Teratoma formation assay

Immunodeficient NOD/SCID mice were purchased from Charles River Laboratory (Frederick, MD, USA). iPSC in culture were treated with Y-27632 (Sigma-Aldrich) at 10 μM for 3 h and harvested into Primate ES Cell Culture Medium (ReproCELL) with 10 μM of Y-27632. The iPSC colonies were broken into small clumps by pipetting up and down several times. Per injection site, approximately 4 × 10⁶ cells were suspended in 200 μl of the above medium containing 50% Matrigel (BD Biosciences),⁵⁶ followed by subcutaneous injection into the dorsal flank of two NOD/SCID mice per iPSC line. When cystic masses grew rapidly, cystic fluid was drained with a sterile syringe so that solid parts would grow for several days⁵⁶ before mice were euthanized at 7 weeks after injection. When solid masses grew slowly, mice were maintained through 21 weeks after injection. All animal experiments and maintenance conformed to the guidelines of the Animal Care and Use Committee and of the American Association of Laboratory Animal Care. Resected teratoma tissues were fixed in 10% formalin, embedded in paraffin blocks, sectioned at a thickness of 5 μm, and mounted on glass slides. Staining with hematoxylin and eosin was performed using standard procedures.