lines with genetic defects through somatic cell nuclear transfer (SCNT) These diseased and genetically abnormal hESC lines could produce an unlimited quantity of diseased cell types that will be an invaluable resource and model to study the disease phenotype and its genetic basis Several technical hurdles need to be bypassed before the enormous implications of ES cell technology for understanding and curing human diseases can be realized hESC research opens up new vistas in the fields of medicine For instance, treating hESCs with the right combination of growth factors may induce the formation of dopaminergic neurons or perhaps insulin secreting beta cells of the pancreas The specialized neurons and beta cells derived from precursor hESCs can then be returned to patients suffering from Parkinson’s disease or diabetes to correct the defects of the malfunctioning organ or tissue However, cellular therapy using hESC derived specialized cell types will very likely be useful for treating only certain human diseases Diseases that involve and affect multiple cell types and organs may not be treatable using a cell based therapeutic approach Thus, Parkinson’s disease and Type I diabetes are often singled-out as the two most promising targets for a cell based therapeutic approach Such treatments from stem cell research will be “cell-based therapies” Presently, doctors administer fluids (injections), solids (pills) or surgical intervention For the first time, treatment may be by the administration of cells directly into the body Thus, several added precautions have to be taken before cell-based therapeutic products can be released into the market Firstly, stringent tests have to be conducted to ensure that the specialized cell types, which are returned to the patient, are totally pure No contaminating undifferentiated hESCs should be present because they have the potential to divide and replicate and produce a tumor if an undetected renegade hESC is accidentally injected into the patient Secondly, specialized cell types derived from hESCs must be rigorously tested in vitro and in animal models in vivo to show that they can restore normal physiological function in disease models Thirdly, several hurdles in the manipulation and differentiation of hESCs must be overcome before the technology can be successfully transferred to the bedside Cell replacement therapies require the growth of large numbers of hESCs Thus, large-scale hESC culture strategies using bioreactors need to be developed to generate sufficient numbers of cells High efficiency directed differentiation strategies via spontaneous, co-culture or genomics approaches, safer and purer populations of hESCs and their differentiated progeny, clinically compliant xeno-free hESC lines and xeno-free storage systems are very urgent areas that need investigation The studies in this thesis address some of these urgent issues, more specifically the derivation and propagation of xeno-free hESC lines, the xeno-free storage of hESCs and the understanding of the molecular genetics of hESCs that can help identify genes involved in the maintenance of pluripotency and commitment to differentiation events LITERATURE REVIEW Regeneration in invertebrates and vertebrates Man has long been fascinated by the regenerative abilities of certain animals Regeneration is a remarkable physiological process in which remaining tissues organize to reform a missing body part All species possess the ability to regenerate damaged tissues, the degree of regeneration, however, varies considerably among species Such differences in regenerative capacity are perhaps indicative of specific mechanisms that control the different types of regeneration Several invertebrates like the Planarian flatworm and the Hydra regenerate tissues with speed and precision Planarians are spectacular examples of whole body regeneration by an invertebrate; a planarian sliced into 50 pieces will regenerate 50 new planarians from each piece The majority of higher vertebrates are incapable of any form of whole organ regeneration, even though they had all the necessary instructions and machinery to generate the tissue during embryonic development (Wolpert et al 1971, Brockes 1997) Of the higher vertebrates, mammals appear to have limited regenerative ability, a tradeoff perhaps for more proficient wound healing ability The most striking examples of whole organ regeneration in mammals are that of antler regeneration in Elks, and in humans, liver regeneration after partial hepatectomy (Kiessling & Anderson 2003) Most tissue repair events in mammals are dedifferentiation independent events resulting from the activation of pre-existing stem cells or progenitor cells In contrast, some vertebrates like the salamanders regenerate lost body parts through the dedifferentiation of specialized cells into new precursor cells These dedifferentiated cells then proliferate and later form new specialized cells of the regenerated organ Stem cells or progenitor cells are the common denominator for nearly all types of regeneration They are either already pre-existing, as in the case for mammals or created by the process of dedifferentiation The process of retina and limb regeneration in urodele amphibians involves complex dedifferentiation and redifferentiation events Following limb amputation, the wound is quickly covered by an epithelium that provides the necessary signals for the underlying tissues to dedifferentiate, proliferate, and form the blastema also known as the amphibian regeneration bud Blastema tissues then undergo redifferentiation to form muscle, bone and other mesodermal tissues to enable the reconstruction of the amputated limb Major cell signaling pathways activated in the blastema during this process are the fibroblast growth factor (FGF) and transforming growth factor (TGF) pathways (Tsonis 1996) Additionally, the blastema appears to express the phosphorylated version of the tumor suppressor gene, retinoblastoma (Rb) that is found highly expressed in many diverse tumors Cancer cells share similarities with blastema cells in that they are both dedifferentiated and pluripotent An animal with powerful regenerative capabilities is often refractory to spontaneous or experimentally induced cancer; this is true for the amphibia Spontaneous tumors are difficult to find in this class of vertebrates (Tsonis and Del Rio-Tsonis, 1988) Studies in the Hydra have identified a family of Wnt proteins, produced during Hydra budding and at the tip of a decapitated Hydra when its head starts to regrow (Hobmayer et al 2000) Thus, the FGF, TGF and Wingless-Type Mmtv Integration Site Family (Wnt) signaling pathways appear to play important and overlapping roles in developmental, cancer, regeneration and stem cell biology Plant meristems Plants but not most animals have the remarkable capacity to regenerate from vegetative parts Many terminally differentiated plant organs, tissues and cells retain their capacity 10 Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells Cell 113: 643-655 Chen J, Sun M, Lee S, Zhou G, Rowley JD, Wang SM (2002) Identifying novel transcripts and novel genes in the human genome by using novel SAGE tags Proc Natl Acad Sci USA 99: 12257–12262 Cheng L, Hammond H, Ye Z, Zhan X, Dravid G (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture Stem Cells 21:131-142 Chiang MK, Melton DA (2003) Single-cell transcript analysis of pancreas development Dev Cell 4: 383-393 Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, Pera RA (2004) Spontaneous differentiation of germ cells from human embryonic stem cells in vitro Hum Mol Genet 13: 727-723 Clark AT, Rodriguez RT, Bodnar MS, Abeyta MJ, Cedars MI, Turek PJ, Firpo MT, Reijo Pera RA (2004) Human STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma Stem Cells 22: 169-179 Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U, Frisen J (2000) Generalized potential of adult neural stem cells Science 288: 16601663 Clarke GN (1999) Sperm cryopreservation: is there a significant risk of crosscontamination? Hum Reprod 14: 2941-2943 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA (2004) Derivation of embryonic stem-cell lines from human blastocysts N Engl J Med 350: 1353-1356 Devireddy RV, Bischof JC (2003) Recent advances in cryobiology using calorimetry In: Kakac S, Smirnov H, Mila MR, eds Low Temperature and Cryogenic Refrigeration Dordrecht, The Netherlands; Kluwer Academic Publishers, 265-294 Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation Nature 429: 41-46 Draper JS, Andrews PW (2002) Embryonic stem cells: advances toward potential therapeutic use Curr Opin Obstet Gynecol 14: 309-315 Draper JS, Pigott C, Thomson JA, Andrews PW (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture J Anat 200(Pt 3): 249258 193 Draper JS, Fox V (2003) Human embryonic stem cells: multilineage differentiation and mechanisms of self-renewal Arch Med Res 34: 558-564 Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, Meisner L, Zwaka TP, Thomson JA, Andrews PW (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells Nat Biotechnol 22: 53-54 Du Z, Cong H, Yao Z (2001) Identification of putative downstream genes of Oct-4 by suppression-subtractive hybridization Biochem Biophys Res Commun 282: 701–706 Edwards BE, Gearhart JD, Wallach EE (2000) The human pluripotent stem cell: impact on medicine and society Fertil Steril 74: 1-7 Edwards RG (2001) IVF and the history of stem cells Nature 413: 349-351 Edwards RG, Pool TB (2002) Human chimaeras and understanding early development Reprod Biomed Online 5: 103 Edwards RG (2002) Personal pathways to embryonic stem cells Reprod Biomed Online 4: 263-278 Edwards RG (2004) Stem cells today: A Origin and potential of embryo stem cells Reprod Biomed Online 8: 275-306 Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells Curr Biol 11: 514-518 Eiges R, Benvenisty N (2002) A molecular view on pluripotent stem cells FEBS Lett 529: 135-141 Englund U, Bjorklund A, Wictorin K, Lindvall O, Kokaia M (2002) Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry Proc Natl Acad Sci U S A 99: 17089-17094 Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood Br J Haematol 109:235–242 Evans MJ & Kaufman M (1981) Establishment in culture of pluripotential stem cells from mouse embryos Nature 292: 151-156 Fong CY, Bongso A, Ng SC, Kumar J, Trounson A, Ratnam S (1998) Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in vitro fertilization and understanding implantation Hum Reprod 13: 2926-2932 Fong CY, Bongso A, Sathananthan H, Ho J, Ng SC (2000) Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties Human Reprod 16: 540-546 194 Parati EA, Pozzi S, Ottolina A, Onofrj M, Bez A, Pagano SF (2003) Neural stem cells: An overview Circulation Research 92: 598-602 Galli R, Borello U, Gritti A, Minasi MG, Bjornson C, Coletta M, Mora M, De Angelis MG, Fiocco R, Cossu G, Vescovi AL (2000) Skeletal myogenic potential of human and mouse neural stem cells Nat Neuroscience 3: 986-991 Gardner RL (2002) Stem cells: potency, plasticity and public perception J Anat 200: 277-282 Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS (2004) Differences between human and mouse embryonic stem cells Dev Biol 269: 360-380 Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, et al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical cord blood from HLA-identical sibling N Engl J Med 321:1174–1178 Gluckman E Current status of umbilical cord blood hematopoietic stem cell transplantation Exp Hematol 2000;28:1197–1205 Hansis C, Grifo JA, Krey LC (2000) Oct-4 expression in inner cell mass and trophectoderm of human blastocysts Mol Hum Reprod 6: 999-1004 Henderson JK, Draper JS, Baillie HS, Fishel S, Thomson JA, Moore H, Andrews PW (2002) Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens Stem Cells 20: 329-337 Hosler BA, Rogers MB, Kozak CA, Gudas LJ (1993) An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells Mol Cell Biol 13: 2919-2928 Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L (2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells Hum Reprod 18: 1404-1409 Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Scholer HR (2003) Derivation of oocytes from mouse embryonic stem cells Science 300: 1251-1256 Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, Lee BC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY (2004) Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst Science 303: 1669-1674 Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR (2002) A stem cell molecular signature Science 298: 601-604 195 Jackson KA, Mi T & Goodell MA (1999) Hematopoietic potential of stem cells isolated from murine skeletal muscle Proc Natl Acad Sci 96:14482-14486 Jahner D, Jaenisch R In: Razin A, Cedar H, Riggs A, eds (1984) DNA Methylation: Biochemistry and Biological Significance New York: Springer-Verlag 189-219 Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM (2002) Pluripotency of mesenchymal stem cells derived from adult marrow Nature 418: 41-49 Kal AJ, van Zonneveld AJ, Benes V, van den Berg M, Koerkamp MG, Albermann K, Strack N, Ruijter JM, Richter A, Dujon B, Ansorge W, Tabak HF (1999) Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources Mol Biol Cell 10: 18591872 Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes J Clin Invest 108: 407-414 Kelly DL, Rizzino A (2000) DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells Mol Reprod Dev 56: 113-123 Kiessling AA, Anderson SC Human embryonic stem cells Boston: Jones and Bartlett, 2003 Kim TE, Lee HS, Lee YB, Hong SH, Lee YS, Ichinose H, Kim SU, Lee MA (2003) Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes in the Nurr1overexpressing neural stem cells Biochem Biophys Res Commun 305: 1040-1048 Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ (2001) Multi-organ, multi-lineage engraftment by a single bone-marrow derived stem cell Cell 105: 369-377 Kuleshova, LL & Lopata, A (2002) Vitrification can be more favorable than slow cooling Fertil Steril 78: 449-454 Laird PW, Jaenisch R (1996) The role of DNA methylation in cancer genetic and epigenetics Annu Rev Genet 30: 441-464 Lal A, Lash AE, Altschul SF, Velculescu V, Zhang L, McLendon RE, Marra MA, Prange C, Morin PJ, Polyak K, Papadopoulos N, Vogelstein B, Kinzler KW, Strausberg RL, Riggins GJ (1999) A public database for gene expression in human cancers Cancer Res 59: 5403-5407 Lanza R & Rosenthal N The stem cell challenge Scientific American June 2004 196 Lash AE, Tolstoshev CM, Wagner L, Schuler GD, Strausberg RL, Riggins GJ, Altschul SF (2000) SAGEmap: a public gene expression resource Genome Res 10: 1051-1060 Lebkowski JS, Gold J, Xu C, Funk W, Chiu CP, Carpenter MK (2001) Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications Cancer J Suppl 2: S83–S93 Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R (2002) Endothelial cells derived from human embryonic stem cells Proc Natl Acad Sci USA 99: 4391-4396 Levsky JM, Shenoy SM, Pezo RC, Singer RH (2002) Single-cell gene expression profiling Science 297: 836-840 Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ (2002) Potential importance of vitrification in reproductive medicine Biol Reprod 67: 16711680 Liker MA, Petzinger GM, Nixon K, McNeill T, Jakowec MW (2003) Human neural stem cell transplantation in the MPTP-lesioned mouse Brain Research 971: 168-177 Livak, KJ, Schmittgen, TD (2001) Analysis of relative gene expression using quantitative PCR and the 2∆∆CT method Methods 25: 402-408 Mann JR (2001) Imprinting in the germ line Stem Cells 19: 287-294 Mayani H & Lansdorp PM (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells Stem Cells 16:153–165 Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells Cell 113: 631-642 Miyamoto K, Hayashi K, Suzuki T, Ichihara S, Yamada T, Kano Y, Yamabe T, Ito Y (2004) Placenta feeder layers support undifferentiated growth of primate embryonic stem cells Stem Cells 22: 433-440 Moody, DE (2001) Genomics techniques: An overview of methods for the study of gene expression J Anim Sci 79(E Suppl.):E128–E135 Moss EG, Tang L (2003) Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites Dev Biol 258: 432-442 Mummery C, Ward D, van den Brink CE, Bird SD, Doevendans PA, Opthof T, Brutel de la Riviere A, Tertoolen L, van der Heyden M, Pera M (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells Circulation 107: 2733-2734 197 Nakahata T, Ogawa M (1982) Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors J Clin Invest 70:1324–1328 Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4 Cell 95: 379-339 Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells Nat Genet 24: 372-376 Okano M, Xie S, Li E (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases Nat Genet 19: 219-220 Okuda A, Fukushima A, Nishimoto M, Orimo A, Yamagishi T, Nabeshima Y, Kuro-o M, Nabeshima Y, Boon K, Keaveney M, Stunnenberg HG, Muramatsu M (1998) UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells EMBO J 17: 2019-2032 Pagano SF, Impagnatiello F, Girelli M, Cova L, Grioni E, Onofri M, Cavallaro M, Etteri S, Vitello F, Giombini S, Solero CL, Parati EA (2000) Isolation and Characterization of Neural Stem Cells from the Adult Human Olfactory Bulb Stem Cells 18: 295-300 Palmer TD, Schwartz PH, Taupin P, Kaspar B, Stein SA, Gage FH (2001) Progenitor cells from human brain after death Nature 411: 42-43 Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, Stanley EG, Ward-van Oostwaard D, Mummery C (2004) Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin J Cell Sci 117(Pt 7): 1269-1280 Pera MF, Bennett W, Cerretti DP (1997) Expression of CD30 and CD30 ligand in cultured cell lines from human germ-cell tumors Lab Invest 76:497-504 Pera MF, Blasco Lafita MJ, Mills J (1987) Cultured stem-cells from human testicular teratomas: the nature of human embryonal carcinoma, and its comparison with two types of yolk-sac carcinoma Int J Cancer 40: 334-343 Pera MF, Blasco-Lafita MJ, Cooper S, Mason M, Mills J, Monaghan P (1988) Analysis of cell differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells Differentiation 39: 139-149 Pera MF, Filipczyk AA, Hawes SM, Laslett AL (2003) Isolation, characterization, and differentiation of human embryonic stem cells Methods Enzymol 365: 429-446 Pera MF, Herszfeld D (1998) Differentiation of human pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2 Reprod Fertil Dev 10: 551-555 198 Pera MF, Reubinoff B, Trounson A (2000) Human embryonic stem cells J Cell Sci 113: 5-10 Pera MF, Trounson AO (2004) Human embryonic stem cells: prospects for development Development 131: 5515-5525 Pera MF (2004) Unnatural selection of cultured human ES cells? Nat Biotechnol 22 : 42-43 Pera MF (2001) Human pluripotent stem cells: a progress report Curr Opin Genet Dev 11: 595-599 Pesce M, Wang X, Wolgemuth DJ, Scholer H (1998) Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation Mech Dev 71: 89-98 Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, Galli R, Del Carro U, Amadio S, Bergami A, Furlan R, Comi G, Vescovi AL, Martino G (2003) Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis Nature 422: 688-694 Rao M (2004a) Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells Dev Biol 275: 269-286 Rao M (2004b) Stem and precursor cells in the nervous system J Neurotrauma 21: 415-427 Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA (2002) “Stemness": transcriptional profiling of embryonic and adult stem cells Science 298: 597-600 Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T (2000) Neural progenitors from human embryonic stem cells Nat Biotechnol 19: 11341140 Reubinoff BE, Pera MF, Vajta G, Trounson AO (2001) Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method Hum Reprod 10: 2187-2194 Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro Nat Biotechnol 18: 399404 Richards M, Fong CY, Tan S, Chan WK, Bongso A (2004) An efficient and safe xenofree cryopreservation method for the storage of human embryonic stem cells Stem Cells 22: 779-89 Richards M, Tan SP, Tan JH, Chan WK, Bongso A (2004) The transcriptome profile of human embryonic stem cells as defined by SAGE Stem Cells 22: 51-64 199 Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells Stem Cells 21:546-556 Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells Nat Biotechnol 20: 933-936 Riess P, Zhang C, Saatman KE, Laurer HL, Longhi LG, Raghupathi R, Lenzlinger PM, Lifshitz J, Boockvar J, Neugebauer E, Snyder EY, McIntosh TK (2002) Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury Neurosurgery 51: 1043-1052 Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF (2001) Purification of a pluripotent neural stem cell from the adult mouse brain Nature 412: 736-739 Rizzino A (2002) Embryonic stem cells provide a powerful and versatile model system Vitam Horm 64: 1-42 Rubinstein P, Dobrila L, Rosenfield RE, Adamson JW, Migliaccio G, Migliaccio AR, Taylor PE, Stevens CE (1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution Proc Natl Acad Sci USA 92:10119–10122 Ruijter JM, Van Kampen VHC, Baas F (2002) Statistical evaluation of SAGE libraries: consequences for experimental design Physiol Genomics 11: 37-44 Saitou M, Barton SC, Surani MA (2002) A molecular programme for the specification of germ cell fate in mice Nature 418: 293-300 Saha S, Sparks AB, Rago C, Akmaev V, Wang CJ, Vogelstein B, Kinzler KW, Velculescu VE (2002) Using the transcriptome to annotate the genome Nat Biotechnol 20: 508-512 Salahuddin SZ, Markham PD, Ruscetti FW, Gallo RC (1981) Long-term suspension cultures of human cord blood myeloid cells Blood 58:931–938 Sathananthan, H, Pera MF, Trounson AO (2002) The fine structure of human embryonic stem cells J Reprod BioMed Online 4: 56-61 Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse Dev Biol 260: 404-413 Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells Proc Natl Acad Sci U S A 97: 11307-11312 200 Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, Benvenisty N (2001) Induced neuronal differentiation of human embryonic stem cells Brain Res 913: 201-205 Schuldiner M, Benvenisty N (2003) Factors controlling human embryonic stem cell differentiation Methods Enzymol 365: 446-461 Schuldiner M, Itskovitz-Eldor J, Benvenisty N (2003) Selective ablation of human embryonic stem cells expressing a "suicide" gene Stem Cells 21: 257-265 Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y, Cheng L, Gearhart JD (2001) Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro Proc Natl Acad Sci USA 98: 113-118 Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells Proc Natl Acad Sci U S A 95: 13726-13731 Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV (2000) Islet transplantation in seven patients with type diabetes mellitus using a glucocorticoid-free immunosuppressive regimen N Engl J Med 343: 2308 Shihabuddin LS, Horner PJ, Ray J, Gage FH (2000) Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus Journal of Neuroscience 20: 8727-8735 Short B, Brouard N, Occhiodoro-Scott T, Ramakrishnan A, Simmons PJ (2003) Mesenchymal Stem Cells Archives of Medical Research 34: 565-571 Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides Nature 336: 688-690 Smith AG (2001) Embryo-derived stem cells- of mice and men Ann Rev Cell Dev Biol 17: 435-462 Smith S & Broxmeyer HE (1986) The influence of oxygen tension on the long-term growth in vitro of haematopoietic progenitor cells from human cord blood Br J Haematol 63:29–34 Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO, Thomson JA (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors Proc Natl Acad Sci U S A 100: 13350-13355 Spradling A, Drummond-Barbosa D, Kai T (2001) Stem cells find their niche Nature 414: 98-104 201 Stappenbeck TS, Mills JC, Gordon JI (2003) Molecular features of adult mouse small intestinal epithelial progenitors Proc Natl Acad Sci USA 100: 1004-1009 Stern MD, Anisimov SV, Boheler KR (2003) Can transcriptome size be estimated from SAGE catalogs? Bioinformatics 19: 443-448 Steyaert, SR, Leroux-Roels, GG, Dhont, M (2000) Infections in IVF: review and guidelines Hum Reprod Update 6: 432-441 Stoll M, Jacob HJ (2001) Genetic rat models of hypertension: relationship to human hypertension Curr Hypertens Rep 3: 157-164 Sukumaran R, Richards M, Tan SP, Tan JH, Chan WK and Bongso A (2005) A Cluster Analyses of the SAGE expression profiles of Human Embryonic Stem Cells indicate possible role of POU5F1 in cell cycle progression and identifies a robust set of candidate genes for stemness In preparation Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM, Patterson KG (1995) Hepatitis B transmission from contaminated cryopreservation tank Lancet 346: 137-140 Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW (2002) Bones marrow cells adopt the phenotype of other cells by spontaneous fusion Nature 416: 542-545 Thirumala S, Ferrer MS, Al-Jarrah A, Eilts BE, Paccamonti DL, Devireddy RV (2003) Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents Cryobiology 47:109-124 Thomas PQ, Johnson BV, Rathjen J, Rathjen PD (1995) Sequence, genomic organization, and expression of the novel homeobox gene Hesx1 J Biol Chem 270: 3869-3875 Thomas PQ, Rathjen PD Johnson BV (1995) Genomic organization and expression of the novel homeobox gene Hesx1 J Biol Chem 270: 3869-3875 Thomas PQ, Rathjen PD (1992) HES-1, a novel homeobox gene expressed by murine embryonic stem cells, identifies a new class of homeobox genes Nucleic Acids Res 20: 5840 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts Science 282: 1145-1147 Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP (1995) Isolation of a primate embryonic stem cell line Proc Natl Acad Sci 92: 78447848 202 Thompson JR, Gudas LJ (2002) Retinoic acid induces parietal endoderm but not primitive endoderm and visceral endoderm differentiation in F9 teratocarcinoma stem cells with a targeted deletion of the Rex-1 (Zfp-42) gene Mol Cell Endocrinol 195: 119133 Tomlinson M, Sakkas D (2000) Is a review of standard procedures for cryopreservation needed? Hum Reprod 15: 2460-2463 Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression Science 270: 484-487 Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE Jr, Hieter P, Vogelstein B, Kinzler KW (1997) Characterization of the yeast transcriptome Cell 88: 243-251 Velculescu VE, Madden SL, Zhang L, Lash AE, Yu J, Rago C, Lal A, Wang CJ, Beaudry GA, Ciriello KM, Cook BP, Dufault MR, Ferguson AT, Gao Y, He TC, Hermeking H, Hiraldo SK, Hwang PM, Lopez MA, Luderer HF, Mathews B, Petroziello JM, Polyak K, Zawel L, Kinzler KW, et al (1999) Analysis of human transcriptomes Nat Genet 23: 387-388 Velculescu VE, Vogelstein B, Kinzler KW (2000) Analysing uncharted transcriptomes with SAGE Trends Genet 16: 423-425 Villa A, Snyder EY, Vescovi A, Martinez-Serrano A (2000) Establishment and properties of a growth factor dependent perpetual neural stem cell line from the human CNS Exp Neurol 161: 67-84 Vogel G (2002) Stem cells Are any two cell lines the same? Science 295: 1820 Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E (1995) Allogeneic sibling umbilical-cord blood transplantation in children with malignant and nonmalignant disease Lancet 346:214–219 Williams BP, Daniels GL, Pym B, Sheer D, Povey S, Okubo Y, Andrews PW, Goodfellow PN (1998) Immunogenetics 27: 322-329 Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP,Wagner EF, Metcalf D, Nicola NA, Gough NM (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential embryonic stem cells Nature 336: 684-687 Wolpert L, Hicklin J, Hornbruch A (1971) Positional information and pattern regulation in regeneration of hydra Symp Soc Exp Biol 25: 391-415 Wright WE, Shay JW (2000) Fundamental differences in human and mouse telomere biology Nature Med 6: 849-851 203 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2002) Feeder-free growth of undifferentiated human embryonic stem cells Nat Biotechnol 19: 971–974 Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast Nat Biotechnol 20: 1261-1264 Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiatied proliferation of human ES cells Nature Methods 2(3): 185-190 Ying QL, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion Nature 2002 416: 545-548 Yoshimizu T, Sugiyama N, De Felice M, Yeom YI, Ohbo K, Masuko K, Obinata M, Abe K, Scholer HR, Matsui Y (1999) Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice Dev Growth Differ 1999 41: 675-684 Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 19: 1129-1133 Zhu SE, Sakurai T, Edashige K, Machida T, Kasai M (1996) Cryopreservation of zonahatched mouse blastocysts J of Reprod and Fertil 107: 37-42 204 Appendix I: List of reagents and chemicals used in hESC cell culture High glucose DMEM without sodium pyruvate, without L-glutamine 1X (GIBCO, Carlsbad, CA; cat no 11960-044), store at 40C L-Glutamine 200 mM, 100X liquid (GIBCO; cat no 25030-081), aliquot and store at –200C Penicillin-Streptomycin liquid containing 5000 units of penicillin and 5000 mg of streptomycin/ml (GIBCO; cat no 15070-63) aliquot and store at –200C MEM non-essential amino acids 10mM, (100X) (GIBCO; cat no.11140050), wrap in foil to protect from light and store at 40C Defined fetal bovine serum (Hyclone, Logan, UT; cat no SH30070-03) aliquot and store at –200C Qualified fetal calf serum (GIBCO; cat no 10099) aliquot and store at –200C Dulbecco’s phosphate buffered saline, PBS (GIBCO; cat no 14040-133), store at 40C Dulbecco’s phosphate buffered saline, Ca2+, Mg2+ free PBS (without Ca2+ and Mg2+) (GIBCO; cat no 14190-144), store at 40C Sterile water, tissue culture grade (GIBCO; cat no 15230-162), store at 40C 10 Porcine gelatin (Sigma, St Louis, MO; cat no G1890), store at 40C 11 Insulin-transferrin-selenium growth supplement (GIBCO; cat no 41400045), store at 40C 205 12 0.05% Trypsin-EDTA (1X) (GIBCO; cat no 25300-062), aliquot and store at –200C 13 β-Mercaptoethanol (GIBCO; cat no 21985-023), store at 40C 14 Dispase (GIBCO; cat no 17105-041), store at 40C 15 Collagenase IV (GIBCO; cat no.17104-019) 16 Knockout™ D-MEM (GIBCO; cat no 10829-018) 17 KnockoutTM Serum Replacement (GIBCO; cat no.10828-028) 18 Basic fibroblast growth factor (bFGF), human recombinant (GIBCO; cat no.13256-029) 19 1M HEPES solution (GIBCO; cat no 15630-080), store at 40C 20 Ethylene glycol (Sigma; cat no E9129), store at room temperature 21 Dimethyl sulfoxide, hybridoma tested, x 10 ml in flamed sealed ampules (Sigma; cat no D2650), store at room temperature and protect from light 22 Mitomycin C, cell culture grade (Sigma; cat no M4287), store at 40C 23 1-well dish, 60-mm diameter, well area: 2.89 cm2 (BD, Franklin Lakes, NJ; cat no 353652) 24 4-well plate, well area: 1.39 cm2 (BD; cat no 353653) 25 4-well culture slides, well area: 1.7 cm2 (BD; cat no 354114) 26 75 cm2 canted neck, vented tissue culture flask (BD; cat no 353136) 27 175 cm2 canted neck, vented tissue culture flask (BD; cat no 353112) 28 15-ml conical centrifuge tubes, high-clarity polypropylene (BD; cat no 352196) 29 50-ml conical centrifuge tubes, high-clarity polypropylene (BD; cat no 352070) 206 30 1-ml individually wrapped serological pipet (BD; cat no 357522) 31 5-ml individually wrapped serological pipet (BD; cat no 357543) 32 10-ml individually wrapped serological pipet (BD; cat no 357551) 33 25-ml individually wrapped serological pipet (BD; cat no 357525) 34 500-ml Stericup-GP filter unit (Millipore, Billerica, MA; cat no SCGP U05 RE) 35 Sterivex-GP 2000 filling bell filter unit (Millipore; cat no SVGP B10 10) 36 33-mm Millex-GP filter unit (Millipore; cat no SLGP 033 RS) 37 Nunc System 100 cryogenic vials with silicon gasket (Nalgene, Rochester, NY; cat no 5000-1012) 38 Glass capillaries, 1.0-mm OD (Clark Electromedical Industries, Kent, UK; cat no GC100T-15) 39 Sterile 30G hypodermic needles (BD; cat no 511252/511256) 40 Vector Red Alkaline Phosphatase Substrate Kit I, SK-5100 (Vector Labs, Inc., Bulingame, CA; cat no SK-5100) 41 KaryoMAX Colcemid solution, liquid (10µg/ml) in PBS (Invitrogen; cat no 15212-012) 42 TRIzolTM reagent (Invitrogen; cat no 15596-026) 43 Ambion DNA-freeTM reagent (Ambion; cat no 1906) 44 SuperScriptTM III first-strand synthesis system for RT-PCR (Invitrogen; cat no 18680051) 45 TaqManTM probes were purchased from Applied BioSystems (ABI) Assay on DemandTM and Assay by DesignTM service 207 ... in the body There are four classes of pluripotent stem cells in humans, other primates and mice These are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells and recently the. .. stem cells versus embryonic stem cells The general differences between the characteristics of adult and embryonic stem cells are summarised in Table The contention that somatic stem cells alone... cells make up the human body viz., germ cells, somatic cells and stem cells Somatic cells include the bulk of the cells that make up the human adult and each of these cells in their differentiated