Isolation, Culture And Identification Of Stem-like Cells Derived From Sheep Skin

Recent years, More and more rearches have been done in adult stem cells (ASCs) .Among various ASCs, Stem cells in skin play a important role in cell therapy and transgenic nulear transfer. The focus of this dissertation work is to investigate the isolation, culture and evaluation of stem-like cells in sheep, and to search for a new source of the somatic nuclear transfer donor cells . Meanwhile, the work also has researched the isolation and culture of cells derived from sheep skin.We get the fibroblast cells from skin tissue and established a stable culture system.3 months sheep's ear tips and 90 days sheep fetal skin tissues have been used as the source of the stem like cells. The method of 0.25% trpysin digest and the tissues adherence were used to get primary cells.Condition medium(Additional 10 ng/ml EGF,5μg/ml Insulin,0. 5μg/ml HICE,l. 8 ×10^-4 mol/L Adenine, 10%FCS in high glucose DMEM medium) and feeder cells have been used to culture 3D-colony cells. The 3D-colony can be passaged 10 times, and the cell doubling time is about 70-90. The result has shown that the 3D-colony cells might have high multiplivation capability as stem cells..The result of AKP test showed that the 3D-colony cells taken the russety colour .The other cells including feeder cells and around cells have not been dyed. It suggests that the 3D-colony cells should have a high activity of AKP, might be in the non-differentiation or low differentiation state. Immunostaining in 3D-colony cells showed neither labeling for vimentin nor labeling for cytokeratin. Immunostaining in the cells from the same passage of the 3D-colony cells showed uniform labeling for vimentin and no labeling for cytokeratin, suggesting that these cells were fibroblast cells.Monoclonal anti- β -1 Intrgrin has been used to lable the 3D-colony cells, and none positive can be observed.This avidence indicate that the 3D-colony cells should not be ESCs. Advanced tests should be done to identify which kind of stem cell it is.In conclusion, this dissertation study showed that it should be viable to isolated the 3D-colony cells which is characteristic to stem cell .And preliminary tests have showed that the 3D-colony cells might be a kind of stem cell. But, the completely identification need more tests to prove.