Vascular Repair Function And Differentiation Mechanism Of Porcine IPSC-ECs

Cardiovascular disease(CVD)is one of the most important pathogenic factors leading to patient death worldwide.Vascular endothelial cells are closely related to CVD,and existing drug and surgical treatment options cannot fundamentally improve the damaged endothelial cell function.With the development of stem cell technology,cell transplantation therapy provides the possibility for vascular endothelial repair.Induced pluripotent stem cells(iPSCs)can effectively avoid ethical issues related to embryonic stem cells(ESCs)and provide more convenience for cell transplantation therapy instead of ESCs.Studies on the directed differentiation of iPSCs into vascular endothelial cells focus on humans and mice.Pigs are large mammals,The similarity of pigs to humans in body size,physiology,immune system and nutrient metabolism.It has certain advantages in determining the safe dose of drugs in pathological toxicological tests and gene editing,and its reproductive cost is relatively low.Hence,Pigs are more suitable as human biomedical model animals.In recent years,pigs have become very popular as animal models of cardiovascular disease.In recent years,pigs have become very popular as animal models of cardiovascular disease.However,there are very few reports on the directed differentiation of porcine induced pluripotent stem cells(piPSCs)into endothelial cells.In 2013,American scientists first reported the method of inducing porcine iPSCs to differentiate into vascular endothelial cells by embryoid differentiation.In 2020,our laboratory established a method for directional differentiation of monolayer piPSCs into vascular endothelial cells with relatively simple culture medium components,serum-free and feeder-free.The vascular endothelial cells obtained by this method have good comparability with the vascular endothelial cells derived from in vivo in terms of gene expression,morphological structure and angiogenesis ability in vitro and in vivo.However,considering that the differentiation efficiency of our induction system is not high and largely depends on flow sorting and purification.In this study,we conducted the following three parts of researches:(1)The repair effect of porcine iPSCs-derived vascular endothelial cells(piPSC-ECs)in a mouse hindlimb ischemia model;(2)Analysis of major molecular events during the differentiation of piPSC-ECs by high-throughput sequencing and bioinformatics;(3)The role of TGF-β signaling pathway in the differentiation of piPSC-ECs was studied.In this study,we used the existing pig induced pluripotent stem cell line Pilw4 as the source cell,and obtained pig iPSCs-derived vascular endothelial cells(piPSC-ECs)by monolayer cell induction method to evaluate the effect of cell transplantation.piPSC-ECs,porcine fetal fibroblasts(PFFs)and equal volume endothelial cell culture medium EGM-2 were injected into the ischemic site of mouse lower limb chronic ischemia model.After the cells were transplanted,they were subjected to live cell tracking detection.Found that the cell signal decayed rapidly;After 28 days of cell transplantation,the histomorphological detection of the injection site was performed by hematoxylin-eosin staining,immunohistochemistry,apoptosis and other techniques.The results showed that the piPSC-ECs transplantation group had the most complete tissue and the highest number of blood vessels in vivo.We collected samples for single-cell transcriptome sequencing at multiple time points in the induced differentiation process.Bioinformatics analysis suggested that TGF-β signaling pathway may play an important role in the differentiation of piPSC-ECs.In response to this finding,we set up 8 experimental groups,adding the small molecule inhibitor SB431542 to inhibit the TGF-β signaling pathway at different time periods during the differentiation of piPSC-ECs.The expression and phosphorylation levels of TGF-β signaling intracellular transduction proteins Smad2 and Smad3 were detected by western blotting;the expression of mesoderm and endothelial genes in each treatment group was detected by Real time-PCR technology,and the positive rate of CD31 in each treatment group was detected by flow cytometry,so as to analyze the role of TGF-β signaling pathway in the whole induction process.In summary,Our piPSC-ECs obtained by serum-free feeder-free monolayer cell differentiation method have the ability to repair lower limb ischemia in mice.During the differentiation of piPSC-ECs,the secretion level of TGF-β1 and the phosphorylation level of Smad2/3 showed dynamic changes.The activation of this signal can promote the differentiation of piPSCs to mesoderm and inhibit the further endothelialization of cells.This study provides detailed experimental evidence for an in-depth understanding of the role of the TGF-β signaling pathway in the regulation of piPSCs directional differentiation into endothelial cells.And laid the foundation for the optimization of the piPSC-ECs induction system.The acquisition of porcine vascular endothelial cells and their functional studies can provide experimental data for human vascular endothelial cell transplantation.It provides a useful reference for the establishment of cardiovascular drug screening platform and the mechanism study of vascular endothelial differentiation and dysfunction.