Small Molecules Directed Differentiation Of Kidney Organoids Based On Human Pluripotent Stem Cells

Pluripotent stem cells(PSCs)induced directed differentiation is one of the main methods to construct kidney organoids.Preliminary research mainly used cytokines to promote the differentiation of pluripotent stem cells.Small molecules have received more and more attention in recent years due to their high efficiency and stability.Therefore,this paper takes induced pluripotent stem cells as the research object to investigate the effects of RhoA-ROCK and PI3K-AKT pathway inhibitors on the differentiation of pluripotent stem cells into the two lineages of metanephric mesenchyme and ureteral buds in kidney organoids,and Further explore the synergistic effect of dual pathway inhibitors on differentiation.The main research contents and results are as follows:(1)Culture of human induced pluripotent stem cell(i PSC)cell lines and design of qPCR primers: first,i PSC cell lines were obtained by feeding layer culture,and the cells were transferred to matrix glue for feeding layer free culture before the experiment.The expression of specific markers of pluripotent stem cells(SSEA4,Na NOG,etc.)and the highly active alkaline phosphatase activity demonstrated that cultured i PSCs maintained good pluripotency.(2)The effect of Rho A-Rock pathway inhibitors on differentiation: Firstly,the cytotoxicity of single inhibitors Cyto D and BMS-3 in the co-existence of Activin A/BMP4 and Activin A+BMP4 was determined,and the appropriate inhibitor concentration was determined according to the morphology and activity of cells to further establish the program suitable for differentiation culture: Activin A+BMP4+10 n M Cyto D culture.During the differentiation process,qPCR and flow analysis were used to analyze the expression of T and LHX1(Post-primitive period markers),SIX2,WT1,HOXD11 and GDNF(Metanephric mesenchymal markers),OSR1 and PAX2(Intermediate mesoderm markers).The differentiation effect of Activin A+BMP4+10 n M Cyto D was slightly improved compared with that of Activin A+BMP4.(3)The effect of PI3K-AKT pathway inhibitors on differentiation: Two inhibitors of metformin and GSK were used as the research objects,and the concentration of the inhibitor was screened according to the morphology and viability of the cells,to determine the appropriate inhibitor concentration,and further establish A program suitable for differentiation culture.Studies have shown that adding 20 n M GSK to BMP4 can improve cell survival.During the differentiation process,qPCR and flow analysis were used to analyze the expression of T and LHX1(Post-primitive period markers),SIX2,WT1,HOXD11 and GDNF(Metanephric mesenchymal markers),OSR1 and PAX2(Intermediate mesoderm markers).he results showed that the differentiation effect of BMP4+20 n M GSK was better than that of BMP4+Activin A.In addition,one day treatment with BMP4+20 n M GSK had better effect than two days treatment.In addition,the 2-NBDG absorption assay further demonstrated that the cells differentiated by BMP4+20 n M GSK had the ability to absorb glucose.(4)The effect of dual-channel small molecule inhibitors on the differentiation of kidney organoids: The small molecule inhibitors CYTO D,BMS-3,U0126 and PD of the MEK-ERK and RhoA-ROCK pathways are used as the research objects.Firstly,it was found through cell survival rate that the addition of other small molecules to BMP4 and GSK inhibited cell survival to a certain extent.The analysis of ECAD and PAX2 by flow cytometry showed that the addition of small molecules to BMP4 and GSK increased the content of PAX2+ECAD+ positive epithelial cells in organoids.However,the mechanism of the action sites of small molecules in different pathways and other aspects still need to be further explored.