Optimization Of Culture Condition For Porcine Induced Pluripotent Stem Cells

Compared with mouse model,pig has a longer lifespan and shares more similarities in anatomy and physiology with human.Therefore pig is an excellent animal model for human disease and regeneration medicine.Theoretically,porcine pluripotent stem cells can be used to simulate human cell therapy and evaluate its safety in the clinical.However,there is no authentic naive porcine embryonic stem cells(pESCs)which limited its application in biomedicine,gene modification and related fields.Porcine induced pluripotent stem cells(piPSCs)bring new inspiration for pESCs research.The piPSCs now become an excellent alternative resource of pESCs,which have potential application prospect in regeneration medicine,cell therapy and molecular breeding field and so on.However,piPSCs are failed to generate chimera with germline transmission through embryo injection,the quality of piPSCs needs to be further promoted.The main reason is attributed to the absence of suitable culture condition for piPSCs.Therefore,optimization of culture condition for piPSCs is beneficial to generate high quality piPSCs,and it also lays a foundation for establishment naive pESCs.Currently,piPSCs were induced by human embryonic stem cells(hESCs)culture medium or mouse embryonic stem cells(mESCs)culture media.But the study about the effects of these two culture conditions on piPSCs is limited.In this study,using defined mESCs and hESCs culture conditions,two different types of piPSCs were generated respectively.The piPSCs derived from mESCs culture condition were morphologically similar to mESCs(here called mpiPSCs);piPSCs derived from hESCs culture condition resembled hESCs(here called hpiPSCs).The differences between mpiPSCs and hpiPSCs were showed as following.The mpiPSCs and hpiPSCs expressed different surface markers.The mpiPSCs expressed SSEA-1;hpiPSCs expressed SSEA-4,TRA-1-81 and TRA-1-60.Gene expression profiles analysis and signaling pathway inhibition results suggested that mpiPSCs depended on JAK/STAT3 and BMP signaling pathways;hpiPSCs depended on FGF signaling pathway.Importantly,the mpiPSCs performed embryonic incorporation more efficiently than the hpiPSCs did.Additionally,the mpiPSCs showed immature feature of mitochondria and lipid droplets accumulation;hpiPSCs have mature structure of mitochondria without lipid droplets in cytoplasm.Lipid droplets accumulation and gene expression pattern indicated that mpiPSCs resembled porcine inner cell mass(ICM).Lipid droplets were found to be accumulated in the porcine oocytes.Fatty acid metabolism was activated during the porcine embryo development.These suggested that the lipid compositions in the porcine embryo may improve porcine pluripotency.In this study,AlbuMAX,a kind of lipid supplement,in combination with mESCs medium significantly improved piPSCs induction.The piPSCs derived from this lipid-content culture condition(here called LpiPSCs)were mESCs-like style,which expressed pluripotent markers SSEA-1,OCT4,SOX2 and NANOG.LpiPSCs formed embryoid bodies in vitro and developed teratoma in immunodeficient mouse,and differentiated into three germ layers.LpiPSCs were able to incorporate into porcine parthenogenetic blastocysts and mouse early embryos,which indicated that LpiPSCs have embryonic incorporation ability.The transcriptome sequencing data analysis showed that the genes related with fatty acid metabolism were up-regulated in the LpiPSCs,which were similar with porcine early embryos.AlbuMAX also promoted cell proliferation for piPSCs maintaining.This study also suggested that AlbuMAX up-regulated cytoplasmic cAMP level and promoted mesenchymal-epithelial transition through cAMP/PKA/CREB signaling pathway by improving E-cadherin expression,which finally enhanced the reprogramming efficiency.In conclusion,results suggested that culture condition affects gene expression pattern and metabolism of piPSCs.The mpiPSCs derived from mESCs culture condition were similar with porcine ICM.AlbuMAX in combination with mESCs medium improved efficiency of piPSCs induction and also promoted piPSCs proliferation.This study provides research foundation and theoretical basis for high quality piPSCs generation and pESCs establishment.