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Cytotechnology

, Volume 62, Issue 6, pp 509–518 | Cite as

A whole-mechanical method to establish human embryonic stem cell line HN4 from discarded embryos

  • Bin Li
  • Lan Xu
  • Wei-Ying Lu
  • Wen Xu
  • Mei-Hong Wang
  • Ke Yang
  • Juan Dong
  • Xiao-Yan Ding
  • Yuan-Hua Huang
Method in Cell Science

Abstract

Since the first human embryonic stem cell (hESC) line was generated by Thomson et al. (in Science 282:1145–1147, 1998), hundreds of hESC lines have been reported by different labs, providing resources for basic research and regenerative medicine as well. However it has been widely recognized that hESC lines varied on their properties, in terms of gene expression profile, epigenetic modify profile, and differentiation tendency. Generation of more hESC lines will largely enhance our knowledge of hESCs innate character. In this current work, we reported the generation of HN4, a hESC line derived from grade III IVF human embryo by using a mixture of human foreskin fibroblast (HFF) and mouse embryonic fibroblast (MEF) as feeder layers, and a whole-mechanical method in inner cell mass (ICM) isolation. HN4 satisfied the criteria of hESCs pluripotency, with high expression of hESC surface markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81), transcription factors (OCT-4, NANOG, REX-1), and alkaline phosphatase. It is able to differentiate to three germ layer derivatives when cultured in vitro, or in teratoma formation. Moreover, it displayed promising potential in neural differentiation under a proper culture condition, suggesting the advantage of HN4 in further investigation. Additionally, the whole-mechanical protocol for ICM isolation facilitates hESC line generation for its ease to handle.

Keywords

Human stem cells ICM mechanical isolation Mixed feeder layer Neural differentiation 

References

  1. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R et al (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68:2150–2156CrossRefGoogle Scholar
  2. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R (2000) Blastocyst quality affects the success of blastocyst-stage embryo transfer. Fertil Steril 74:282–287CrossRefGoogle Scholar
  3. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS (2001) Entrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172:383–397CrossRefGoogle Scholar
  4. Carpenter MK, Rosler E, Rao MS (2003) Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 5:79–88CrossRefGoogle Scholar
  5. 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 21:131–142CrossRefGoogle Scholar
  6. Choo A, Padmanabhan J, Chin A, Fong WJ, Oh SK (2006) Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions. J Biotechnol 122:130–141CrossRefGoogle Scholar
  7. Dvash T, Ben-Yosef D, Eiges R (2006) Human embryonic stem cells as a powerful tool for studying human embryogenesis. Pediatr Res 60:111–117CrossRefGoogle Scholar
  8. Familari M, Selwood L (2006) The potential for derivation of embryonic stem cells in vertebrates. Mol Reprod Dev 73:123–131CrossRefGoogle Scholar
  9. Findikli N, Kahraman S, Akcin O, Sertyel S, Candan Z (2005) Establishment and characterization of new human embryonic stem cell lines. Reprod Biomed Online 10:617–627CrossRefGoogle Scholar
  10. Heins N, LindahI A, Karlsson U, Rehnström M, Caisander G, Emanuelsson K et al (2006) Clonal derivation and characterization of human embryonic stem cell lines. J Biotechnol 122:511–520CrossRefGoogle Scholar
  11. Hong S, Kang UJ, Isacson O, Kim KS (2008) Neural precursors derived from human embryonic stem cells mainstain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons. J Neurochem 104:316–324Google Scholar
  12. Hovatta O, Mikkola M, Gertow K, Strömberg AM, Inzunza J, Hreinsson J et al (2003) A culture system using foreskin fibroblasts as a feeder cells allow production of human embryonic stem cells. Hum Reprod 18:1404–1409CrossRefGoogle Scholar
  13. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M et al (2000) Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med 6:88–95Google Scholar
  14. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K et al (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25:4694–4705CrossRefGoogle Scholar
  15. Lerou PH, Daley GQ (2005) Therapeutic potential of embryonic stem cells. Blood Rev 19:321–331CrossRefGoogle Scholar
  16. Lerou PH, Yabuuchi A, Huo H, Miller JD, Boyer LF, Schlaeger TM et al (2008) Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos. Nat Protoc 3:923–933CrossRefGoogle Scholar
  17. Li B, Peng QP, Lu WY, Xu W, Jin YX, Huang YH (2008) Growth state of human embryonic stem cells on mixed feeder layers with mouse embryonic fibroblasts and human foreskin fibroblasts at different ratios. CRTER 12:424–428Google Scholar
  18. Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS, Mattson MP (2008) Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev Biol 8:90CrossRefGoogle Scholar
  19. Mandal A, Tipnis S, Pal R, Ravindran G, Bose B, Patki A et al (2006) Characterization and in vitro differentiation potential of a new human embryonic stem cell line, ReliCellhES1. Differentiation 74:81–90CrossRefGoogle Scholar
  20. Mummery C (2004) Stem cell research: immortality or healthy old age? Eur J Endocrinol 151(Suppl. 3):U7–U12CrossRefGoogle Scholar
  21. Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG et al (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69:2007–2014CrossRefGoogle Scholar
  22. Qian K, Chen H, Zhang SM, Zhu GJ (2006) Human fibroblast cell supporting undifferentiated human embryonic stem cells proliferation in vitro. Acta Med Univ Sci Technol Huazhong 35:462–464Google Scholar
  23. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404CrossRefGoogle Scholar
  24. Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cells masses and embryonic stem cell lines. Nat Biotechnol 20:933–936CrossRefGoogle Scholar
  25. Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21:546–556CrossRefGoogle Scholar
  26. Robertson EJ (1987) Embryo-derived stem cell lines. In: Robertson EJ (ed) Teratocarcinomas and embryonic stem cells. IRL Press, Oxford, pp 71–112Google Scholar
  27. Shin S, Mitalipova M, Noggle S, Tibbitts D, Venable A, Rao P et al (2006) Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem Cells 24:125–138CrossRefGoogle Scholar
  28. Srivastava D, Ivey KN (2006) Potential of stem cell-based therapies for heart disease. Nature 441:1097–1099CrossRefGoogle Scholar
  29. Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J et al (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23:306–314CrossRefGoogle Scholar
  30. Ström S, Inzunza J, Grinnemo KH, Holmberg K, Matilainen E, Strömberg AM et al (2007) Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Human Reprod 22:3051–3058CrossRefGoogle Scholar
  31. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147CrossRefGoogle Scholar
  32. Urban SV, Kiss J, Vas V, Kovacs J, Uher F (2006) Stem cell therapy for diabetes mellitus: progress, prospects and challenges. Orv Hetil 147:791–797Google Scholar
  33. Van de Stolpe A, van den Brink S, van Rooijen M, Ward-van Oostwaard D, van Inzen W, Slaper-Cortenbach I et al (2005) Human embryonic stem cells: towards therapies for cardiac disease. Derivation of a Dutch human embryonic stem cell line. Reprod Biomed Online 11:476–485CrossRefGoogle Scholar
  34. 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 19:1129–1133CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Bin Li
    • 1
    • 3
  • Lan Xu
    • 2
  • Wei-Ying Lu
    • 1
  • Wen Xu
    • 1
  • Mei-Hong Wang
    • 1
  • Ke Yang
    • 2
  • Juan Dong
    • 2
  • Xiao-Yan Ding
    • 2
  • Yuan-Hua Huang
    • 1
  1. 1.Reproductive Medical Center of Hainan ProvinceHainan Medical CollegeHaikouChina
  2. 2.Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.Obstetrics & Gynecology Hospital of Fudan UniversityShanghaiChina

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