Figure 2. LepR⁺ cells contain most of the CFU-F in adult bone marrow and are the major source of new bone in adult mice.

(A) Percentage of all CFU-F colonies that were labeled by conditional reporter expression when cultured from enzymatically dissociated bone marrow of the indicated genotypes. Macrophage colonies were excluded by staining with anti-CD45 antibody in all experiments. (n=3–11 mice/genotype from at least 3 independent experiments).

(B) Percentage of bone marrow cells expressing each marker that formed CFU-F colonies in culture (n=3–5 mice/genotype from at least 3 independent experiments).

(C) Percentage of non-hematopoietic (CD45⁻Ter119⁻) bone marrow cells expressing each marker that formed CFU-F colonies in culture (n=3–11 mice/genotype from at least 3 independent experiments). Two-tailed Student’s t-tests were used to assess statistical significance. *P<0.05, **P<0.01, ***P<0.001.

(D) The percentage of CFU-F colonies that arose from Tomato⁺CD45⁻Ter119⁻CD31⁻ cells from Lepr-cre; tdTomato mice that gave rise to Oil-red O⁺ adipocytes, Toluidine blue⁺ chondrocytes, and/or Alizarin-red S⁺ osteoblasts (n=3 mice from 3 independent experiments).

(E) Development of bone and hematopoiesis in ossicles formed by individual CFU-F colonies that arose from LepR⁺ stromal cells from 4 Col2.3-GFP; Lepr-cre; tdTomato mice.

(F–I) Representative femur sections from Lepr-cre; tdTomato; Col2.3-GFP mice of different ages showing the increasing generation of Tomato⁺Col2.3-GFP⁺ osteoblasts with age (3–5 mice/age from at least 4 independent experiments).

(J) The frequency of Tomato⁺CD45⁻Ter119⁻CD31⁻Col2.3-GFP⁻ bone marrow stromal cells in the femurs of Lepr-cre; tdTomato; Col2.3-GFP mice did not change with age. Cells from ~6-month-old Col2.3-GFP; tdTomato mice were negative controls (CON). Data in all remaining panels represent mean±SD from 3–5 mice/age from at least 3 independent experiments.

(K) Percentage of Col2.3-GFP⁺ osteoblasts that were also Tomato⁺ in enzymatically dissociated bone from Lepr-cre; tdTomato; Col2.3-GFP mice of different ages. Osteoblasts from age-matched Col2.3-GFP or Col2.3-GFP; tdTomato mice were used as negative controls in each experiment (CON). Two-tailed Student’s t-tests were used to assess statistical significance among consecutive ages. *P<0.05, **P<0.01, ***P<0.001.

(L and M) Percentage of osteoblasts (L) and osteocytes (M) that were Tomato⁺ in bone sections from Lepr-cre; tdTomato; Col2.3-GFP mice.