Primary Study On Establishment Of Embryonic Stem Cells From Kunming Mouse

Embryonic stem cells(ESCs)can self-renew and proliferate indefinitely in vitro,which retain their capability to differentiate into all cell types.By now, various kinds of animals and human ESC lines had been established. KUNMING(KM)mouse is used most-widely for biological experiment in our country.Therefore in this experiment the KM mouse was chosen to explore the isolation methods of mouse ESCs(mESCs)line to provide feasible methods and theories for Buffalo ESCs isolation.The study included two parts.The first part focused on the effects of embryonic stages and passage methods on putative mESCs isolation.The second part focused on the detection of putative mESCs.The alkaline phosphatase(AKP)staining,RT-PCR analysis of Oct4 and Sox2,karyotype analysis and differentiation capability assays were used to testify the charaterastics of putative mESCs.The differentiation capability assays were carried out by two approachs.One was spontaneous and the other was inducing putative mESCs to differentiate into neural cells.The results showed that:1)The isolation efficiencies of the expanded blastocysts was higher than that of the hatched blastocysts(P＜0.05),although there were not significant differences in the attachment and the primary colonies formation rates between the expanded and hatched blastocysts(P＞0.05), putative mESCs were not successfully obtained from the hatched blastocysts;2) Judging from the putative mESCs morphology and AKP staining,there wasn't significant difference by the mechanical and trypsinized passage methods after passage five,which revealed that both of the passage methods were suitable to passage putative mESCs;3)putative mESCs remained undifferentiated culturing to passage 25 in vitro,showed the high activity of AKP,expressed Oct4 and Sox2,and had a normal karyotype 40XY,which indicated that the isolated putative mESCs had the general feature of putative mESCs;4)During suspension culture in the absence of feeder layers,LIF and bFGF,putative mESCs formed embryoid bodies(EB).When the EB were cultured in the absence of LIF and bFGF,which was called spontaneous differentiation,they were able to differentiate into various cell types.When the EB were cultured in the absence of LIF and serum with additional 20 ng/mL bFGF,which was called induced differentiation,they differentiated into neural cells.The AKP staining showed both the differentiated cells of two approachs were negative and the immunofluorescence assay showed the cells derived from induced differentiation expressed Nestin,which demonstrated that putative mESCs had the ability to differentiate into somatic cells in vitro.In conclusion,putative mESCs were successfully isolated from the expanded blastocytes in this study,and were pluripotent,which might lay the foundations for further study of Buffalo ESCs in our lab.