Sources and Derivation of Human Embryonic Stem Cells

  • Michal Amit
Part of the Methods in Molecular Biology book series (MIMB, volume 997)


Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass (ICM) of the developing embryo. hESCs culture as cell lines in vitro and possess great potential in such research fields as developmental biology and cell-based therapy, as well as such industrial purposes as drug screening and toxicology. When ESCs were first derived by Thomson and colleagues, traditional methods of immunostaining and culturing, using primary mouse embryonic fibroblasts and medium supplemented by serum were used. Considerable efforts have since led to improved methods for isolating new lines in defined and reproducible conditions. This chapter discusses sources for embryos for ESC isolation, commonly used methods for deriving hESC lines, and a number of possible culture systems.

Key words

Human Pluripotent stem cells Derivation Sources 



The author thanks Mrs. Hadas O’Neill for editing the manuscript. The research conducted was partly supported by Technion Research and Development Foundation (TRDF).


  1. 1.
    Stevens LC, Little CC (1954) Spontaneous testicular teratomas in an inbred strain of mice. Proc Natl Acad Sci USA 40:1080–1087PubMedCrossRefGoogle Scholar
  2. 2.
    Pierce GB, Dixon FJ (1959) Testicular teratomas. II. Teratocarcinoma as an ascetic tumor. Cancer 12:584–589PubMedCrossRefGoogle Scholar
  3. 3.
    Pierce GB, Verney EL (1961) An in vitro and in vivo study of differentiation in teratocarcinomas. Cancer 14:1017–1029PubMedCrossRefGoogle Scholar
  4. 4.
    Kleinsmith LJ, Pierce GB (1964) Multipotentiality of single embryonal carcinoma cells. Cancer Res 24:1544–1551PubMedGoogle Scholar
  5. 5.
    Gardner RL (1998) Contribution of blastocyst micromanipulation to the study of mammalian development. Bioessays 20:168–180PubMedCrossRefGoogle Scholar
  6. 6.
    Brinster RL (1974) The effect of transferred into to the mouse blastocyst on subsequent development. J Exp Med 140:1049–1056PubMedCrossRefGoogle Scholar
  7. 7.
    Mintz B, Illmensee K (1975) Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc Natl Acad Sci USA 72:3585–3589PubMedCrossRefGoogle Scholar
  8. 8.
    Andrews P (2002) From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 357:405–417PubMedCrossRefGoogle Scholar
  9. 9.
    Martin GR, Evans MJ (1975) Multiple differentiation of clonal teratocarcinoam stem cells following embryoid body formation in vitro. Cell 6:467–474CrossRefGoogle Scholar
  10. 10.
    Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156PubMedCrossRefGoogle Scholar
  11. 11.
    Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638PubMedCrossRefGoogle Scholar
  12. 12.
    Evans MJ (1972) The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratocarcinoma cells. J Embryol Exp Morphol 28:163–196PubMedGoogle Scholar
  13. 13.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  14. 14.
    Thomson JA et al (1995) Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 92:7844–7848PubMedCrossRefGoogle Scholar
  15. 15.
    Thomson JA et al (1996) Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 55:254–259PubMedCrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Steptoe PC, Edwards RG (1978) Birth after the reimplantation of a human embryo. Lancet 2:366PubMedCrossRefGoogle Scholar
  18. 18.
    Gardner DK, Lane M, Calderon I, Leeton J (1996) Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril 65:349–353PubMedGoogle Scholar
  19. 19.
    Gardner DK (1994) Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int 18:1163–1179PubMedCrossRefGoogle Scholar
  20. 20.
    Reubinoff BE, Pera MF, Fong C, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404PubMedCrossRefGoogle Scholar
  21. 21.
    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
  22. 22.
    Verlinsky Y, Strelchenko N, Kukharenko V, Rechitsky S, Verlinsky O, Galat V, Kuliev A (2005) Human embryonic stem cell lines with genetic disorders. Reprod Biomed Online 10:105–110PubMedCrossRefGoogle Scholar
  23. 23.
    Mateizel I, De Temmerman N, Ullmann U, Cauffman G, Sermon K, Van de Velde H, De Rycke M, Degreef E, Devroey P, Liebaers I, Van Steirteghem A (2006) Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21:503–511PubMedCrossRefGoogle Scholar
  24. 24.
    Lanzendorf SE, Boyd CA, Wright DL, Muasher S, Oehninger S, Hodgen GD (2001) Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines. Fertil Steril 76:132–137PubMedCrossRefGoogle Scholar
  25. 25.
    Vrana KE, Hipp JD, Goss AM, McCool BA, Riddle DR, Walker SJ, Wettstein PJ, Studer LP, Tabar V, Cunniff K, Chapman K, Vilner L, West MD, Grant KA, Cibelli JB (2003) Nonhuman primate parthenogenetic stem cells. Proc Natl Acad Sci USA 100:11911–11916PubMedCrossRefGoogle Scholar
  26. 26.
    Revazova ES, Turovets NA et al (2007) Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 9:432–449PubMedCrossRefGoogle Scholar
  27. 27.
    Revazova ES et al (2008) HLA homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 10:11–24PubMedCrossRefGoogle Scholar
  28. 28.
    Hwang WS et al (2005) Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308:1777–1783PubMedCrossRefGoogle Scholar
  29. 29.
    Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R (2006) Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439:216–219PubMedCrossRefGoogle Scholar
  30. 30.
    Giritharan G, Ilic D, Gormley M, Krtolica A (2011) Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles. PLoS One 6:e26570PubMedCrossRefGoogle Scholar
  31. 31.
    Kim K, Zhao R, Doi A, Ng K, Unternaehrer J, Cahan P, Huo H, Loh YH, Aryee MJ, Lensch MW, Li H, Collins JJ, Feinberg AP, Daley GQ (2011) Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol 29:1117–1119PubMedCrossRefGoogle Scholar
  32. 32.
    Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark AT, Plath K, Lowry WE, Benvenisty N (2010) Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7:521–531PubMedCrossRefGoogle Scholar
  33. 33.
    Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448(7151):313–317PubMedCrossRefGoogle Scholar
  34. 34.
    Solter D, Knowles BB (1975) Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 72:5099–5102PubMedCrossRefGoogle Scholar
  35. 35.
    Amit M, Itskovitz-Eldor J (2002) Derivation and spontaneous differentiation of human embryonic stem cells. J Anat 200:225–232PubMedCrossRefGoogle Scholar
  36. 36.
    Turetsky T, Aizenman E, Gil Y, Weinberg N, Shufaro Y, Revel A, Laufer N, Simon A, Abeliovich D, Reubinoff BE (2007) Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum Reprod 23:46–53PubMedCrossRefGoogle Scholar
  37. 37.
    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
  38. 38.
    Amit M, Margulets V, Segev H, Shariki C, Laevsky I, Coleman R, Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68:2150–2156PubMedCrossRefGoogle Scholar
  39. 39.
    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
  40. 40.
    Simón C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, Asensi A, Sánchez E, Esplugues J, Fisher S, Pellicer A (2005) First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertil Steril 83:246–249PubMedCrossRefGoogle Scholar
  41. 41.
    Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, Caceres E, McMaster M, McDonagh S, Li Y, Mandalam R, Lebkowski J, Fisher SJ (2005) Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 83:1517–1529PubMedCrossRefGoogle Scholar
  42. 42.
    Tecirlioglu RT, Nguyen L, Koh K, Trounson AO, Michalska AE (2010) Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium. In Vitro Cell Dev Biol Anim 46:231–235PubMedCrossRefGoogle Scholar
  43. 43.
    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
  44. 44.
    Inzunza J, Gertow K, Stromberg MA, Matilainen E, Blennow E, Skottman H, Wolbank S, Ahrlund-Richter L, Hovatta O (2005) Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23:544–549PubMedCrossRefGoogle Scholar
  45. 45.
    Ström S, Holm F, Bergström R, Strömberg AM, Hovatta O (2010) Derivation of 30 human embryonic stem cell lines. In Vitro Cell Dev Biol Anim 46:337–344PubMedCrossRefGoogle Scholar
  46. 46.
    Aguilar-Gallardo C, Poo M, Gomez E, Galan A, Sanchez E, Marques-Mari A, Ruiz V, Medrano J, Riboldi M, Valbuena D, Simon C (2010) Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6 M, -7, -8, and -9) on human feeder. In Vitro Cell Dev Biol Anim 46:317–326PubMedCrossRefGoogle Scholar
  47. 47.
    Ilic D, Giritharan G, Zdravkovic T, Caceres E, Genbacev O, Fisher SJ, Krtolica A (2009) Derivation of human embryonic stem cell lines from biopsied blastomeres on human feeders with minimal exposure to xenomaterials. Stem Cells Dev 18:1343–1350PubMedCrossRefGoogle Scholar
  48. 48.
    Valbuena D, Galánm A, Sánchez E, Poo ME, Gómez E, Sánchez-Luengo S, Melguizo D, García A, Ruiz V, Moreno R, Pellicer A, Simón C (2006) Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL 3 -4 -5) on human feeder and in serum-free conditions. Reprod Biomed Online 13:875–886PubMedCrossRefGoogle Scholar
  49. 49.
    Ellerström C, Strehl R, Moya K, Andersson K, Bergh C, Lundin K, Hyllner J, Semb H (2006) Derivation of a xeno-free human embryonic stem cell line. Stem Cells 24:2170–2176PubMedCrossRefGoogle Scholar
  50. 50.
    Crook JM, Peura TT, Kravets L, Bosman AG, Buzzard JJ, Horne R, Hentze H, Dunn NR, Zweigerdt R, Chua F, Upshall A, Colman A (2007) The generation of six clinical-grade human embryonic stem cell lines. Cell Stem Cell 1:490–494CrossRefGoogle Scholar
  51. 51.
    Prathalingam N, Ferguson L, Young L, Lietz G, Oldershaw R, Healy L, Craig A, Lister H, Binaykia R, Sheth R, Murdoch A, Herbert M (2012) Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture. Stem Cell Res Ther 3:12PubMedCrossRefGoogle Scholar
  52. 52.
    Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R (2005) Human embryonic stem cells derived without feeder cells. Lancet 365:1636–1641PubMedCrossRefGoogle Scholar
  53. 53.
    Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24:185–187PubMedCrossRefGoogle Scholar
  54. 54.
    Amit M, Chebath J, Marguletz V, Laevsky I, Miropolsky Y, Shariki K, Peri M, Revel M, Itskovitz-Eldor J (2010) Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells. Stem Cell Rev 6:248–259PubMedCrossRefGoogle Scholar
  55. 55.
    Amit M, Laevskym I, Miropolsky Y, Shariki K, Peri M, Itskovitz-Eldor J (2011) Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat Protoc 6:572–579PubMedCrossRefGoogle Scholar
  56. 56.
    Olmer R et al (2010) Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem Cell Res 5:51–64PubMedCrossRefGoogle Scholar
  57. 57.
    Singh H, Mok P, Balakrishnan T, Rahmat SN, Zweigerdt R (2010) Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. Stem Cell Res 4:165–179PubMedCrossRefGoogle Scholar
  58. 58.
    Steiner D et al (2010) Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28:361–364PubMedCrossRefGoogle Scholar
  59. 59.
    Amit M, Itskovitz-Eldor J (2012) Atlas on human pluripotent stem cells—derivation and culturing. Series: stem cell biology and regenerative medicine. In: Turksen (ed), Chapter 1, Fig 1.11 a, Humana PressGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Michal Amit
    • 1
  1. 1.Department of Obstetrics and GynecologyTechnion Israel Institute of Technology, Rambam Medical CenterHaifaIsrael

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