P-25: Characterization of Sheep Ovarian Multipotent Theca Stem Cells

Background Theca cells play important roles during folliculogenesis. They synthesize androgens, provide crosstalk with granulosa cells and oocytes during development, and provide structural support of the growing follicle to produce a mature and fertilizable oocyte. In children patients who are going to be ovariectomy, follicogenesis is at early stage and thus the follicles need to be mature in vitro. However, maturation of primordial follicles is almost impossible in vitro. Co-culture of early stage follicles with naturally surrounding cells like theca cells may significantly improve the current methods toward mature follicles. MaterialsAndMethods In this study, we successfully isolated and characterized theca stem cells (TSCs) from sheep ovary. Adherent TSCs were morphologically similar to those of sheep adult fibroblast cells (AFs) Results Cell cycle analysis indicated that the TSCs have a higher proliferative capacity than AFs. TSCs were positive for the mesenchymal stromal cells (MSCs) markers including CD29, CD44, CD73 and CD105. The isolated TSCs also expressed stemness markers including OCT4 and KLF4. Nanog was expressed at very low level and SOX2 was not expressed by TSCs.To further confirmation, TSCs were induced to differentiate into osteocytes and adipocytes. Differentiation of osteocytes and adipocytes were validated by staining with Cell cycle analysis indicated that the TSCs have a higher proliferative capacity than AFs. TSCs were positive for the mesenchymal stromal cells (MSCs) markers including CD29, CD44, CD73 and CD105. The isolated TSCs also expressed stemness markers including OCT4 and KLF4. Nanog was expressed at very low level and SOX2 was not expressed by TSCs.To further confirmation, TSCs were induced to differentiate into osteocytes and adipocytes. Differentiation of osteocytes and adipocytes were validated by staining with alizarin red and oil red respectively. Moreover, real time PCR was investigated for analysis of specific markers of the differentiated cells. Osteocalcin and col1 and were induced in osteocytes. Adipocytes were positive for LPL and PPAR alfa. TSCs resembled those of ovarian stromal cells and showed only moderate potential to differentiate towards lineages of mesenchymal origin Conclusion Result of this study can be used for obtaining theca cells which further can be used for co-culture with primary stage follicules in order to gain mature follicules in vitro.