The Preliminary Study Of The Trans-Differentiation From Goat Bone Marrow Derived Mesenchymal Stem Cells To Putative Germ Cells

Germ cells carry genetic information and pass it down to the next generation, which allows the good merits to be inherited among human or livestock. Therefore germ cell genesis has become one of the most heated topics in developmental biology. However, little knowledge about the germ cells is available. To study the germ cells in vitro, stem cells have proved to be effective and efficient in vitro models. Bone marrow harbors the bone marrow mesenchymal stem cells (BMSCs) which are self-renewable and able to proliferate constantly. The plasticity of BMSCs has been demonstrated by its ability to trans-differentiate into many lineage of cells of three different germ layers in many species. Compared with embryonic stem cells (ESCs), the BMSCs wield many advantages. They are easier to isolate, more convenient to culture in vitro, faster to proliferate and with lower immunogenicity, all of which render BMSCs the ideal candidate for studying the germ cell differentiation in vitro. Hence, the research adopted the goat BMSCs as the in vitro model, and studied the ability of the exogenous factors (e.g., retinoic acid [RA] and BMP4 signalling molecules) and intrinsic factor expression (e.g., DAZL gene ectopic expression) to trigger the trans-differentiate to putative germ cells, laying out a foundation for future research into mechanism underlying the mammal germ cells.This research is divided into three sections. The first section is about the trans-differentiation from goat BMSCs to putative germ cells by RA and BMP4 induction; the second section is about the over expression of DAZL in goat BMSCs to promote the trans-differentiation; the third part is about the construction of goat germ cell specific reporting vector pVASA-GFP. The results of each section are as mentioned below.1 We treated goat BMSCs with different doses of RA (0.01,0.1,0.5,1,5,10,50uM) and BMP4 (0.01,0.1,1,5,25,50, and 100 ng/ml). We optimized the concentration of RA and BMP4 based on the expression of germ cell markers MVH and DAZL. The results showed that, MVH and DAZL expression was significantly increased when BMP4 reached 25 ng/ml; the expression of those two genes was also drastically improved when RA reached 1μM. Therefore, the optimal concentration of RA and BMP4 is 1μM and 25ng/ml respectively.Subsequently, we treated goat BMSCs in three groups, with 1μM RA,25ng/m BMP4 and 1μM RA+25ng/mlBMP4 respectively. In terms of the morphology, the cells in RA and RA+BMP4 treated groups turned round with increased nuclear-plasmic ratio, while little morphological changes were observed in BMP4 treated group. The RT-PCR resuts showed that MVH、DAZL、C-KIT、NANOG、STELLA and OCT4 were all expressed in the three treated groups, but only the RA and RA+BMP4 groups could induce the expression of STRA8、SCP3、PIWIL2 and RNF17. In this section, we successfully trans-differentiate the goat BMSCs to putative germ cells with RA and BMP4, and the putative germ cells derived expressed the germ cell markers, which facilitates the generation of post-meiotic germ cells.2 In terms of an endogenous factor, a germ cell specific gene, deleted in Azoospermia-like (DAZL), was over-expressed by plasmid and mRNA techniques. We adopted the RA-treated goat BMSCs as control to compare the induction potential between exogenous and endogenous factors. The results showed that the mRNA level of both DAZL and MVH both significantly increased, and the mRNA method proved more efficient. The expression of SCP3 showed growth but not siginificantly. To futher confirm the results, we performed the immunofluorescence to detect the protein level of DAZL and MVH. Based on the signal intensity, the expression of MVH and DAZL was both enhanced, with the mRNA technique displaying more efficiency.Moreover, to investigate the role of DAZL in germ cell differentiation, we knocked down the DAZL in RA-treated goat BMSCs. The results showed that all the four shRNA vectors significantly down-regulated the expression of DAZL and MVH in mRNA level, the SCP3 expression declined significantly by SH2 and SH3, while NANOG expression climbed up significantly by SH2 and SH4. The immunofluorescence results demonstrated the significant down-regulation of DAZL and MVH. Our date illustrated the pivotal role of DAZL in germ cell differentiation.3. We successfully cloned the promoter of VASA, including 834 bp of 5’non-coding sequence and 210 bp of the coding sequence. Then we linked the cloned sequence into the N1 vector, and constructed the germ cell specific report system VASA-EGFP. Subsequently, we transfected the vectors into the cells treated with RA for 4 days, green fluorescence was observed. The successful construction of the vector provides good method to isolate, purify and characterize the germ cells differentiated in vitro.