Fig. 4.

Irreversible loss of bmMSC osteogenic potential in the absence of initial angiocrine support. (A) MSCs were implanted into nude mice with or without ECFCs and BMP-2 for 28 d. von Kossa staining was used for the grafts. Top panels are montages of contiguous pictures capturing the entire cross-section of the explants, with dashed black lines delineating the border of each mounted picture. (Insets) Macroscopic views of the explants. (B) Immunohistochemical analysis of osteogenic differentiation. Osteoblasts express osterix (red). Human MSC-derived cells express h-vimentin (green). Yellow arrowheads: human osteoblasts. White arrowheads: murine osteoblasts. (C) Quantification of tissue mineralization. (D) Percentage of human osteoblasts (osterix⁺/h-vimentin⁺). (E) bmMSCs were transplanted into primary GFP-SCID mice for 7 d and then retrieved and transplanted with ECFCs into secondary nude mice for 28 d. von Kossa and immunofluorescence staining was used for BMP-2–stimulated secondary grafts. Yellow arrowheads: human osteoblasts. White arrowheads: murine osteoblasts. (F) mRNA expression of osteogenic factors. All primers were human-specific (SI Appendix, Table S1). Data are normalized to human β-actin (ACTB). [Scale bars: 1 mm (A, Upper); 200 μm (A, Lower; E, Upper); 3 mm (A, Insets); 50 μm (B, Upper); and 10 μm (B and E, Lower).] Bars represent mean ± SEM (n = 4 mice per group). *P < 0.05. **P < 0.01 compared with grafts with MSCs alone.