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. 2017 Jul 10;7:4928. doi: 10.1038/s41598-017-05401-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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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.