Osteogenic Factor Runx2 Marks a Subset of Leptin Receptor-Positive Cells that Sit Atop the Bone Marrow Stromal Cell Hierarchy

Abstract

Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFP^low sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP^- population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR⁺Runx2-GFP^low cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR⁺ population without osteoblastic commitment, and the LepR⁺Runx2-GFP^low stromal cells sit atop the BM stromal hierarchy.

Figure 1

LepR⁺ cells in the bone marrow cavity express Runx2. (A–H) Z-stack confocal (A,D–F and H) and confocal (B,C and G) images of thick bone sections of Runx2-GFP mice (5–6 weeks old). The images were taken from whole bone tissue (A), endosteum (B,C and F), epiphyseal cartilage (D) and bone marrow (E,G and H). Bone tissues are stained with osteocalcin (Red) (B), SOST (Red) (C), Type-2 collagen (Col2) (Red) (D), CD45 and Ter119 (Red) (E), VE-cadherin (VE-Cad) and CD31 (Red) (F), Perilipin (White) (G), and Leptin receptor (LepR) (Red) (H) antibodies. Nuclei are visualized with Hoechst 33342 (blue) and propidium iodide (PI) (Red). Arrows: Osteoblasts (B), Osteocytes (C), and LepR⁺ cells (H).

Figure 2

LepR⁺ cells contain Runx2-GFP^low and Runx2-GFP⁻ sub-populations. (A) Representative FACS plots (gated on live CD45⁻Ter119⁻CD31⁻ cells) showing the expression of LepR in Runx2-GFP⁺ stromal populations from 5 week-old Runx2-GFP mice. Left panel showing representative FACS plot of WT control (gated on live CD45⁻Ter119⁻CD31⁻ cells). Black and red lines represent the isotype control and specific antibody against LepR, respectively. n = 3. (B) Representative FACS plots (gated on live cells) showing frequency of Runx2-GFP⁺ population in the CD45⁻Ter119⁻CD31⁻LepR⁺ cell population (right panel) from 5–6 week-old Runx2-GFP mice. Left panel showing negative control for LepR antibody (gated on live cells). Black and red lines represent the WT control and Runx2-GFP mice, respectively (right panel). n = 3. (C and D) Quantification of the frequency (C) and absolute number (D) of Runx2-GFP^low and Runx2-GFP⁻ sub-populations in LepR⁺ cells (CD45⁻Ter119⁻CD31⁻). n = 3. *P < 0.05, **P < 0.01. Data are represented as mean ± SD.

Figure 3

Stromal stem cell activity in BM is enriched in LepR⁺Runx2-GFP^low population. (A) Z-stack confocal images of thick bone sections of 6 week-old LepR-Cre/Tomato/Runx2-GFP mice. Arrows: LepR-Cre-derived Tomato⁺ (LepR/Tomato⁺)/Runx2-GFP^low cells. Arrowheads: LepR/Tomato⁺/Runx2-GFP⁻ cells. *: Runx2-GFP^high bone-lining mature osteoblasts. Nuclei were visualized with Hoechst 33342 (blue). (B) Z-stack confocal images of thick bone sections of 6 week-old LepR-Cre/Tomato mice stained with Runx2 (left panel, green) and control IgG (right panel). Arrows: LepR/Tomato⁺/Runx2⁺cells. Nuclei are visualized with To-PRO-3 (blue). (C) Representative FACS plots (gated on live cells) showing the percentages of Runx2-GFP-positive (designated as Runx2-GFP^low) and -negative (designated as Runx2-GFP⁻)cells (middle panel) in the CD45⁻Ter119⁻CD31⁻LepR/Tomato⁺ stromal population (left panel) from 6 week-old LepR-Cre/Tomato/Runx2-GFP mice. Right panel showing representative FACS plot of control (gated on live CD45⁻Ter119⁻CD31⁻LepR/Tomato⁺ cells) in 6 week-old LepR-Cre/Tomato mice. (D–O) CD45⁻Ter119⁻CD31⁻ stromal cells (gated on live cells) were sorted based on expression of LepR-Cre/Tomato and Runx2-GFP from 6-7 week-old LepR-Cre/Tomato/Runx2-GFP mice, and percentage of CFU-F (D) and clonal sphere (mesensphere) formation (G) were determined. Representative image of CFU-F colony (E; Giemsa staining, F; Tomato and GFP fluorescence). n = 3 independent experiments. Arrows: LepR/Tomato and Runx2-GFP double-positive cells. Representative image of mesensphere formation (H; bright field, Tomato, and GFP fluorescence). n = 3 independent experiments. Differentiation phenotypes of LepR/Tomato⁺/Runx2-GFP^low cells shown by Alizarin Red S: osteoblasts (I), lipid droplets and staining with FABP4 antibody: adipocytes (J), and Alcian Blue: chondrocytes (K). Expression levels of LepR (L), PDGFRa (M), CXCL12 (N) and Runx2 (O) were measured by quantitative real-time PCR. n = 3–5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are represented as mean ± SD.

Figure 4

LepR⁺Runx2-GFP^low cells differentiate into osteoblasts through multilayered cell formation in response to PTH-induced anabolic effects. (A–F) Z-stack confocal images of thick bone sections of 6 week-old LepR-Cre/Tomato/Runx2-GFP mice (A and B), tamoxifen-administered iOsx/Tomato/Runx2-GFP mice (C and D), and tamoxifen-administered iOsx/Tomato/Col1(2.3)-GFP mice (E and F) with vehicle (A,C and E) and hPTH(1–34) (B,D and F) intermittent treatment. Arrows: LepR-Cre-derived Tomato⁺(LepR/Tomato⁺)Runx2-GFP⁺ cells (A and B), iOsx/Tomato⁺Runx2-GFP⁺ cells (D) and iOsx/Tomato⁺Col1(2.3)-GFP⁺ cells (F). Arrowheads: iOsx/Tomato⁻Runx2-GFP⁺ cells (C and D) and iOsx/Tomato⁺Col1(2.3)-GFP⁻ cells (F). *: Bone-lining mature osteoblasts. Nuclei were visualized with To-PRO-3 (blue).

Figure 5

Differentiation model of LepR⁺Runx2-GFP^low cells into osteoblasts. LepR⁺Runx2-GFP^low cells form multilayered structures along the bone surface in response to intermittent PTH treatment. In this process, the LepR⁺Ranx2-GFP^low cells increase the expression levels of Runx2, Osx and Col1 sequentially, and eventually differentiate into mature osteoblasts.