Functional Study On SOX15 In The Pluripotency Regulation Of Porcine Embryo-derived Stem Cells

Embryonic stem cells(ESCs)derived from the inner cell mass of mammalian blastocysts,have the potential to differentiate into all cell types while maintaining pluripotency and hold immense promise in livestock production and clinical therapy.Pigs,as important livestock and model animals,have played a crucial role in biological development.However,a na?ve-state pig embryonic stem cell line with germline chimeric competency has not been established due to incomplete understanding of the regulation of pluripotency networks during pig embryonic development.Previous research by our team found that pig E7-8 EPI(Embryonic day 7-8;Epiblast)may correspond to the mouse E4.5 EPI,suggesting that pig E7-8 EPI has the characteristics of a na?ve pluripotent state,with specific high expression of SOX15 at this stage.SOX15,a member of the SOX family of G-group genes,has a conserved HMG(SRY-related HMG Box)domain.It has been found to be highly expressed in undifferentiated ESCs,with high similarity to SOX2,and can bind to OCT4 to play a regulatory role.Based on this evidence,it was hypothesized that SOX15 is a key factor in the pluripotency regulatory network and a pluripotency hallmark gene specific to the na?ve state in pigs.This study analyzed and compared the sequence of SOX15 to show its high similarity to the HMG domain of SOX2 across species.The transcriptome sequencing data of pig embryos at different developmental stages showed that SOX15 gradually increased from the 2-cell stage and was specifically highly expressed in the late blastocyst stage.Real-time quantitative PCR(q PCR)was used to confirm the consistency of the expression of SOX15 during pig embryonic development.SOX15 was also found to be highly expressed in cells with high expression of pluripotency factors by q PCR and immunofluorescence,indicating its association with pluripotency in pig ESCs.To investigate the effect of SOX15 on pluripotency in pig ESCs,we generated a SOX15 knockdown system using the p LCDM and p EPSC cell lines with different SOX15 expression levels.The results showed that SOX15 knockdown resulted in smaller cell clones,decreased alkaline phosphatase(AP)positivity,increased cell doubling time,and overall downregulation of pluripotency-related genes.This suggests that knocking down SOX15 in pig ESCs leads to a decrease in pluripotency levels.To further explore the effect of SOX15 on the spontaneous differentiation ability of the cells,we examined the spontaneous differentiation ability of the cells after SOX15 knockdown.The results showed that the diameter of the embryoid body was significantly smaller than that of the control group,and the endoderm marker gene expression was increased,while the ectoderm marker gene GAFP expression was decreased.These results indicate that SOX15 affects cell spontaneous differentiation.In conclusion,this study investigated the expression pattern of SOX15 in early-stage pig embryos and pig ESCs and explored its effect on pluripotency by constructing a SOX15 knockdown system.The results show that knocking down SOX15 in pig ESCs leads to a decrease in pluripotency levels and provide data reference for understanding the establishment and regulation mechanism of pig ESCs as well as guiding the identification of specific markers for the na?ve state in pigs.