Extended Data Fig. 2: HFOs recapitulate patterns of early cardiomyogenesis.

a, Paraffin sections from different parts of the HFO stained for cardiac troponin T (cTnT) showing that the cTnT-positive myocardial layer encloses the cTnT-negative inner core on its back side. b, Proportion of NKX2.5-eGFP/ ISL1-double-positive (second heart field-like) cells among all NKX2.5-eGFP-positive cells in HFOs from d7 – d13 of differentiation. n = 3 (d7, d13) or 4 (d10) derived from 3 independent experiments; data are presented as mean ± SEM. (c–j) Patch clamp analysis of HFO-derived NKX2.5-eGFP-positive cardiomyocytes in whole-cell current clamp mode. c–e, Representative traces for three different action potential (AP) phenotypes. Left panels show spontaneous electrical activity of the cells; right panels depict subsequently evoked APs of the same cell. Here, the cell membrane was hyperpolarized by application of negative holding currents (from −2 to −50 pA) to achieve comparable conditions for each cell mimicking physiological resting membrane potentials of around −80 mV. Short current pulses (1 ms, 400 – 1500 pA) were applied to evoke APs. The classification of the phenotypes was based on the AP duration of these traces: Cells were classified as ventricular-like when they showed APs with a plateau phase longer than 200 ms as measured at 50% repolarization level (APD₅₀) (c). Cells were classified as atrial-like when they displayed a triangular shape with APD₅₀ values of 20 – 200 ms (d). Cells showing APs without overshoot were termed atypical (e). f, Distribution of cardiac subtypes in HFOs based on the classification in c–e. (g–j) Additional AP parameters for ventricular-like cells isolated from HFOs (g: MDP/RMP, maximal diastolic potential/ resting membrane potential; h: APD₅₀; i: AP amplitude; j: upstroke velocity). Spontaneous AP: n = 36 cells, evoked AP: n = 35 cells isolated from 3 HFOs from one experiment; data are presented as mean ± SEM. Scale bars: a: 200 µm.