Abstract

Background: Our previous study showed that cardiomyocytes (CMs) differentiated from hiPSCs with cyclin-D2 overexpression ( CCND2-OE hiPSC-CMs) induced the proliferation to repopulate > 50% of the scar in immunodeficient mouse hearts. To reduce the immunogenicity of CCND2-OE hiPSCs, we knocked out the HLA classes -I and -II ( KO/OE hiPSCs) and assessed the therapeutic efficacy and mechanism of KO/OE hiPSCs-derived CMs ( KO/OE hiPSC-CMs) for myocardial infarction (MI) treatment. Methods: KO/OE hiPSC-CM or wild-type hiPSC-CM ( WT hiPSC-CM) spheroids were differentiated in shaking flasks, purified, characterized, and injected into pig hearts post ischemia/reperfusion (I/R), while controls received medium injection only. Cardiac function was evaluated using cardiac magnetic resonance imaging (cMRI). CM proliferation was assessed through immunostaining and single-nucleus RNA sequencing (snRNAseq). Results: KO/OE hiPSC-CM spheroids secreted abundant follistatin compared to WT hiPSC-CM spheroids (30.3±4.13 vs 16.6±1.43 ng/mL, p=0.0056). Follistatin, which interacts with HIPPO/YAP pathway, stimulated WT hiPSC-CM proliferation and increased cell number by 28.3% over 16 days in vitro and promoted adult mice CM proliferation after MI in vivo . cMRI assessments indicated better cardiac function and scar sizes in KO/OE hiPSC-CM spheroid treated pig hearts compared to medium or WT hiPSC-CM spheroid treated pigs. Although the injected cells were only identified at 1 week, cell-cycle activity was significantly higher in the CMs of KO/OE hiPSC-CM spheroid treated pig hearts compared to the other two groups. A cluster of cycling CMs, enriched for five genes associated with cell proliferation according to snRNAseq data, was more prevalent in the KO/OE hiPSC-CM spheroid (3.65%) compared to the medium (0.89%) or WT hiPSC-CM spheroid treated (1.33%) hearts at 1 week. Pathways linked to cardiac regeneration—MAPK, HIPPO/YAP, and TGFB—were significantly upregulated in pig CMs treated with KO/OE hiPSC-CM spheroids. Furthermore, YAP protein levels and nuclear localization in CMs were higher in the KO/OE hiPSC-CM spheroid treated pig hearts compared to controls. Thus, follistatin secreted by implanted KO/OE hiPSC-CM spheroids appear to target the HIPPO/YAP pathway to promote pig CM proliferation. Conclusions: KO/OE hiPSC-CM spheroids significantly improved cardiac function and reduced infarct size in pig hearts after I/R by secreting follistatin which promoted pig CM proliferation through YAP signaling.