Differentiation Of Porcine Induced Pluripotent Stem Cells Into Endothelial Cells

Cardiovascular disease(CVD)is a kind of disease involving the heart or blood vessels which become one of the serious diseases leading to death.Coronary artery disease(CAD)is a kind of cardiovascular disease,mainly caused by vascular injury or loss of vascular function caused by endothelial dysfunction,thereby reducing blood flow and ultimately leading to myocardial cell death and myocardial infarction.These diseases cause great harm to mankind while the traditional methods such as drug treatment have limitations so that they have not yet been fully cured.Cell transplantation therapy has become a concern.Embryonic stem cells(ESCs)and induced pluripotent stem cells(i PSCs)can be sources of cells for cell transplantation therapy.Transplantation therapy with ESCs,always being allogenetic,involves ethical and immune rejection issues,while autologous transplantation of i PSCs,generated from the patient,facilitates the treatment of vascular injury disorders.There are already many scientists conducted detailed research on the differentiation of i PSCs into endothelial cells focusing on human and mouse.As an important agricultural livestock,pig is similar in size,physiology and anatomy to human.It also have the advantages of fast growth cycle and long life.Thus,using pig as a model to study and treat cardiovascular diseases is more advantageous than using mice.Studies on establishment and culture of porcine i PSCs and differentiating porcine i PSCs to endothelial cells will be able to ensure for the creation of a model of cardiovascular disease in pigs.At present,many groups have established porcine i PSCs via different methods.There was poorly research on the differentiation of i PSCs into endothelial cells in porcine.The first study using porcine i PSCs to differentiate into endothelial cells was reported by Joseph et.al in 2013.In this study,porcine i PSCs were cultured into embryoid bodies(EBs)under suspension conditions for differentiating,however,it took longer and was less efficient.Here,we want to solve the above issues,and this parper conducted the following four parts.(1)We induced four porcine stem cell lines,including porcine embryo derived stem cell lines Penk6 and Pe WCHX,and porcine i PSCs Pilw4 and M10,differentiation via a two-dimensional(2D)feeder-free cultures method.The expression kinatics of the lineage-specific markers were measured in the early stage of differentiation and the differentiation potential to mesoderm of the four different cell lines was assessed.(2)Different combinations of cytokines or chemical molecules were used in inducing the porcine i PSCs Pilw4 cell lines to mesoderm,in order to screen for a method with the highest differentiation efficiency into mesoderm.(3)A method for the induction the porcine i PSCs into endothelial cells in a 2D feeder-free cultures was established.(4)Induced human ESCs and porcine i PSCs into endothelial cells using the established differentiation methods and compared the differentiation differences between the two stem cell lines.Our research found the porcine embryo derived stem cell lines Penk6 and Pe WCHX have no significant changes in cell morphology during induction,while porcine i PSCs Pilw4 and M10 lost their original clone morphology and showed partial apoptosis.The measurement of lineage marker genes indicated that the Penk6,Pe WCHX and M10 did not initiate the differentiation into the primitive streak and the expression of mesoderm marker genes changed irregularly.However,during the induction of Pilw4,the primitive streak,endoderm and mesoderm marker genes were upregulated and then decreased,reflecting the initiation differentiation of Pilw4.So,we selected pi PSCs Pilw4 as the source cells for differentiation into endothelial cells in subsequent experiments.We used four pre-existing methods for the differentiation of human ESCs into endothelial cells to induce pi PSCs and detected the expression of lineage-specific genes during differentiation.One of the methods was proved to be effective in promoting the differentiation of pi PSCs into mesoderm,which was then optimized.In this process,we studied the effect of CHIR99201 and BMP4 in the early stage of differentiation(day 0-4).Treating cells in 6 groups: F(day 0-2 null and day 3-4 FGF2),FB(day 0-2 null and day 3-4 FGF2+BMP4),BFB(day 0-2 BMP4 and day 3-4 FGF2+BMP4),CF(day 0-2 CHIR99021 and day 3-4 FGF2),CFB(day 0-2 CHIR99021 and day 3-4 FGF2+BMP4),CBFB(day 0-2 CHIR99021+BMP4 and day 3-4 FGF2+BMP4).After 4 days of treatment,VEGF and BMP4 were added in day 5-7 and then switch to commercial endothelial cell culture medium in day 8-10.In day 11 of induction,CD31 positive cells were flow-sorted and the endothelial cells markers were detected.The results showed the expression of primitive streak markers in B,FB and BFB groups were low in the early stage of differentiation and the ratios of CD31 positive cells in the three groups were only 3.12%-5.16%.In CF,CFB and CBFB groups,the expression of the primitive streak and endoderm markers increased and then decreased,and the mesoderm markers were continued upregulated during the early induction.CFB groups resulted in a higher ratio of CD31 positive cell(21.3%),compared with that of CF group(12.2%)or CBFB group(17.1%).These results indicated that CHIR99201 plays an important role in the early stage of differentiation(day0-4)from porcine i PSCs into endothelial cells,and the addition of BMP4 in the middle of differentiation(day 5-7)can significantly increase the differentiation efficiency of endothelial cells.We utilized the CFB group as the method for differentiating porcine i PSCs into endothelial cells,and obtained functional endothelial cells from porcine i PSCs(pi PS-ECs).The pi PS-ECs showed similar morphology,gene expression patterns and functional characteristics to the porcine aortic endothelial cells(AOCs).Next,we induced human ESCs through CBFB induction method.The results showed that the induction efficiency of porcine i PSCs was higher than that of human ESCs via the induction method,which may be related to differences in gene expression patterns in the lineage-specifc markers during induction.Above all,we have established a two-dimensional(2D)feeder-free cultures method,which is suitable for differentiating pi PSCs into functional endothelial cells.This study provides a possibility to evaluate the autologous transplantation of vascular endothelial cells in large animals and creative porcine cardiovascular disease model.