Figure 3. Structural and functional characterization of hPSC-CMs cultured in 2D-SG, 3D-SG and 3D-MG conditions.

(A) Structural analysis of hPSC-CMs. Cells were dissociated, replated and stained for sarcomeric α-actinin (green) and DAPI (blue). Overall appearance of myofibrillar structure was categorized into 3 different levels: Score 1 cells are α-actinin^pos but without clear sarcomeric striations; Score 2 cells have diffuse punctate staining pattern and some patterned striations in partial cell area; and Score 3 cells have highly organized and well-defined myofibrillar structure with distinct paralleled bands of z-discs distributed throughout the cell area. Percentage of the cells by the scores was generated by counting 102 and 136 cells for 3D-SG and 3D-MG, respectively. Note that 3D-MG produced CMs with higher levels structural maturation. Scale bar = 25 μm. (B) Calcium transients of hPSC-CMs cultured in 3D-SG and 3D-MG conditions. Representative traces of hPSC-CMs field-stimulated at 1 Hz were acquired by optical fluorescence imaging. Measurements of calcium transients are presented as mean ± SD of n = 37 and 40 line scans for 3D-SG and 3D-MG culture conditions, respectively. **P < 0.01; ****P < 0.0001. (C) Representative patch clamp recording and summary of action potential parameters. Electrophysiological parameters measured: N, cell number; dV/dtmax, maximum action potential upstroke velocity; MDP, maximum diastolic potential; APA, action potential amplitude; APD50, action potential duration at 50% of repolarization; APD90, action potential duration at 90% of repolarization. Data are presented as mean ± SD.