Use of oncostatin M for mouse embryonic stem cell culture
Theriogenology 41:336, 1994 USE O F O N C O S T A T I N M FOR M O U S E E M B R Y O N I C S T E M CELL C U L T U R E E. Wolf, R. Kramer, H. Thoenen an...
Theriogenology 41:336, 1994 USE O F O N C O S T A T I N M FOR M O U S E E M B R Y O N I C S T E M CELL C U L T U R E E. Wolf, R. Kramer, H. Thoenen and G. Brem Department of Molecular Animal Breeding Ludwig-Maximilian University 80539 Munich FRG
Pluripotential embryonic stem (ES) cells are routinely cultured in the presence of leukemia inhibitory factor (LIF) which is provided by feeder cells, conditioned media, or as recombinant protein. The cytokine oncostatin M (OSM) is structurally and functionally related to LIF and binds the high-affinity LIF receptor. We first investigated the effects of OSM on ES cell proliferation and differentiation in vitro. Then ES cells cultured for 4 weeks with OSM as the only exogenous differentiation inhibiting agent were injected into blastocysts (C57BL/6, Balb/c) to demonstrate their ability to form chimeric mice. D3 (Doetschman et al., 1985, J. EmbryoL Exp. Morph. 87, 27-45) and HM-1 (Magin et al., 1992, Nucleic Acids Res. 20, 3795-96) ES cell cultures were started with 103 cells/cm 2 in DMEM/15% FCS/10 4 M 2-mercaptoethanol supplemented with OSM, LIF, Buffalo rat liver (BRL) cell-conditioned medium, or without exogenous differentiation-inhibiting factors. ES cells were cultured in 35 mm tissue culture dishes without feeder cells or gelatin coating. After 7 days the percentage of undifferentiated colonies was determined by morphological criteria and by immunofluorescence for the stage-specific mouse embryonic antigen (SSEA-1). Cells were counted using a Neubauer hemocytometer. OSM inhibited differentiation and 10 100 stimulated proliferation of ES cells in a dose-dependent manner (Fig. 1). Used at a concentration of 10 ng/ml, OSM ._~ 80 ¢-¢0 was as effective as LIF at the same ,_O dose. BRL cell-conditioned medium -6 ~" 0 60stimulated ES cell proliferation very I1) markedly, but less than 50% of the -4 "~ ~¢ - 40Q) colonies were completely undifferenE tiated. Omission of all of these factors -2 20"O from the culture medium resulted in t<3 complete differentiation of ES cells. • 0 Very similar effects were observed 0 0.1 1 10 50 with both ES cell lines investigated. concentration of OSM [nglml] To perform the most reliable test Fig. 1. Dose-response for O S M on of their pluripotency, ES cells were growth and differentiation of ES cells injected into blastocysts after longterm culture in the presence of OSM. Chimeric mice were obtained from D3 ES cells (10/12 born animals) and HM-1 ES cells (5/9 born animals) following injection into C57BL/6 or Balb/c blastocysts. Coat chimerism ranged from 10% to 90% and between 20% and 50% in chimeras originating from D3 and HM-1 ES cells, respectively. These mice are currently under mating to evaluate germ-line colonization by ES cells. In summary, our results demonstrate that OSM - like LIF - can preserve the pluripotency of mouse ES cells in culture.