Generation of ANP- and BNP-deficient human iPSC-derived cardiomyocytes reveals natriuretic peptide-dependent angiogenic paracrine effects.

BACKGROUND

Cell therapy using pluripotent stem cell-derived cardiomyocytes (PSC-CMs) has emerged as a promising strategy for cardiac regeneration. Although transplanted PSC-CMs can contribute to remuscularization, accumulating evidence suggests that paracrine mechanisms also play an important role in functional recovery. Among potential paracrine factors, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are highly expressed in PSC-CMs; however, whether PSC-CM-derived ANP and BNP contribute to cardiac repair after transplantation remains to be determined.

METHODS

In this study, we generated human induced pluripotent stem cell (hiPSC) lines deficient in ANP (NPPA knockout), BNP (NPPB knockout), or both peptides using CRISPR-Cas9 genome editing. These iPSCs were differentiated into cardiomyocytes, and their characteristics and paracrine effects were then examined in vitro using endothelial cell assays and co-culture with cardiac fibroblasts.

RESULTS

ANP- and/or BNP-deficient hiPSC-derived cardiomyocytes (hiPSC-CMs) showed comparable cardiac differentiation efficiency, cell size, proliferative capacity, and susceptibility to apoptosis compared with wild-type hiPSC-CMs. In trans-well migration and Matrigel tube formation assays, conditioned medium from wild-type hiPSC-CMs significantly enhanced endothelial cell migration and tube formation compared with medium from double-knockout hiPSC-CMs lacking both ANP and BNP, suggesting that natriuretic peptides contribute to the pro-angiogenic effects of hiPSC-CMs in vitro. In contrast, co-culture with hiPSC-CMs attenuated TGF-β1-induced myofibroblast gene expression in cardiac fibroblasts regardless of natriuretic peptide deficiency, possibly reflecting minimal expression of the natriuretic peptide receptor NPRA in fibroblasts under in vitro conditions.

CONCLUSIONS

These findings suggest that ANP and BNP are involved in the pro-angiogenic paracrine activity of hiPSC-CMs in vitro. This study provides genetically modified hiPSC-CM models that may serve as a platform for future in vivo transplantation studies to determine whether PSC-CM-derived natriuretic peptides contribute to cardiac repair after transplantation.