Effects Of Cardiotrophin-1on Cardiac Differentiation Of Mouse Induced Pluripotent Stem Cells

BackgroundSeveral types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of several stem cell transcriptional factors. Such induced pluripotent stem cells, termed as iPSCs, was firstly gained by Yamanaka in2006. These iPSC shares the similar properties with embryonic stem cells (ESCs), such as self-renewal and multipotent differentiation, while overcomes the ethical problem caused by the embryos studies. Studies have shown that iPSC could differentiate into all three germ layer cells including cardiomyocytes and could be a possible source for cell therapy of myocardial infarction or congestive heart failure. Therefore, iPSC shows great potential in the area of stem cell research. However, the low cardiac differentiaton efficiency hindered its application in cardiac regeneration research. Cardiotrophin-1, which shows high level of expression during the embryonic cardiac development, could promote the cardiac differentiation of ESCs as well as BMMSCs. But no case of its effect on miPSC cardiac differentiation was reported till now.Objectives This study was aimed to investigate the effect of cardiotrophin-1on cardiac differentiation of mouse induced pluripotent stem cells.Methods1. Isolation of mouse embryonic fibroblasts and the preparation of feeder cells for iPSC culture.2. iPSC cultivation (Thawing, passaging and cryopreservation).3. Identification of sternness factors Oct-4by Immunofluorescence (IF) staining.4. Culture embryoid bodies which produced by Hanging Drop method in iPS differentiaton medium with or without the existence of CT-1.5. Detect the expressions of cardiac specific markers of EBs at different time points using real-time PCR analysis.6. Using IF staining and Flow Cytometry (FCM) to examine the expression of cardiac specific cell marker cTnI.7. Observe the cellular ultra-microstructure of iPS-derived cardiomyocytes (iPS-CMs) using Transmission Electron Microscope (TEM).Results1. The iPS clone was in compactly oval or round shape with strong three-dimensional sense and distinct boundaries, and was Oct-4positive in IF staining.2. Compared with control group, the gene expressions of various cardiac specific cell markers in CT-1group significantly increased at different time points: Cardiac mesodermal marker, flk-1, increased (1.61±0.03) folds at day7and (2.01±0.01) folds at day10, respectively (P<0.05). Cardiac progenitor cell marker, Nkx2, locus5increased (2.08±0.08) folds at day7,(2.12±0.15) folds at day10and (1.99±0.06)folds at day14, respectively (P<0.01). Tbx5, a T-box transcriptional factor that expresses at the early stage of cardiac development, increased (1.61±0.05) folds at day7and (1.86±0.04) at day10, respectively (P<0.05). Finally, the cardiac specific cell marker, cTnT increased (1.75±0.04) folds and (1.78±0.05) folds at day14, respectively (P<0.05). 3. Immunofluorescence staining showed cTnⅠ positive cells in both groups and flow cytometry analysis showed that the rates of cTnⅠ positive cells were28.5%and56.4%, respectively (P<0.05).4. Compared with those in control group, more organized myofibril, mitochondria and cell junction formation were observed in CT-1group.ConclusionCT-1significantly promotes the cardiac differentiation of miPS cells and iPS-CM maturity by up-regulating the expressions of cardiac mesodermal marker flk-1and cardiac progenitor markers Nkx2.5and Tbx5. These Findings provide a potent evidence for iPSC-based cardiac cell therapy for myocardial infarction or congestive heart failure.