Di Benedetto, Giorgia (2024) Identification and characterization of new molecules that act as boosters for direct cardiac reprogramming, via the induction of cardiovascular precursors. [Tesi di dottorato]

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Abstract

The development of therapeutic approaches based on Direct Cardiac Reprogramming (DCR) of fibroblasts into induced-Cardiomyocytes (iCMs) is emerging as an attractive strategy to repair the injured myocardium. Chemical-induced Direct Cardiac Reprogramming (CiDCR) stands out among strategies for its potential in non-genetic transdifferentiation, offering a safe avenue by avoiding alterations to the genetic set-up of the target cell. Despite recent progress in DCR, further improvement in reprogramming efficiency and a deeper understanding of underlying mechanisms are required. This study contributes to the advancement of CiDCR by addressing heterogeneity, identifying novel reprogramming factors, elucidating epigenetic barriers and employing innovative tools for cell identity tracing. Culture conditions optimization became imperative due to a significant degree of phenotypic heterogeneity observed among the reprogramming cells. Molecular characterization of our reprogramming process revealed that fibroblasts may undergo a CardioVascular Precursor (CVP) phase before attaining a full cardiomyocytic phenotype. Given the intrinsic ability of CVPs to grow in suspension and form cardiospheres, we developed growth conditions which allow to isolate homogeneous cell populations undergoing reprogramming and enrich mature iCMs population. The inhibition of pro-inflammatory pathways and promotion of antioxidant activity have recently been correlated with an increase in reprogramming efficiency. Based on this evidence, we have started collaborating with Arterra Bioscience to evaluate the eff ects on CiDCR efficiency of 11 Mixtures with anti-inflammatory or anti-oxidant activity, derived from extracts of plant cell cultures, agricultural production waste, plants and fruits and microalgae. In the meantime, we started investigating the role of Plant Homeodomain Finger 10 (PHF10 / BAF45a) in DCR, which is part of the PBAF complex belonging to the family of epigenetic regulators SWI / SNF, that often works in opposition to Polycomb. Downregulation of PHF10 expression during DCR perturbs the efficacy of the process, as demonstrated by the reduction of both multipotent induced-Cardiovascular Precursor Cells (iCPCs) and terminally differentiated iCMs generation at different stages of reprogramming. Finally, we integrated the logical design of synthetic cis-regulatory DNA (LSD) within our system as a promising tool for genetically tracing cell identities and monitoring state changes. In conclusion, these findings significantly contribute valuable insights towards refining and advancing CiDCR by mitigating the heterogeneity inherent in the protocol, identifying new potential reprogramming factors and gaining a deeper understanding of reprogramming mechanisms.

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