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A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones

Nature Cell Biology volume 16, pages 1157–1167 (2014)Cite this article

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Abstract

The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow haematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors before Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C–X–C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2–creER, as well as Sox9–creER and aggrecan (Acan)–creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals.

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Figure 1: Fate mapping of Col2–cre+ and Osx–cre+ cells during endochondral ossification.
Figure 2: Col2–creER marks cells earlier than Osx–creER+ cells in the osteoblast lineage in fetal mice.
Figure 3: Col2–creER+ cells encompass early cells of the osteoblast lineage in postnatal mice.
Figure 4: Col2–creER+ cells generate multiple mesenchymal lineages in postnatal growing bones.
Figure 5: Relationship of growth skeletal progenitor cells and adult bone marrow stromal/mesenchymal progenitor cells.
Figure 6: Sox9–creER+ and Acan–creER+ cells encompass early growth skeletal progenitor cells in postnatal growing bones.

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References

  1. Kronenberg, H. M. Developmental regulation of the growth plate. Nature 423, 332–336 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Maes, C. et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev. Cell. 19, 329–344 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Akiyama, H. et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc. Natl Acad. Sci. USA 102, 14665–14670 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Frenette, P. S., Pinho, S., Lucas, D. & Scheiermann, C. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu. Rev. Immunol. 31, 285–316 (2013).

    Article  PubMed  Google Scholar 

  5. Mendez-Ferrer, S. et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Morikawa, S. et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J. Exp. Med. 206, 2483–2496 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhou, B. O., Yue, R., Murphy, M. M., Peyer, J. G. & Morrison, S. J. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sacchetti, B. et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324–336 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Wang, W. et al. Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I. Hum. Mol. Genet. 20, 3910–3924 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jacob, A. L., Smith, C., Partanen, J. & Ornitz, D. M. Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev. Biol. 296, 315–328 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ford-Hutchinson, A. F. et al. Inactivation of Pten in osteo-chondroprogenitor cells leads to epiphyseal growth plate abnormalities and skeletal overgrowth. J. Bone Miner. Res. 22, 1245–1259 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Hilton, M. J., Tu, X. & Long, F. Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage. Dev. Biol. 308, 93–105 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ovchinnikov, D. A., Deng, J. M., Ogunrinu, G. & Behringer, R. R. Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis 26, 145–146 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Kalajzic, Z. et al. Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters. Bone 31, 654–660 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Rodda, S. J. & McMahon, A. P. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133, 3231–3244 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Nakashima, K. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Ono, N. et al. Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage. Dev. Cell 29, 330–339 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Otto, F. et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765–771 (1997).

    Article  CAS  PubMed  Google Scholar 

  20. Ara, T. et al. Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny. Immunity 19, 257–267 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Omatsu, Y., Seike, M., Sugiyama, T., Kume, T. & Nagasawa, T. Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation. Nature 508, 536–540 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Liu, Y. et al. Osterix-cre labeled progenitor cells contribute to the formation and maintenance of the bone marrow stroma. PLoS ONE 8, e71318 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chen, J. et al. Osx-Cre targets multiple cell types besides osteoblast lineage in postnatal mice. PLoS ONE 9, e85161 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Greenbaum, A. et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495, 227–230 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nakamura, E., Nguyen, M. T. & Mackem, S. Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Dev. Dyn. 235, 2603–2612 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Soeda, T. et al. Sox9-expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons. Genesis 48, 635–644 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Henry, S. P. et al. Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage. Genesis 47, 805–814 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Mizoguchi, T. et al. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev. Cell 29, 340–349 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mignone, J. L., Kukekov, V., Chiang, A. S., Steindler, D. & Enikolopov, G. Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469, 311–324 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Balordi, F. & Fishell, G. Mosaic removal of hedgehog signaling in the adult SVZ reveals that the residual wild-type stem cells have a limited capacity for self-renewal. J. Neurosci. 27, 14248–14259 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pitulescu, M. E., Schmidt, I., Benedito, R. & Adams, R. H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank D. Rowe (University of Connecticut, USA) for Col2.3–GFP and Oc–GFP mice and S. Mackem (National Cancer Institute, USA) for Col2a1–creERT2 mice, H. Akiyama (Kyoto University, Japan) and B. de Crombrugghe (MD Anderson Cancer Center, USA) for Sox9–creERT2 and Acan-creERT2 mice, and L. Prickett, K. Folz-Donahue and M. Weglarz of Harvard Stem Cell Institute/Massachusetts General Hospital flow cytometry core and T. Diefenbach of Ragon Institute imaging core for their expert assistance. This work was supported by National Institutes of Health Grants DE022564 to N.O. and DK056246 to H.M.K.

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Authors and Affiliations

  1. Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    Noriaki Ono, Wanida Ono & Henry M. Kronenberg

  2. Department of Orthodontics and Pediatric Dentistry, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA

    Noriaki Ono & Wanida Ono

  3. Department of Immunobiology and Hematology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

    Takashi Nagasawa

  4. Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0076, Japan

    Takashi Nagasawa

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  1. Noriaki Ono
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Contributions

N.O. and H.M.K. conceived the project and designed the experiments. T.N. provided mice. N.O. and W.O. performed the experiments. N.O. and W.O. analysed the data. N.O. and H.M.K. wrote the manuscript; T.N. critiqued the manuscript.

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Correspondence to Henry M. Kronenberg.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fate mapping of Col2-cre+ and Osx-cre+ cells during endochondral ossification.

a. E11.5 femur sections of Col2-cre; R26RTomato mice were stained for nuclei and CD31. Shown is mesenchymal condensation. Perichondrium is on top. Red represents tdTomato, blue represents Alexa633 and gray represents DAPI. Scale bars: 100 μm. b,d. P3 femur sections of Col1-GFP; Col2-cre; R26RTomato (b) or Col1-GFP; Osx-cre; R26RTomato (d) mice were stained for nuclei. Shown is trabecular bone. Arrowheads: Col1-GFP+Tomato+ osteoblasts. Green: EGFP, red: tdTomato, gray: DAPI and DIC (differential interference contrast). Scale bars: 50 μm. c,e. P3 femur sections of Cxcl12-GFP; Col2-cre; R26RTomato (c) or Cxcl12-GFP; Osx-cre; R26RTomato (e) mice were stained for nuclei. Shown are diaphyseal endocortices and bone marrows. Arrows: Cxcl12-GFP+Tomato+ stromal cells. Green: EGFP, red: tdTomato, gray: DAPI and DIC (differential interference contrast). Scale bars: 50 μm. f. E14.5 femur sections of Osx-cre::GFP; R26RTomato mice were stained for nuclei and EdU, which was administered 3 hours prior to analysis. Shown are higher magnification views of Figure 1e. Arrowheads: proliferating Osx+ cells in the perichondrium. Green: EGFP, red: tdTomato, blue: Alexa647, gray: DAPI. Scale bars: 50 μm. g. Shown is the percentage of EdU+ cells among tdTomato+ cells in the E14.5 perichondrium of Col2-cre; R26RTomato (blue dots) and Osx-cre::GFP; R26RTomato (red dots). n = 3 mice for Col2-cre; R26RTomato, n = 4 mice for Osx-cre::GFP; R26RTomato.

Supplementary Figure 2 Col2-creER+ cells encompass early cells of the osteoblast lineage and generate multiple mesenchymal lineages.

a. Pregnant mice received 1mg tamoxifen at E11.5, and Col2-creER; R26RTomato mice were chased until postnatal day 21 (P21). Shown are femur sections with the distal side on the left. Col2creER-E11.5 represents descendants of Col2-creER+ cells at E11.5. White indicates tdTomato. Scale bars: 500 μm. b. Pregnant mice received 1mg tamoxifen at E13.5, and Osx-creER; R26RTomato mice were chased until P0 (left panel), P7 (middle panel) or P14 (right panel). Shown are femur sections with the distal side on the left. OsxcreER-E13.5 represents descendants of Osx-creER+ cells at E13.5. Red indicates tdTomato. Scale bars: 500 μm. c. Lineage-tracing of postnatal Osx-creER+ cells was performed by injecting 0.1mg tamoxifen into P3 mice. Pulsed Osx-creER; R26RTomato mice were chased for a month (left panel), two months (middle panel) and six months (right panel). Shown are femur sections with the distal side on the left. OsxcreER-P3 represents descendants of Osx-creER+ cells at P3. Red indicates tdTomato. Scale bars: 500 μm. d, e No tamoxifen controls of Col2-creER; R26RTomato at P5 (d) and P10 (e). month of age (g, i) or 1 year of age (h, j) are shown. Shown are femur sections with the distal side on the left. White indicates tdTomato. Scale bars: 100 μm. f Oc-GFP; Col2-creER; R26RTomato mice received 0.1mg tamoxifen at P3 and were chased for 2 months. Shown are metaphyseal spongiosa. Femur sections were stained for nuclei and LipidTOX deep red. Asterisks: Tomato+ osteoblasts, arrowheads: Tomato+ adipocytes. Green: EGFP, red: tdTomato, blue: LipidTOX Deep Red, gray: DAPI. Scale bars: 100 μm (left panel) or 20 μm (right panel).

Supplementary Figure 3 Colony-forming unit fibroblasts (CFU-F) assay and flow cytometry analysis of CD45−Ter119−CD140a+Sca1+ (PαS) fraction.

a, b. Colony-forming unit fibroblast (CFU-F) assay was performed using unfractionated P5 bone marrow cells plated at a clonal density (106 cells per 9.6 cm2) and cultured for 10 days. Cells were harvested from Col2-cre; R26RTomato (a; left panel), Osx-cre; R26RTomato (a; left panel) mice, Col2-creER; R26RTomato (b; left panel) or Osx-creER; R26RTomato mice that received 0.1mg tamoxifen at P3 (b; right panel). Cells were stained for Wheat Germ Agglutinin (WGA)-Alexa488 conjugate. Red: tdTomato, green: Alexa488. The number of colonies (>50 cells) of each color was also shown. Scale bars: 2 mm. c. Scheme to identify a CD45−Ter119−CD140a+Sca1+ (PαS) fraction. Dissociated bone cells were stained for CD45, Ter119, PDGFRα (CD140a) and Sca1, or CD45, Ter119 and isotype control antibodies, and subjected to flow cytometry analysis. First, cells were gated on forward scatter and scatter profiles to identify single cells (left three subpanels). Second, cells were gated for a CD45−Ter119− fraction to analyze a non-hematopoietic fraction (left fourth subpanel, red box). This fraction was expanded for CD140a and Sca1. Red box designates PαS fraction (upper right panel; upper left and lower right subpanels indicate single-color staining, and lower and left subpanel indicates isotype control). d–f. Flow cytometry analysis was performed using dissociated bone cells harvested from Cxcl12-GFP mice at P14 (left panels), P32 (middle panel) and 8 weeks old (right panels). Cells were stained for CD45, Ter119, PDGFRα (CD140a) and Sca1 or their corresponding isotype controls. Representative dot plots of CD45−Ter119− fraction are shown on the leftmost panels. (d) Histograms of CD45− Ter119−Cxcl12-GFPhigh fraction developed for Sca-1; blue lines: isotype control. (e) Histograms of CD45−Ter119−CD140a+Sca1+ (PαS) fraction developed for Cxcl12-GFP; blue lines: GFP− controls. (f) Dot plots of CD45−Ter119−Cxcl12-GFPhigh fraction developed for CD140a and Sca1. CD45−Ter119−CD140a+Sca1+ (PαS) and CD45−Ter119−CD140a+Sca1+ (PαS−) fractions are boxed. n = 4 mice for P14 and P32, n = 3 mice for 8 weeks old mice. All data are represented as mean ± SD.

Supplementary Figure 4 Relationship of growth skeletal progenitor cells and adult bone marrow stromal/mesenchymal progenitor cells.

a. Representative dot plots of cells from Nes-GFP; Col2-cre; R26RTomato (left panel) and histograms of Nes-GFP+ fraction (right panel). X-axis represents tdTomato, and Y-axis represents GFP (left panel) or percentage of maximum (right panel). n = 5 mice per group. All data are represented as mean ± SD. b. Flow cytometry analysis was performed using dissociated bone cells harvested from P7 Cxcl12-GFP; Col2-creER; R26RTomato mice after administration of tamoxifen at E13.5 (1mg into pregnant mice). Cells were stained for CD45, Ter119, PDGFRα (CD140a) and Sca1, and CD45−Ter119− fraction (left panel) was gated for PDGFRα (CD140a) and Sca1 (PαS, right panel). X-axis represents tdTomato, and Y-axis represents GFP. c. Flow cytometry analysis was performed using dissociated bone cells harvested from Cxcl12-GFP; Nes-creER; R26RTomato mice that received 0.1 mg tamoxifen at P3 and were chased for indicated periods. CD45− fraction was gated for GFP. Left subpanel: 2 days chase, middle subpanel: 4 weeks chase. X-axis represents tdTomato, and Y-axis represents GFP. Percentage of NescreER-P3 cells (purple line) among CD45−Cxcl12-GFPhigh stromal cells during the chase was plotted. X-axis represents the duration of the chase, and Y-axis represents the percentage of Tomato+GFP+ cells among total GFP+ cells. n = 3 mice for 2 days chase and 4 weeks chase, n = 4 mice for 1 week chase. All data are represented as mean ± SD.

Supplementary Figure 5 Sox9-creER+ and Acan-creER+ cells encompass early growth skeletal progenitor cells in postnatal growing bones.

a,b. Sox9-creER; R26RTomato mice received 0.1 mg tamoxifen at P3 and were chased for 48 hours (a) or a week (b). Shown are distal halves of growth cartilages at the junction of growth plate and primary spongiosa. Perichondrium is on top. Femur sections were stained for nuclei and CD31. Asterisks: Tomato+ chondrocytes, sharps: Tomato+ perichondrial cells, arrows: Tomato+ cells in primary spongiosa, arrowheads: a group of Tomato+ cells directly under the growth plate and beneath the perichondrium. Red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 100 μm. c,d. Col1-GFP; Acan-creER; R26RTomato (c) or Acan-creER; R26RTomato (d) received 0.1mg tamoxifen at P3 and were chased for 48 hours (c) or a week (d). Femur sections were stained for nuclei and CD31. Asterisks: Tomato+ chondrocytes, sharps: Tomato+ perichondrial cells, arrows: Tomato+ cells in primary spongiosa, arrowheads: a group of Tomato+ cells directly under the growth plate and beneath the perichondrium. Red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars represent 100 μm.

Supplementary Figure 6 A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones.

Diagram of a growing endochondral bone is shown. Shown on the left is growth plate, and shown on the right is diaphysis. As bones rapidly grow (to the left in this diagram), a subset of Sox9+Col2+Acan+ cells self-renews and provides a stable source of Osx+ precursors in the metaphysis. While Osx+ precursors become Col1+ differentiated osteoblasts on the bone surface and Cxcl12+ stromal cells in bone marrow, these precursors are transient and not indefinitely self-renewing in this actively growing portion of bones. A subset of these chondrogenic cells also provides a source of bone marrow mesenchymal stromal progenitor cells (BMSCs), which are highly enriched among bone marrow pericytes such as PDGFRα+Sca1+ “PαS” cells and Nes-GFP+ cells. LepR+ stromal cells are likely to overlap with Cxcl12+ stromal cells and PαS cells.

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Ono, N., Ono, W., Nagasawa, T. et al. A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones. Nat Cell Biol 16, 1157–1167 (2014). https://doi.org/10.1038/ncb3067

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