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

Nature Biotechnology volume 35, pages 1202–1210 (2017)Cite this article

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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.

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Figure 1: Complexity of the BM microenvironment.
Figure 2: Osteoblasts and skeletal cells.
Figure 3: Vascular and perivascular cells.
Figure 4: Neuronal cells.
Figure 5: Extracellular matrix.
Figure 6: Stromal cells.

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Acknowledgements

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

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Author notes

  1. Daniel L Coutu and Konstantinos D Kokkaliaris: These authors contributed equally to this work.

Authors and Affiliations

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

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

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  1. Daniel L Coutu
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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.

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Correspondence to Timm Schroeder.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Display of colocalization analysis results.

The Coloc module of Imaris was used for voxel colocalization analyses. The software tools were used to select thresholds for two given markers (marker A and B here), which are indicated by the red box. The value given by x represents the percentage of voxels with a fluorescence intensity for marker A above the threshold that also have a fluorescence intensity for marker B above threshold. Inversely, the value given by y represents the percentage of voxels with a fluorescence intensity for marker B above the threshold that also have a fluorescence intensity for marker A above threshold. The colocalization data presented is derived from the full or partial image (see figure legends for details) shown in the same figure panel.

Supplementary Figure 2 Identification of distinct anatomical locations and cell types in adult mouse femurs without landmark staining.

a) Examples showing how to identify trabecular (i) and cortical (ii) bone surfaces, as well as growth plate (iii) and articular (iv) cartilage using only a vascular marker (collagen 1 is here shown to validate proper identification). i) Trabecular bone in the metaphyseal area can be identified by the complete absence of vascular sinusoids (dotted lines) although it can be irrigated by rare arterioles or capillaries (arrowheads). ii) Similarly, cortical bone shows a complete absence of sinusoids but is traversed by some arterioles (not shown). At the bone surface (dotted line), vasculature consists of capillaries running parallel to the long axis of the bone (arrowheads), as opposed to the central marrow where mainly sinusoids are present and radiate axially from the center of the marrow cavity. iii, iv) Growth plate and articular cartilage show a complete absence of vasculature and are located in very specific anatomical locations. b) Adipocytes and megakaryocytes can be difficult to distinguish by inexperienced researchers without specific staining. However, adipocytes are typically bigger and rounder than megakaryocytes. Also, a cytoplasmic staining in adipocytes clearly shows a lack of staining in the large lipid droplet (upper left) whereas a lipid droplet staining shows a smaller spherical staining (lower left). Cytoplasmic (upper right) or membrane (lower right) stainings in megakaryocytes shows cells with irregular shapes, in many of which we can observe multiple/complex nuclei (arrowheads).

Supplementary Figure 3 Some Nes-GFP-expressing cells are ALP+CD31− osteoblastic cells.

Images show a zoom of data presented in Figure 2b and only four optical sections (total thickness 9.96μm). Near the distal growth plate of the femur, Nes-GFP expressing cells (green, white arrowheads) are closely associated with CD31+ blood vessels (red), but are osteoblastic cells expressing ALP (grey). Scale bars: 20μm

Supplementary Figure 4 Osx-CreERT (tdTomato) reporter shows a similar expression pattern to that of the Osx-GFP reporter.

7 weeks old female Osx-CreERT mice received 2mg 4-hydroxytamoxifen intraperitoneally and femurs were harvested three days later. Expression of the tdTomato reporter at day 3 post-4OHT recapitulates that of the Osx-GFP reporter (see figure 2c). Scale bar of detail 70μm.

Supplementary Figure 5 Antibody staining for osteocalcin partially overlaps with OC-YFP expression.

Images show a zoom of data presented in Figure 2e and only four optical sections (total thickness 9.96μm). Near the distal growth plate of the femur, OC-YFP expressing cells (green, white arrowheads) line collagen 1+ (grey) trabecular bone surfaces and stain positive for osteocalcin antibody (red). Osteocalcin antibody also detects OC+ matrix away from YFP+ cells in trabecular bone and adjacent to the growth plate (black arrowheads). Scale bars: 30μm

Supplementary Figure 6 Expression of mesenchymal-progenitor-cell markers near the distal growth plate.

Images show a zoom of data presented in Figure 2f and only four optical sections (total thickness 9.96μm). We can observe CD140a+Sca1+ cells lining CD31+ blood vessels (white arrowheads), whereas bone lining cells are either FGFR2+ (black arrowheads) or FGFR2+CD140a+ (white arrows). Scale bars 20 μm.

Supplementary Figure 7 CD105 is not a panendothelial marker.

(i) Most arterioles do not express CD105, but only CD31 and Sca1 (white arrowheads) potentially marking distinct arteriolar sub-types. (ii) Diaphyseal arteries marked by SM22 expression are CD105-CD31+Sca1+. (iii) Strong CD105 expression in the endothelial wall of the central sinus.

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Coutu, D., Kokkaliaris, K., Kunz, L. et al. Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules. Nat Biotechnol 35, 1202–1210 (2017). https://doi.org/10.1038/nbt.4006

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