F344 njcl rnu rnu rats

The vascular bed was induced in male F344/NJcl-rnu/rnu rats weighing 200–380 g (Clea Japan) as described above. On day 21, a three-layer human cardiomyocyte sheet was transplanted onto the vascular bed and encapsulated with an EVOH membrane. The following day, another three-layer sheet was transplanted onto the first three-layer sheet, and the construct was encapsulated with an EVOH membrane. The cell sheets were incubated in vivo for 7 days, following which the rat was euthanized with an overdose of pentobarbital.

After 4 weeks of osteoblast differentiation, iPSCs-osteoblasts were dissociated with 0.5 mg/mL collagenase type IV for 30 min and 0.25% trypsin–EDTA for 5 min at 37⁰C. These cells (2 × 10⁵) were transplanted in atelocollagen (AteloCell^®, Atelocollagen honeycomb sponge, KOKEN, Tokyo, Japan) as a scaffold for the cells. Ten-week-old male F344/NJclrnu/rnu rats were obtained from Clea Japan, Inc. (Tokyo, Japan). After anesthesia induction with 4% sevoflurane (Maruishi Pharmaceutical Co. Ltd., Osaka, Japan), the rats were further anesthetized by intraperitoneal injection with sodium pentobarbital (30 mg/kg body weight; somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan). After the calvarial bone was exposed, critical-sized bone defects (diameter = 5 mm) were created in the dorsal area. The scaffold/iPSC–osteoblast complex was transplanted into the bone defects. After transplantation, the periosteal flap was closed by suture.^{20 (link)} The rats receiving transplantation were euthanized at 4 weeks and assessed using radiographical and histological analyses.

In vivo tumorigenicity study using animals was approved by the IRB of the Foundation for Biomedical Research and Innovation (FBRI), the Committee for Animal Experiments of the FBRI, and the animal care committee of RIKEN BDR. Subretinal transplantation was conducted in RIKEN BDR. Female nude rats were used in this study to reduce fighting between rats bred in a same cage^{71 (link)}. Six-week-old female nude rats (F344/NJcl-rnu/rnu rats; CLEA Japan) were used for the transplantation and underwent general condition observations and weight measurements during the observation period. Surgical stress of subretinal transplantation in rats was evaluated in sham-operated eyes (Supplementary Fig. 5e,f). Fundus imaging and OCT analysis were performed using RS-3000 Advance (Nidek) and Micron IV and OCT (Phoenix research labs) according to the manufacturers’ instructions. After necropsy, the eyeballs were fixed at 4 °C in SUPERFIX (KY-500; Kurabo Japan), embedded in paraffin, and sliced with a microtome at 3-µm thickness. One in every five sections was used for HE staining and histopathological analysis, and the other sections were used for immunohistochemistry.