Generation of lamin A/C haploinsufficient hiPSC-CMs. (A) Predicted effect of the LMNA R225X mutation on the two splicing products lamin A and C. (B) Sanger sequencing of LMNA exon 4 in hiPSCs with heterozygous R225X mutation (top), or in hiPSCs obtained after CRISPR/Cas9-based scarless correction of the mutation (bottom). (C) Schematic of the protocol for step-wise directed differentiation of hiPSC-CMs. CHIR, CHIR-99021; AA, ascorbic acid. (D) Quantification of cardiac differentiation efficiency by flow cytometry for TNNT2 and NKX2-5 on hiPSC-CMs at day 14 of differentiation. (E) RT-qPCR analyses at the indicated stages of hiPSC-CM differentiation (see panel C). Differences versus mutant were calculated by two-way ANOVA with post hoc Holm–Sidak binary comparisons (*, P < 0.05; ***, P < 0.001; n = 3 differentiations; average ± SEM). (F) Representative Western blot for A- and B-type lamins and differentiation markers during iPSC-CM differentiation. (G) Quantification of lamin A/C expression in hiPSC-CMs from Western blot densitometries. Differences versus mutant were calculated by one-way ANOVA with post hoc Holm–Sidak binary comparisons (**, P < 0.01; ***, P < 0.001; n = 3 differentiations; average ± SEM). Throughout the figure (and in all other figures), Mut or Mutant indicates LMNA R225X hiPSCs, and Corr.1/2 or Corrected 1/2 indicates the two isogenic corrected control LMNA R225R hiPSC lines.