Abstract Body (Do not enter title and authors here): Background: Our previous study showed that cardiomyocytes (CMs) differentiated from hiPSCs with cyclin-D2 overexpression (^CCND2-OEhiPSC-CMs) induced the proliferation to repopulate > 50% of the scar in immunodeficient mouse hearts. To reduce the immunogenicity of ^CCND2-OEhiPSCs, we knocked out the HLA classes -I and -II (^KO/OEhiPSCs) and assessed the therapeutic efficacy and mechanism of ^KO/OEhiPSCs-derived CMs (^KO/OEhiPSC-CMs) for myocardial infarction (MI) treatment.
Methods: ^KO/OEhiPSC-CM or wild-type hiPSC-CM (^WThiPSC-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/OEhiPSC-CM spheroids secreted abundant follistatin compared to ^WThiPSC-CM spheroids (30.3±4.13 vs 16.6±1.43 ng/mL, p=0.0056). Follistatin, which interacts with HIPPO/YAP pathway, stimulated ^WThiPSC-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/OEhiPSC-CM spheroid treated pig hearts compared to medium or ^WThiPSC-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/OEhiPSC-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/OEhiPSC-CM spheroid (3.65%) compared to the medium (0.89%) or ^WThiPSC-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/OEhiPSC-CM spheroids. Furthermore, YAP protein levels and nuclear localization in CMs were higher in the ^KO/OEhiPSC-CM spheroid treated pig hearts compared to controls. Thus, follistatin secreted by implanted ^KO/OEhiPSC-CM spheroids appear to target the HIPPO/YAP pathway to promote pig CM proliferation.
Conclusions: ^KO/OEhiPSC-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.