The Primary Investigation Of Establishment Of Pig Embryo Derived Stem Cells

Embryonic stem cells (ESC) and trophoblast stem cells (TSC) are derived from inner cell mass (ICM) of blastocyst or the intact blastocyst. Up to now ESCs have been successfully derived from mice, human, monkeys, and rats, and TSCs have been established from mice, humans, rabbits, rhesus and monkeys. Despite these success, it has been notoriously difficult to establish either ESCs or TSCs from ungulates. Since its similarity of body size and physiology character to human, pig is increasingly becoming the model for humandesease research.The aim of the study is to try to establish the pig trophoblast stem cell line with the whole blastocysts. We also established the STO cell lines transfected with the exogenous porcine (leukemia inhibitor factor) LIF gene, this cell line will be used as the feeder layers for porcine ESCculture. Meanwhile, we constructed the expression vector of porcine derived transcription factors, these vectors will be used as assistance for the establishment of porcine ESCs.ObjectiveWe try to establish the pig trophoblast stem cell line with the in vitro fertilization derived intact blastocysts; then we establish the STO cell lines transfected with the exogenous LIF gene. We also construct the expression vectors of porcine derived transcription factors.Method1. The blastocysts were derived from in vitro fertilization, followed by pronase treatment to remove the zona. Then the intact blastocysts, including both ICM and trophectoderm, were seeded on the feeder cells of STO. The traditional FGF culture system was used as culture medium. The cells were passaged mechanically by splitting them into small pieces. When the quantity of the cells was enough, we took the alkaline phosphates (AP) staining to characteriz the pluripotency of the ESCs. The Karyotype of the cells was identified.2. The STO cells were transfected by lipofectin transfection with the pig LIF eukaryotic expression vector pCAG-pLIF and screened by G418. The expression level of the insertedexogenous LEF gene was tested by RT-PCR and QPCR together with western blot. The cell lines which expressed pig LIF efficiently were used as the feeder cells to culture the rat induced pluripotency stem cells(iPSCs). The growth curve and the alkaline phosphates expressions detection as well as the immunocytofluorescent were undertaken to identify the pluripotency of the rat iPSCs.3. The cDNA sequences of transcription factors Oct4、SOX2、REX1、LIN28、DPPA5 was choned from porcine embryonic fibroblasts(PEF) through overlap PCR and transfected into the TET-ON induced expression plasmid, followed by plasmid extraction and restriction endonuclease digestion to verify the correct construction of the recombinant vector. Then we transfected the recombinant vector together with the pEFla-Tet3G plasmid into the PEFs. We also use the QPCR to check the expression level of the exogenous transcriptionfactors in PEFs.Results1. Several porcine trophoblast stem cells populations have been obtained and one of them was passeged 9 times. The other cell populayion stopped before 9 passages. All these cells were abundant with lip droplets and the lipid became lager gradually, which resulted in the cells stopping growing. Because the cells can only be passaged for 8-9 times, so we cannot get enough cells for stem cell identification and good karyotype figure.2. The pLIF transgene STO cells have been obtained. The expression level of the inserted exogenous LIF gene of the STO-pLIF cell lines was higher than that of the STO (p<0.05), and the riPSCs cultured with the STO-pLIF were close to the traditional riPSCs, and had difference with the riPSCs cultured in N2B27 without LIF(P<0.05).3. We have constructed the induced expression vectors of porcine derived transcription factors. The QPCR results showed that the expression of the exogenous genes were higher than that of the wild type cells.Conclusion1. It is still necessary to optimize the cell line establishment method, although the porcine trophoblaststem cells population could be derived with intact porcine blastocyst and current FGF culturesystem.2. The stable STO cell lines expressing porcine recombinant porcine leukemia inhibitory factor effectively were established successfully and these cells can maintainthe pluripotency and selfrenew of rat iPSCs as the feeder cells.3. The recombinant induced expression vectors of porcine transcription factors can raise the epression level of the exogenous transcription factors, these vectors will be used for the future research.