Optimization Of Human Cardiac Reprogramming Factors Using Engineered Human Fibroblasts
Abstract: Many investigators have demonstrated differentiation of cardiomyocytes through human- and mouse- derived induced pluripotent stem cells (iPSCs) using specific cardiac reprogramming factors. We have discovered a non-coding RNA termed Cardiac Inducing RNA (CIR), that can induce differentiation of human iPSCs into cardiac muscle – like cells. Using iPSC – derived cardiomyocytes has several drawbacks: teratoma formation, cost ineffectiveness, and not suitable for patient specific generated cardiomyocytes. In contrast, reprogramming patient-derived fibroblasts may provide a specific and robust approach to the aforementioned iPSC drawbacks. A cocktail of three transcription factors, i.e., Gata4, Mef2c, Tbx5 (GMT) has been demonstrated to be effective in reprogramming mouse embryonic fibroblasts into cardiomyocytes. However, in the human counterparts these factors, CIR and GMT, were found insufficient and showed little differentiation capabilities indicating human cardiomyocyte differentiation requires additional reprogramming factors. In our current studies, we have generated engineered fibroblast cell lines in vitro that contain the reporter protein – enhanced green fluorescent protein (EGFP), under the control of cardiac specific protein promoters which produce α-myosin heavy chain (αMHC) and cardiac troponin T (cTnT). By retrovirally integrating constitutively expressed GMT, we can supplement our engineered cells with CIR. Using these identified factors and our unique engineered cell lines, we can determine if CIR reprograms and transforms fibroblasts into cardiomyocyte – like cells. To analyze the cells we will use: western blots to determine the proteins expressed and immunofluorescent microscopy to stain and image for contractile proteins found within the cells. By determining how effectively CIR and GMT can reprogram fibroblasts into contracting heart muscle cells, we can subsequently use this method as a novel treatment approach on damaged heart tissue to recover normal heart function vastly improving the quality of life for many patients.
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