| Pigs are important livestock for meat,recognized as large medical model animals in the medical community,and the best candidates for allogeneic organ transplantation donors.Pluripotent stem cells have important applications in biomedicine and livestock biobreeding because of their long-term passaging capacity and potential to differentiate into all types of tissue cells derived from all three germ layers.Although the research on pluripotent stem cells,represented by mouse embryonic stem cells(mESC)and mouse induced pluripotent stem cells(miPSC),has made rapid progress in recent years,the research on pluripotent stem cells in large mammals,represented by pigs,has progressed very slowly.Pluripotent stem cells with all the biological properties of mESC have not been obtained yet.The conservativeness and specificity of pluripotency regulatory mechanisms among different species still need to be studied in depth.The establishment of mESC and miPSC is the starting point of mammalian pluripotent stem cell research.mESC initiated the process of human knowledge and exploration of the biological properties and translational applications of mammalian embryonic stem cells,while the establishment of miPSC opened a new pathway for the acquisition of pluripotent stem cells and greatly contributed to the progress of research on the regulatory mechanisms of mammalian pluripotency.Given that porcine pluripotent stem cells with biologically equivalent characteristics to mESC and miPSC have not yet been established,an in-depth comparative study of the similarities and differences between mouse mESC and miPSC at the transcriptome level would be an essential reference for exploring porcine pluripotent stem cell lineage building strategies.Therefore,we first conducted a systematic study of transcriptomic data from seven different sources of mESC and miPSC using the Meta-analysis strategy to explore the common features and differences in gene expression and regulatory networks of mouse pluripotent stem cells established by two different pathways at the molecular level.The results of the study are as follows: Unsupervised clustering analysis and transcriptome principal component analysis showed differential clustering between mESC and miPSC,and together they showed significant differences relative to somatic cells when compared to somatic cells.2131 differentially expressed genes between mESC and miPSC as ensembles and somatic cells.The differentially expressed genes between mESC and miPSC and the differentially expressed genes between stem cells and somatic cells were intersected to obtain a total of 497 differentially expressed genes.Systematic analysis of mouse MII stage oocyte and somatic cell transcriptome data identified 11 candidate genes that may have a critical role in reprogramming.q PCR validation experiments supported the results of this analysis.To date,porcine pluripotent stem cell lines equivalent to mESC with germline chimeric developmental capacity have not been obtained.In order to explore the biological characteristics and regulatory mechanisms of porcine pluripotent stem cell lines,we established porcine embryonic-derived pluripotent stem cell lines(PeWV1)and exogenous transcription factor-induced porcine pluripotent stem cell line(PiPSC)with our collaborative research team.The biological characteristics and pluripotency analysis of the two cell lines are as follows: The PeWV1 clones showed a flattened morphology,which was similar to that of human embryonic stem cell clones.PeWV1 was positive in alkaline phosphatase(AP)staining and expressed pluripotency genes such as OCT4,SOX2 and NANOG,as well as the trophectoderm-specific gene CDX2.PeWV1 suspension cultures can formEmbryoid bodies and can spontaneously differentiate into multiple cell types,which could express tridermic marker genes in endoderm,mesoderm and ectoderm.The PiPSC clone had a bulging morphology and showed characteristics between mouse embryonic stem cell morphology and human embryonic stem cell clone morphology;PiPSC was also positive in AP staining and expressed pluripotency genes such as OCT4,SOX2 and NANOG,but the expression level of NANOG was relatively low.PiPSC suspension cultures can formEmbryoid bodies and can spontaneously differentiate into multiple cell types,which could express express tridermic marker genes in endoderm,mesoderm and ectoderm.The exogenously induced genes SOX2,KLF4 and C-MYC were consistently expressed in PiPSC,while the exogenously induced gene OCT4 was silenced in PiPSC.The porcine embryonic-derived pluripotent stem cells and induced pluripotent stem cells cultured in this study,although possessing a certain degree of pluripotency characteristics,still cannot achieve the full biological characteristics of mouse pluripotent stem cells.To explore the key molecular regulatory network bottlenecks that hinder the establishment of porcine pluripotency state,we analyzed the transcriptome data of porcine and mouse pluripotent stem cells and inner cell mass(ICM)of embryos by cross-species comparison,hoping to provide valuable information for the establishment of porcine pluripotent stem cell lines.The main findings are as follows: In comparison with porcine ICM,a total of 6196 differentially expressed genes were identified in PeWV1 and a total of 4737 differentially expressed genes were identified in PiPSC.In comparison with mouse ICM,a total of 84 differentially expressed genes were identified in mESC,and a total of 420 differentially expressed genes were identified in miPSC.The differential genes between these two porcine pluripotent stem cells and ICM and the differential genes between mouse pluripotent stem cells and ICM were intersected,99 cross-species co-differentially expressed genes were identified,of which 76 were co-up-regulated across species and 23 were up-regulated in porcine stem cells and downregulated in mouse stem cells.Cross-species cross-tabulation analysis of differential genes between porcine and mouse embryonic-derived stem cells and ICM revealed that 3 genes were co-up-regulated in PeWV1 and mESC,4 genes were co-down-regulated.4 genes were upregulated in PeWV1 and down-regulated in mESC,2 genes were down-regulated in PeWV1 and up-regulated in mESC.Cross-species cross-tabulation analysis of differential genes between porcine and mouse induced pluripotent stem cells and ICM revealed 31 genes co-upregulated in PiPSC and miPSC,10 genes up-regulated in PiPSC and down-regulated in miPSC,and 45 genes down-regulated in PiPSC and up-regulated in miPSC.In summary,we cultured two different sources of porcine pluripotent stem cells and characterized their biological characteristics.The pluripotency characteristics of PeWV1 and PiPSC were different from those of mESC.By analyzing the transcriptome data of pluripotent stem cells from different sources,we found that miPSC had more specific high-expression genes than mESC.This might be related to the insufficient somatic cell reprogramming.By analyzing the transcriptome data of pluripotent stem cells from different sources across species,we found that the number of differentially expressed genes between pig pluripotent stem cells and ICM was significantly higher than that between mouse pluripotent stem cells and ICM.Cross-analysis of these differential genes revealed important candidate genes related to the regulation of pluripotency that are common across species.This study provides new data for the in-depth understanding of the mechanism of the establishment and regulation of mammalian pluripotency,and lays a foundation for the establishment of porcine pluripotency stem cells with germinal chimerism in the future. |
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