Figure 2.

Characterization of the hiPSC-CM differentiation protocol

(A) Schematic representation of the cardiac differentiation protocol. hiPSCs were differentiated to CMs in a monolayer cardiac differentiation protocol using chemical Wnt signaling mediators (CHIR99021 and IWR-1) and, eventually, further matured into 3D EHT constructs based on fibrinogen and thrombin polymerization.

(B) Representative example of 2D monolayer-based cardiac differentiation (left panel) and 3D mini-EHT construct between two flexible microposts positioned 7 mm from each other (right panel).

(C) Normalized gene expression ratios for isoforms of Myosin Heavy Chain (MYH7/MYH6), Myosin Light Chain (MYL2/MYL7), and Cardiac Troponin I (TNNI3/TNNI1) after 15 and 30 days of differentiation. Data are representative of three independent experiments (n = 3), and values are expressed as mean ± SEM. Significance of the difference is indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

(D) Immunostaining of cardiac troponin T (cTnT) -positive CMs (cTnT, red and Hoechst, blue) in monolayer-based cardiac differentiation (2D) or EHT constructs (3D). White dotted lines indicate the borders of the 3D EHT constructs. Scale bar: 50 μm.

(E) Voltage-current relation curve of the L-type Ca²⁺ current (pA/pF) assessed after whole-cell patch-clamp configuration. Each data point indicated biological replicates (DMD: N = 13, DMD isogenic: N = 7, healthy: N = 11), and values are expressed as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 (DMD versus DMD isogenic control) or ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, and ^$$$$p < 0.0001 (DMD versus healthy control).

(F) Representative AP recordings. DMD hiPSC-CMs displayed arrhythmogenic firing patterns including DADs and OPPs.

(G) Patch-clamp recordings at day 24 of differentiation for mean APD90 (ms). Additional measurements were performed with di-4-ANEPPS (gray dots).

(H) Patch-clamp recordings for AP amplitude (mV), RMP (mV), cell capacitance (pF), and beating frequency (Hz). Each data point indicates biological replicates (DMD: N = 19, DMD isogenic: N = 7, healthy: N = 8). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.