Human Embryonic Stem Cell Derivation and Directed Differentiation
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.
Keywords
Embryonic Stem Cell Human Embryonic Stem Cell Preimplantation Genetic Diagnosis Embryoid Body Inner Cell MassPreview
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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 227:271–278PubMedCrossRefGoogle Scholar
- Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M (2001) Insulin production by human embryonic stem cells. Diabetes 50:1691–1697PubMedGoogle 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 17:126–140PubMedCrossRefGoogle Scholar
- Cerdan C, Rouleau A, Bhatia M (2004) VEGF-A165 augments erythropoietic development from human embryonic stem cells. Blood 103:2504–2512PubMedCrossRefGoogle 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 102:906–915PubMedCrossRefGoogle 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 21:131–142PubMedCrossRefGoogle Scholar
- Conley BJ, Trounson AO, Mollard R (2004) Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther 19:218–223PubMedCrossRefGoogle 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 350:1353–1356PubMedCrossRefGoogle 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 22:770–778PubMedCrossRefGoogle Scholar
- Denham M, Trounson A, Mollard R (2002) Respiratory lineage differentiation of embryonic stem cells in vitro. Keystone Symposia — stem cells, Colorado, USAGoogle 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 11:514–518PubMedCrossRefGoogle 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 77Google 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 13:421–435PubMedCrossRefGoogle 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 3:31–36PubMedGoogle 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 100:15625–1630PubMedCrossRefGoogle 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 7:281–287PubMedCrossRefGoogle 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 16:2650–2661PubMedCrossRefGoogle 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 22:367–376PubMedCrossRefGoogle Scholar
- Herzog EL, Chai L, Krause DS (2003) Plasticity of marrow-derived stem cells. Blood 102:3483–3493PubMedCrossRefGoogle 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 18:1404–1409PubMedCrossRefGoogle 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 6:88–95PubMedGoogle 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 418:41–49PubMedCrossRefGoogle Scholar
- Jones GM (2000) Growth and viability of human blastocysts in vitro. Reprod Med Rev 8:241–287CrossRefGoogle 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 10:143–147PubMedCrossRefGoogle 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 98:10716–1021PubMedCrossRefGoogle 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 99:1580–1585PubMedCrossRefGoogle 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 108:407–414PubMedCrossRefGoogle 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 6:217–245PubMedGoogle 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 87Google 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 99:4391–4396PubMedCrossRefGoogle Scholar
- Loebel DA, Watson CM, De Young RA, Tam PP (2003) Lineage choice and differentiation inmouse embryos and embryonic stem cells. Dev Biol 264:1–14PubMedCrossRefGoogle 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 292:1389–1394PubMedCrossRefGoogle 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 21:111–117PubMedCrossRefGoogle 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 21:521–526PubMedCrossRefGoogle 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 100:5828–5833PubMedCrossRefGoogle 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 200:233–242PubMedCrossRefGoogle 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 107:2733–2740PubMedCrossRefGoogle 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 69:2007–2014PubMedCrossRefGoogle 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 104Google Scholar
- Pera MF, Trounson AO (2004) Human embryonic stem cells: prospects for development. Development 131:5515–5525PubMedCrossRefGoogle Scholar
- Pera MF, Reubinoff B, Trounson A (2000) Human embryonic stem cells. J Cell Sci 113:5–10PubMedGoogle 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 117:1269–1280PubMedCrossRefGoogle Scholar
- Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA (2003) Insulin staining of ES cell progeny from insulin uptake. Science 299:363PubMedGoogle Scholar
- Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA (2002) “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 298:597–600PubMedCrossRefGoogle Scholar
- Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK (2003) Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 12:1–11PubMedGoogle 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 18:399–404PubMedCrossRefGoogle 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 19:1134–1140PubMedCrossRefGoogle 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 20:933–936PubMedCrossRefGoogle Scholar
- Rossant J (2001) Stem cells from the Mammalian blastocyst. Stem Cells 19:477–482PubMedCrossRefGoogle 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 225Google Scholar
- Sathananthan H, Pera M, Trounson A (2001) The fine structure of human embryonic stem cells. Reprod Biomed Online 4:56–61CrossRefGoogle 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 10:55–63PubMedCrossRefGoogle 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 95:13726–13731PubMedCrossRefGoogle 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 100:13350–13355PubMedCrossRefGoogle 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 28Google 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 22:790–797PubMedCrossRefGoogle Scholar
- Stojkovic M, Lako M, Strachan T, Murdoch A (2004b) Derivation, growth and applications of human embryonic stem cells. Reproduction 128: 259–267PubMedCrossRefGoogle 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 163Google 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 12:1921–1928PubMedCrossRefGoogle 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 92:7844–7848PubMedCrossRefGoogle 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 55:254–259PubMedCrossRefGoogle 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 282:1145–1147PubMedCrossRefGoogle Scholar
- Trounson AO (2001) The derivation and potential use of human embryonic stem cells. Reprod Fertil Dev 13:523–532PubMedCrossRefGoogle Scholar
- Trounson A (2005) Derivation characteristics and perspectives for mammalian pluripotential stem cells. Reprod Fertil Dev 17:135–141PubMedCrossRefGoogle Scholar
- Trounson A (2004) Stem cells, plasticity and cancer — uncomfortable bed fellows. Development 131:2763–2768PubMedCrossRefGoogle 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 22:2–11PubMedCrossRefGoogle 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 166Google Scholar
- Watanabe D, Suetake I, Tada T, Tajima S (2002) Stage-and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev 118:187–190PubMedCrossRefGoogle 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 19:971–974PubMedCrossRefGoogle Scholar
- Xu C, Police S, Rao N, Carpenter MK (2002a) Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 91:501–508PubMedCrossRefGoogle 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 20:1261–1264PubMedCrossRefGoogle 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 19:1129–1133PubMedCrossRefGoogle Scholar
- Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321PubMedCrossRefGoogle Scholar