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

Abstract

Background: When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes (^CCND2-OEhiPSC-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-OEhiPSC-CMs (^KO/OEhiPSC-CMs) to reduce the cells' immunogenicity and then assessed the therapeutic efficacy of ^KO/OEhiPSC-CMs for the treatment of myocardial infarction.

Methods: ^KO/OEhiPSC-CM and wild-type hiPSC-CM (^WThiPSC-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/OEhiPSC-CM spheroids than in animals treated with medium or ^WThiPSC-CM spheroids. ^KO/OEhiPSC-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/OEhiPSC-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/OEhiPSC-CM spheroid-treated hearts (3.65%) than in the hearts from the medium (0.89%) or ^WThiPSC-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/OEhiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from ^KO/OEhiPSC-CM spheroids than ^WThiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, P=0.0056). Treatment with follistatin increased ^WThiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice.

Conclusions: ^KO/OEhiPSC-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.

Keywords: cardiomyocytes; cyclins; follistatin; induced pluripotent stem cells; myocardial infarction.