Isolation,culture and identification of murine embryonic stem cells of Kunming species / 重庆医科大学学报

ABSTRACT

Objective:By cocculture with mesenchymal stem cells as feeder cells,we observe whether we can isolate and culture embryonic stem cells(ES cells)which maintain undifferentiated and multipotential. Methods:tissues and cell types were used in basical research to cure disease. The blastocysts of 3.5 d from Kunming species mice were collected and cultured on mesenchymal stem cells layers. The embryonic stem cell colonies were preliminarily identified by light microscope AKP staining and 0ct-4 staining in morphology and biological character. Results:The embryonic stem cell colonies by cocculture with mesenchymal stem cells as feeder cells were identified. The results showed that 1. all the ES cells had its typical morphological characteristics such as nestlike colonies,round or elliptic,with clear edge and smooth surface,strong refraction ,significant swelling growth,compactness of cell aggragation and indistinction between two edges of neighbour cells within a colony,relatively bigger nuclear compared with little cytoplasm,i.e.,high ratio of the nuclear- cytoplasm; 2.after the AKP staining,all of the ES cells showed strong AKP activity as same as the ES cells of F0,and on the contrary,the embryonic fibroblast cells had no expression of AKP showing the lost of staining;3. after the Oct-4 staining,all of the ES cells showed strong Occt-4 activity. Conclusion:By cocculture with mesenchymal stem cells as cells,we can isolate and culture embryonic stem cells (ES cells) which maintain undifferentiated and multipotential.