Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche

Abstract

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation1, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal2,3,4, differentiation, ageing, niche5, and diversity6,7,8. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5+ HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7–35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5+) are directly attached to VE-cadherin+ cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Multi-step unbiased screening identifies Hoxb5 as a candidate LT-HSC marker.
Figure 2: Hoxb5 distinguishes between LT-HSC and non-LT-HSC.
Figure 3: Previously defined HSC immunophenotypes contain Hoxb5 cells.
Figure 4: LT-HSCs exhibit near-homogenous attachment to VE-cadherin+ cells.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data was deposited at GEO under accession number GSE77078.

References

  1. Spangrude, G. J., Heimfeld, S. & Weissman, I. L. Purification and characterization of mouse hematopoietic stem cells. Science 241, 58–62 (1988)

    Article  ADS  CAS  Google Scholar 

  2. Guo, W. et al. Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation. Nature 453, 529–533 (2008)

    Article  ADS  CAS  Google Scholar 

  3. Yilmaz, O. H. et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441, 475–482 (2006)

    Article  ADS  CAS  Google Scholar 

  4. Ito, K. et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 431, 997–1002 (2004)

    Article  ADS  CAS  Google Scholar 

  5. Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014)

    Article  ADS  CAS  Google Scholar 

  6. Dykstra, B. et al. Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell 1, 218–229 (2007)

    Article  CAS  Google Scholar 

  7. Beerman, I. et al. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc. Natl Acad. Sci. USA 107, 5465–5470 (2010)

    Article  ADS  CAS  Google Scholar 

  8. Lu, R., Neff, N. F., Quake, S. R. & Weissman, I. L. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nature Biotechnol. 29, 928–933 (2011)

    Article  CAS  Google Scholar 

  9. Uchida, N. & Weissman, I. L. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J. Exp. Med. 175, 175–184 (1992)

    Article  CAS  Google Scholar 

  10. Morrison, S. J. & Weissman, I. L. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1, 661–673 (1994)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  12. Gazit, R. et al. Fgd5 identifies hematopoietic stem cells in the murine bone marrow. J. Exp. Med. 211, 1315–1331 (2014)

    Article  CAS  Google Scholar 

  13. Hills, D. et al. Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis. Blood 117, 3521–3528 (2011)

    Article  CAS  Google Scholar 

  14. Seita, J. et al. Gene expression commons: an open platform for absolute gene expression profiling. PLoS ONE 7, e40321 (2012)

    Article  ADS  CAS  Google Scholar 

  15. Chan, C. K. F. et al. Identification and specification of the mouse skeletal stem cell. Cell 160, 285–298 (2015)

    Article  CAS  Google Scholar 

  16. Chan, C. K. F. et al. Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells. Proc. Natl Acad. Sci. USA 110, 12643–12648 (2013)

    Article  ADS  CAS  Google Scholar 

  17. Hosen, N. et al. Bmi-1-green fluorescent protein-knock-in mice reveal the dynamic regulation of bmi-1 expression in normal and leukemic hematopoietic cells. Stem Cells 25, 1635–1644 (2007)

    Article  CAS  Google Scholar 

  18. Yamamoto, R. et al. Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells. Cell 154, 1112–1126 (2013)

    Article  CAS  Google Scholar 

  19. Fathman, J. W. et al. Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential. Stem Cell Reports 3, 707–715 (2014)

    Article  CAS  Google Scholar 

  20. Oguro, H., Ding, L. & Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102–116 (2013)

    Article  CAS  Google Scholar 

  21. Herzenberg, L. A., Tung, J., Moore, W. A., Herzenberg, L. A. & Parks, D. R. Interpreting flow cytometry data: a guide for the perplexed. Nature Immunol. 7, 681–685 (2006)

    Article  CAS  Google Scholar 

  22. Kiel, M. J. et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005)

    Article  CAS  Google Scholar 

  23. Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014)

    Article  CAS  Google Scholar 

  24. Christensen, J. L. & Weissman, I. L. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc. Natl Acad. Sci. USA 98, 14541–14546 (2001)

    Article  ADS  CAS  Google Scholar 

  25. Osawa, M., Hanada, K., Hamada, H. & Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273, 242–245 (1996)

    Article  ADS  CAS  Google Scholar 

  26. Wright, D. E., Wagers, A. J., Gulati, A. P., Johnson, F. L. & Weissman, I. L. Physiological migration of hematopoietic stem and progenitor cells. Science 294, 1933–1936 (2001)

    Article  ADS  CAS  Google Scholar 

  27. Forsberg, E. C. et al. Molecular signatures of quiescent, mobilized and leukemia-initiating hematopoietic stem cells. PLoS ONE 5, e8785 (2010)

    Article  ADS  Google Scholar 

  28. Fleming, W. H. et al. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J. Cell Biol. 122, 897–902 (1993)

    Article  CAS  Google Scholar 

  29. Passegué, E., Wagers, A. J., Giuriato, S., Anderson, W. C. & Weissman, I. L. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J. Exp. Med. 202, 1599–1611 (2005)

    Article  Google Scholar 

  30. Jaiswal, S. et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138, 271–285 (2009)

    Article  CAS  Google Scholar 

  31. Moraga, I. et al. Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Cell 160, 1196–1208 (2015)

    Article  CAS  Google Scholar 

  32. Wu, J., Anczuków, O., Krainer, A. R., Zhang, M. Q. & Zhang, C. OLego: fast and sensitive mapping of spliced mRNA-Seq reads using small seeds. Nucleic Acids Res. 41, 5149–5163 (2013)

    Article  CAS  Google Scholar 

  33. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012)

    Article  CAS  Google Scholar 

  34. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge N. Neff and G. Mantalas for advice regarding RNA sequencing; B. Yu and A. Beel for providing critical input on imaging data; H. Nishikii for advice regarding imaging data; S. Karten for help in editing the manuscript; L. Jerabek and T. Storm for laboratory management; A. McCarty and C. Wang for animal care; P. Lovelace and J. Ho for FACS facility management; H. Zeng, Y. Li, and C. Wang for collaboration in generating the mouse model; and Y. Sato for technical assistance in Imaris software analysis. The authors would like to acknowledge ongoing support for this work: NCI and NHLBI of the NIH under award numbers R01 CA086065, U01 HL099999, and R01 HL058770, and by the Virginia and D. K. Ludwig Fund for Cancer Research (I.L.W.); Stanford University Medical Scientist Training Program (T32 GM007365) and NHLBI Ruth L. Kirschstein National Research Service Award (F30-HL122096) (J.Y.C.); and Human Frontier Science Program Long-Term Fellowships, the Uehara Memorial Foundation Research Fellowship, Toyobo Biotechnology Foundation Research Fellowship, and Kanzawa Medical Research Foundation Overseas study grants (M.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.

Author information

Authors and Affiliations

Authors

Contributions

J.Y.C. and M.M. contributed equally to this work, and either has the right to list himself first in bibliographic documents. M.M. and J.Y.C. conceived, performed, analysed, and oversaw the experiments, with suggestions from I.L.W. M.M. and J.Y.C. identified Hoxb5 as a LT-HSC marker, and made and characterized the Hoxb5–tri-mCherry mouse. S.K.W. and K.S.K. performed experiments and prepared figures under the supervision of M.M. and J.Y.C. S.Y. generated CUBIC data and evaluated the association with VE-cadherin vasculature. R.S. designed and performed RNA-seq and associated data analysis. J.S. and D.S. designed the gene expression commons for microarray analysis. D.S. provided critical advice regarding combined analysis of microarray and RNA-seq data. M.M., J.Y.C., S.K.W., K.S.K., and I.L.W. wrote the manuscript. H.N. and R.S. provided comments on the manuscript.

Corresponding authors

Correspondence to Masanori Miyanishi or Irving L. Weissman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 GEXC expression of previously reported HSC markers in mouse bone marrow.

a, Ideal expression pattern of HSC-specific genes (pink represents increased expression, blue represents decreased expression). b, Relative expression of Hoxb5 (top left), α-catulin/Ctnnal1 (top middle), Fgd5 (top right), CD150/Slamf1 (bottom left), Hoxb4 (bottom middle), Gfi-1 (bottom right) in haematopoietic and stromal populations as determined by microarray analysis.

Extended Data Figure 2 Gating scheme for HSC and progenitors.

a, Representative flow cytometry gating to isolate pHSCs, MPPs, and oligopotent progenitors from mouse bone marrow. Panels gated as shown after exclusion of doublets and dead cells. b, Hoxb5 reporter expression (red) in Flk2+ MPPs, megakaryocyte erythrocyte progenitor (MEP), granulocyte monocyte progenitor (GMP), common myeloid progenitor (CMP), and common lymphoid progenitor (CLP) populations compared to wild-type controls (blue). Values indicate the percentage of mCherry+ cells ± s.d. in each fraction for n = 3 mice.

Extended Data Figure 3 Hoxb5 is not expressed in CD45 bone marrow.

Hoxb5 reporter expression in the CD45 compartment within bone marrow of wild-type (red) and three Hoxb5–tri-mCherry mice (blue, orange, and green, n = 3 mice).

Extended Data Figure 4 FMO gating for Hoxb5+ signal.

Representative flow cytometry gating to separate mCherry (Hoxb5)-high, -low, and -negative populations in both wild-type and Hoxb5–tri-mCherry mice.

Extended Data Figure 5 Hoxb5 distinguishes between LT-HSCs and non-LT-HSCs.

a, Reconstitution kinetics in primary recipients 4, 8, and 12 weeks after receiving ten Hoxb5neg (n = 9 mice), Hoxb5lo (n = 13 mice), or Hoxb5hi (n = 18 mice) pHSCs. Each column represents an individual mouse. b, Reconstitution kinetics 4, 8, and 12 weeks after whole bone marrow secondary transplant. c, Reconstitution kinetics in primary recipients receiving three Hoxb5neg (n = 11 mice), Hoxb5lo (n = 12 mice), or Hoxb5hi (n = 14 mice) pHSCs. Each column represents an individual mouse. d, Reconstitution kinetics following secondary transplant of 100 sorted LSK Hoxb5 (n = 14 mice) or Hoxb5+ (n = 9 mice) cells and 2 × 105 supporting cells.

Extended Data Figure 6 Limiting dilution analysis of Hoxb5+ and Hoxb5 pHSCs.

Limiting dilution results of ten- and three-cell transplants of Hoxb5hi (red, n = 18 mice for ten-cell and n = 14 mice for three-cell), Hoxb5lo (green, n = 13 mice for ten-cell and n = 12 mice for three-cell), and Hoxb5neg (blue, n = 9 mice for ten-cell and n = 11 mice for three-cell). Frequency of LT/ST-HSCs by limiting dilution analysis is 1 in 2.1 for Hoxb5hi, 1 in 2.4 for Hoxb5lo, and 1 in 16.1 for Hoxb5neg cells.

Extended Data Figure 7 Previously defined HSC immunophenotypes contain Hoxb5 cells.

Representative HSC gating strategy for various HSC definitions after exclusion of doublets and dead cells. a, CD11a (LSK CD150+CD34−/loCD11a)21. b, HSC-1 (LSK CD150+CD48−/loCD229−/loCD244)20. c, Fraction I (LSK CD150+CD34−/loCD41)18. d, CD150+CD48CD41 cells22 (n = 5 mice).

Extended Data Figure 8 Specificity of Hoxb5 as a single marker for LT-HSCs.

a, Flow cytometry plots of wild type (top row) and Hoxb5–tri-mCherry (bottom row) after excluding doublets, dead cells, autofluorescence, and gating on Hoxb5+ events. Frequencies shown are percentage in gate ± s.d. in each fraction (n = 3 mice).

Extended Data Figure 9 Comparison of processing methods on pHSC and Hoxb5+ LT-HSC yield.

a, b, Relative frequency of pHSCs (a) and Hoxb5+ LT-HSCs (b) in tibial plugs (flushed) (n = 6 mice) compared to tibial plugs plus bones (crushed) (n = 6 mice).

Extended Data Figure 10 Hoxb5+ HSCs are evenly distributed in the tibia.

a, Distribution of Hoxb5+ cells (red and arrows) in bone marrow in 3D-reconstructed images. Nuclei are counterstained with DAPI (blue) wild-type (top panel) Hoxb5–tri-mCherry (middle and bottom panel). Scale bar, 100 μm. b, Cartoon representing the location of the proximal, medial, and distal sampling. c, Representative 3D-reconstructed images of Hoxb5+ cells (red) in proximal (left column), medial (middle column), and distal (right column) regions of the tibia. Scale bar, 150 μm. Nuclei are counterstained with DAPI (blue); n = 3 mice.

Supplementary information

Supplementary Table 1

The file shows the marker definitions of the cell populations used in gene microarray profiling. (XLSX 30 kb)

Supplementary Table 2

This file contains a list of HSC-specific genes found by screening the haematopoietic sub-fractions in the adult mouse bone marrow. (XLSX 33 kb)

Supplementary Table 3

This file contains the RNA expression of α-catulin in Hoxb5 subsets within the pHSC gate. (XLSX 9 kb)

Supplementary Table 4

Tis file contains a list of flow cytometry reagents used in this study, including, antigen, clone, fluorophore, and manufacturer. (XLSX 10 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, J., Miyanishi, M., Wang, S. et al. Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 530, 223–227 (2016). https://doi.org/10.1038/nature16943

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature16943

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing