168 CHARACTERIZATION AND DIFFERENTIATION INTO OOCYTE-LIKE CELL MASSES OF PORCINE MESENCHYMAL STEM CELLS DERIVED FROM OVARIAN THECA CELLS

Abstract

Recent findings have shown that ovaries after birth have germ line stem cells, which were considered as an alternative for the production of an animal model. The present study was therefore aimed to characterise ovarian theca cells and generate oocyte-like cell masses in vitro in porcine. Theca cells isolated from ovarian follicle were cultured in A-DMEM supplemented with 10% FBS at 38.5°C in a humidified atmosphere of 5% CO₂ in air. The cells were evaluated the expression of transcriptional factors (Oct3/4, Nanog, and Sox2) by immunocytochemical staining and RT-PCR, and followed by differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions. Differentiation of multiple mesenchymal lineages was confirmed by RT-PCR and specific marker staining. Differentiated cells into osteocytes, adipocytes, and chondrocytes were characterised by von Kossa and Alizarin Red staining, Oil red O staining, and Alcian Blue staining, respectively. The specific genes of osteocytes (Osteonectin, Osteocalcin and Runx2) and adipocytes (aP2) were analysed by RT-PCR. In vitro oogenesis was induced in DMEM/F12 by the previously described method (Dyce et al. 2006) for 48 days. Expression of transcriptional factors (Oct4, Sox2, and Nanog) and oocyte-specific markers (c-Mos and GDF9b) was analysed by RT-PCR in these differentiated cells. At 48 days of differentiation, the oocyte-like cell masses were further cultured in TCM-199 supplemented with 0.5 μL mL^–1 FSH and 0.5 μL mL^–1 LH for 15 days. Induced cells were morphologically observed following Hoechst 33342. Expression of Oct3/4 was analysed by immunocytochemical staining in these cells. Among the transcriptional factors, only Sox2 was detected by immunocytochemical staining and RT-PCR in the theca cells. Differentiation to osteocytes, adipocyte, and chondrocytes was confirmed by specific-marker staining and gene expression by RT-PCR, respectively. The morphology of oocyte-like cell masses was distinct by 40 days of differentiation. Granulosa or cumulus-like cells were distributed through the whole surface of oocyte-like cell masses. Transcriptional factors, c-Mos, and GDF9b were detected in the cell masses by RT-PCR. After being transferred oocyte-like cell masses to TCM-199, zona pellucida-like structure was formed around the edge of the cell mass. After 15 days of culture in TCM-199, the morphology of cells was changed into blastocyst-like structure, which surrounded cumulus-like cells. Oct3/4 was expressed by immunocytochemical staining in a blastocyst-like structure. These observations demonstrated that ovarian theca cells have similar characteristics to mesenchymal stem cells in view of multilineage differentiation. Theca cells can be differentiated into oocyte-like cell masses, which expressed oocyte-specific markers. These cell masses were further developed to a blastocyst-like structure, which expressed Oct3/4. Further studies are required to evaluate in vivo differentiation to oocyte-like cells.