Establishment Of The System For Inducing Embryonic Stem Cells To Differentiate Into Vascular Endothelial And Hematopoietic Cells In Vitro

Background:In the process of angiogenesis and remodeling,vascular endothelial cells(EC)originate from hemangioblast(HA).However,due to the lack of in-depth understanding of many details of this development process,it is very difficult for mature vascular endothelium to expand in vitro,which greatly limits its functional research and clinical application.In recent years,many studies have shown that there is a group of"vascular endothelial stem cells(VESC)"with specific molecular phenotype in mammalian adult vascular endothelium,expressing Kit(also known as CD117),Procr(also known as EPCR,CD201)and other molecules,which can form stable clones in vitro and generate tens of millions of progeny endothelial cells.In addition,VESC has the bidirectional potential to differentiate into primary endothelial cells and peripheral cells,and can produce functional blood vessels in vivo,which provides a new research direction for tissue engineering blood vessels and clinical treatment.The origin of hematopoietic cells(HC)is closely related to vascular endothelium.At present,the mainstream point of view is that hematopoietic cells are derived from mesoderm-derived,functionally specialized hematopoietic endothelium(HE)and evolved through endothelial-hematopoietic transition(EHT)differentiation to produce primitive hematopoiesis and directional hematopoiesis in multiple sites and time nodes in early embryo,with obvious spatiotemporal specificity.Mouse embryonic hematopoietic cells express endothelial cell markers CD31,CD144,etc.Hematopoietic cells derived from peripheral blood CD34~+can be differentiated into endothelial cells through in vitro culture.These studies also indirectly confirm that hematopoietic cells and endothelial cells have the same developmental origin.Embryonic stem cells(ESC)are a kind of all-round stem cells that can differentiate into almost all cell types of the body and has strong proliferation ability.They are ideal seed cells for tissue engineering research.In recent years,a number of studies have shown that in terms of stem cell factor(SCF),vascular endothelial growth factor(VEGF),interleukin-3(IL-3),erythropoietin(EPO)and other cytokines,ESC can be induced to differentiate into endothelial stem and progenitor cells(ESPC)in vitro,and further differentiate into endothelial cells,vascular parietal cells(peripheral cells and vascular smooth muscle)and further pitted into arteries and veins;Or differentiate into hematopoietic stem and progenitor cells(HSPC),and further differentiate into myeloid progenitor,megakaryocyte-erythroid progenitor cells(MEP),and other downstream blood cells.These findings have laid a theoretical foundation for the in vitro induction and editing of ESC.However,due to the limitations of induction methods,the unclearness of regulatory network,and the lack of microenvironment,the differentiation system of ESC in vitro directed endothelial cells and hematopoietic cells is still not very mature.Objective:In this study,embryonic stem cells are directionally induced to differentiate into endothelial cells and hematopoietic cells by controlling various cytokines and conditions,using their developmental totipotency and induced plasticity,so as to construct a complete in vitro directional differentiation system.Through flow detection,immunohistochemistry and other means to verify the surface molecular characteristics of the two cells after induction,to explore whether there are intermediate states of"stem and progenitor cells"and their surface molecular characteristics in the induction process,and to explore the correlation between endothelial and hematopoietic development,so as to provide experimental basis for optimizing stable and mature in vitro induced differentiation system and further conducting large-scale tissue engineering application research.Methods:According to the origin of endothelial and hematopoietic cells in embryonic stage,in the serum-free StemPro culture system,BMP4(bone morphogenetic protein 4)and Activin A,which can induce stem cells to differentiate into mesoderm,FGF-Basic(fibroblast growth factor basic)and VEGF,which can induce proliferation and migration of endothelial cells,were all used in this study to induce mouse embryonic stem cell line R1/E to form embryoid bodies(EB),simulating mesoderm development process.SCF,VEGF,SCF,flt3-ligand(FLt3L),IL-3 were used to induce EB to differentiate into EC and HC,respectively,and to establish a differentiation system of embryonic stem cells targeting vascular endothelial and hematopoietic cells in vitro.Results:After 4 days of differentiation induced by the four-factors system,ESC was successfully induced into a large number of EB with full morphology and clear clone boundary.After 6 days of continuous directed differentiation into endothelium and hematopoiesis,several endothelial tubular structures and hematopoietic colonies were successfully induced.Through flow detection analysis,the proportion of CD31~+endothelial cells are 1.35±0.05%.Further analysis of CD31~+CD144~+CD45~-population show that 3.0±0.2%of the cell phenotype are Kit~+CD201~+,suggesting that this part of cells may be ESPC upstream of differentiation.The proportion of hematopoietic cells with CD45~+is 35.0±0.5%,of which 0.35±0.05%are Kit~+CD201~+,suggesting that this part of cells may be HSPC upstream of differentiation.The number of these two groups of cells are very limited,but they have very typical stem progenitor cell characteristic markers,and their phenotypes are similar,which indirectly confirms that endothelial cells and hematopoietic cells have the same fate and origin,and coincides with previous studies.Conclusion:This study successfully established a system for differentiation of embryonic stem cells into endothelial cells and hematopoietic cells in vitro,indirectly verifying that the two may have a common embryonic origin,and capturing cells with molecular characteristics of endothelial cells and hematopoietic stem progenitor cells through flow detection,which can be used as a good in vitro induced differentiation system,laying an experimental foundation for in-depth exploration of the development regulation mechanism of endothelial cells and hematopoietic cells and large-scale induced differentiation of embryonic stem cells in vitro.