| Embryonic stem cells(ESCs)originate from the inner cell mass of blastocyst and can maintain stable self-renewal pluripotency in vitro under certain conditions.Embryonic stem cells can proliferate indefinitely in vitro and differentiate into cells of all three germ layers.These unique properties make them exceptionally valuable for drug discovery and regenerative medicine.Embryonic stem cells have different pluripotency characteristics: Na?ve pluripotency,with comprehensive differentiation potential and chimeric contribution ability,which is represented by mouse ESCs;while Primed pluripotency has limited differentiation ability and chimeric contribution potential,which is represented by human and primate ESCs.Na?ve pluripotent ESCs have not been established in other large animals such as pigs.The first mouse ESC line has been established under the condition containing fetal bovine serum(FBS)and either feeder cells or leukemia inhibitory factor(LIF).However,under this condition,only mouse embryos from 129/Sv strain can efficiently give rise to ESCs and most strains of mice are refractory to ESCs generation.In 2008,taking advantage of 2i culture system,containing inhibitors of MEK and GSK3,a series of ESCs from different m ouse strains were generated.Meanwhile,rat ESCs have also been successfully obtained in this system.Furthermore,by screening the factors that can maintain the activity of distal enhancer of OCT4,5i/L/A culture system containing LIF,Activin A and five different kinase inhibitors has been established,which can support the self-renewal of human Na?ve ESCs and transform Primed human ESCs to Na?ve state.However,the systems seem to have no effect on establishment of ESCs with naive pluripotency from other species.In 2007,through chemical screening,a medium consisting of human LIF、CHR99021、(S)-(+)-dimethindene maleate(Di M)and minocycline hydrochloride(Mi H),termed LCDM,has been established.The condition can derive pluripotent stem cells(PSCs)from human and mouse embryos conferring both embryonic and Ex Em chimeric competency.These cells are designated as extended pluripotent stem(EPS)cells.Due to the advantage of cross species PSC lines establishment,the study opens a path toward capturing plur ipotent stem cells with bi-potent chimeric competency from a variety of other mammalian species.The pig is a main source of meat.It is an ideal animal model for biomedical research,as it is very similar to humans in metabolism,immunity and physiology.And because the size of organs is similar to that of humans,pigs are potential donors for xenotransplantation.Therefore,porcine Therefore,the naive porcine PSCs are highly desirable.In the study,we tried to obtain porcine PSCs using in vivo blastocysts under the LCDM condition,and compared the expression patterns of pluripotent genes and the activity of pluripotency-associated pathways with human and mouse EPS cells.The results are as follows: Under LCDM system,there is no obvious difference in th e efficiency of establishment of pluripotent stem cells using MEF and STO as feeder layer cells.The cell clones established under LCDM system showed flat morphology and compact state.The cells were AP positive and expressed core transcription factors OCT4,SOX2,NANOG,and SSEA3 and SSEA4,and can differentiate in vitro.Low input high throughput sequencing compared the expression of the Na?ve and Primed genes among the PSCs obtained from mouse,human and pig.We found that EPS cells or PSCs were different from ESCs cells or ICM cells in mouse,human and pig deriving from LCDM system.Although there is also expression of core transcription factors,there are still some differences in the expression of Na?ve and Primed related genes.In addition,the activity of the signaling pathways related to pluripotency was analyzed.The results showed that TGFβ signaling pathway was activated in the pig PSCs,which may limit the pluripotency of the cell.Based on this result,we added TGFβ signaling pathway inhibitor SB431542 into LCDM medium.The results showed that compared with the p PSC-LCDM cell line,the cells showed higher expression of core transcription factors and proliferation ability.Gene expression profiles during the transition from ICM to p PSC-LCDMs were established by low input high throughput sequencing.The results showed that the transcription groups of ICMs,blastocyst outgrowths(BOs)and PSCs were quite different,and BO5 is a key turning point in the process of obtaining PSCs.From the above results,we can draw the following conclusions:(1)Under LCDM condition,the efficiency of pluripotent stem cell lines establishment from porcine in vivo embryos can reach to 50%,and MEF and STO,as feeder layer cells,(MEF/STO)had no obvious effect on the efficiency of pluripotent stem cell lines establishment.(2)The LCDM system can establish porcine stem cell line with certain pluripotency.(3)Under LCDM system,the inhibition of TGFβ signaling pathway effectively enhanced the expression of core transcrip tion factors and promoted cell proliferation.(4)Analysis of gene expression profiles showed that ICMs,BOs and PSCs transcripts in ICM transformed to porcine pluripotent cells were quite different.Moreover,MAPK,JAK/STAT,TGFβ,WNT and other signal pathways involved in the process of ICM to p PSCs may be the key signaling pathways affecting the establishment and pluripotent of porcine ESCs. |
