Studies Related To Factors Affecting The Growth Of Mouse Embryonic Stem Cells

Factors affecting the in vitro growth of mouse embryonic stem cells were investigated in this study. Treatment of MEF by 5 g/ml mytomycin C for 4h, 10 g/ml for l~4h, or 20 g/ml for 1-2.5h could suppress their proliferation and maintain their viability. Irradiation of MEF by 14, 21, 28GRAY -rays could also suppress the proliferation of MEF and maintain their viability. When ES cells were seeded and grown on feeder or 0.1% gelatin coated plate, addition of exogenous LIF to the medium could increase the AKP staining positive level (feeder layer culture: 17.80 3.26, 19.58 3.19 , 21.8 3.17 vs 2.73 0.22; p<0.05; feeder-free culture: 14.14 2.35 , 17.87 2.38, 19.21 2.92 vs 0.053 0.0125) and total clone formation rate (29.1%, 31.8%, 33.5% vs 6.9%; p<0.05; 24.7%, 27.8%, 31.2%vs 0.5%, p<0.05) in spite of the feeder presence or absence. In addition, feeder layer could also increase AKP staining positive level (2.73 0.22 vs 0.053 0.0125)and total clone formation rate (6.9% vs 0.5%, p<0.05) in absence of LIF. Purification of Milli-Q water by sub-boiling distilled and employment of qualified FBS could increase the AKP staining positive level and total clone formation rate of mouse ES cells.In conclusions, (1) Treatment of MEF with appropriate of mytomycin C for optimum duation or certain intensity of y-radial can suppress their proliferation and maintain their viability.(2) Feeder layer has certain action in suppressing ES cell differentiation in culture, while exogenous LIF play predominant role in maintaining ES cells undifferentiation. (3) Purification of Milli-Q water by sub-boiling distilled and employment of qualified FBS can improve the ability of ES cells to maintan their undifferentaion state in vitro.