Stem Cells Dev. 2019 Feb 15. doi: 10.1089/scd.2018.0149. [Epub ahead of print]

Induction of mouse peritoneum mesenchymal stem cells into germ cell-like cells using follicular fluid and cumulus cells conditioned media.

Mirzaeian L^1,2, Eftekhari-Yazdi P³, Esfandiari F^4,5, Eivazkhani F⁶, Rezazadeh Valojerdi M^7,8, Moini A^9,10,11, Fathi R¹².

Abstract

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell like cells using human follicular fluid (HFF) and human cumulus conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in DMEM. After passage 3, purified PMSCs were assessed for morphology, proliferation rate and cell viability. Then isolated PMSCs underwent two characterization procedures: 1) differentiation to mesodermal lineage and 2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh) and theca cell (Lhr) markers were assessed using real-time PCR and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal and epithelial cell specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed germ and oocyte cell specific markers and this expression increased in the 3rd week of culture. In both groups of AP and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl and Amh genes under just HCCM induction showed upregulation significantly in on the 21st day of culture compared to day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.