Effects Of Apelin On Cardiac Differentiation Of Human Induced Pluripotent Stem Cells

BackgroundInduced pluripotent stem cells, commonly abbreviated as iPSCs, are a type ofpluripotent stem cell artificially derived from adult somatic cell by reprogramming. MouseiPSCs were first generated from mouse fibroblasts in2006.Human iPS cells (hiPSC) wereestablished in2007by the transduction of four defined transcription factors.Theproduction of iPSCs represents an unlimited source of cardiomyocytes because of theirgreat potential for differentiation and poses fewer legal and ethical issues than does thegeneration of human embryonic stem cells (ESC).Therefore, hiPSCs are one of the mostpromising sources of cells for cardiac regenerative therapy. Nevertheless, many importantproblems, especially the high efficiency of differentiation of iPS cells to avoid teratomaformation, must be addressed before hiPSCs can be used clinically.Apelin is a new peptide identified in1998which regulates cardiovascular systemfunction. In the zebrafish and mouse embryos, it is the key factor to ensure normal cardiacdevelopment. It is recently proved that apelin could enhance the cardiac differentiation ofESCs.ObjectivesThis study was aimed to investigate the effect of apelin on cardiac differentiation of human induced pluripotent stem cells.Methods1. Isolation of mouse embryonic fibroblasts and preparation of feeder cells forhiPSC culture.2. hiPSC culture and passaging.3. Using immunofluorescence (IF) staining to identify stemness factors Oct4andNanog.4. Adopting suspension method in differention medium to let hiPSC form embryoidbodies (EBs). Experiment groups were designed into control group and apelintreatment.5. qPCR analysis was applied to measure the expressions of cardiac specificmarkers of EBs at different time points.6. Evaluation the expressions of cardiac specific proteins of EBs at different timepoints by Western blot.7. Using IF staining to testify the expression of cardiac specific cell marker cTn Iand connexin43.8. Flow Cytometry (FCM) was applied to testify the portion of cTn I positive cells.9. Using Transmission Electron Microscope (TEM) to observe the cellularultrastructure of hiPSC-derived cardiomyocytes (hiPSC-CMs).Results1. The hiPSC clone was in round shape in the background of feeder cells withstrong three-dimensional sense and clear boundaries. Suspension method wasused to allow hiPSCs form EBs. Oct4and Nanog were positive in IF stanning.2. Compared with control group, the gene expressions of pluripotency markers inapelin group decresed and cardiac specific cell markers increased at differenttime points: The expression of Oct4and Nanog decresed, which are(63.2±10.1)%and（51.7±1.6）%of control group, respectively.(P<0.01).Theexpression of cardiac progenitor cells marker Nkx2.5and cardiac specific marker cTn I at day7increased (1.62±0.23) folds and (2.50±0.22), respectively (P<0.05,P<0.01).3. Compared with control group, protein Nanog expression was gradually decresedwith days of apelin treatment, which was observed most obvious at da y15.Protein Mef2c was upregulated at day15.The protein expression of cTn T andcTn I were significantly increased at day7and day15, respectively.4. Immunofluorescence staining showed that cTn I positive and connexin43positive cells were more in apelin group.5. Flow cytometry analysis showed that the rates of cTn I positive cells were（9.3±5.2）%in control group and（52.2±6.7）%in apelin treatment(P<0.05).6. Compared with control group, more organized myofibril, mitochondria wereobserved in apelin group.ConclusionIn this study, embryoid bodies formation assay is used to generate cardiomyocytesfrom hiPSC. We demonstrate apelin enhances hiPSCs cardaic differentiation directly in thepresence of fetal bovine serum.This effect may depend on the increased expression ofNkx2.5and Mef2c which are both key factors in heart development in-vivo. And theultra-microstructure of hiPSC derived cardiomyocytes suggests apelin could also promotethe formation of basic cardiac structure to generate function.