Study On Culture And Differentiation Of Pig Spermatogonialstem Cells In Vitro

Pig spermatogonial stem cells (pSSCs), locating on the basement membrane of seminiferous tubule, can self-renew to maintain their quantity and direct differentiation to produce other adult stem cells as well, and they are the sole cells that can pass genetic materials to offspring in vivo of males. Due to the its plasticity, SSCs can replace embryonic stem cells (ESCs) for the treatment and genetic modification, without involving ethical and immune rejection problems, which opens up a broad prospect for its applications. However, because the mechanisms of germ cell differentiation and the interaction of key genes with inducing agent that regulate cell differentiation remain unknown, SSCs show poor reproducibility and low efficiency in induce differentiation. Besides, it is difficult to obtain fully functional somatic cells by inducing SSCs in vitro, which as a result, can not meet the needs of researched and production practices. Currently, the system of isolation, culture and induce differentiation of mouse SSCs has been relatively mature, but studies on large animals still lag behind. Therefore, this study was conducted by isolating, cultivating, identificating and inducing differentiation of Jiangquhai pig SSCs. Also, the expression of key genes during differentiation was detected in order to establish the culture system of pig SSCs in vitro and lay the foundation for exploring the regulation mechanisms of key genes related to induce differentiation.1、This study used Jiangquhai pigs as the cell source materials.4-7day-old testicles were harvested to isolate cell suspension and then SSCs and sertoli cells were obtained by differential adherent separation. The results showed that, these two cells could be better separated through three times of differential adhesion and the differential times were2h,1h and1h, respectively. The isolated sertoli cells were used as feeder layer cells.2、To better long-term culture pSSCs in vitro, we compared the two passage methods of SSCs:picked clone passage and trypsin digestion passage. The results proved that picked clone passage helped prevent the feeder layer from growing excessively rapid, thus contributing to the proliferation of SSCs. The effect of feeder layer between the mitomycin-C-treated Sertoli cells and untreated Sertoli cells were compared, we determined the mitomycin-C-treated cultured Sertoli cells as feeder layer, ultimately. Simultaneously, replacing with new ones after the feeder layer cells had been used2-3times was more conducive to the proliferation of SSCs. We once cultivated SSCs up to six months in vitro and passaged to the20th generation.3. The isolated SSCs formed clonal cell clusters both on the feeder layer cells and in the medium containing factors, and then continue to proliferate after passaging. The immune identification showed that Jiangquhai pig SSCs could express intergrin-β1, Dazl, SOX2and SSEA1stem cell-specific proteins, which proved that these SSCs could well maintain in an undifferentiated state.4. Since the SSCs had been passaged to the third generation, different chemical inducers were added in order to differentiate SSCs into neuron-like cells, adipocytes and osteoblasts, respectively. Then, we observed daily and made records. As the results showed, SSCs were successfully induced into neuron-like cells on the8th day and toluidine blue staining and NSE antibody identification both showed positive results; SSCs were induced into osteoblasts on the16th day and ALP, Von Kossa staining and Collagon1antibody identification all showed positive results; SSCs were induced into adipocytes on the22ed day and ipid droplets were stained red by oil red o staining. qRT-PCR results showed that, Nestin and P-tubulin, Cbfal and Osterix, PPARy and C/EBPa had their highest expression on about the6th day, the12th day and the18th day, respectively.In this study, we cultivated pSSCs in vitro culture system that contained factors added culture medium and sertoli cells used as the feeder layers through the methods of picked clone passage and replacement of fresh feeder layers. The results showed that pSSCs could survive and proliferate for six months, which provides a reference for future culture and proliferation of pSSCs in vitro. In addition, this experiment successfully induced SSCs into neuron-like cells, adipocytes and osteoblasts, and detected the expression of key genes during the differentiation, which lays the foundation for exploring the regulation mechanism of related genes in vitro differentiation of pSSCs.