Fig 2: In Vitro Differentiation Potential of ISL1+ HVPs(A–D) Day 15+ HVPs were analyzed for ventricular markers: (A) MLC2v and cTnT; (B) cTnI; (C) pacemaker marker HCN4; and (D) atrial marker MLC2a. Scale bar, 20 µm. (E and F) Optical recordings of spontaneous (E) and paced (F) day 18 HVP cells. Isochrome maps at 20-ms intervals show spontaneous action potential initiation. The white asterisk represents the origin of point electrical stimulation. (G and H) Optical recordings of spontaneous (G) and paced (H) calcium transients in day 18 HVP cells. The white asterisk represents the origin of point electrical stimulation. (I and J) Patch clamp recordings of day 15+ (I) and day 39+ (J) HVPs showed typical ventricular-like action potentials. (K) Whole cell patch clamp recordings of day 21 and day 40 HVP-derived ventricular cardiomyocytes. Cultures demonstrated that there were no significant differences in various AP parameters between the two time points, suggesting that in vitro, HVPs reached maturity by day 18. (L) Representative If traces in the presence of 1 mM Ba2+. (M) Patch clamp recording comparison of If in ES03- and HES2-derived ventricular cardiomyocytes. Steady-state I-V (left) and steady-state activation relations (right) for If (n = 12). (N and O) Whole cell patch clamp recording of INa in ES03. (N) Representative INa traces during activation (left) and inactivation (right). (O) Plots of peak INa density I-V relationship (left) and steady-state activation/inactivation (right), n = 5. Histograms and traces are shown as mean ± SEM.

Fig 3: In Vivo Transplantation of HVPs into Healthy and Injured Hearts(A) White dashed box denotes the location of the HVP graft 8 months following intramyocardial injection into a healthy heart. (B) Immunofluorescence staining showed that HVP ventricular grafts in the heart expressed NKX2.5-GFP (green), MLC2v, connexin 43, and MHY11; a minority of cells co-expressed MLC2a and was negative for HCN4 and ISL1. CD31 was only present outside the graft. (C) The white dashed box identifies the localization and the engraftment of the xeno tissue 2 months after injection of the HVPs into the border zone of MI hearts. Red asterisks denote infarction site. (D) Immunofluorescence staining showed that HVP ventricular grafts in post-MI hearts co-expressed NKX2.5-GFP (green), connexin 43, human nuclei, MLC2v, MHY11, human fibronectin, and laminin. A minority of cells was positive for ki67; MLC2a, ISL1, and HCN4 staining was not observed. Endothelial marker CD31 was detected in close proximity to the HVP graft. (E) High-resolution imaging of the border zone of post-MI HVP grafts demonstrated engrafted, differentiated cardiomyocytes of human origin, with well-organized sarcomeres (stains of cell membrane [WGA, green], human mitochondria [red], and F-actin [orange]). (F) MRI assessment of post-MI hearts 2 months following HVP transplantation showed improved global and local function in comparison with placebo. Representative short axis images show wall thickness at the site of induced infarction (arrows). LV, left ventricle; RV, right ventricle. (G) Mean measurements ± SD of wall thickening, ejection fraction, and volume during end diastole (EDV) and end systole (ESV). *p < 0.05; **p < 0.01; n/s, not significant.