Resource

Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules

Received:
Accepted:
Published online:

Abstract

The bone marrow (BM) microenvironment contains many types of cells and molecules with roles in hematopoiesis, osteogenesis, angiogenesis and metabolism. The spatial distribution of the different bone and BM cell types remains elusive, owing to technical challenges associated with bone imaging. To map nonhematopoietic cells and structures in bone and BM, we performed multicolor 3D imaging of osteoblastic, vascular, perivascular, neuronal and marrow stromal cells, and extracellular-matrix proteins in whole mouse femurs. We analyzed potential interactions between cells and molecules on the basis of colocalization of markers. Our results shed light on the markers expressed by different osteolineage cell types; the heterogeneity of vascular and perivascular cells; the neural subtypes innervating marrow and bone; the diversity of stromal cells; and the distribution of extracellular-matrix components. Our complete imaging data set is available for download and can be used in research in bone biology, hematology, vascular biology, neuroscience and extracellular-matrix biology.

  • Subscribe to Nature Biotechnology for full access:

    $250

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    , , & Structure and function of bone marrow hemopoiesis: mechanisms of response to ionizing radiation exposure. Cancer Biother. Radiopharm. 17, 405–426 (2002).

  2. 2.

    & Postnatal bone growth: growth plate biology, bone formation, and remodeling. in Pediatric Bone: Biology & Diseases (eds. Glorieux, F.H. et al.) (Elsevier, 2012).

  3. 3.

    & The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).

  4. 4.

    & Hematopoietic stem cell trafficking. StemBook (2008).

  5. 5.

    , & Bone regeneration: the stem/progenitor cells point of view. J. Cell. Mol. Med. 14, 103–115 (2010).

  6. 6.

    et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience 113, 155–166 (2002).

  7. 7.

    , & Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim. Biophys. Acta 1840, 2506–2519 (2014).

  8. 8.

    & Hemopoietic stromal microenvironment. Am. J. Hematol. 15, 195–203 (1983).

  9. 9.

    , , & Multicolor quantitative confocal imaging cytometry. Nat. Methods (2017).

  10. 10.

    Advances in the osteoblast lineage. Biochem. Cell Biol. 76, 899–910 (1998).

  11. 11.

    & Genetic mouse models for bone studies: strengths and limitations. Bone 49, 1242–1254 (2011).

  12. 12.

    , & Genetic control of bone formation. Annu. Rev. Cell Dev. Biol. 25, 629–648 (2009).

  13. 13.

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

  14. 14.

    et al. Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317 (2006).

  15. 15.

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

  16. 16.

    et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643 (2013).

  17. 17.

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

  18. 18.

    et al. Visualizing levels of osteoblast differentiation by a two-color promoter-GFP strategy: type I collagen-GFPcyan and osteocalcin-GFPtpz. Genesis 43, 87–98 (2005).

  19. 19.

    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).

  20. 20.

    et al. The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129, 3783–3793 (2002).

  21. 21.

    , , & The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 20, 349–356 (2005).

  22. 22.

    The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma in rats. Anat. Rec. 186, 161–184 (1976).

  23. 23.

    & The structure of developing bone marrow sinuses in extramedullary autotransplant of the marrow in rats. Anat. Rec. 171, 477–494 (1971).

  24. 24.

    , & The microcirculation of the bone marrow. Anat. Rec. 168, 55–68 (1970).

  25. 25.

    Vital microscopy of bone marrow in rabbit. Scand. J. Clin. Lab. Invest. 11 Supp 38, 1–82 (1959).

  26. 26.

    , , , & Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa. J. Biol. Chem. 260, 11300–11306 (1985).

  27. 27.

    et al. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J. Cell Biol. 118, 1511–1522 (1992).

  28. 28.

    et al. Expression of the CD34 gene in vascular endothelial cells. Blood 75, 2417–2426 (1990).

  29. 29.

    & Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line. J. Immunol. 141, 1925–1933 (1988).

  30. 30.

    et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274 (2009).

  31. 31.

    , , , & Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier. Dev. Dyn. 202, 325–332 (1995).

  32. 32.

    , , , & Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin. Blood 93, 165–175 (1999).

  33. 33.

    , & Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014).

  34. 34.

    , & Expression of CD34 in endothelial cells, hematopoietic progenitors and nervous cells in fetal and adult mouse tissues. Eur. J. Immunol. 25, 1508–1516 (1995).

  35. 35.

    , & Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J. Histochem. Cytochem. 54, 385–395 (2006).

  36. 36.

    , , & Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).

  37. 37.

    , , , & Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168 (2014).

  38. 38.

    et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 147, 1146–1158 (2011).

  39. 39.

    , , , & Bone marrow innervation regulates cellular retention in the murine haemopoietic system. Br. J. Haematol. 98, 569–577 (1997).

  40. 40.

    , & Noradrenergic and peptidergic innervation of the mouse femur bone marrow. Acta Histochem. 98, 453–457 (1996).

  41. 41.

    , & Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells Tissues Organs 181, 176–188 (2005).

  42. 42.

    , , & Biological basis of bone formation, remodeling, and repair-part III: biomechanical forces. Tissue Eng. Part B Rev. 14, 285–293 (2008).

  43. 43.

    & Markers for characterization of bone marrow multipotential stromal cells. Stem Cells Int. 2012, 975871 (2012).

  44. 44.

    et al. Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis. Biogerontology 2, 165–171 (2001).

  45. 45.

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

  46. 46.

    & Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors. J. Exp. Med. 150, 919–937 (1979).

  47. 47.

    et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat. Cell Biol. 15, 533–543 (2013).

  48. 48.

    et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015).

  49. 49.

    & Reproducibility: standardize antibodies used in research. Nature 518, 27–29 (2015).

  50. 50.

    Reproducibility crisis: blame it on the antibodies. Nature 521, 274–276 (2015).

  51. 51.

    , , , & Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469, 311–324 (2004).

  52. 52.

    et al. An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart. Development 135, 3599–3610 (2008).

  53. 53.

    & Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133, 3231–3244 (2006).

  54. 54.

    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).

  55. 55.

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

  56. 56.

    et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature 502, 232–236 (2013).

  57. 57.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

Download references

Acknowledgements

This study was supported in part by the SystemsX StemSysMed grant to T.S.

Author information

Author notes

    • Daniel L Coutu
    •  & Konstantinos D Kokkaliaris

    These authors contributed equally to this work.

Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.

    • Daniel L Coutu
    • , Konstantinos D Kokkaliaris
    • , Leo Kunz
    •  & Timm Schroeder

Authors

  1. Search for Daniel L Coutu in:

  2. Search for Konstantinos D Kokkaliaris in:

  3. Search for Leo Kunz in:

  4. Search for Timm Schroeder in:

Contributions

D.L.C. developed the method with K.D.K. and L.K. D.L.C. and K.D.K. designed the project. K.D.K., D.L.C. and L.K. performed the experiments. D.L.C. performed the analyses. D.L.C., K.D.K. and T.S. wrote the manuscript, on which all authors commented. T.S. obtained funding and supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Timm Schroeder.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1–3

  2. 2.

    Life Sciences Reporting Summary