Caspase inhibitor z vad fmk

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.