FIG. 1.

Osteogenic induction of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs). (A) Cell culture and osteogenic induction methods for MSCs and iPSCs. Clonal MSCs and iPSCs were established from the C57BL/6J mouse strain using bone marrow and gingival tissues, respectively. “MSC medium” and “ES medium” are growth media for MSCs and iPSCs, respectively. “EB culture” represents floating culture of iPSCs forming embryoid bodies. RA, all-trans retinoic acid. “Day 0” refers to the day when osteogenic induction commences. The “osteogenic induction medium” contained the osteogenic factors dexamethasone, β-glycerophosphate, and ascorbate-2-phosphate. (B-I) MSC and iPSC cultures during osteogenic induction. (B, C) Phase-contrast photomicrographs on day 5 (scale bars: 100 μm). (B) MSC culture showed elongated fibroblastic cells in confluence. (C) iPSC culture showed fibroblastic cells (arrowheads) that migrated from the attached EBs (asterisks). (D, E) Scanning electron microscopic images on day 10 (scale bars: 50 μm). (D) MSC culture showed elongated spindle-like cells in monolayer. (E) iPSC culture showed multilayer cells and many rounded cells on the surface of the layers. (F–I) von Kossa staining images on day 20 (F, G) and 30 (H, I) (scale bars: 100 μm). (F, H) MSC culture on day 20 exhibited a von Kossa-positive area (asterisks) (F), which evenly spread over the cell culture on day 30 (H). (G, I) iPSC culture on day 20 exhibited studded von Kossa-positive areas (asterisks) concentrated on and around cell aggregates (G), which are more apparent on day 30 (I). Insets: von Kossa staining results in six-well culture plates.