Effects of Leukemia Inhibitory Factor, Stem Cell Factor, and Basic Fibroblast Growth Factor on the Proliferation of Bovine ICM Cells In Vitro

Abstract

Experiments were conducted to determine the effects of leukemia inhibitory factor (LIF), stem cell factor (SF), and basic fibroblast growth factor (bFGF) on the proliferation of undifferentiated primitive ectodermal cells isolated from bovine inner cell masses (ICM). ICMs of bovine hatched blastocysts produced at 10 days post-insemination (dpi) were immunosurgically isolated. Either one or three ICMs were cultured on mouse embryonic fibroblasts (MEF) in a 100 μl droplet of control medium with or without several combinations of murine recombinant LIF (1,000 units/ml), human recombinant SF (40 ng/ml), and human recombinant bFGF (40 ng/ml) as follows: 1) LIF, 2) SF, 3) bFGF, 4) LIF+SF, 5) LIF+bFGF, 6) SF+bFGF and 7) LIF+SF+bFGF at 37°C under 5% CO₂ in air. Attachment of ICMs to a feeder layer and proliferation of ectodermal cells were observed daily. After 6 to 7 days of culture, the primary ectodermal cells outgrown from ICM clumps were mechanically isolated with a 26G needle and mechanically disaggregated by pipetting through a fine pipette to make small clumps of cells. The primary ectodermal cell clumps were cultured and serially passaged every 6 to 7 days. The attachment of isolated ICMs started 1 day after culture in each treatment, and the rates of attachment ranged from 74 to 94%. The rates of ectodermal cells outgrowing from 1 ICM and 3 ICMs (37 and 56% respectively) were higher in the LIF+SF containing medium than those in the control medium and bFGF containing medium (16 and 17%; 22 and 22% respectively). When primary ectodermal cells that had been isolated from ICMs were cultured in the LIF+SF containing medium, the undifferentiated ectodermal cells were serially passaged (6 passages) during 2 months in culture. The results of this study demonstrate that 1) groups of 3 ICMs gave superior results to single ICM for the formation of ectodermal cell colonies from the ICM clumps and 2) LIF and SF are important factors in supporting the primary outgrowth and serial passage of ectodermal cells, as well as for maintaining an undifferentiated state as determined by a histochemical staining of alkaline phosphatase.