(a) von Kossa staining and counterstaining with methylene blue. Most inner areas of the osteogenically induced GF-iPSC construct showed calcification (asterisks). Some noncalcified fibrous areas were observed (arrow heads). Scale bar: 100 μm. (b) Magnification of the dotted square in panel (a). The calcified inner area (asterisks) was surrounded by a strongly calcified zone (arrow heads). Arrows indicate osteoid-like tissues. Scale bar: 100 μm. (c) EDX analyses of the calcified layer area. The yellow, blue, and red dots represent the elemental distribution of calcium, phosphorous, and carbon. Scale bar: 10 μm. (d) Energy peaks in the EDX graph correspond to elemental phosphorous (P), calcium (Ca), carbon (C), and oxygen (O). (e) TEM image of bone nodules formed in the calcified area (scale bar: 1 μm). (f) SAED pattern (002, 211, and 310 rings) of an electron-dense area (asterisk in panel (e)) was indicative of hydroxyapatite.