Study of direct reprogramming of human fibroblasts into cardiomyocytes in a mechanically stimulated-3D microenvironment

Myocardial infarction is the leading cause of death worldwide and it causes cardiomyocyte loss, formation of a non-functional fibrotic scar and, eventually, a pathological remodelling of the heart wall and deterioration of cardiac functions. Traditional therapeutic strategies are unable to restore myocardium functionality and heart transplant, the only procedure that guarantees a full cardiac recovery, is affected by several problems, such as the need for lifelong immunosuppression and shortage of organ donors. Innovative research, based on regenerative medicine’s approach, aims at obtaining mature cardiomyocytes in vitro by inducing stem cell differentiation or somatic cell reprogramming, including the direct reprogramming of cardiac fibroblasts into cardiomyocytes. Direct reprogramming can be achieved by different strategies including the forced expression of transcription factors and/or microRNAs, and/or the use of chemical agents. However, cardiomyocyte-like cells are obtained at low efficiency and are generally immature. This Master thesis was aimed at investigating whether a 3D culture system integrated with mechanical stimulation is able to enhance miRNA-mediated direct reprogramming efficiency and maturation of human cardiac fibroblasts into induced cardiomyocytes. To this purpose cells transfected with reprogramming miRNAs through novel lipoplexes were culture in 3D hydrogels in static and mechanically stimulated conditions. Culture conditions enhancing the expression of cardiac markers and decreasing the expression of fibroblast markers were found and critically discussed, outlining the next steps in the field. The work has been carried out within the BIORECAR ERC project (grant agreement 772168).