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. Author manuscript; available in PMC: 2015 Aug 7.
Published in final edited form as: Cell Stem Cell. 2014 Jun 19;15(2):154–168. doi: 10.1016/j.stem.2014.06.008

Figure 4. LepR+Col2.3-GFP− cells are quiescent under normal physiological conditions in adult bone marrow but go into cycle to regenerate bone after injury.

Figure 4

(A–C) BrdU incorporation (14 day pulse) (A and B) or Hoechst staining (C) by various stromal cell fractions from enzymatically dissociated bone and bone marrow obtained from 2-month-old Lepr-cre; tdTomato; Col2.3-GFP mice. Unless otherwise indicated, data in all remaining panels represent mean±SD from 3–4 mice in 3 independent experiments, with statistical significance assessed by two-tailed Student’s t-tests. ns, not significant, *P<0.05, **P<0.01, ***P<0.001.

(D) Percentage of Tomato+Col2.3-GFP−CD45−Ter119−CD31− bone marrow stromal cells that incorporated a 14 day pulse of BrdU.

(E) Percentage of Tomato+CD45−Ter119−CD31− bone marrow stromal cells that incorporated a 14 day pulse of BrdU in femurs and tibias from Lepr-cre; tdTomato mice and Lepr-cre; tdTomato; iDTR mice two weeks after diphtheria toxin (DT) treatment.

(F) Bone marrow cellularity in the femurs and tibias of Lepr-cre; tdTomato; iDTR mice at the indicated time points after DT treatment.

(G) Number of Tomato+CD45−Ter119−CD31− stromal cells in bone marrow from Lepr-cre; tdTomato; iDTR mice after DT treatment.

(H) Number of CD150+CD48−Lineage−Sca-1+c-kit+ HSCs in the femurs and tibias 7 days after DT treatment.

(I and J) Quantification of adipocyte number per 7 μm femur section (I) and the percentage of adipocytes that were Tomato+ (J) 14 days after DT treatment.

(K) Percentage of Col2.3-GFP+ osteoblasts that were also Tomato+ in enzymatically dissociated bone 2 weeks after DT treatment.

(L) Percentage of Col2.3-GFP+ osteoblasts that were also Tomato+ at metaphyseal (Met) and diaphyseal (Dia) bones 2 weeks after diphtheria toxin treatment.

(M) Representative femur sections from Lepr-cre; tdTomato; Col2.3-GFP; iDTR mice at 2 weeks after DT treatment. Note the formation of ectopic trabecular bone by Tomato+Col2.3-GFP+ cells in the diaphyseal bone marrow cavity (ii).

(N and O) Bone formation rate in Lepr-cre; iDTR and control mice after DT treatment. Two doses of calcein were injected at day 0 and 7 after DT treatment then the distance between calcein bands was measured at 14 days after DT treatment.

(P) Osteoclasts (arrow) were not labeled by Tomato in Lepr-cre; tdTomato mice.

(Q) Percentage of Tomato+CD45−Ter119−CD31− bone marrow stromal cells that incorporated a 14 day pulse of BrdU in Lepr-cre; tdTomato mice at various times after irradiation. (n=3–5 mice/time point from 3 independent experiments)