Generation And Characterization Of Porcine Partially Reprogrammed Induced Pluripotent Stem Cells

Pigs hold great promises in modeling human disease, and induced pluripotent stem cells (iPSCs) provides a new valuable material for the research of genetic modified animals and cell replacement therapy (CRT), while further utilization of iPSCs is limited by many unsolved problems, such as inmmunorejection and tumorigenicity. Nonetheless, the method of cellμLar reprogramming has offered us a revolutionary tool for creating more ideal material in this field. Herein, porcine partially reprogrammed induced pluripotent stem cells (pPiPSCs) were obtain from porcine adipose-derived stem cells (pADSCs) using the method of iPSCs generation, and the characterization of them was present, new celluar material was expected to be obtained for both agriculture and human medium. These study consisted of three parts:Experiment 1 Generation and identification of porcine partially reprogrammed iPSCs. By using feeder-free system, porcine adipose-derived stem cells (pADSCs) were reprogrammed by lentiviral vector with four human transcription factors (Oct4, Sox2, c-Myc and Klf4), and porcine partially reprogrammed iPSCs (pPiPSCs), holding similar morphology and proliferation activity to porcine fully reprogrammed iPSCs (pFiPSCs), were finally obtained. Compared to pFiPSCs, pPiPSCs were negative for AP and Nanog, and the mRNA expression of Oct4, Sox2, Dnmt3b and CDH1 in pPiPSCs were lower, and little expression of ESSRB, Lin28 and Dppa2 was observed. Meanwhile, differentiation efficiency and ability of embryoid body (EB) formed by pPiPSCs were lower than pFiPSCs derived EB in vitro, and no teratoma could be formed in NOD/SCID mouse. These results suggested that we successfully obtained pPiPSCs, which held limited redifferentiation potential.Experiment 2 Biocharacteristic of porcine partially reprogrammed iPSCs. The expression of CD29, CD90, CD44 and CD 105 in pPiPSCs and pFiPSCs were analyzed by flow cytometry. We found that only CD29 was detected in pPiPSCs. However, only one of the two lines of pFiPSCs expressed the single marker of CD29, their positive rate and expression intensity were significantly lower than pPiPSCs. For mRNA level of immunogenicity genes, expression of MHCIwas observed in pPiPSCs, while not in pFiPSCs. Co-stimμLatory molecules (CD40 and CD86) did not express in both type of cells. As to the genes of immunosuppression, low level of Gatectinl was observed in both pPiPSCs and pFiPSCs, while the expression level of MMP8 in pPiPSCs was higher than pFiPSCs. When factors that inhibit the differentiation of iPSCs were withdrawn, plastic-adherent cells were gained from both types of cells, pPiPSC-derived cells (pPiPSC-DCs) showed spindle-like morphology, and could be expanded to more than 20 passages without significant senescence. In contrast, epithelial-like morphology and stationary proliferation in 5 passages was found for pFiPSC-derived cells (pFiPSC-DCs). Differentiation capacity of pPiPSC-DCs and pFiPSC-DCs was then analyzed by adipogenesis and osteogenesis. For adipogenic differentiation, the dosage of triglyeride deposition in pPiPSC-DCs was significantly higher than pFiPSC-DCs. In terms of osteogenic differentiation, many mineralization nodules could be found in pPiPSC-DCs group, while no obviously nodules was observed in pFiPSC-DCs group. Furthermore, flow cytometry results revealed that high levels of CD29, CD44 and CD90 were expressed in pPiPSC-DCs, while their expression rate and intensity in pFiPSC-DCs were significantly lower than pPiPSC-DCs. Partial genes correlated with immunogenicity could be observed in the both types of cells, while the expression level of genes related to immunosuppression is higher in pPiPSC-DCs than pFiPSC-DCs. Immunosuppression ability of pPiPSC-DCs and pFiPSC-DCs was further estimated functionally, each cell line at passage one suppressed the proliferation of mouse spleen lymphocytes, and this effect became higher as the number of cells co-cultured with lymphocytes increased; but in group of the same number of cells co-cultured with lymphocytes, no significant difference between pPiPSC-DCs and pFiPSC-DCs was found. However, when cells were expanded to passage three, this effect was higher in pPiPSC-DCs than pFiPSC-DCs. These results revealed that, compared to pFiPSCs, pPiPSCs possessed high level of reprogramming memory.We further analyzed the development ability of reconstructive embryos which were produced with SCNT method that was donated with differentiated cells. To evaluate the SCNT efficiency and blastocyst quality, pPiPSC-DCs and pFiPSC-DCs were severed as donor cells for nuclear transfer to produce cloned embryos. There is no significant difference between each cell line-derived reconstructive embryos, while the blastocyst rate of 19DCs (one cell line of pPiPSC-DCs)-derived reconstructive embryos was significant higher than others. For blastocyst quality, results had shown that the total cell number of 19DCs-derived blastocyst was significant higher than 30DCs (one cell line of pFiPSC-DCs)-derived blastocyst. These results indicated that, pPiPSC-DCs were more favorable for the development of cloning embryos in comparison to pFiPSC-DCs.In all, pPiPSCs were firstly established from pADSCs in feeder-free condition in present study. And we further revealed the events during reprogramming form pADSCs and biological characteristics of pPiPSCs, new material was provided for researches including mechanism of reprogramming, cell replacement therapy and production of cloning pigs.