Characterization and MSC phenotype of bone-derived FGFR1/2⁺ cells in vitro. (A) Left to right: Bone-derived cells expanded with FGF-2 stimulation could produce adipocytes (Oil Red O stain), osteoblasts (Alizarin Red S stain), and chondrocytes (Alcian Blue stain) in vitro and maintained this capacity for at least 10 low-density passages (passage 6 is shown). Scale bar indicates 50 μm. (B) Left to right: Undifferentiated MSCs expanded with FGF-2 expressed FGFR1/2 and PTHrP; MSC-derived Nile Red⁺ adipocytes lost expression of FGFRs; MSC-derived Runx2⁺ osteoblasts lost FGFR2 but maintained low FGFR1 expression in some cells; and MSC-derived Sox9⁺ chondrocytes lost FGFR2 expression but up-regulated FGFR3 (inset shows chondrocyte-specific collagen 2 and expression of FGFR1 in rare undifferentiated cells). Scale bar indicates 25 μm in all panels except the far-right, which indicates 50 μm. (C) FACS analysis of passage-2 MSCs expanded with FGF-2 showing the absence of macrophage (CD11b), endothelial (CD31), and hematopoietic (CD45) cell markers and expression of the C57Bl/6 mesenchymal stem–cell markers CD105, CD90, CD73, CD44, and CD34. These cells also expressed MHC class I (H2-K^b). (D) Top panel: When used as third-party cells in a MLR, FGFR1/2⁺ MSCs efficiently suppressed T-cell activation (detected as IL-2 production by ELISA) in a dose-dependent manner. Bottom panel: FGFR1/2⁺ MSCs could process exogenous antigens (in this case, OVA) and cross-present them on MHC I to OVA-responsive T cells. (E) FGFR1/2⁺ MSCs expanded with FGF-2 for 6 passages were implanted subcutaneously on HA/TCP ceramic particles for 8 weeks. Histological analysis revealed the presence of adipocytes (top left, H&E), osteoblasts (top right, Von Kossa/Toluidine Blue), fibrous tissue (bottom left, H&E), and hematopoietic marrow irrigated by large sinusoids (bottom right, H&E). Scale bar indicates 50 μm.