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Cardiomyocyte progenitors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodelling after experimental myocardial infarction

ABSTRACT: Both naturally occurring and artificially engineered multipotent cardiac progenitor cells (CPCs) are associated with highly variable cardiomyocyte (CM) differentiation efficiencies, resulting in unpredictable and/or low CM yields. Therefore, CM-committed progenitors may be a more reliable cell product for therapeutic use. Here, we demonstrate that unipotent progenitors are part of the phenotype spectrum arising during direct reprogramming of neonatal cardiac fibroblasts towards CMs using lentiviral transduction of Gata4, Mef2c, Tbx5, and Myocd. Induced CM progenitors (iCMPs) simultaneously express CPC- and CM-specific proteins and can be selected via hairpin oligonucleotide molecular beacons targeting Myh6/7-mRNA–expressing cells. Purified iCMPs can be expanded extensively with preserved phenotype using ascorbic acid and give rise to CM-like cells in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevent CM loss, attenuate fibrotic scarring, and preserve ventricular function. Given the clinical failure of conventional cardiac cell therapy using adult stem cells, further translational development of this iCMP approach is warranted.

ORGANISM(S): Mus musculus

PROVIDER: GSE159315 | GEO | 2023/06/20

REPOSITORIES: GEO

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Project description:Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine–based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.

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Cardiomyocyte progenitors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodelling after experimental myocardial infarction

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