| 1. Mouse fetal tissues could be cut into uniform small pieces by scissors cutting and needle grinding, which grew well in tissue culture and great amounts of cells grew from the attached small tissue clump to form primary mouse embryonic fibroblasts (MEF) monolayer. Treatment of MEF by 5ug/ml mytomycin C for 2.5-4h, 10ug/ml for l-4h, or 20ug/ml for 1-2.5h could suppress their proliferation and maintain their viability. Irradiation of MEF by 14, 21, 28GRAY r -rays could also suppress the proliferation of MEF and maintain their viability.2. At 48h after thawed and cultured on MEF feeder layer in complete medium supplied with LIF (1000U/ml), mouse ES cells formed clones with clear edge, smooth surface. The cell boundary was disappeared within clones and no differentiated cells were found in the edge of clones. At this time, Oct-4 ES cells expressed strong green fluorescence of Oct-4-GFP, AKP staining shown strong positive and RT-PCR analysis confirmed that these cells expressed high levels of Oct-4 mRNA, indicating that these ES cells kept in undifferentiated status well. If ES cells were cultured in feeder-free system for 48h, they appeared presented differentiated morphology. These results indicate that feeder layer can control the differentiation of ES cells thawed during culture.3. After cultured on feeder for 2-3 passages, 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 2.69 + 0.26; feeder-free culture: 14.14 + 2.35, 17.87 + 2.38, 19.21+2.92 vs 0.016 + 0.0005, P<0.01)and total clone formation rate (29.1%, 31.8%, 33.5% vs 6.5%; 24.7%, 27.8%, 31.2% vs 0.8%,p<0.01) in spite of the feeder presence or absence. In addition, feeder layer could also increase AKP staining positive level (2.69 + 0.26 vs 0.016 + 0.0005) and total clone formation rate (6.5% vs 0.8%, p<0.01). These results indicate that feeder layer has certain action in suppressing ES cell differentiation in culture, while exogenous LIF play predominant role in maintaining ES cells undifferentiation.4. Basic media and supplement of glutamine, sodium pyruvate and MEM NEAA did not affect the growth of ES cells cultured in vitro, but water and serum quality had a significant influence on ES cells cultured in vitro. 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.5. The neural differentiation process of mouse ES cells induced by monolayer differentiation and embryoid differentiation was investigated. When monolayer differentiation method was employed by culturing ES cell in serum free DMEM/F12+N2B27 medium, ES cells differentiated graduallyinto neural stem cells, glia and neurons as evidence that the specific marker of neural stem cell, Soxl began expressed on D3 and reached high level from D4 to D8 in 46C cells, and neuronal special protein Tau expressed since D7 in TK23 cells. Since D3 of onset culture in Serum free medium, differentiated cells moved away from the edges of ES cell clones and reached 200u.m far away from the edges of clones on D8. On D13, many neuronal fibers grew up to 1000um and formed a complex network to each other. Among differentiated neural cells, B-tubulin III positive neuron cells were predominated and some were nestin positive neural stem cells and GFAP positive glial cells. When embryoid differentiation method was employed by 4-/4+RA treatment and trypsinization of cellular aggregates before culture in serum free medium, Soxl-GFP still expressed at D13 in 46C cells. Nestin positive cells were more than GFAP positive. B -tubulin III positive cells were still dominated which gave multiplex morphology and some of them had very long axons. The results indicated that both methods can be employed to induce the neural differentiation of mouse ES cells effectively. But B -tubulin...
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