Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction

Abstract

Background: Human-induced pluripotent stem cells (hiPSCs) with normal or upregulated levels of CCND2 expression were differentiated into cardiomyocytes (CCND2 WT CMs or CCND2 OE CMs, respectively) and injected into infarcted pig hearts. Methods: Acute myocardial infarction (AMI) was induced via a 60-minute occlusion of the left-anterior descending coronary artery. Immediately after reperfusion, CCND2 WT CMs or CCND2 OE CMs (3×10 7 cells each), or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. Results: The number of the engrafted CCND2 OE CMs exceeded that of the engrafted CCND2 WT CMs from 6 to 8-fold, rising from 1 week to 4 weeks post-implantation. In contrast to the treatment with the CCND2 WT CMs or the delivery vehicle, the administration of CCND2 OE CM was associated with significantly improved left-ventricular function, as revealed by magnetic resonance imaging (MRI). This correlated with the reduction of infarct size, fibrosis, ventricular hypertrophy, CM apoptosis, and the increase of vascular density and arterial density, as per the histological analysis of the treated hearts. Expression of the cell proliferation markers (e.g., Ki67, phosphorylated histone 3 [PH3] and Aurora Kinase B [Aurora B]) was also significantly upregulated in the recipient CMs from the CCND2 OE CM-treated than from the CCND2 WT CM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured hiPSC-CMs were significantly greater after their treatment with exosomes isolated from the CCND2 OE CMs (CCND2 OE Exos) than from the CCND2 WT CMs (CCND2 WT Exos). As demonstrated by our study, CCND2 OE Exos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2 OE Exos vs. CCND2 WT Exos identified 206 and 91 miRNAs that were significantly upand down-regulated, respectively. Gene ontology (GO) enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. Conclusions: We have demonstrated that an enhanced potency of the CCND2 OE CMs promoted myocyte proliferation in both grafts and the recipient tissue in a large mammal acute myocardial infarction (AMI) model. These results suggest that the CCND2 OE CMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Gut Bacterial Composition and Functional Potential of Tibetan Pigs Under Semi-Grazing

“…Compared with rodents, swine was considered as a more ideal model for studying human diseases due to its narrow disparities in organ characteristics compared to those of humans ( Gehrig et al, 2019 ). Porcine models were especially adopted for studies of lung cystic fibrosis in humans ( Lu and Kolls, 2021 ) and were also used in the field of pathogenesis of neurodegenerative ( Yan et al, 2018 ) and cardiovascular ( Hinkel et al, 2015 ; Fan et al, 2020 ; Zhao et al, 2021 ) diseases. Furthermore, pigs’ omnivorous eating behavior contributed to its role in the study of gut microbiota.…”

Extracellular vesicles fail to trigger the generation of new cardiomyocytes in chronically infarcted hearts

“…We acknowledge that our data are at variance with those reported in other studies. However, these discrepancies could be explained by important differences in the design of the experiments, including (1) the usual delivery of EV at the time of injury 27 - 29 (and not at the more chronic stage, like in our protocol) with expected differences in local cardio-instructive cues, (2) the use of cells of a different origin (the epicardium) 29 , cultured under hypoxic conditions 30 or genetically engineered 27 , and (3) the use of outcome measures failing to provide direct evidence for cytokinesis 30 . Of note, in the study of Balbi et al 28 where exosomes secreted by CPC from human atrial explants induced cardiomyocyte proliferation by a periostin-dependent mechanism, the exosome-induced improvement in left ventricular function was unchanged after periostin inhibition, thereby suggesting a minimal, if any, contribution of the exosome-induced cardiomyocyte cell cycling in the preservation of cardiac function.…”

Cardiac Cell Therapy with Pluripotent Stem Cell-Derived Cardiomyocytes: What Has Been Done and What Remains to Do?

“…Harnessing this proliferative capacity by transcriptional manipulation may accentuate the potential of PSC-CM therapy to repopulate damaged myocardium. Indeed, it was recently shown that overexpression of cyclin D2, a protein involved in cell cycle regulation, led to significantly increased graft sizes and subsequent improvements in left ventricular function in both small and large animal models [51,52].…”