Summary
The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.
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Axelrod HR (1984) Embryonic stem cell lines derived from blastocysts by a simple technique. Dev Biol 101:225–228
Axelrod HR, Lader E (1983) A simplified method for obtaining embryonic stem cell lines from blastocysts. In: Silver LM, Martin GR, Strickland S (eds) Teratocarcinoma stem cells' Cold Spring Harbor Conferences on Cell Proliferation, vol 10. Cold Spring Harbor Press, New York, pp 665–670
Bernstine EG, Hooper ML, Grandchamp S, Ephrussi B (1973) Alkaline phosphatase activity in mouse teratoma. Proc Natl Acad Sci USA 70:3899–3903
Bradley A (1987) Production and analysis of chimaeric mice. In: Robertson E (ed) Teratocarcinomas and embryonic stem cells: a practical approach. IRL Press, Oxford, pp 113–151
Bradley A, Evans MJ, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256
Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:255–262
Coon HG (1966) Clonal stability and phenotypic expression of chick cartilage cells in vitro. Proc Natl Acad Sci USA 55:66–73
Coon HG (1968) Clonal culture of differentiated rat liver cells. J Cell Biol 39:29a
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 87:27–45
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Gardner RL, Rossant J (1979) Investigation of the fate of 4.5 day post-coitum mouse inner cell mass cells by blastocyst injection. J Embryol Exp Morphol 52:141–152
Handyside AH, Barton SC (1977) Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts. J Embryol Exp Morphol 37:217–226
Handyside AH, Hunter S (1986) Cell division and death in the mouse blastocyst before implantation. Roux's Arch Dev Biol 195:519–526
Handyside AH, Hooper ML, Kaufman MH, Wilmut I (1987) Towards the isolation of embryonal stem cell lines from the sheep. Roux's Arch Dev Biol 196:185–190
Heath JK, Rees AR (1985) Growth factors in mammalian embryogenesis. In: Evered D, Nugent J, Whelan J (eds) Growth factors in biology and medicine, CIBA Foundation Symp, vol 116. Pitman, London, pp 3–22
Hooper M, Hardy K, Handyside A, Hunter S, Monk M (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326:292–295
Koopman P, Cotton RGH (1984) A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Exp Cell Res 154:233–242
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638
Martin GR, Evans MJ (1975) Differentiation of clonal teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci USA 72:1441–1445
Melton DW (1987) HPRT gene organisation and expression. In: Maclean N (ed) Oxford surveys of eukaryotic genes, vol 4. Oxford University Press, Oxford, pp 34–76
Nesbitt MN, Franke U (1973) A system of nomenclature for band patterns of mouse chromosomes. Chromosoma 41:145–158
Quinn P, Barros C, Whittingham DG (1982) Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fert 66:161–168
Robertson E (1987) Embryo-derived stem cell lines. In: Robertson E (ed) Teratocarcinomas and embryonic stem cells: a practical approach. IRL Press, Oxford, pp 71–112
Solter D, Damjanov I (1979) Teratocarcinoma and the expression of oncodevelopmental genes. In: Fishman WH, Busch H (eds) Oncodevelopmental antigens. Methods in cancer research, vol 18. Academic Press, New York, pp 277–332
Smith TA, Hooper ML (1983) Medium conditioned by feeder cells inhibits the differentiation of embryonal carcinoma cell cultures. Exp Cell Res 145:458–462
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 121:1–9
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 56:313–321
Whittingham DG (1971) Culture of mouse ova. J Repr Fert [Suppl] 14:7–21
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Handyside, A.H., O'Neill, G.T., Jones, M. et al. Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line. Roux's Arch Dev Biol 198, 48–56 (1989). https://doi.org/10.1007/BF00376370
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DOI: https://doi.org/10.1007/BF00376370