Effects Of Different Small Molecule Inhibitors On The Pluripotency Of Bovine Embryonic Stem Like Cells

Embryonic stem cells(ESCs)are pluripotent stem cells derived from blastocyst ICMs after in vitro culture by inhibiting differentiation,due to their unlimited growth ability and multi-directional differentiation ability,ESCs have been shown broad application prospects in developmental biology,regenerative medicine and disease research in recent years.To date,ESCs from mice,humans,rats,and monkeys have been successfully cultured,but the isolation and culture of livestock animal ESCs remains to be further studied.The study of livestock animal ESCs is helpful to elucidate the regulation mechanism of early embryonic development and embryonic cell pluripotency,and is of great value in animal breeding,especially in production of genetically modified animal combined with gene editing.In this study,mTseR1 stem cell culture medium was used as the basic culture medium,and L Wnt-3A/MEF mixed cells were used as feeder layers.The inner cell mass cells isolated by immunosurgery from bovine in vitro fertilized blastocysts were culture in mTseR1 with different combinations of 5-AzaC(DNA methyltransferase inhibitor),CHIR99021(GSK3? inhibitor),and PD032901(MEK inhibitors),and theeffect of different culture systems on the growth and the pluripotency maintenance of bovine embryonic stem like cells(bESLCs)were investigated.The results are as follows:1.Compared to the basic culture system which supported development to passage 8,all the three combined culture systems,5AMT(5-AzaC),2iMT(CHIR99021 + PD032901),3iMT(CHIR99021 + PD032901 + 5-AzaC),increased the passage number of bESLCs,the highest number of passages reached the 15,12,and 17,respectively.The culture system supplemented with 5-AzaC significantly increased the adherence rate of ICM cells and the primary colony formation rate.The cell clones cultured in the 5AMT,2iMT,and 3iMT culture systems maintained undifferentiated morphology for longer time.The cell clones have a flat morphology,clear cell boundaries and normal karyotypes.All the b ESLCs cultured in the four culture systems mentioned above were positive for alkaline phosphatase staining.The bESLCs from 3iMT culture systems expressed NANOG,OCT4,SOX2,CDX2,SSEA-1,TRA-1-60,TRA-1-81,and were able to differentiated into embryoid bodies,which expressed the differentiation specific proteins of the three germ layers.The above results show that bESLCs had basic features of ESCs.To investigate the pluripotency of b ESLCs and the effects of different small molecule inhibitors,the expression of pluripotency-related genes in bESLCs at passage 5 was examined by real-time quantitative PCR,results showed that the expression of OCT4,NANOG,SOX2 and STAT3 genes in b ESLCs from 5AMT and3 iMT,which supplemented with 5-AzaC,was significantly increased,and SOX2 expression was also significantly increased in 2iMT culture system.Subsequently,the DNA methylation status in promoter regions of NANOG and SOX2 genes was detected by BSP(bisuilfite sequencing PCR)method in bESLCs derived from four culture systems,results showed that compared with control group,there was no significant difference in the methylation status of the NANOG promoter region among three treatment groups,but DNA promoter region of the SOX2 was significantly demethylated,the difference was significant.The above results indicatethat 2i(CHIR99021+PD032901)and 5-AzaC can effectively promote the pluripotency-related gene expression in bESLCs.After culture and passage,bESLCs formed different clone morphologies in the above culture system,the following study were conducted to compared the effects of clone morphology and passage methods on cell pluripotency.There were obvious morphological differences between the CMt(central multilayer)cells and PMn(peripheral monolayer)cells in the bESLCs clones,and the alkaline phosphatase activity presented overt increased in the CMt region.Results from real-time quantitative PCR showed that the expression of pluripotency genes OCT4,NANOG,and SOX2 were significantly higher in CMt than in PMn region,especially for NANOG,while the expression of PRDM14,OTX2 and the trophoblast cell marker gene CDX2 were opposite,therefore,only CMt cells were used for passage in this study.Compared the clone morphology of bESLCs from different passages,it was shown that the cell clones became transparent and the borders were blurred as passage number increased,the expression of NANOG,SOX2 and C-MYC were significantly decreased,and the expression of CDX2,STAT3,EZH2 were significantly increased,while OCT4 stably expressed.In addition,the pluripotency-related gene expressions of bESLCs with dome-shaped or flat-shaped clones were compared.Real-time quantitative PCR results showed that between the two morphologically clone,no difference in the expression levels of OCT4,GATA6 and C-MYC,but the expression levels of NANOG,SOX2,CDX2,CDH1 and FGFR2 were lower in dome-shaped clone than that in flat clone,whereas the expression of STAT3 and CDH2 was higher in dome-shaped clone than that in flat-shaped clone.In summary,this study shows that the bESLCs cultured in 5-AMT,2iMT,and3 iMT culture systems have the characteristics as ESCs.5-AzaC can improve the efficiency of bESLCs isolation,and 2i inhibitors and 5-AzaC can effectively promote bESLCs pluripotent marker expression and inhibit differentiation.In terms of cell growth status,passage numbers and pluripotency gene expression,the 3iMT culture system is more suitable for in vitro culture and pluripotency maintenance of bESLCs.