Figure 4.
Fetal C-KIT+ Cells Contributed to Bone Formation and Regeneration in Adult Bone Marrow
All KitMerCreMer; R26tdTomato; Col2.3-GFP and KitMerCreMer; R26tdTomato mice were tamoxifen treated at E12.5 and 14.5.
(A) Representative confocal images of femur sections from 2-month-old KitMerCreMer; R26tdTomato; Col2.3-GFP mice. Osteoblasts were revealed by Col2.3-GFP fluorescence; adipocytes were revealed by anti-perilipin staining. Note that TOMATO was also expressed by hematopoietic cells in these mice but levels of TOMATO expression in stromal cells were ∼10- to 100-fold higher than in hematopoietic cells. Therefore, short-exposure images showed mainly TOMATO fluorescence in stromal cells (n = 3 mice from three independent experiments).
(B) Representative flow cytometry plots of enzymatically dissociated femurs showed that 46% of Col2.3-GFP+ osteoblasts were derived from C-KIT+ cells in KitMerCreMer; R26tdTomato; Col2.3-GFP mice (n = 3 mice from three independent experiments).
(C) Representative confocal images of the callus region of KitMerCreMer; R26tdTomato; Col2.3-GFP mice at 2 weeks after femur fracture (n = 3 mice from three independent experiments).
(D–F) Frequencies of CD45−TER119−CD31−PDGFRα+ bone marrow stromal cells (D), Col2.3-GFP+ osteoblasts (E), and perilipin+ adipocytes (F) that expressed TOMATO in 2-, 6-, and 12-month-old KitMerCreMer; R26tdTomato; Col2.3-GFP mice. All data represent mean ± SD of 3 mice per age from three independent experiments. The statistical significance of differences among different ages was measured by repeated measures one-way ANOVAs with Greenhouse-Geisser correction and Tukey's multiple comparison tests (∗p < 0.05).
(G) BrdU incorporation (14-day pulse) by TOMATO+ bone marrow stromal cell from 2-month-old KitMerCreMer; R26tdTomato mice (n = 3 mice from three independent experiments).
(H) Percentage of TOMATO+ bone marrow stromal cells from 2-month-old KitMerCreMer; R26tdTomato mice that were DAPI+Annexin V+ (n = 3 mice from three independent experiments).