The Establishment And Pluripotency Improvement Of Porcine Pluripotent Stem Cells Derived From Porcine Embryos

The pig is an important farm animal with few ethical restrictions and it is considered an useful model of human disease and an ideal animal for the preclinical testing of cell transplantation and regenerative medicine, bearing similar properties with human in physiology, the organ anatomical structure, genetic and immunology. But the utility of these models has been hampered by a lack of genuine porcine embryonic stem cells. The researchers had widely explored the culture system of porcine pluripotent stem cells according to the research experience of mouse embryonic stem cells and human embryonic stem cells in the past twenty years.The first porcine pluripotent stem cell line derived from porcine embryos had been reported by Evans et al, Strojek et al. and Piedrahita et al in 1990. They all used the porcine blastocysts as primary materials based on the culture system of mouse embryonic stem cells, and they also discussed the effects of feeder cells, embryonic age, the whole embryos and the isolated inner cell mass on the formation of primary colonies and described the characterisation of the putative procine embryonic stem cells. The researchers performed comprehensive studies on the various factors affecting the establishment of porcine embryonic stem cells, including the embryonic age, the feeder cell type, the isolation methods of the inner cell mass, the source of embryos and the components in the culture medium and so on. However, there still existed several questions:(1) All the cell lines were the putative procine embryonic stem cells;(2) There were no uniform characterization standards;(3) The establishment efficiency was low for ineffective culture system;(4) The in vivo and in vitro differentiation ability was weak, and it was difficult to form teratomas;(5) Transferring exogenous genes into porcine ESC-like cells was inefficient, and the genes were not stably expressed;(6) There were no germline chimeric aninals.To solve these problems, we had carried on the exploration of the follo wing four parts:(1) explore the influence factors of porcine embryonic stem cells;(2) explore the pluripotency characteristic and the in vitro and in vivo differentiated abilities of porcine embryonic stem cells;(3) explore the feasibility of gene editing in porcine embryonic stem cells;(4) explore the feasibility of acquiring somatic nuclear transfer animals and chimeric animals using porcine embryonic stem cells as donor cells.Study on the content, we found that:(1) We derived a porcine pluripotent stem cell(p PSCs) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The p PSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the p PSCs were alkaline phosphatase positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. in vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the p PSCs could differentiate into cells of the three germ layers. The p PSCs transfected with fuw-Ds Red(pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when p PSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts. When p PSC-FDs were injected into day 4.5 blastocysts, they became involve d in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients.(2) The composition of MXV medium is too complex to further make studies on porcine specific molecular mechanism of pluripotency regulation. Therefore, we optimized the MXV medium and obtained a chemically defined and serum-free culure medium, namely KO medium. We also derived a new porcine pluripotent stem cell line(p PSC-KOs) from day 5.5 in vitro fertilization porcine blastocysts in KO medium. The p PSC-KOs possessed normal pluripotency and could spontaneously differentiate into cells of the three germ layers in vitro and also directional differentiate into neurocyte and bone nodule. The pPSC-KOs could be passaged with a stable expression of both e GFP and pluripotent markers and contributed to the in vitro embryonic development of the chimeric blastocysts. Above all, the se findings indicated that the p PSCs and pPSC-KOs were porcine pluripotent cells that would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.According to our studies, the following contents can be got:(1) We derived a porcine pluripotent stem cell(p PSCs) line through optimizing the influence factors of porcine embryonic stem cells, such as the oxygen concentration, the culture medium and the embryonic age. The pPSCs could be stably passaged and had a similar pluripotent characteristics with human embryonic stem cells and mouse embryonic stem cells.(2) The p PSCs could differentiate into cells of the three germ layers in vitro and in vivo and undertake gene editing operation and stably carry the exogenous genes. When p PSC-FDs were used as donor cells for somatic nuclear transfer, the reconstructed embryos could develope into blastocysts. When the pPSC-FDs were injected into day 4.5 blastocysts, they contributed to the viscera of foetuses and placenta.(3) We got a chemically defined and serum-free culure medium(KO medium) by withdrawing the composition of MXV medium one by one, and also established a new porcine pluripo tent stem cell line(p PSC-KOs) with a more similar pluripetency with mouse embyronic stem cells.(4) The pPSC-KOs could spontaneously differentiate into cells of the three germ layers and directional differentiate into neurocyte and bone nodule. The pPSC-KOs transfected with fuw-eGFP could be passaged with a stable expression of exogenous genes and the reconstructed embryos could develope into blastocysts that used p PSC-KO-FEs as donor cells.(5) The porcine pluripotent stem cell lines(p PSCs and p PSC-KOs) established from different culture medium(MXV medium and KO medium) relied on different signalling pathway, the p PSCs are FGF signalling and LIF signalling dependent, however, the p PSC-KOs tends to depend on LIF signalling.