Stem Cell Reviews and Reports

, Volume 7, Issue 4, pp 775–781 | Cite as

A Survey of Parameters Involved in the Establishment of New Lines of Human Embryonic Stem Cells

  • Ana Maria Fraga
  • Érica Sara Souza de Araújo
  • Raquel Stabellini
  • Naja Vergani
  • Lygia V. PereiraEmail author


Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998, more than 1,200 different hESC lines have been established worldwide. Nevertheless, there is still a recognized interest in the establishment of new lines of hESC, particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998, when human LIF (hLIF) was used for maintenance of pluripotency. However, there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries, and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines, our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines, indicating that optimum conditions have not been consolidated yet, and thus, hESC establishment is still an evolving field of research.


Human embryonic stem cells Cell line derivation Pluripotency Human embryo Cell therapy 



This work was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico/Departamento de Ciência e Tecnologia do Ministério da Saúde (CNPq/MS/DECIT). AMF, RS and ESSA have/had Ph.D. fellowships from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The authors declare no conflicts of interest.

Supplementary material

12015_2011_9250_MOESM1_ESM.xls (178 kb)
Supplementary Table 1 Original data on the derivation of hESC lines (XLS 178 kb)


  1. 1.
    Evans, M. J., & Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature, 292, 154–156.PubMedCrossRefGoogle Scholar
  2. 2.
    Guasch, G., & Fuchs, E. (2005). Mice in the world of stem cell biology. Nature Genetics, 37(11), 1201–1206.PubMedCrossRefGoogle Scholar
  3. 3.
    Pera, M. F., & Trounson, A. O. (2004). Human embryonic stem cells: prospects for development. Development, 131(22), 5515–5525.PubMedCrossRefGoogle Scholar
  4. 4.
    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.PubMedCrossRefGoogle Scholar
  5. 5.
    Osafune, K., Caron, L., Borowiak, M., et al. (2008). Marked differences in differentiation propensity among human embryonic stem cell lines. Nature Biotechnology, 26(3), 313–315.PubMedCrossRefGoogle Scholar
  6. 6.
    Silva, S. S., Rowntree, R. K., Mekhoubad, S., & Lee, J. T. (2008). X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proceedings of the National Academy of Science (USA), 105(12), 4820–4825.CrossRefGoogle Scholar
  7. 7.
    Drukker, M., & Benvenisty, N. (2004). The immunogenicity of human embryonic stem-derived cells. Trends in Biotechnology, 22, 136–141.PubMedCrossRefGoogle Scholar
  8. 8.
    Nakajima, F., Tokunaga, K., & Nakatsuji, N. (2007). Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in cell transplantation therapy. Stem Cells, 25, 983–985.PubMedCrossRefGoogle Scholar
  9. 9.
    Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology, 18(4), 399–404.PubMedCrossRefGoogle Scholar
  10. 10.
    Hasegawa, K., Pomeroy, J. E., & Pera, M. F. (2010). Current technology for the derivation of pluripotent stem cell lines from human embryos. Cell Stem Cell, 6(6), 521–531.PubMedCrossRefGoogle Scholar
  11. 11.
    Amit, M., Carpenter, M. K., Inokuma, M. S., et al. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology, 227(2), 271–278.PubMedCrossRefGoogle Scholar
  12. 12.
    Cowan, C. A., Klimanskaya, I., McMahon, J., et al. (2004). Derivation of embryonic stem-cell lines from human blastocysts. The New England Journal of Medice, 350, 1353–1356.CrossRefGoogle Scholar
  13. 13.
    Park, S. P., Lee, Y. J., Lee, K. S., et al. (2004). Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Human Reproduction, 19, 676–684.PubMedCrossRefGoogle Scholar
  14. 14.
    Inzunza, J., Gertow, K., Strömberg, M. A., et al. (2005). Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells, 23(4), 544–549.PubMedCrossRefGoogle Scholar
  15. 15.
    Darnfors, C., Flodin, A., Andersson, K., et al. (2005). High-resolution analysis of the subtelomeric regions of human embryonic stem cells. Stem Cells, 23(4), 483–488.PubMedCrossRefGoogle Scholar
  16. 16.
    Chavez, S. L., Meneses, J. J., Nguyen, H. N., Kim, S. K., & Pera, R. A. (2008). Characterization of six new human embryonic stem cell lines (HSF7, 8, −9, −10, −12, and −13) derived under minimal-animal component conditions. Stem Cells and Development, 17(3), 535–546.PubMedCrossRefGoogle Scholar
  17. 17.
    Chen, A. E., Egli, D., Niakan, K., et al. (2009). Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell, 4(2), 103–106.PubMedCrossRefGoogle Scholar
  18. 18.
    Chung, Y., Klimanskaya, I., Becker, S., et al. (2008). Human embryonic stem cell lines generated without embryo destruction. Cell Stem Cell, 2(2), 113–117.PubMedCrossRefGoogle Scholar
  19. 19.
    Lerou, P. H., Yabuuchi, A., Huo, H., et al. (2008). Human embryonic stem cell derivation from poor-quality embryos. Nature Biotechnology, 26(2), 212–214.PubMedCrossRefGoogle Scholar
  20. 20.
    Liu, W., Yin, Y., Long, X., et al. (2009). Derivation and characterization of human embryonic stem cell lines from poor quality embryos. Journal of Genetics and Genomics, 36, 229–239.PubMedCrossRefGoogle Scholar
  21. 21.
    Andrews, P. W., Stacey, G. N., & Sato, J. D. (editors) (2010). Special issue on novel human embryonic stem cell lines and related resources. In Vitro Cellular & Developmental Biology. Animal, 46(3–4), 167–402.Google Scholar
  22. 22.
    Borstlap, J., Luong, M. X., Rooke, H. M., et al. (2010). International stem cell registries. In Vitro Cellular & Developmental Biology. Animal, 46, 242–246.CrossRefGoogle Scholar
  23. 23.
    Pennings, G. (2003). New Belgian Law on Research on Human Embryos: trust in progress through medical science. Journal of Assisted Reproduction and Genetics, 20(8), 343–346.PubMedCrossRefGoogle Scholar
  24. 24.
    Fraga, A. M., Sukoyan, M., Rajan, P., et al. (2010). Establishment of a Brazilian line of human embryonic stem cells in defined medium—implications for cell therapy in an ethnically diverse population. Cell Transplantation, doi: 10.3727/096368910X522261.
  25. 25.
    Snyder, E. Y., & Loring, J. F. (2006). Beyond fraud—stem-cell research continues. The New England Journal of Medicine, 354(4), 321–324.PubMedCrossRefGoogle Scholar
  26. 26.
    Laurent, L. C., Nievergelt, C. M., Lynch, C., et al. (2010). Restricted ethnic diversity in human embryonic stem cell lines. Nature Methods, 7(1), 6–7.PubMedCrossRefGoogle Scholar
  27. 27.
    Mosher, J. T., Pemberton, T. J., Harter, K., et al. (2010). Lack of population diversity in commonly used human embryonic stem-cell lines. The New England Journal of Medicine, 362(2), 183–185.PubMedCrossRefGoogle Scholar
  28. 28.
    Greely, H. T. (2006). Moving human embryonic stem cells from legislature to lab: remaining legal and ethical questions. PLoS Medicine, 3(5), e143.PubMedCrossRefGoogle Scholar
  29. 29.
    Klimanskaya, I., Chung, Y., Becker, S., Lu, S. J., & Lanza, R. (2006). Human embryonic stem cell lines derived from single blastomeres. Nature, 444(7118), 481–485.PubMedCrossRefGoogle Scholar
  30. 30.
    Feki, A., Bosman, A., Dubuisson, J. B., et al. (2008). Derivation of the first Swiss human embryonic stem cell line form a single blastomere of an arrested four-cell stage embryo. Swiss Medical Weekly, 138(37–38), 540–550.PubMedGoogle Scholar
  31. 31.
    Solter, D., & Knowles, B. B. (1975). Immunosurgery of mouse blastocyst. Proceedings of the National Academy of Science (USA), 72, 5099–5102.CrossRefGoogle Scholar
  32. 32.
    Schiewe, M. C., Hansen, C. T., & Schmidt, P. M. (1992). Lack of antibody specificity by mouse trophectoderm during immunosurgery. Theriogenology, 38(1), 21–32.PubMedCrossRefGoogle Scholar
  33. 33.
    Fernandes, A. M., Meletti, T., Guimarães, R., et al. (2010). Worldwide survey of published procedures to culture human embryonic stem cells. Cell Transplantion. doi: 10.3727/096368909X485067.Google Scholar
  34. 34.
    Horii, T., Nagao, Y., Tokunaga, T., & Imai, H. (2003). Serum-free culture of murine primordial germ cells and embryonic germ cells. Theriogenology, 59(5–6), 1257–1264.PubMedCrossRefGoogle Scholar
  35. 35.
    Khoo, M. L., McQuade, L. R., Smith, M. S., Lees, J. G., Sidhu, K. S., & Tuch, B. E. (2005). Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor. Biology of Reproduction, 73(6), 1147–1156.PubMedCrossRefGoogle Scholar
  36. 36.
    Skottman, H., & Hovatta, O. (2006). Culture conditions for human embryonic stem cells. Reproduction, 132, 691–698.PubMedCrossRefGoogle Scholar
  37. 37.
    Martin, M. J., Muotri, A., Gage, F., & Varki, A. (2005). Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nature Medicine, 11, 228–232.PubMedCrossRefGoogle Scholar
  38. 38.
    Dahéron, L., Opitz, S. L., Zaehres, H., et al. (2004). LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells, 22(5), 770–778.PubMedCrossRefGoogle Scholar
  39. 39.
    Winston, R. M. (2007). Does government regulation inhibit embryonic stem cell research and can it be effective? Cell Stem Cell, 1(1), 27–34.PubMedCrossRefGoogle Scholar
  40. 40.
    Hanna, J., Cheng, A. W., Saha, K., et al. (2010). Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proceedings of the National Academy of Science (USA), 107(20), 9222–9227.CrossRefGoogle Scholar
  41. 41.
    Lengner, C. J., Gimelbrant, A. A., Erwin, J. A., et al. (2010). Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell, 141(5), 872–883.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ana Maria Fraga
    • 1
  • Érica Sara Souza de Araújo
    • 1
  • Raquel Stabellini
    • 1
    • 2
  • Naja Vergani
    • 1
  • Lygia V. Pereira
    • 1
    Email author
  1. 1.Laboratório Nacional de Células-Tronco Embrionárias and Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular, Departamento de Genética e Biologia Evolutiva, Instituto de BiociênciasUniversidade de São PauloSão PauloBrazil
  2. 2.Programa de Transtorno Bipolar (PROMAN), Instituto de PsiquiatriaUniversidade de São PauloSão PauloBrazil

Personalised recommendations