The Role Of Oleanolic Acid In The Maturation Of Cardiomyocytes Derived From HiPSCs

Part Ⅰ Detection of hiPSC-CMs biological characteristicsObjective:To observe the changes of cell morphology and markers and the changes of mitochondria morphology between hiPSCs and hiPSC-CMs.Methods: The special medium was used to maintain the stemness of hiPSCs and cell expansion.When hiPSCs reached a high cell density,we added medium containing GSK3 inhibitor(CHIR99021)on day 0,changed the medium to B27 medium without insulin on day 2,added medium containing Wnt pathway inhibitor(IWP2)on day 3,changed the medium to B27 medium without insulin on day 5,changed the medium to B27 medium with insulin on day 7 to induce hiPSCs into hiPSC-CMs,and we purified hiPSC-CMs with the medium containing sodium lactate on day14,then the re-inoculation was performed on day 17.Immunofluorescence was used to detect the stemness factors of hiPSCs and myocardial related markers of hiPSC-CMs.RT-q PCR was used to detect the expression of troponin TNNI1,TNNI3 and myosin MYH6,MYH7 in hiPSC-CMs.Mito Traker was used to investigate the morphology and distribution of mitochondria in hiPSCs and hiPSC-CMs.JC-1 was used to detect the mitochondrial membrane potential of hiPSCs and hiPSC-CMs.The ultrastructure of mitochondria in hiPSCs and hiPSC-CMs were detected by transmission electron microscope(TEM).We investigated cell morphology and expression of different markers,as well as changes in mitochondrial morphology and membrane potential in hiPSCs and hiPSC-CMs.Results: hiPSCs was small and round with a nucleus in the center,and the cell clumps were formed of tightly arranged cells and soomth on the edge.hiPSCs expressed stemness factors Nanog and SOX2.hiPSC-CMs started beating on day 7,and the single cells’ beat could be observed after re-inoculation.Immunofluorescence and RT-q PCR results showed that hiPSC-CMs expressed myocardial related markers c Tn T,Cx43,α-actinin,troponin TNNI1,TNNI3,myosin heavy chain MYH6,MYH7.Mito Traker indicated that hiPSCs mitochondria were scattered and dotted in the cytoplasm and the mitochondria of hiPSC-CMs changed from dots to linear or networked around the nucleus.JC-1 results showed that the red fluorescence of mitochondrial membrane potential increased in hiPSC-CMs.Electron microscopy results showed that the mitochondria of hiPSCs were small and nearly round with loose cristae and the mitochondria in hiPSC-CMs was increased in the volume and length with more cristae.Conclusion: We successfully used hiPSCs induce hiPSC-CMs,and the two type of cells had different morphology and mitochondrial structure.Part Ⅱ Oleanolic acid promotes the maturation of hiPSC-CMs by pyruvate kinase isoform conversionObjective: To explore the role of oleanolic acid in the maturation of hiPSC-CMs.Methods: CCK8 was used to detect the cell viability of hiPSC-CMs after adding OA and WB was used to detect the expression of PKM1 and PKM2 to find out the optimal drug concentration for subsequent experiments.hiPSC-CMs were divided into three groups: Control group,DMSO group and OA group,and treated with optimal drug concentration and the same volume of DMSO for subsequent experiments.Immunofluorescence staining with α-actinin was performed in three groups of cells to analysis cell size,morphology and sarcomere length.WB was used to detect the expression of PKM1,PKM2 and MFN2 in the three groups of cells.RT-q PCR was used to detect the gene expression of TNNI3,MYH6 and MYH7.Mito Traker was used to observe the morphological structure of mitochondria.JC-1 was used to observe cell mitochondrial membrane potential.TEM was used to investigate the ultrastructure of mitochondria.EDU was used to detect cell proliferation.Flow cytometry was used to detect cell cycle changes.Results: Cell viability detection and WB detection of PKM1 and PKM2 protein expression determined that 10umol/L was the optimal drug concentration for OA.Immunofluorescence results showed that after adding OA the cell area and the sarcomere length of hiPSC-CMs increased,but the cell roundness index decreased.WB results showed that the ratio of PKM1/PKM2 and the expression of MFN2 increased in hiPSC-CMs adding OA group.RT-q PCR results showed that the gene expression of TNNI3,MYH6 and MYH7 in hiPSC-CMs adding OA group increased.The results of Mito Traker showed that the mitochondria of the control group were mostly distributed in linear,while the mitochondria of the hiPSC-CMs adding OA formed a network structure.The results of JC-1showed that the mitochondrial membrane potential of hiPSC-CMs adding OA group was enhanced.The TEM results showed that compared with the control group,the mitochondria of hiPSC-CMs adding OA were longer and the mitochondrial fusion was observed.EDU results showed that cell proliferation decreased in hiPSC-CMs adding OA group.The cell cycle results showed that the proportion of multinucleated cells in hiPSC-CMs adding OA group increased,while the proportion of monocytes decreased.Conclusion: After adding OA,the cell morphology and sarcomere structure of hiPSC-CMs were more mature,and the expression of myocardial related proteins and gene also indicated that the cells were more mature.The changes in mitochondrial morphology and structure also indicated that OA can promote the development of hiPSC-CMs mitochondria.EDU results showed that cell proliferation decreased in hiPSC-CMs adding OA group,but the cell cycle results showed that the number of multinucleated cells in hiPSC-CMs adding OA group increased,indicating that hiPSC-CMs adding OA group cell multinucleation increased.These results all indicated that the addition of OA promotes the maturation of hiPSC-CMs.