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Clean-Up Human Embryonic Stem Cell Lines Using Humanized Culture Condition

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.

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References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7.

    Article  CAS  PubMed  Google Scholar 

  2. Kim HS, Oh SK, Park YB, Ahn HJ, Sung KC, Kang MJ, et al. Methods for derivation of human embryonic stem cells. Stem Cells. 2005;23:1228–33.

    Article  PubMed  Google Scholar 

  3. Trounson A. The production and directed differentiation of human embryonic stem cells. Endocr Rev. 2006;27:208–19.

    Article  PubMed  Google Scholar 

  4. Engle SJ, Puppala D. Integrating human pluripotent stem cells into drug development. Cell Stem Cell. 2013;12:669–77.

    Article  CAS  PubMed  Google Scholar 

  5. Oh SK, Kim HS, Park YB, Seol HW, Kim YY, Cho MS, et al. Methods for expansion of human embryonic stem cells. Stem Cells. 2005;23:605–9.

    Article  CAS  PubMed  Google Scholar 

  6. Ellerstrom C, Strehl R, Moya K, Andersson K, Bergh C, Lundin K, et al. Derivation of a xeno-free human embryonic stem cell line. Stem Cells. 2006;24:2170–6.

    Article  PubMed  Google Scholar 

  7. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med. 2005;11:228–32.

    Article  CAS  PubMed  Google Scholar 

  8. Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol. 2000;227:271–8.

    Article  CAS  PubMed  Google Scholar 

  9. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol. 2002;20:933–6.

    Article  CAS  PubMed  Google Scholar 

  10. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, et al. Human feeder layers for human embryonic stem cells. Biol Reprod. 2003;68:2150–6.

    Article  CAS  PubMed  Google Scholar 

  11. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19:971–4.

    Article  CAS  PubMed  Google Scholar 

  12. Li Y, Powell S, Brunette E, Lebkowski J, Mandalam R. Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol Bioeng. 2005;91:688–98.

    Article  CAS  PubMed  Google Scholar 

  13. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA. Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006;3:637–46.

    Article  CAS  PubMed  Google Scholar 

  14. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8:424–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol. 2004;22:53–4.

    Article  CAS  PubMed  Google Scholar 

  16. Mitalipova MM, Rao RR, Hoyer DM, Johnson JA, Meisner LF, Jones KL, et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol. 2005;23:19–20.

    Article  CAS  PubMed  Google Scholar 

  17. Desai N, Ludgin J, Goldberg J, Falcone T. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line. J Assist Reprod Genet. 2013;30:609–15.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, et al. Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions. PLoS ONE. 2010;5:e14099.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Irie A, Koyama S, Kozutsumi Y, Kawasaki T, Suzuki A. The molecular basis for the absence of N-glycolylneuraminic acid in humans. J Biol Chem. 1998;273:15866–71.

    Article  CAS  PubMed  Google Scholar 

  20. Muchmore EA, Diaz S, Varki A. A structural difference between the cell surfaces of humans and the great apes. Am J Phys Anthropol. 1998;107:187–98.

    Article  CAS  PubMed  Google Scholar 

  21. Baharvand H, Jafary H, Massumi M, Ashtiani SK. Generation of insulin-secreting cells from human embryonic stem cells. Dev Growth Differ. 2006;48:323–32.

    Article  CAS  PubMed  Google Scholar 

  22. Sundberg M, Andersson PH, Akesson E, Odeberg J, Holmberg L, Inzunza J, et al. Markers of pluripotency and differentiation in human neural precursor cells derived from embryonic stem cells and CNS tissue. Cell Transplant. 2011;20:177–91.

    Article  CAS  PubMed  Google Scholar 

  23. Pal R, Mamidi MK, Das AK, Bhonde R. Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions. J Biosci Bioeng. 2013;115:200–6.

    Article  CAS  PubMed  Google Scholar 

  24. Desai N, Rambhia P, Gishto A. Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems. Reprod Biol Endocrinol. 2015;13:9.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet. 2005;365:1636–41.

    Article  CAS  Google Scholar 

  26. Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, et al. Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril. 2005;83:1517–29.

    Article  PubMed  Google Scholar 

  27. Couture LA. Scalable pluripotent stem cell culture. Nat Biotechnol. 2010;28:562–3.

    Article  CAS  PubMed  Google Scholar 

  28. Zhu H, Yang J, Wei Y, Chen HH. Development of a xeno-free substrate for human embryonic stem cell growth. Stem Cells Int. 2015;2015:621057.

    PubMed  PubMed Central  Google Scholar 

  29. Chen KG, Mallon BS, McKay RD, Robey PG. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell. 2014;14:13–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rodin S, Domogatskaya A, Strom S, Hansson EM, Chien KR, Inzunza J, et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol. 2010;28:611–5.

    Article  CAS  PubMed  Google Scholar 

  31. Prowse AB, Doran MR, Cooper-White JJ, Chong F, Munro TP, Fitzpatrick J, et al. Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media. Biomaterials. 2010;31:8281–8.

    Article  CAS  PubMed  Google Scholar 

  32. Soteriou D, Iskender B, Byron A, Humphries JD, Borg-Bartolo S, Haddock MC, et al. Comparative proteomic analysis of supportive and unsupportive extracellular matrix substrates for human embryonic stem cell maintenance. J Biol Chem. 2013;288:18716–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Melkoumian Z, Weber JL, Weber DM, Fadeev AG, Zhou Y, Dolley-Sonneville P, et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat Biotechnol. 2010;28:606–10.

    Article  CAS  PubMed  Google Scholar 

  34. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000;18:399–404.

    Article  CAS  PubMed  Google Scholar 

  35. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 2003;18:1404–9.

    Article  PubMed  Google Scholar 

  36. Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells. 2005;23:306–14.

    Article  CAS  PubMed  Google Scholar 

  37. Meng G, Liu S, Krawetz R, Chan M, Chernos J, Rancourt DE. A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture. Stem Cells Dev. 2008;17:413–22.

    Article  CAS  PubMed  Google Scholar 

  38. Rajala K, Hakala H, Panula S, Aivio S, Pihlajamaki H, Suuronen R, et al. Testing of nine different xeno-free culture media for human embryonic stem cell cultures. Hum Reprod. 2007;22:1231–8.

    Article  CAS  PubMed  Google Scholar 

  39. International Stem Cell Initiative C, Akopian V, Andrews PW, Beil S, Benvenisty N, Brehm J, et al. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim. 2010;46:247–58.

    Article  Google Scholar 

  40. Meng G, Liu S, Li X, Krawetz R, Rancourt DE. Extracellular matrix isolated from foreskin fibroblasts supports long-term xeno-free human embryonic stem cell culture. Stem Cells Dev. 2010;19:547–56.

    Article  CAS  PubMed  Google Scholar 

  41. Beattie GM, Lopez AD, Bucay N, Hinton A, Firpo MT, King CC, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells. 2005;23:489–95.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This research was supported by the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012M3A9C6049722).

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Authors and Affiliations

  1. Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, 71, Ihwajang-gil, Jongno-gu, Seoul, 03087, Korea

    Jin Ah Baek, Hye Won Seol, Juwon Jung, Hee Sun Kim, Sun Kyung Oh & Young Min Choi

  2. Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea

    Hee Sun Kim & Young Min Choi

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  1. Jin Ah Baek
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  2. Hye Won Seol
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  3. Juwon Jung
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  4. Hee Sun Kim
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Correspondence to Young Min Choi.

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Ethical statement

This study was approved by the Ethics Committee of the Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University (IRB No. 219932-201510-ER-05-01-01).

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Baek, J.A., Seol, H.W., Jung, J. et al. Clean-Up Human Embryonic Stem Cell Lines Using Humanized Culture Condition. Tissue Eng Regen Med 14, 453–464 (2017). https://doi.org/10.1007/s13770-017-0053-2

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  • DOI: https://doi.org/10.1007/s13770-017-0053-2

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