Valganciclovir

NOG (NOD/Shi‐scid IL2Rgnull) mice, aged 6–8 weeks, were maintained under specific‐pathogen‐free conditions in the Animal Center of the Central Institute for Experimental Animals in accordance with the guidelines of the facility. The recipient mice were anesthetized by inhalation of isoflurane (Dainippon Pharmaceutical Co., Ltd., Japan, Osaka). For subcutaneous transplantation, HSV‐TK knockin hiPSCs (TK#1–3, 3–5 × 10⁵ cells) suspended in 0.1 ml of a 1:1 mixture of growth factor reduced Matrigel (BD Bioscience, San Jose, CA, USA) and Williams' Medium E (Thermo Fisher Scientific), were subcutaneously inoculated into mice. The mice were monitored daily and the teratomas were measured using calipers. Teratoma volume (TV) was calculated using the formula TV = ½ × A × B² (A: length [mm]; B: width [mm]). The criteria for successive engraftment were as follows: progressive nodule growth at the site of injection and TV values exceeding 10 mm³. After 5 weeks, 0.2 mg/ml of valganciclovir (Sigma–Aldrich, St. Louis, MO, USA) or saline (Otsuka Pharmaceutical, Tokyo, Japan) was added to the drinking water. At 21–25 days after GCV or vehicle administration, teratomas were recovered from the sacrificed mice.

Female FNRG recipients were withdrawn from NTBC 4 to 5 d prior to surgery. Male TK-NOG mice 7 wk of age received 48 h of 0.1 mg/mL valganciclovir (Sigma Aldrich) in drinking water 14 d prior to transplantation (19 (link)). uPA^+/− mice were transplanted 8 d after retrorsine injection when they were 20 to 21 d old. Human cells were transplanted as described previously (11 ). Briefly, under sterile conditions and using isoflurane anesthesia, the skin was cleaned with povidone iodine, and a 0.5- to 1-cm incision was created over the left flank, after which the peritoneum was mobilized and opened. The ventral tip of the spleen was mobilized onto the peritoneum, and human cells were injected into the spleen using a 28-gauge insulin syringe. After hemostasis was achieved by applying pressure to the injection site, the peritoneum was closed using 4.0 Vicryl suture (Ethicon) and the skin was closed using metal Mikron Autoclips (BD Biosciences). Mice received two doses of postoperative buprenorphine (Abbot Animal Health) 0.05 mg/kg for analgesia. Metal clips were removed 14 d after surgery.