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Human Embryonic Stem Cell Derivation and Directed Differentiation

  • Conference paper
The Promises and Challenges of Regenerative Medicine

Part of the book series: Ernst Schering Research Foundation Workshop ((SCHERING FOUND,volume 54))

Abstract

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine.

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References

  • Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278

    Article  PubMed  CAS  Google Scholar 

  • Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M (2001) Insulin production by human embryonic stem cells. Diabetes 50:1691–1697

    PubMed  CAS  Google Scholar 

  • Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17:126–140

    Article  PubMed  CAS  Google Scholar 

  • Cerdan C, Rouleau A, Bhatia M (2004) VEGF-A165 augments erythropoietic development from human embryonic stem cells. Blood 103:2504–2512

    Article  PubMed  CAS  Google Scholar 

  • Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M (2003) Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102:906–915

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Conley BJ, Trounson AO, Mollard R (2004) Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther 19:218–223

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Daheron L, Opitz SL, Zaehres H, Lensch WM, Andrews PW, Itskovitz-Eldor J, Daley GQ (2004) LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 22:770–778

    Article  PubMed  CAS  Google Scholar 

  • Denham M, Trounson A, Mollard R (2002) Respiratory lineage differentiation of embryonic stem cells in vitro. Keystone Symposia — stem cells, Colorado, USA

    Google Scholar 

  • Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11:514–518

    Article  PubMed  CAS  Google Scholar 

  • Galat V, Strelchenko N, Ozen S, Sky S, Kukharenko V, Verlinsky Y (2004) Human embryonic stem cells from embryos affected by genetic diseases (abstract 115). International Society for Stem Cell Research 2nd Annual Meeting, Boston, USA pp 77

    Google Scholar 

  • Gertow K, Wolbank S, Rozell B, Sugars R, Andang M, Parish CL, Imreh MP, Wendel M, Ahrlund-Richter L (2004) Organized development from human embryonic stem cells after injection into immunodeficient mice. Stem Cells Dev 13:421–435

    Article  PubMed  Google Scholar 

  • Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trombetta C, Boudjema E (2002) The role of preimplantation diagnosis for aneuploidies. Reprod Biomed Online 4 Suppl 3:31–36

    PubMed  Google Scholar 

  • Green H, Easley K, Iuchi S (2003) Marker succession during the development of keratinocytes from cultured human embryonic stem cells. Proc Natl Acad Sci U S A 100:15625–1630

    Article  PubMed  CAS  Google Scholar 

  • Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E, Reubinoff BE (2003) Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 7:281–287

    Article  PubMed  CAS  Google Scholar 

  • Hanna LA, Foreman RK, Tarasenko IA, Kessler DS, Labosky PA (2002) Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev 16:2650–2661

    Article  PubMed  CAS  Google Scholar 

  • Heins N, Englund MC, Sjoblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C, Semb H (2004) Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22:367–376

    Article  PubMed  Google Scholar 

  • Herzog EL, Chai L, Krause DS (2003) Plasticity of marrow-derived stem cells. Blood 102:3483–3493

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6:88–95

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Jones GM (2000) Growth and viability of human blastocysts in vitro. Reprod Med Rev 8:241–287

    Article  Google Scholar 

  • Katz-Jaffe MG, Trounson AO, Cram DS (2004) Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability. Mol Hum Reprod 10:143–147

    Article  PubMed  CAS  Google Scholar 

  • Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA (2001) Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 98:10716–1021

    Article  PubMed  CAS  Google Scholar 

  • Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F, Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N, Sasai Y (2002) Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 99:1580–1585

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Klimanskaya I, Hipp J, Rezai KA, West M, Atala A, Lanza R (2004) Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells 6:217–245

    PubMed  CAS  Google Scholar 

  • Kukharenko V, Strelchenko N, Galat V, Sky O (2004) Panel of human embryonic stem cell lines (abstract 143). International Society for Stem Cell Research 2nd Annual Meeting, Boston, USA pp 87

    Google Scholar 

  • Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R (2002) Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 99:4391–4396

    Article  PubMed  CAS  Google Scholar 

  • Loebel DA, Watson CM, De Young RA, Tam PP (2003) Lineage choice and differentiation inmouse embryos and embryonic stem cells. Dev Biol 264:1–14

    Article  PubMed  CAS  Google Scholar 

  • Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292:1389–1394

    Article  PubMed  CAS  Google Scholar 

  • Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA (2003) High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21:111–117

    Article  PubMed  CAS  Google Scholar 

  • Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A, Stice S (2003) Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21:521–526

    Article  PubMed  CAS  Google Scholar 

  • Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya M, Nishiyama A, Arakawa A, Suemori H, Nakatsuji N, Kawasaki H, Murakami F, Sasai Y (2003) Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci U S A 100:5828–5833

    Article  PubMed  CAS  Google Scholar 

  • 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 (2002) Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 200:233–242

    Article  PubMed  CAS  Google Scholar 

  • Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, de la Riviere AB, Passier R, Tertoolen L (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107:2733–2740

    Article  PubMed  CAS  Google Scholar 

  • Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG, Yoon HS (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69:2007–2014

    Article  PubMed  CAS  Google Scholar 

  • Pebay A, Wong R, Pitson S, Peh G, Koh K, Tellis I, Nguyen L, Pera M (2003) Maintenance of human embryonic stem cells by sphingosine-1-phosphate and platelet-derived growth factor in a serum-free medium. First National Stem Cell Centre Scientific Conference — Stem Cells and Tissue Repair 2003, pp 104

    Google Scholar 

  • Pera MF, Trounson AO (2004) Human embryonic stem cells: prospects for development. Development 131:5515–5525

    Article  PubMed  CAS  Google Scholar 

  • Pera MF, Reubinoff B, Trounson A (2000) Human embryonic stem cells. J Cell Sci 113:5–10

    PubMed  CAS  Google Scholar 

  • Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, Stanley EG, Wardvan Oostwaard D, Mummery C (2004) Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci 117:1269–1280

    Article  PubMed  CAS  Google Scholar 

  • Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA (2003) Insulin staining of ES cell progeny from insulin uptake. Science 299:363

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK (2003) Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 12:1–11

    PubMed  Google Scholar 

  • Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404

    Article  PubMed  CAS  Google Scholar 

  • Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T (2001) Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:1134–1140

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Rossant J (2001) Stem cells from the Mammalian blastocyst. Stem Cells 19:477–482

    Article  PubMed  CAS  Google Scholar 

  • Sathananthan AH (2003) Origins of human embryonic stem cells and their spontaneous differentiation. First National Stem Cell Centre Scientific Conference — Stem Cells and Tissue Repair, Melbourne, Australia 2003, pp 225

    Google Scholar 

  • Sathananthan H, Pera M, Trounson A (2001) The fine structure of human embryonic stem cells. Reprod Biomed Online 4:56–61

    Article  Google Scholar 

  • Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10:55–63

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Stamp LA, Crosby HA, Hawes SM, Strain AJ, Pera MF (2003) Characterisation of GCTM5, a putative marker for early liver cells. First National Stem Cell Centre Annual Conference — Stem Cells and Tissue Repair, Melbourne, Australia 2003, pp 28

    Google Scholar 

  • Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong L, Evans J, Herbert M, Hyslop L, Ahmad S, Murdoch A, Strachan T (2004a) Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22:790–797

    Article  PubMed  Google Scholar 

  • Stojkovic M, Lako M, Strachan T, Murdoch A (2004b) Derivation, growth and applications of human embryonic stem cells. Reproduction 128: 259–267

    Article  PubMed  CAS  Google Scholar 

  • Strelchenko N, Kukharenko V, Verlinksy O (2004) Human ES-cells derived from different embryo stages (abstract 359). International Society for Stem Cell Research 2nd Annual Meeting 2004, pp 163

    Google Scholar 

  • Tanaka TS, Kunath T, Kimber WL, Jaradat SA, Stagg CA, Usuda M, Yokota T, Niwa H, Rossant J, Ko MS (2002) Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 12:1921–1928

    Article  PubMed  CAS  Google Scholar 

  • 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 U S A 92:7844–7848

    Article  PubMed  CAS  Google Scholar 

  • Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP (1996) Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 55:254–259

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Trounson AO (2001) The derivation and potential use of human embryonic stem cells. Reprod Fertil Dev 13:523–532

    Article  PubMed  CAS  Google Scholar 

  • Trounson A (2005) Derivation characteristics and perspectives for mammalian pluripotential stem cells. Reprod Fertil Dev 17:135–141

    Article  PubMed  CAS  Google Scholar 

  • Trounson A (2004) Stem cells, plasticity and cancer — uncomfortable bed fellows. Development 131:2763–2768

    Article  PubMed  CAS  Google Scholar 

  • Vallier L, Rugg-Gunn PJ, Bouhon IA, Andersson FK, Sadler AJ, Pedersen RA (2004) Enhancing and diminishing gene function in human embryonic stem cells. Stem Cells 22:2–11

    Article  PubMed  CAS  Google Scholar 

  • Verlinksy Y, Strelchenko N, Kukharenko V, Galat V (2004) Preimplantation genetic diagnosis: as a source of human embryonic stem cell lines (abstract 370). International Society for Stem Cell Research 2nd Annual Meeting., Boston, USA 2004, pp 166

    Google Scholar 

  • Watanabe D, Suetake I, Tada T, Tajima S (2002) Stage-and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev 118:187–190

    Article  PubMed  CAS  Google Scholar 

  • Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974

    Article  PubMed  CAS  Google Scholar 

  • Xu C, Police S, Rao N, Carpenter MK (2002a) Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 91:501–508

    Article  PubMed  CAS  Google Scholar 

  • Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA (2002b) BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20:1261–1264

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Monash Immunology and Stem Cell Laboratories, Monash University and Australian Stem Cell Centre, Wellington Road, Clayton, Victoria, 3800, Australia

    A. Trounson

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  1. A. Trounson
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Editors and Affiliations

  1. Nihon Schering K.K. Research Center, BMA 3F, 1-5-5- Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    J. Morser

  2. The Center For Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    S. I. Nishikawa

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Trounson, A. (2005). Human Embryonic Stem Cell Derivation and Directed Differentiation. In: Morser, J., Nishikawa, S.I. (eds) The Promises and Challenges of Regenerative Medicine. Ernst Schering Research Foundation Workshop, vol 54. Springer, Berlin, Heidelberg . https://doi.org/10.1007/3-540-37644-5_3

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