(a) Subcutaneous transplantation of osteogenically induced GF-iPSC constructs into immunodeficient mice resulted in tumor formation (upper panel: arrowhead) at the fourth week. Lower panel: an extracted tumor (scale bar: 1 cm). (b) H&E staining of the extracted transplants indicates teratoma, where the transplanted cell construct (arrowhead) was surrounded by extensive osseous tissue (asterisk). Scale bar: 1 mm. Inset: magnification of the dotted square. Arrows indicate aligned cells on the osseous tissue (asterisk). Scale bar: 50 μm. (c) von Kossa staining and counterstaining with methylene blue. The transplanted calcified cell construct (circle) and the ectopically formed bone region (asterisk) exhibited robust calcification. Arrowheads indicate sparse calcium deposition in the teratoma. Scale bar: 500 μm. Inset: magnification of the dotted square. Arrows indicate aligned cells in direct contact with the calcified tissue surface (asterisk) (scale bar: 50 μm).