Study On Induced Differentiation Of Human Pluripotent Stem Cells Into Germ Cells

Objective:Construct the human embryonic stem cell line which carried germ cells specific gene VASA fluorescent report system, use the stable system to optimize germ cell differentiation in vitro.Methods:Constrcut the chHES137-VASA-EGFP fluorescence report gene cell line by lentivirus infection,further use monoclonal method to screen sublines which share the same gene type. Subjective the successful construced cell lines to the study of differentiation into primordial germ cells. Pick the completely undifferentiated chHES137 VASA-EGFP clones 5-6 days after passage to prepare for embryoid bodies (EB).Induce EBs to differentiate into primordial germ cells by the combination of Wnt3a (30ng/ml) and BMP4 (100ng/ml). Take the 12day-induced cells to Flow cytometric analysis for EGFP positive cells, and collect the sorted EGFP positive and negative groups, then detect the expression of germ cell development related genes by RT-PCR and VASA in situ staining. Furthermore, utilize the successfully constructed cell lines to compare the influence of different induction base medium on the differentiation efficiency.Results:In the process of induction,chHES137-VASA-EGFP shows green fluorescent protein expression. Flow cytometric analysis shows that the positive rate of 12day-induced cells is 9.8±0.2, germ cell development related genes VASA,DAZL and SCP3 are all expressed in Flow cytometric analysis EGFP positive cells, while no expression are detected in the negative group. In situ staining of the 12day-induced cells reveals that EGFP green fluorescent expression cells are partly overlapped with red fluorescent expression cells marked by VASA antibody, suggesting that EGFP green fluorescent expression areas are namely endogenous VASA expression positions. FACS detect the germ cell differentiation efficiency of different base medium system, EGFP positie rate of D/F12-EB group is 9.7±1.2, which is significantly higher than the other three culture system, and germ cell development related genes expressed in D/F12-EB group,while in other groups it is not obvious.Conclusion:The constucted chHES137-VASA-EGFP report gene cell line can effectively trace the primordial germ cells during the differentiation process in vitro. Compared with other medium, D/F12-EB base medium is more likely to support the differentiation of germ cell in vitro. Combination of Wnt3a and BMP4 can effectively induce the formation of primordial germ cells. Chapter 2 Comparative study of differentiation into germ cells of the pluripotent stem cells from premature ovarian failure(POF-iPS) and normal cells (hEFs -iPS),as well as human embryonic stem cells.Objective:Construct the VASA-EGFP fluorescent reporter cell line of POF-iPS, hEFs-POF,and compare the differentiation capacity of chHES137-VASA-EGFP to germ cells.Methods:Construct the POF-iPS-VASA-EGFP and hEFs-iPS-VASA-EGFP fluorescence report gene system by lentivirus infection.Use the differentiation system of chHES 137-VASA-EGFP to germ cells to compare the differentiation capacity of three different cell lines of POF-iPS-VASA-EGFP, hEFs-iPS-VASA-EGFP and chHES 137-VASA-EGFP to germ cells.Take the 12day-induced cells to Flow cytometric analysis, detect the induction rate.Results:POF-iPS-VASA-EGFP, hEFs-iPS-VASA-EGFP and chHES137-VASA-EGFP all express green fluorescent in the induction process.The induction efficiencies of three kinds of cell lines into germ cells are different,The positive cells rates of POF-iPS-VASA EGFP,hEFs-iPS-VASA-EGFP and chHES137-VASA-EGFP are 3.2%,4.6% and 7.6%,respectively.Conclusion:The differentiation capacities of POF-iPS -VASA EGFP,hEFs -iPS-VASA-EGFP and chHES137-VASA- EGFP into germ cells are different.