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Roux's archives of developmental biology

, Volume 201, Issue 3, pp 134–141 | Cite as

Bovine embryonic stem cell-like cell lines cultured over several passages

  • S. Saito
  • N. Strelchenko
  • H. Niemann
Original Articles

Summary

A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 μg/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mm mercaptoethanol, 4.5 g/l glucose and 20 mm HEPES-buffer (=passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-banding on the chromosomes of ES-like cells was in accordance with the diploid set of the bovine genome. ES-like cells were fused to in vitro matured bovine oocytes and, upon successful fusion, cultured in vitro over 5 days. Successful fusion was observed in 79.8% (67/84), 31.3% initiated cleavege and 10.4% reached the 8–16 cell stage at termination of culture.

Key words

Bovine demi-blastocysts In vitro culture Chromosomes Stem cells Nuclear transfer 

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References

  1. Axelrod HR (1984) Embryonic stem cell lines derived from blastocysts by a simplified technique. Dev Biol 101:225–228Google Scholar
  2. Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256Google Scholar
  3. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87:27–45Google Scholar
  4. Doetschman T, Williams P, Maeda N (1988) Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev Biol 127:224–227Google Scholar
  5. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156Google Scholar
  6. Evans MJ, Kaufman MH (1983) Pluripotential cells grown directly from normal mouse embryos. Cancer Surveys 2:185–207Google Scholar
  7. Evans MJ, Notarianni E, Laurie S, Moor RM (1990) Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts. Theriogenology 33:125–128Google Scholar
  8. Handyside A, Hooper ML, Kaufman MH, Wilmut I (1987) Towards the isolation of embryonal stem cell lines. Roux's Arch Dev Biol 196:185–190Google Scholar
  9. Hare WCD, Mitchell D, Betteridge KJ, Eaglesome MD, Randall GCB (1976) Sexing two-week old bovine embryos by chromosomal analysis prior to surgical transfer: Preliminary methods and results. Theriogenology 5:243–253Google Scholar
  10. Hassan-Hauser C, Schellander K, Korb H, Knaus E, Schleger W, Mayr B (1990) Langzeitkultivierung von Rinderembryonen im Hinblick auf die Erstellung totipotenter embryonaler Stammzellen. Zuchthygiene 25:22–32Google Scholar
  11. Hooper ML, Hardy K, Handyside A, Hunter S, Monk M (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by culture cells. Nature 326:292–295Google Scholar
  12. King WA, Linares T, Gustavsson I, Bane AA (1979) A method for preparation of chromosomes from bovine zygotes and blastocysts. Vet Sci Commun 3:51–56Google Scholar
  13. Kuzan FB, Wright RW (1982) Observation on the development of bovine morulae on various cellular and noncellular substrates. J Anim Sci 54:811–816Google Scholar
  14. Lin CC, Newton DR, Church RB (1977) Identification and nomenclature for G-banded bovine chromosomes. Can. J. Genet Cytol 19:271–283Google Scholar
  15. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cell. Proc Natl Acad Sci USA 78:7634–7638Google Scholar
  16. Martin GR, Evans MJ (1975) Differentiation of clonal lines of teratocarcinoma stem cells: Formation of embryoid bodies in vitro. Proc Natl Acad Sci USA 72:1441–1445Google Scholar
  17. Piedrahita JA, Anderson GB, BonDurant RH (1990a) Influence of feeder-layer type on the efficiency of isolation of porcine embryo-derived cell lines. Theriogenology 34:865–877Google Scholar
  18. Piedrahita JA, Anderson GB, BonDurant RH (1990b) On the isolation of embryonic stem cells: comparative behaviour of murine, porcine and ovine embryos. Theriogenology 34:879–901Google Scholar
  19. Rastan S, Robertson EJ (1985) X-chromosome deletions in embryo-derived (EK) cell lines associated with a lack of X-chromosome inactivation. J Embryol Exp Morphol 90:379–388Google Scholar
  20. Robertson EJ (1987) Embryo-derived stem cell lines. In: Robertson EJ (ed) Teratocarcinomas and embryonic stem cells, a practical approach. IRL Press, Oxford, pp 71–112Google Scholar
  21. Robertson EJ, Kaufman MH, Bradley A, Evans MJ (1983) Isolation properties and karyotype analysis of pluripotential (EK) cell lines from normal and parthenogenetic embryos. In: Silver LM, Martin GR, Strichland S (eds) Tetracarcinoma stem cells. Cold Spring Harbour Laboratory, New York, pp 647–664Google Scholar
  22. Rossant J, Papaioannou VE (1984) The relationship between embryonic, embryonal carcinoma and embryo-derived stem cells. Cell Differ 15:155–161Google Scholar
  23. Smith TA, Hooper ML (1983) Medium conditioned by feeder cells inhibits the differentiation of embryonal carcinoma cultures. Exp Cell Res 145:458–462Google Scholar
  24. Smith AG, Hooper ML (1987) Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol 121:1–9Google Scholar
  25. Smith AG, Heath JK, Donaldson DD, Wong GW, Moreau J, Stahl M, Rogers D (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690Google Scholar
  26. Stringfellow DA, Gray BW, Toivio-Kinnucan M, Galik P, Riddell KP, Brock KV, Kemppainen RJ (1991) A continuous cell line established from a preimplantation bovine embryo (abstract). Theriogenology 35:275Google Scholar
  27. Thompson S, Clarke AR, Pow AM, Hooper ML, Melton DW (1989) Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56:313–321Google Scholar
  28. Voelkel SA, Amborski GF, Hill KG, Godke RA (1985) Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos. Theriogenology 24:271–281Google Scholar
  29. Ware CB, First NL (1988) Development of embryonic stem cell lines from farm animals (abstract). Biol Reprod 38 (Suppl 1):129Google Scholar
  30. Willadsen SM (1986) Nuclear transplantation in sheep embryos. Nature 320:63–65Google Scholar
  31. Williams TJ, Elsden RP, Seidel GR Jr (1984) Pregnancy rates with bisected bovine embryos. Theriogenology 22:521–531Google Scholar
  32. Williams RL, Hilton DJ, Pease S, Wilson TA, Steward CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NH (1988) Myeloid leucaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687Google Scholar
  33. Wilmut I, Archibald AL, Harris S, McClenaghan M, Simons P, Whitelaw CBA, Clark AJ (1990) Methods of gene transfer and their potential use to modify milk composition. Theriogenology 33:113–123Google Scholar
  34. Wintenberger-Torres S, Popescu PC (1980) Transfer of cow blastocysts after sexing. Theriogenology 14:309–318Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • S. Saito
    • 1
  • N. Strelchenko
    • 1
  • H. Niemann
    • 1
  1. 1.Institut für Tierzucht und Tierverhalten (FAL)Neustadt 1Federal Republic of Germany

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