PhD Scholar Indian Institute of Technology Guwahati, Assam India Guwahati, Assam, India
Abstract: The ever-increasing burden of cardiovascular disorders in today's world necessitates the need to find a suitable regenerative therapy for the generation of functional cardiomyocytes (CMs). Existing methods for the same involve the use of viral vectors, which eventually integrate within the genome of resulting myocytes, rendering them unfit for cell therapy. The quest for a minimal yet efficient cardiac reprogramming cocktail has thus motivated us to investigate the efficacy of using transcription factors (TFs) in the form of recombinant proteins to generate integration-free functional CMs by reprogramming human fibroblasts. The domain of cardiac reprogramming using recombinant proteins offers multiple advantages over the use of integrative methods and is yet to be fully explored to unravel its immense potential. To achieve this, we have generated a recombinant protein toolbox consisting of a unique combination of six cardiac-specific TFs, which can potentially replace their viral counterparts in the direct cardiac reprogramming paradigm. Firstly, the TFs have been expressed and purified under native conditions from a bacterial system with maximum purity. We have then validated the transduction ability and the functionality of the purified proteins in mammalian cells using various assays. Following this, each purified cardiac-specific recombinant protein was applied to fibroblasts, substituting its respective lentiviral vector in the reprogramming factor combination. This substitution successfully induced the upregulation of cardiac-specific markers, facilitating the generation of CMs from fibroblasts. Simultaneously, induced pluripotent stem cells (iPSCs) were differentiated into beating CMs, further enriched via lactate enrichment to give a homogenous population of cells. Overall, this unique toolbox of recombinant fusion proteins will thus serve as a safe alternative for the prospective derivation of integration-free functional CMs, thereby bringing cell therapy closer to clinic. Further, the knowledge gained about the crucial TFs and signalling pathways would broaden our horizon for a better understanding of the cardiovascular milieu in general.
