The Derivation And Gene Modification Of Mouse Oogonial Stem Cells

Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary result in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days, then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs.The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feerder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse.The objective of our study is to test and establish genetic modification on mOSCs using CRISPR-Cas9 system, and thus to investigate the function and their regulatory mechanism of genes related with mOSCs sternness and differentiation to oocyte. mOSCs as the precursor of oocyte have a significance to the development of reproductive biology and obstetrics-gynecology, however, the mechanism of maintaining stemness is still unclear which leads to difficult isolation and derivation of mOSC cell line; In addition, it is also unclear for the process and its mechanism of differentiation of mOSCs to oocytes, so it is necessary to investigat these two scientific issues. Genes related with OSC stemness such as DDX4, Fragilis, Dazl and their function should play important roles in sustaining stemness, and genes relevant to differentiation to oocyte such as BMP4 have been reported to promote primordial germ cells (PGCs) to differentiate to oocyte, which genes'function on differentiation of mOSCs is unclear. To observe the function of these genes, it is necessary to knock out these genes in mOSCs by CRISPR-Cas9 system, and thus to exanmine their real function and related regulatory pathways by their changed phenotypes. CRISPR-Cas9 as current popular technology for gene alteration is suitable to be used in our study to realize the goals described above because this technology only uses one sgRNA-plasmid to introduce the Cas9 protein to and cleavage the target DNA sequence resulting in the gene mutation. Initially, we tested the modification of GRIN2B gene on 293T cells by CRISPR-Cas9, and tested Tet2 gene on mOSCs because these two genes have been reported to be altered successfully in previous studies. Finally, we knock out the YAP gene on mOSCs using the same system. The results revealed that we successfully modified the genes, GRIN2B, Tet 2, and YAP respectively in 293T or mOSCs, and that comparing to wild-type mOSCs, YAP-/--mOSCs showed significantly lower expression of YAP protein by western blot analysis. The mutation of YAP apparently inhibited the ability of proliferation of mutaed mOSCs and its mechanism was related with apoptosis displayed by lower expression of Bcl-2 by western blot. In addition, the related pathway was focused on PI3K-mTOR by western blot analysis. In a word, we have successfully established the platform of genetic modification of mOSCs by CRISPR-Cas9, and found that YAP mutation led to increase of apoptosis and inhibition of proliferation, and described their related pathways.