Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지

Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells

인간태아 섬유아세포와 생쥐배아 섬유아세포를 기저세포로 활용한 인간 배아줄기세포의 확립

  • Cho, Hye Won (Department of Obstetrics and Gynecology, College of Medicine, Pusan National University) ;
  • Ko, Kyoung Rae (Yalemari Wemen's Clinic) ;
  • Kim, Mi Kyoung (Infertility Clinic, Pusan National University Hospital) ;
  • Lee, Jae Ik (Infertility Clinic, Pusan National University Hospital) ;
  • Sin, Su Il (Department of Obstetrics and Gynecology, College of Medicine, Pusan National University) ;
  • Lee, Dong Hyung (Department of Obstetrics and Gynecology, College of Medicine, Pusan National University) ;
  • Kim, Ki Hyung (Department of Obstetrics and Gynecology, College of Medicine, Pusan National University) ;
  • Lee, Kyu Sup (Department of Obstetrics and Gynecology, College of Medicine, Pusan National University)
  • 조혜원 (부산대학교 의과대학 산부인과학교실) ;
  • 고경래 (예일마리 산부인과) ;
  • 김미경 (부산대학교병원 불임크리닉) ;
  • 이재익 (부산대학교병원 불임크리닉) ;
  • 신수일 (부산대학교 의과대학 산부인과학교실) ;
  • 이동형 (부산대학교 의과대학 산부인과학교실) ;
  • 김기형 (부산대학교 의과대학 산부인과학교실) ;
  • 이규섭 (부산대학교 의과대학 산부인과학교실)
  • Published : 2005.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

Keywords

References

  1. Doetschman TC, Eistetter H, Kata M, Schmidt W, Kemler R. The in vitro development of blastocyst derived embryonic stem cell lines: formation of visceral york sac, blood islands and myocardium. J Embryo Exp Morphol 1985; 87: 27-45
  2. Peta MF, Reubinoff B, Trouson A. Human embryonic stem cell. J Cell Sci 2000; 13: 5-10
  3. Smith AG. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988; 336: 688-90 https://doi.org/10.1038/336688a0
  4. Williams RL. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stems. Nature 1988; 336: 684-7 https://doi.org/10.1038/336684a0
  5. Bongso A, Fong CY, Ng SC, Ratnam SS. The growth of inner cell mass cells from human blastocysts (abstract). Theriogenology 1994; 41: 161 https://doi.org/10.1016/S0093-691X(05)80071-8
  6. Bongso A, Fong CY, Ng SC, Ratnam SS. Isolation and culture of inner cell mass cells from human blastocysts. Hum. Reprod. 1994; 9: 2110-7
  7. Thomson JA, Itskovitz Eldor J, Shapiro SS, Waknitz MA, Swiergiel 11, Marshall VS, Jones JM. Embryonic stem cell lines from human blastocysts. Science 1998; 282: 1145-7 https://doi.org/10.1126/science.282.5391.1145
  8. Reubinoff BE, Pera M, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnol 2000; 18: 399-404 https://doi.org/10.1038/74447
  9. Martin GR. Isolation of a pluripotent stem cell line from early mouse embryos cultured in medium conditioned by teratocacinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 7634-8
  10. Evans MJ, Kaufman M. Establishment in culture of pi uri potential stem cell lines from mouse embryos. Nature 1981; 292: 151-6
  11. Doetschman TC, Williams P, Maeda N. Establishment of hamster blastocyst derived embryonic stem cells. Dev Bioi 1988; 127: 224-7 https://doi.org/10.1016/0012-1606(88)90204-7
  12. Graves KH, Moreadith MH. Drivation and characterization of putative pluripotential embryo stem cells from preimplantation rabbit embryos. Mol Reprod Dev 1993; 36: 424-33 https://doi.org/10.1002/mrd.1080360404
  13. Wheeler MB. Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev 1994; 6: 563-8 https://doi.org/10.1071/RD9940563
  14. Cibelli JB, Stice SL, et aI. Transgenic bovine chimeric offspring produced from somatic cell derived stem-like cells. Nature Biotechnol 2000; 16: 642-6 https://doi.org/10.1038/nbt0798-642
  15. Geron. 1999. http://www.geron.com
  16. Solter D, Geartart J. Putting stem cells to work. Science 1999; 283: 1468-70 https://doi.org/10.1126/science.283.5407.1468
  17. Fishel SB, Edwards RG, Evans CJ. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 1984; 223: 816-8 https://doi.org/10.1126/science.6546453
  18. Edwards RG. IVF and history of stem cells. Nature 2001; 413: 349-51 https://doi.org/10.1038/35096649
  19. Edwards RG. Personal pathways to embryonic stem cells. Reprod Biomed Online 2002; 4: 236-78
  20. Richards M, Fong C-Y, Chan W-K, Wong P-C, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nature Biotechnol 2002; 20: 933-6 https://doi.org/10.1038/nbt726
  21. Hovatta 0, Mikkola M, Gertow K, Stromberg A-M, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M and Ahrlund-Richter L. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003; 18: 1404-9 https://doi.org/10.1093/humrep/deg290
  22. Solter D, Knowles BB. Imrnunosurgery of mouse ？blastocyst. Proc Natl Acad Sci USA 1975; 72: 5099-5102
  23. Reubinoff BE, Pera MF, Vatja G, Trounson AD. Effective cryopreservation human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001; 16: 2187-94 https://doi.org/10.1093/humrep/16.10.2187
  24. Kaufman DS, Hanson ET, Lewis RL, Thomson lA. Hematopoietic colony-forming cells derived from human embryonic stem cells into embryoid bodies compromising the three. 2001
  25. Filippi MD, Porten F, prsteur FL, Schiavon V, Millot GA, Vainchenker W, de Sanvage Fl, Dubart Kupper Schmitt A, Sainteny F. Requirement for mitogenactivated protein kinase activation in the response of embryonic stem cell-derived hematopoietic cells to thrombopoietin in vitro. Blood 2002; 99(4): 1174 -82 https://doi.org/10.1182/blood.V99.4.1174
  26. Andressen C, Stocker E, Klinz FJ, Lenka N, Hescheler J, Fleischmann B, Arnhold S, Addicks K. Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation. Stem Cells 2001; 19(5): 419-24. https://doi.org/10.1634/stemcells.19-5-419
  27. Arnhold S, Lenartz D, Kruttwig K, Klinz Fl, Kolossov E, Hescheler l, Sturm V, Andressen C, Addicks K. Differentiation of green fluorescent protein labeled embryonic stem cell-derived neural precur-SOT cells into Thy-I-positive neurons and glia after transplantation into adult rat striatum. J Neurosurg 2000; 93: 1026-32 https://doi.org/10.3171/jns.2000.93.6.1026
  28. Soria B, Roche E, Bema G, Leon-Quinto T, Reig lA, Martin F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozocininduced diabetic mice. Diabetes 2000; 49: 157-62. https://doi.org/10.2337/diabetes.49.2.157
  29. Assad S, Maor G, Amit M, Itskovitz-eldor l, Skorecki K, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50: 1691-7 https://doi.org/10.2337/diabetes.50.8.1691
  30. Klug MG, Soonpaa MH, Koh GY, Field U. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest. 1996; 98: 216-21 https://doi.org/10.1172/JCI118769
  31. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108: 407-14
  32. Heinrich S, Thenben T, Hescheler J, Lindner M, Brandt MC, Wartenberg M. Characteristics of calcium sparks in cardiomyocytes derived from embryonic stemcells. Am J PhysioI 2001; 281: H411-2
  33. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nature BiotechnoI 2001; 19: 971-4 https://doi.org/10.1038/nbt1001-971