Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation

Abstract

(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.