FIG. 4.

SEM observation of iPSCs during osteogenic differentiation. (A–C) Day 10. (A) Rounded cells (arrowheads) were growing on the multilayered fibroblastic cells (asterisks). Many small globular vesicle structures (arrows) were observed on the multilayered fibroblastic cells. Scale bar: 10 μm. (B) Magnification of the dotted square in (A). Vesicle-like amorphous structures (arrows) were budding from the rounded cells. Scale bar: 5 μm. Inset: The surface of the rounded cells was covered with many tiny microvillus-like projections (scale bar: 0.5 μm). (D–F) Day 30. (D) The rounded cells exhibited a rough surface covered by small granulated accretions. Scale bar: 10 μm. (E) Magnification of the dotted square in (D). The surface of the rounded cells was gritty with ruptured vesicle accretions. Fibrous structures were rarely observed with the accretions. Scale bar: 5 μm. (C, F) EDX analyses of the areas shown in SEM images (A) and (D), respectively. Energy peaks in the EDX graph correspond to elemental phosphorous (P), calcium (Ca), carbon (C), and oxygen (O). Insets: (C) Elemental calcium (yellow dots) and phosphorous (blue dots) were sparsely present on the layered cells on day 10. Accumulation of these elements on the vesicle structures was barely observed. (F) Elemental calcium (yellow dots) and phosphorous (blue dots) intensely accumulated on the surface of the rounded cells by day 30. Color images available online at www.liebertpub.com/scd