Follistatin From hiPSC-Cardiomyocytes Promotes Myocyte Proliferation in Pigs With Postinfarction LV Remodeling

BACKGROUND: When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes ( CCND2-OE hiPSC-CMs) and administered to the infarcted hearts of immunodeficient mice, the cells proliferated after administration and repopulated >50% of the scar. Here, we knocked out human leukocyte antigen class I and class II expression in CCND2-OE hiPSC-CMs ( KO/OE hiPSC-CMs) to reduce the cells’ immunogenicity and then assessed the therapeutic efficacy of KO/OE hiPSC-CMs for the treatment of myocardial infarction. METHODS: KO/OE hiPSC-CM and wild-type hiPSC-CM ( WT hiPSC-CM) spheroids were differentiated in shaking flasks, purified, characterized, and intramyocardially injected into pigs after ischemia/reperfusion injury; control animals were injected with basal medium. Cardiac function was evaluated via cardiac magnetic resonance imaging, and cardiomyocyte proliferation was assessed via immunostaining and single-nucleus RNA sequencing. RESULTS: Measurements of cardiac function and scar size were significantly better in pigs treated with KO/OE hiPSC-CM spheroids than in animals treated with medium or WT hiPSC-CM spheroids. KO/OE hiPSC-CMs were detected for just 1 week after administration, but assessments of cell cycle activity and proliferation were significantly higher in the endogenous pig cardiomyocytes of the hearts from the KO/OE hiPSC-CM spheroid group than in those from the other 2 groups. Single-nucleus RNA-sequencing analysis identified a cluster of proliferating cardiomyocytes that was significantly more prevalent in the KO/OE hiPSC-CM spheroid–treated hearts (3.65%) than in the hearts from the medium (0.89%) or WT hiPSC-CM spheroid (1.33%) groups at week 1. YAP (Yes-associated protein) protein levels and nuclear localization were also significantly upregulated in pig cardiomyocytes after treatment with KO/OE hiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from KO/OE hiPSC-CM spheroids than WT hiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, P =0.0056). Treatment with follistatin increased WT hiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice. CONCLUSIONS: KO/OE hiPSC-CM spheroids significantly improved cardiac function and reduced infarct size in pig hearts after ischemia/reperfusion injury by secreting follistatin, which upregulated HIPPO/YAP signaling and proliferation in endogenous pig cardiomyocytes.