Differentiation Of Mouse Embryonic Stem Cells Into Germ Cells Induced By Mouse Testis Sertoli Cells

Background and objectiveEmbryonic stem cells (ESC) are totipotent and are capable of differentiating into all sorts of cells that a whole organic body needs. Committed differentiation of ES cells is the hot spot in the research field at present, meanwhile differentiation of ES cells into germ cells is a novel field of research. The use of primordial germ cells (PGC) and germ stem cells (GSC) in transplantation studies to restore fertility has been initiated with varied degrees of success, leading scientists to push boundaries even further by using ES cells as the starting point for germ cell differentiation. Till now, five labs have successfully isolated germ cells in ES-derived embryonic bodies (EBs). All of the researchers have established EBs from ES cells, which consist of tissue lineages typical of the early embryo. After adding some revulsant or conditioned medium or just spontaneous differentiation, they could detect markers specificly expressed by germ cells. Our study is to coculture mouse testis sertoli cells and ES cells. Sertoli cells interlace with ES cells, at the same time sertoli cells can secrete some cytokines such as bone morphogenesis protein (BMP-4), which both support differentiation of ES cells into germ cells. We aim to induce ES cells to differentiate into germ cells by simulating the in vivo microenvironment. We cocultured mouse testis sertoli cells and ES cells and detected germ cells specific markers by RT-PCR, in situ hybridization, immunohistochemistry staining and Western Blotting at a definite time point. The expression of mouse vasa homologue (Mvh) indicated differentiation into germ stem cells. In the study we have induced ES cells to differentiate into germ cells by simulating the in vivo microenvironment and established a comparatively stable in vitro differentiation system, which provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitatenuclear transfer technology and infertility treatments.Part I Isolation and identification of Sertoli cells from mouse testis Methods: Testis from male ICR mouse 18 to 20 days old were isolated and cultured by enzymatic digestion. Sertoli cell gene with a zinc finger domain (SERZ) was obtained by RT-PCR and inserted into pcDNA3.0 to construct the recombinant plasimids pcDNA3.0-SERZ. The recombinant plasimids pcDNA3.0-SERZ were cleaved by restriction endonucease Kpnl and Xbal respectively to obtain the linear templets and the probes were prepared further. The expression of SERZ mRNA in the cultured cells was analysed by in situ hybridization. The expression of androgen binding protein (ABP) mRNA in the cultured cells was detected by RT-PCR. In addition, sertoli cells have typical structures under electron microscope, and the cells were observed under electron microscope. Results: Under light microscope, the majority of sertoli cells were polygonal and cells were completely extended like a membrane. The neighbouring cells were interlaced with one another. Under electron microscope, the nuclei were triangular or irregular, and weakly stained with obvious nucleoli. The organelles were well developed, including glycogens, lipid droplets, lysosomes and mitochondria. And tight junctions were observed between neighboring cells. As identified by in situ hybridization, the cultured cells were positive to the probes. RT -PCR on the cultured cells confirmed high expression of ABP. Conclusions: We successfully isolate and identify sertoli cells from mouse testis with the purity over 85%, which provides correct and effective tools for the following studies.Part II Differentiation of mouse embryonic stem cells (ES) into germ cells induced by mouse testis sertoli cellsMethods: Mouse testis sertoli cells were cocultured with mouse ES cells (the coculture group), in contrast, ES cells were solo cultured in ES cell medium supplemented with FCS (the spontaneous differentiation group) and ES cells were cultured in mouse testis sertoli cells-conditioned medium (the conditioned mediuminduced group). Three days and seven days later, the cells were collected respectively, and then sorted by fluorescence activated cell sorter (FACS). ES cells were labeled with green fluorescence protein (GFP), so ES cells colud be separated with other cells by GFP label. After FACS, total RNA of a portion of the cells was extracted using the acid guanidium thiocyanate-phenol-chloroform method and RT-PCR was performed to examine the expression of Mvh, DAZL, c-Kit, Oct-4 and Mil-2, using P-actin as an internal control. At the same time a part of the cells in the co-culture group were lysed and the samples were electrophoresed in SDS polyacrylamide gel. The expression of SSEA-1 was analysed by Western Blotting. Another portion of the cells in the coculture group were harvested to prepare the smears, and the expression of SSEA-1 was analysed by immunohistochemistry staining, and the expression of Mvh was detected by in situ hybridization. Results: RT-PCR on 3d coculture group confirmed positive expression of the germ cell markers Mvh, DAZL, c-Kit, Oct-4 and Mil-2, in 7d cocultuer group the expression of Mvh, DAZL, c-Kit and Mil-2 increased a little, while the expression of Oct-4 decreased a little. The expression of the germ cell markers was also identified in the 3d conditioned medium induced group and the 3d spontaneous differentiation group, and the expression of Mvh, DAZL, c-Kit and Mil-2 in the 7d conditioned medium induced group and the 7d spontaneous differentiation group arised slightly resp., while the expression of Oct-4 deduced slightly resp. Comparison was made between the gene expression at the same stage, the expression of Mvh, DAZL, c-Kit and Mil-2 in the coculture group was slightly higher than that in the conditioned medium induced group resp., and the latter was slightly higher than that in the spontaneous differentiation group resp. The expression of Oct-4 in the coculture group was appreciably lower than that in the conditioned medium induced group, and the latter was appreciably lower than that in the spontaneous differentiation group. The cells were positively stained for SSEA-1 as showed by immunohistochmistry. The expression of SSEA-1 protein was gradually decreased as time goes by, which was revealed by Western Blotting. In situ hybridization of the 7d coculture group showed that a small amount of the cells were positively stained for Mvh, indicating that ES cells differentiated into germ cells with a low differentiationrate. Conclusions: We coculture mouse testis sertoli cells and ES cells. Sertoli cells interlace with ES cells, at the same time sertoli cells can secrete some cytokines such as BMP-4, which both support differentiation of ES cells into germ cells. The differentiation rate, though not high, is briefly higher than that of the conditioned medium induced group and the spontaneous differentiation group. The above results convince that we have successfully induced ES cells to differentiate into germ cells by simulating the in vivo microenvironment.Conclusions1) We have indicated that ES cells can be induced to differentiate into germ cells when cocultured with mouse testis sertoli cells.2) It has been verified that the in vivo microenvironment plays a crucial role on cell differentiation, and the cell inducement and the cytokines can effectively induce committed differentiation of ES cells.