Osteoblast differentiation from MPCs.
MPCs were cultured at 2 × 104/cm2 on FN in DMEM without serum, EGF, or PDGF-BB, but with β-glycerophosphate, 10−7 M dexamethasone, and 10 mM ascorbic acid. Differentiation to osteoblasts was shown by presence after 14 days of osteopontin, bone sialoprotein, and osteonectin by FACS (A; specific antibody, thick line; control IgG, thin line). This was confirmed by immunohistochemistry (B; enlargement = 10 ×; arrows indicate nuclei) and Western blot (C; clones nos. 1, 2, and 3; see also Figure 12; β-actin serves as loading control) on day 14. Positive control on Western blot (C+): HT85 cells were induced to differentiate to osteoblasts. Presence of CaPo4 crystals was shown by Von Kossa staining, and presence of osteoblast differentiation was further confirmed by increased alkaline phosphatase staining (D; enlargement = 10 ×). A representative example of more than 5 experiments is shown.
