Teratoma Formation and Histological Analysis

Teratoma analysis was performed as described previously [72 (link)]. Briefly: iPSCs were trypsinized, washed in PBS, and 10⁶ cells were subcutaneously injected in the hindlimb of athymic NUDE mice (Crl:NU(NCr)-Foxn1nu). After 3–4 weeks, the mice were euthanized, and teratomas were removed and fixed in 4% paraformaldehyde in PBS. Teratomas were cut into pieces with a 5 mm diameter, dehydrated, and paraffinized. The paraffin pieces were then cut into 5 μm slices using a microtome Leica RM2235 (Leica Biosystems, Wetzlar, Germany). The glass-slide attached paraffin slices were dried, rehydrated, and stained with hematoxylin, washed and then stained with eosin. Then they were washed, dehydrated, and mounted with Canadian balsam and coverslip. The teratoma histological sections were analyzed using the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA).