Transcriptome Analysis Of Porcine Epiblast And Key Factors Screening Of Porcine Embryonic Stem Cell Culture System

Embryonic Stem Cell(ESC)can maintain self-renewal and proliferate indefinitely during culture,while retaining multi-directional differentiation potential.ESCs have two pluripotency states,Na(?)ve states,represented by mouse ESCs(m ESCs),and Primed states,represented by human ESCs(h ESCs).Na(?)ve pluripotency has comprehensive differentiation capacity and germline chimeric potential,while Primed pluripotency has limited differentiation capacity and chimeric contribution potential.So far,species other than mice and rats have not successfully established Na(?)ve state ESCs with the ability of germline chimerism.Pig is a large animal model whose physiological structure and immune characteristics are very similar to human,the establishment of porcine ESC is of great significance for the establishment of human genetic disease model and human xenotransplantation donor.Since 1990,the research of porcine ESCs has made great progress.However,there is no porcine pluripotent stem cell(p PSC)that can realize germ-line chimerism.In addition,in the study of p ESC also face several key problems: the early embryonic development period corresponding to Na(?)ve status is unclear,the pluripotency regulation mechanism is unclear,the pluripotency marker genes are uncertain,and the evaluation criteria are unclear.The pluripotency of ESC is derived from the embryo,but after in vitro culture,the maintenance and regulation network of pluripotency of ESCs has changed.For example,m ESC more closely resembles the pre-epiblast(Pre-EPI)than the Inner Cell Mass(ICM).It is of great significance for the identification of pig pluripotent cell lines and the understanding of pluripotent state.The study showed the greatest similarity between mouse embryonic Pre-EPI cells and Na(?)ve m ESC.Therefore,this study first compared the transcriptome data of mouse,human Na(?)ve and Primed ESC and their embryonic cells at each early stage to verify the similarity between Na(?)ve cells and pre-EPI cells.Then,the single-cell transcriptome data of early porcine embryos at different developmental stages were analyzed,and the specific developmental stage in which Pre-EPI appeared in early pig embryos was preliminarily determined by comparison with the transcriptome data analysis results of early mouse and human embryos.On this basis,the signal pathways enriched in porcine Pre-EPI were analyzed,and the expression patterns of key genes of pluripotency related signal pathways in different developmental stages of porcine embryos were compared and analyzed to further clarify the regulatory characteristics of porcine Pre-EPI signal pathways.We compared the pluripotency and transcriptome of various p ESC lines: p PSC(KOFL),p LCDM and porcine expanded pluripotent stem cell(p EPSC),and analyzed to determine the suitable tool cells for screening porcine Na(?)ve PSC culture system.Then,according to the characteristics of the porcine Pre-EPI signaling pathway,the regulatory factors of the key signaling pathway were screened to obtain the porcine Na(?)ve PSC culture system.Our results show that:(1)Transcriptome analysis of mouse,human Na(?)ve and Primed PSCs and embryonic transcriptome data showed that mouse Na(?)ve and Primed PSCs had the highest similarity with E4.5 Pre-EPI,and mouse Primed PSCs had the highest similarity with E5.5 EPI.Compared with ICM,human Na(?)ve PSCs are more similar to EPI of pre-implantation late blastocyst.(2)Principal component analysis(PCA)was performed on the homologous genes of pluripotent cells at each stage of mouse and pig pre-gastrulating embryos.The results showed that the pluripotent matching stage of mouse and pig had broad developmental consistency,and the porcine E7-8 EPI corresponded to the mouse E4.5 Pre-EPI.Therefore,porcine E7-8 EPI was called porcine Pre-EPI in this study.Analysis of X chromosome activity,metabolic pattern and expression of pluripotent marker genes in porcine embryo cells at various stages showed that X chromosome was in double activated state and had two metabolic patterns of glycolysis and oxidative phosphorylation in porcine E7-8 Pre-EPI.Comparison of mouse and human ESC results further confirmed that porcine E7-8 Pre-EPI is Na(?)ve pluripotent.(3)By analyzing the expression patterns of pluripotency marker genes in pigs at various stages,SOX15 is expressed in pluripotent cells of early porcine embryos and is highly specifically expressed in E7-8 Pre-EPI.Therefore,SOX15 can be used as a candidate gene for marker genes of porcine Naive pluripotent state.(4)Analysis of signaling pathways enriched in porcine Pre-EPI found that the pluripotency related signaling pathway and Hedgehog signaling pathway were enriched in E7-8 Pre-EPI.Further comparative analysis of the expression patterns of key genes of pluripotency related signaling pathways in different developmental stages of porcine embryos showed that the expression of related factors of PI3 K,Activin/Nodal,Hedgehog signaling pathway in E7-8 Pre-EPI was up-regulated along with the expression of related factors of BMP,AMPK,WNT signaling pathway and RAS/RAF in MAPK signaling pathway was down-regulated.(5)A comparative analysis of the pluripotency and transcriptome of p ESC lines obtained from various systems in the laboratory.p EPSC has high expression of Primed pluripotent state marker genes,and low expression of marker genes in each germ layer,which can efficiently and spontaneously differentiate into ectodermal / mesodermal / endodermal cells in vitro,without obvious differentiation bias.Thus,p EPSC with stable pluripotent characteristics and extensive developmental potential was selected as a tool for screening porcine Na(?)ve PSC system.(6)According to the signal pathway characteristics of porcine E7-8 Pre-EPI,the regulatory factors of key signal pathways were screened.The LASI+ 3i system containing PI3 K,Activin/Nodal,Hedgehog activator and AMPK,BMP,RAF and WNT inhibitors was established by testing the AP positive rate,doubling time and the expression of known Na(?)ve pluripotency and E7-8 Pre-EPI marker genes in pigs.(7)After p EPSC,pg Epi SC and pig in vitro fertilization(IVF)blastocysts were cultured in vitro with LASI+3i culture system,This system can increase the expression of two p PSC pluripotency marker genes SOX2,REX1,ESRRB and the known pig E7-8 Pre-EPI marker genes SOX15,PRDM14,NODAL,PRDM14,TFCP2L1.The number of ICM and Pre-EPI cells in blastocyst was also significantly increased.(8)In order to make the system more conducive to the establishment of cell lines from scratch,the LASI+ 3i system was further optimized,and the LASI+ 4i culture system was obtained by adding CHIR99021 to promote cell self-renewal.LCMSB59 system was used to derive outgrowth from D8 IVF blastocysts.During outgrowth passage,cells were transferred to LASI+ 4i system to obtain LCMS-LASI4 i cell line,which had a m ESC-like protuberant clone morphology.Compared with p EPSC and p LCDM,the expression levels of core transcription factors SOX2,KLF4 and porcine E7-8 Pre-EPI marker gene SOX15 were significantly increased,and the cells could spontaneously differentiate into ectodermal / mesodermal / endodermal / trophoblast lineages in vitro,showing a wide range of differentiation potential.In summary,we draw the following conclusions:(1)Porcine E7-8 EPI corresponds to Na(?)ve Pre-EPI;(2)SOX15 is highly specifically expressed in E7-8 Pre-EPI,which can be used as a candidate gene for marker genes of Na(?)ve pluripotent state in pigs.(3)p EPSC has the characteristics of Primed PSCs.(4)Based on the characteristics of porcine E7-8 Pre-EPI signal pathway,screening established LASI+ 3i system containing PI3 K,Activin/Nodal,Hedgehog activator and AMPK,BMP,RAF and WNT inhibitors can enhance the expression of known Na(?)ve pluripotent and E7-8 Pre-EPI marker genes in p EPSC and pg Epi SC.The number of ICM and Pre-EPI cells in porcine IVF blastocyst was also significantly increased.(5)The LASI+4i culture system obtained by adding CHIR99021 to LASI+3i.The LCMS-LASI4 i cell line was established in this system using IVF embryos.It has a high level expression of core transcription factors and porcine E7-8 Pre-EPI marker genes,and has the ability to differentiate between three germ layers in vitro,and also has the ability to differentiate into the trophoblast lineage.