Bcl xl bh4

Six-week-old NOD-SCID mice were purchased from the Jackson laboratory (Boston, MA). Mice were housed in compliance with Boston Children’s Hospital guidelines, and all animal-related protocols were approved by the Institutional Animal Care and Use Committee. h-iECs for implantation were expanded for 7 days in vitro after differentiation unless otherwise specified. h-iECs were pretreated with 20 μM caspase inhibitor/Z-VAD-FMK (APExBio, catalog no. A1902) and 0.5 μM BCL-XL-BH4 (Millipore, catalog no. 197217) in h-iEC medium overnight before implantation. Briefly, h-iECs and h-MSCs (2 × 10⁶ total per mouse, 1:1 ratio) or h-iECs alone (1 × 10⁶ cells per mouse) were resuspended in 200 μl of pH-neutral pregel solution containing bovine collagen I (3 mg/ml; Trevigen, catalog no. 3442-050-01), fibrinogen (3 mg/ml), 50 μl of Matrigel (Corning, catalog no. 354234), FGF-2 (1 μg/ml; PeproTech, catalog no. 100-18B), and erythropoietin (1 μg/ml; ProSpec, catalog no. CYT-201). During anesthesia, mice were first injected with 50 μl of thrombin (10 U/ml; Sigma-Aldrich, catalog no. T4648) subcutaneously and then injected with 200 μl of cell-laden pregel solution into the same site. All experiments were carried out in five mice, and explants were harvested after 1 week and 1 month.

Cardiomyocytes used for in vivo studies were cryopreserved on Day 20–22 of differentiation and thawed immediately prior to cell injection, following our previously described protocol (Gerbin et al., 2015 (link); Laflamme et al., 2007 (link)). One day prior to cryopreservation, cells were heat-shocked for 30 minutes at 42°C, treated with 10 μM Y-27632 for 1 hour, and dispersed with 0.25% trypsin in EDTA. Cells were spun down, resuspended in CryoStor (Sigma C2874) at 1×10⁷ cells/mL, and frozen in cryovials. To thaw cryopreserved cells, cryovials were thawed briefly at 37°C, followed by addition of RPMI-B27 + 200 U/mL DNase. Cells were washed and resuspended in an RPMI-based pro-survival cocktail containing 50% (vol/vol) growth factor-reduced Matrigel, 100 μM ZVAD (benzylox-ycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methyl ketone, Millipore 627610), 50 nM Bcl-XL BH4 (cell-permeant TAT peptide, Millipore 197217), 200 nM cyclosporine A (Novartis), 100 ng/mL IGF-1 (Peprotech 100–11), and 50 μM pinacidil (Sigma P154).