Human embryonic stem cell lines isolation, cultivation, and characterization

  • Maria A. Lagarkova
  • Artem V. Eremeev
  • Anatoly V. Svetlakov
  • Nikolay B. Rubtsov
  • Sergei L. Kiselev


A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics, most important of which is the pluripotency, hESC lines vary significantly in their transcriptional profiles, genetic, and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences, the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report, we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria, including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines, namely hESM01–04, were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free, serum-free conditions using mTeSR1 and Matrigel. The fifth line, hESMK05 was derived in feeder-free, serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community.


hESC lines Isolation Characterization 


  1. Adewumi O.; Aflatoonian B.; Ahrlund-Richter L. et al. Characterization of human embryonic stem cell lines by the international stem cell initiative. Nat. Biotechnol. 25: 803–816; 2007. doi:10.1038/nbt1318.CrossRefPubMedGoogle Scholar
  2. Allegrucci C.; Young L. E. Differences between human embryonic stem cell lines. Hum. Reprod. 13: 103–120; 2007.Google Scholar
  3. Amit M.; Shariki C.; Margulets V.; Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70: 837–845; 2004. doi:10.1095/biolreprod.103.021147.CrossRefPubMedGoogle Scholar
  4. Baker D. E.; Harrison N. J.; Maltby E. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25: 207–215; 2007. doi:10.1038/nbt1285.CrossRefPubMedGoogle Scholar
  5. Bielanska M.; Tan S. L.; Ao A. High rate of mixoploidy among human blastocysts cultured in vitro. Fertil. Steril. 78: 1248–1253; 2002. doi:10.1016/S0015-0282(02)04393-5.CrossRefPubMedGoogle Scholar
  6. Cowan C. A.; Klimanskaya I.; McMahon J. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350: 1353–1356; 2004. doi:10.1056/NEJMsr040330.CrossRefPubMedGoogle Scholar
  7. Draper J. S.; Smith K.; Gokhale P. et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22: 53–54; 2004. doi:10.1038/nbt922.CrossRefPubMedGoogle Scholar
  8. Ellerström C.; Strehl R.; Moya K. et al. Derivation of a xeno-free human embryonic stem cell line. Stem Cells 10: 2170–2176; 2006. doi:10.1634/stemcells.2006-0130.CrossRefGoogle Scholar
  9. Gardner D. K.; Lane M.; Schoolcraft W. B. Physiology and culture of the human blastocyst. J. Reprod. Immunol. 55: 85–100; 2002. doi:10.1016/S0165-0378(01)00136-X.CrossRefPubMedGoogle Scholar
  10. Imreh M. P.; Gertow K.; Cedervall J. et al. In vitro culture conditions favoring selection of chromosomal abnormalities in human es cells. J. Cell. Biochem. 99: 508–516; 2006. doi:10.1002/jcb.20897.CrossRefPubMedGoogle Scholar
  11. Inzunza J.; Sahlen S.; Holmberg K. et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol. Hum. Reprod. 10: 461–466; 2004. doi:10.1093/molehr/gah051.CrossRefPubMedGoogle Scholar
  12. Korneev S. A.; Korneeva E. I.; Lagarkova M. A. et al. Novel noncoding antisense RNA transcribed from human anti-NOS2A locus is differentially regulated during neuronal differentiation of embryonic stem cells. RNA 14: 1232–1239; 2008. doi:10.1261/rna.1084308.CrossRefGoogle Scholar
  13. Lagarkova M. A.; Volchkov P. Y.; Lyakisheva A. V. et al. Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle 5: 416–420; 2006.PubMedGoogle Scholar
  14. Lagarkova M. A.; Volchkov P. Y.; Philonenko E. S. et al. CD 30 is a marker of undifferentiated human embryonic stem cells rather than a biomarker of transformed hESCs. Cell Cycle 7: 3475–3480; 2008a.Google Scholar
  15. Lagarkova M. A.; Volchkov P. Y.; Philonenko E. S. et al. Efficient differentiation of hESCs into endothelial cells in vitro is secured by epigenetic changes. Cell Cycle 7: 2929–2935; 2008b.PubMedGoogle Scholar
  16. Ludwig T. E.; Levenstein M. E.; Jones J. M. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24: 185–187; 2006. doi:10.1038/nbt1177.CrossRefPubMedGoogle Scholar
  17. Maitra A.; Arking D. E.; Shivapurkar N. et al. Genomic alterations in cultured human embryonic stem cells. Nat. Genet. 37: 1099–1103; 2005. doi:10.1038/ng1631.CrossRefPubMedGoogle Scholar
  18. Mikkola M.; Olsson C.; Palgi J. et al. Distinct differentiation characteristics of individual human embryonic stem cell lines. BMC Dev. Biol. 6: 40–46; 2006. doi:10.1186/1471-213X-6-40.CrossRefPubMedGoogle Scholar
  19. Mitalipova M. M.; Rao R. R.; Hoyer D. M. et al. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23: 19–20; 2005. doi:10.1038/nbt0105-19.CrossRefPubMedGoogle Scholar
  20. Prokhorovich M. A.; Lagar'kova M. A.; Shilov A. G. et al. Cultures of hESM human embryonic stem cells: chromosomal aberrations and karyotype stability. Bull. Exp. Biol. Med. 144: 126–129; 2007. doi:10.1007/s10517-007-0271-z.CrossRefPubMedGoogle Scholar
  21. Reubinoff B. E.; Pera M. F.; Fong C. Y. et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18: 399–404; 2000. doi:10.1038/74447.CrossRefPubMedGoogle Scholar
  22. Rubtsov N. B.; Karamisheva T. V.; Astakhova N. M. et al. Zoo-fish with region-specific paints for mink chromosome 5q: delineation of inter- and intrachromosomal rearrangements in human, pig, and fox. Cytogenet. Cell Genet. 90: 268–270; 2000. doi:10.1159/000056786.CrossRefPubMedGoogle Scholar
  23. Skottman H.; Hovatta O. Culture conditions for human embryonic stem cells. Reproduction 132: 691–698; 2006. doi:10.1530/rep. 1.01079.CrossRefPubMedGoogle Scholar
  24. Thomson J. A.; Itskovitz-Eldor J.; Shapiro S. S. et al. Embryonic stem cell lines derived from human blastocysts. Science 282; 1998.Google Scholar

Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  • Maria A. Lagarkova
    • 1
  • Artem V. Eremeev
    • 2
  • Anatoly V. Svetlakov
    • 2
  • Nikolay B. Rubtsov
    • 3
  • Sergei L. Kiselev
    • 1
  1. 1.Vavilov Institute of General Genetics RASMoscowRussia
  2. 2.Center for Reproductive MedicineKrasnoyarskRussia
  3. 3.Institute of Cytology and Genetics RASNovosibirskRussia

Personalised recommendations