Figure 1
Differentiation of hiPSC-CM, formation of cardiac microtissues, and definitions of metrics. (A) Timeline shows cardiomyocyte differentiation from human-induced pluripotent stem cells (hiPSCs) in high density 2D culture. Cardiac directed differentiation was achieved with Wnt activation at day 1 and inhibition at day 3 (see “Methods”). Cardiac phenotype, visually confirmed by beating cells, appeared between days 8 and 12. Cardiomyocytes differentiated from hiPSCs were used for the production of microtissues or were further purified with a lactate-based metabolic selection protocol. Microtissues self-assembled in microwells after 14–28 days of differentiation of hiPSC-CMs (i.e., without or with lactate purification) and addition of human cardiac fibroblasts (hCFs), and they were cultured in the presence of 1 Hz electrical field stimulation (estim). (B) Schematic of three-dimensional (3D) cardiac microtissue generation shows non-adhesive agarose gels with cylindrical recesses with hemispherical bottoms that guide self-assembly. Cardiac microtissues were cultured for 6–8 days with 1 Hz pacing. (C) Phase contrast image shows consistent spherical microtissue formation after 5 days of 3D culture in all 35 microwells. Scale bar, 800 μm. (D) Confocal image shows a representative cardiac tissue with hiPSC-CM (green) and hCF (red) stained with CellTracker dyes. Scale bar, 200 μm. (E) Confocal image shows a representative cardiac troponin I (red), vimentin (green), and DAPI stained cryosection (10 μm thick) of a microtissue fixed after 7 days in 3D culture. Scale bar, 50 μm. (F) Fluorescence image of microtissues at 3.2 × magnification was obtained during optical mapping. Typically, the action potentials (APs) from 4–9 microtissues were recorded simultaneously. (G-I) Schematics of the AP metrics of that were defined (with units) as: (G) “excitability” (%) measured from the percentage of captured APs during 10 s duration of recording with 2 s pacing cycle length, (H) “stimulation time delay” (ms; stim delay) between stimulation pulse and evoked AP upstroke (dF/dtmax), “rise time” (ms) of AP, “AP duration” (ms) to 30%, 50%, and 80% repolarization (APD30, APD50, APD80), “APD to the maximum repolarization rate” (ms; APDMxR) defined as time between AP upstroke and the end of rapid repolarization marked by d2F/dt2max, “APD triangulation” (ms; APDtri) defined as APDMxR—APD50, and (I) occurrence of “early afterdepolarization” (EAD) reported as (%) of microtissues showing EADs.
