Induced Pluripotent Stem Cell-derived Cardiovascular Progenitor Cell Engraftment Can Improve Heart Function After Myocardial Ischemia In Mice

PartⅠReprogramming of Induced Pluripotent Stem Cells from Adult Human Adipose Stem Cells under Feeder-free ConditionObjective:Using human adipose stem cells (hASCs) as the starting population for reprogramming induced pluripotent cells (iPSCs) under feeder-free condition.Methods:hASCs were isolated from human adipose tissue and the resulting cells were reprogrammed using lentivirus transfection encoding for four transcription factors. Afterwards, the efficiency of reprogramming was compared between iPSCs generated with and without feeder cells by monitoring cell morphology through microscope. In addition, cells were seeded into 24 wells culture plates at passage 2-3, then cell viability and proliferation at different time points were compared using XTT. The pluripotency of these cells were identified by AP staining and immunostaining, as well as cell differentiation in vivo. Moreover, the genomics of these iPSCs was analyzed by microarray.Results:iPSCs were successfully reprogrammed without feeder cell by use of lentivirus encoding for 4 transcription factors. Despite no significant difference in cell viability and proliferation capacity between iPSCs with or without feed cells, feeder free iPSCs displayed higher efficiency regarding reprogramming. In addition, the resulting iPSCs can give rise to all three germ layers in vivo. Besides that, microarray showed gene expression profile of these cells is similar with human embryonic stem cells (hESCs).Conclusion:Human ASCs can be reprogrammed into iPSCs without feeder cells in relative short time, which may replace fibroblast and offer a new cells source for iPSC reprogramming.PartⅡDifferentiation and reporter gene transfection of hASCs derived iPSCsObjective:To observe the differentiation capacity of iPSCs and verify the effects of two reporter genes transfection on these ASC derived iPSCs.Methods:Two reporter genes were transfected by lentivirus. Experimental groups were divided into group A168H, GFP-Fluc and A168H-GFP-Fluc, blank iPSCs were served as control. Cell morphology was observed by microscope; cell viability and proliferation were evaluated by XTT; cell phenotype was assessed by immunostaining. Cardiac differentiation was induced and identified by immunostaining using cardiac specific antibodies. The correlation between the number of HSV1-A168Htk+/GFP-Fluc+ cells and signals were assessed by BLI in vitro.Results:There was no significant difference with respect to cell morphology between different groups, as well as cell viability and proliferation ability. In addition, the differentiation capacity was similar among different groups. Immunostaining displayed that endoderm was marked as AFP and Sox 17, mesoderm was labeled with a-SMA and ectoderm was recognized with Tuj-1. Cardiac differentiation displayed that these cell can be induced into beating cardiomyocytes. The successful induction was confirmed by immunostaining against a-actinin, c-TnT, NKX2.5 and MEF2c. Moreover, in vitro BLI showed that the intensity of signals were correlated with the number of HSV1-A168Htk+/GFP-Fluc+ cells.Conclusion:iPSCs can give rise to three germ layers in vitro without being affected by reporter genes transfection. In addition, there is a strong correlation between signals and cell number which may use for tracking cell survival in vivo.Part III Induced Pluripotent Stem Cell-derived Cardiovascular Progenitor Cell Engraftment Can Improve Heart Function after Myocardial Infarction in Mice Objective To observe the effect of induced pluripotent stem cell-derived cardiovascular progenitor cells (CPCs) on heart functionalimprovement inmouse myocardial infarction model, and explore the underlying mechanisms.Methods:HSV1-A168Htk expressing human iPSCs weredirected differentiated into CPC. Ex vitro uptake of radiotracer I8F-FHBG was tested by y-counter and PET imaging. Female NOD SCID mice were randomly separated into high cell engraftment group (n=20), low cell engraftment (n=20), PBS group (n=20), hypoxia group (n=10) and control group. CPCs were then transplanted into murine infarction models (1×106 cells/mouse for high cell engraftment group and 0.5×106 cells/mouse for low cell engraftment group). Noninvasive bioluminence imaging (BLI) and PET imaging were used to track hiPS-CPC fate over a four week time period. Echocardiographic, cardiac MRI, and PV loop were used to analyze the heart function after cell engraftment. The mice were sacrificed on day 28, H&E staining, immunostaining and TUNEL staining were tested. Finally, q-PCR and laser capture microdissction (LCM) were used to compare the expression of angiogenic genes in CPCs under normoxia versus hypoxic condition, respectively.Results:Ex vivo y-counter and PET imaging shows the signal of engraft cells goes up in a line with time increasing, and reaches the peak value after 120 minutes of uptaken. In vivo BLI and PET imaging on different time points demonstrated a substantial decline in surviving cells after cell engraftment. Echocardiographic and cardiac MRI analyses revealed significant improvement of left ventricular contractile function (LVFS and LVEF) in high cell engraftment groups compared with low cell engraftment group and PBS group after transplant(P=0.0354 and P=0.0356 on day 28, respectively). PV loop shows significant increase of left ventricular end-systolic volume (ESV, P=0.0041), end-diastolic volume (EDV, P=0.0093) and end-systolic pressure (ESP, P=0.0016) as well as echocardiographic and cardiac MRI analyses. CPC engraftment was assessed using PET imaging data acquired on day 1, and found to correlate linearly with myocardial functional improvement four weeks after cell transplant (R2=0.72), implying a strong "dose-effect" relationship. H&E staining showed thicker heart wall size for the high cell engraftment group compared to low cell engraftment and control groups. Immunohistochemistry of the peri-infarct region by GFP staining also showed the highest cell survival in the high cell engraftment group compared to the other two treated groups. Host myocardial tissue in hearts from the high engraftment group also contained significantly fewer apoptotic cells than hearts in the low engraftment group as assessed by TUNEL staining of the peri-infarct area. hiPS-CPCs exposed to hypoxic conditions demonstrated substantial upregulation of all tested genes related to angiogenesis both ex vivo and in vivo.Conclusion:iPSCs derived CPC can significantly improve the heart function after myocardial infarction. PET reporter gene imaging provides important diagnostic and prognostic information regarding the ultimate success of cell treatment.