Study Onisolation, Culture, Characterization And Committed Differentiation Of Embryonic Stem Cells From Sheep

The major issues regarding embryonic stem cell (ES) culture and construction in alot species are difficult to passage and preserve as well as easy to commitself-differentiation. Research on what induce and regulate ES differentiation is verylimited, with minimum progress for livestock ES cell investigation. Even though someresearch for sheep ES cell has been reported earlier, how to consistently establishsuccessful cell differentiation and derive control of their developmental regulationmechanism is still largely unresolved. Therefore, this study took sheep as the researchanimal, a stable in vitro culture system of separation and preventing differentiation ofsESC-like cells were established and optimized, the biological characteristics of theobtained sESC-like cells were identified initially, and their induction of differentiationculture system were studied systematically. The main results are as follows:1. In vitro fertilization and development culture system of sheep oocytes weresuccessfully established. TCM-199maturation medium, supplemented with10%～15%ESS,10μg/mL FSH,20μg/mL LH,1.5μg/mL17-βE2,100IU/mL penicillin and100IU/mL streptomycin could promote the in vitro maturation of sheep oocytes. Highercleavage rate(76.0%) and blastocyst rate(31.1%) could be obtained when the sheepoocytes matured in vitro for22～24h were fertilized with epididymal semen a, developedin the media of the combination of Sofaa and high sugar Sofaa supplemented with10%FBS or the combination of CM and BM, and cultured at38.5℃, mixed three gases (5%of CO2,5%O2,90%N2) and saturated humidity conditions.2. The isolation system of sESC-like cells was established successfully. Sheepembryonic fibroblasts, mouse embryonic fibroblasts, and their1:1mixed cells couldbetter support the growth of sESC-like cells. When there was no the condition to obtainin vivo embryos, it was an alternative to isolate ESC-like cells using sheep IVF embryos,because they also have better potential of proliferation and passage. The quality andembryo ages of blastocyst affected the attachment and proliferation of sESC-like cells.The similar ICM adherence rate of sheep blastocysts could be obtained with mechanicalcutting (46%) and immunification (53.6%).3. The culture system of sESC-like cells was established successfully, and the cellscould be subcultured to20passages. Mechanical method combined with enzymatic digestion method was suitable for the separation and passage of the sESC-like cells.DMEM high glucose culture medium containing a certain concentration of FBS, cellgrowth factor, insulin and VC could promote the proliferation of sESC-like cells, and itwas suitable for culture of sESC-like cells. The obtained sESC-like cells had the typicalcharacteristics of ES cells. AKP staining was positive. They could express thepluripotency genes of Kit, Rex1, Sox2, Nanog and Oct4, and immunofluorescencestaining showed that the cells were positive for OCT4, SSEA-1, SSEA-3, SSEA-4,TRA-1-60and TRA-1-81. They had normal chromosome ploidy (2n=54). They couldspontaneously differentiate into cystidia and three germinal layer cells in vitro，so thesESC-like cells had the characteristics of self-renewval and differentiation.4. The in vitro differentiation culture system of sESC-like cells was establishedsuccessfully. sESC-like cells could be induced to differentiate into nerve cells in theinducing medium including a certain concentration of retinoic acid or the combination ofretinoic acid, bFGF and Insulin, and neuron specific gene and protein（beta-tubulin IIIand nestin）were expressed in the cells; they also could be induced to differentiate intoosteoblasts in the inducing medium supplemented with beta-glycerol sodium phosphate,DEX and VC salt, meanwile AKP, Alizarin red and Kossa staining for the cells werepositive; DMSO or DMSO in combination with retinoic acid could promoted sESC-likecells to differentiate into cardiomyocyte-like cells, meanwhile the gene expression ofNKX2.5and GATA4and the specific protein of alpha-actin were detected in thedifferentiation cells; in adipocytes-inducing medium they could differentiated intoadipocytes, and o-oil red staining were possitive; a certain concentration of ITS, EGF andNA-inducing medium could promote sESC-like cells to differentiate intoinsulin-secreting cells, and the gene expression of Insulin and PAX4and the specificprotein of Insulin were detected in the differentiation cells. That was to say, the sESC-likecells could be induced to differentiate into the different cells from the three germinallayers.