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 isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. 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.

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 panel left), Alpha-catulin/Ctnnal1 (top panel middle), Fgd5 (top panel right), CD150/Slamf1 (bottom panel left), Hoxb4 (bottom panel middle), Gfi-1 (bottom panel right) in haematopoietic and stromal populations as determined by microarray.

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, MEP, GMP, CMP, CLP compared to wild-type controls (blue). Values indicate the percentage of mCherry-positive 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 CD45-negative compartment within bone marrow. Wild type (Red) and three Hoxb5-tri-mCherry mice (blue, orange, and green, n=3 mice).

Extended Data Figure 4. FMO gating for Hoxb5 positive signal

Representative flow cytometry gating to separate mCherry (Hoxb5) hi, lo, and negative populations in both wild-type and Hoxb5-tri-mCherry

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

a, 4, 8, and 12-week reconstitution kinetics in primary recipients receiving 10 Hoxb5^neg (n=9 mice), Hoxb5^lo (n=13 mice), or Hoxb5^hi (n=18 mice) pHSCs. Each column represents an individual mouse. b, 4, 8, and 12-week reconstitution kinetics following WBM secondary transplant. c, Reconstitution kinetics in primary recipients receiving three Hoxb5^neg (n=11 mice), Hoxb5^lo (n=12 mice), or Hoxb5^hi (n=14 mice) pHSCs. Each column represents an individual mouse. d, Reconstitution kinetics following secondary transplant of 100 sorted Lin⁻cKit⁺Sca1⁺ (LSK) Hoxb5^neg (n=14 mice) or Hoxb5^pos (n=9 mice) cells and 2×10⁵ supporting cells.

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

Limiting dilution results of 10 cell and 3 cell transplants of Hoxb5^hi (red, n=18 mice for 10 cell and n= 14 mice for 3 cell), Hoxb5^lo (green, n=13 mice for 10 cell and n=12 mice for 3 cell), and Hoxb5^neg (blue, n=9 mice for 10 cell and n=11 mice for 3 cell). Frequency of LT-HSC/ST-HSC by limit dilution for Hoxb5^hi is 1 in 2.1, for Hoxb5^lo is, 1 in 2.4 and Hoxb5^neg is 1 in 16.1.

Extended Data Figure 7. Hoxb5-negative cells contaminate previous HSC definitions

Representative HSC gating strategy for various HSC definitions after exclusion of doublets and dead cells. a, CD11a⁻ (LSK CD150⁺CD34^–/loCD11a⁻). b, HSC-1 (LSK CD150⁺CD48^–/loCD229^–/loCD244⁻). c, Fraction I (LSK CD150⁺CD34^–/loCD41⁻). d, CD150⁺CD48⁻CD41⁻ cells (n=5 mice).

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

a, Flow cytometry plots of wild type (top row) and Hoxb5-tri-mCherry (bottom row) after excluding doublets, dead cells, autofluorescence, and gated on Hoxb5-positive events. Frequencies shown are percent gate ± S.D. in each fraction (n=3 mice).

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

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

Extended Data Figure 10. Hoxb5-positive HSC are evenly distributed in the tibia

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

Figure 1. Multi-step unbiased screening identifies Hoxb5 as an LT-HSC marker

a, Microarray heat map depicting relative expression (pink = high, blue = low) of HSC-specific genes in haematopoietic and stromal populations. Each row represents a gene; each subcolumn a replicate microarray; each labeled column a category of cell populations. The 45 genes in the top panel displayed limited activity in all non-HSC populations examined. b, Transcriptional profiling by RNA-seq of the 45 genes from (a). Three genes (top panel) exceeded the estimated threshold for detection (FPKM > 7.0) in HSCs while showing minimal expression (FPKM < 2.5) in MPPa and MPPb populations. c, Heterogeneity of expression for the three remaining candidate genes in HSCs as assessed by single-cell qPCR. d, Venn diagram reflecting the four-step screen that yielded Hoxb5 as an ideal candidate in the HSC transcriptome. e, Targeting strategy to generate a triple-mCherry Hoxb5 knock-in mouse reporter line (Hoxb5-tri-mCherry). f, Hoxb5 reporter expression (red) in immunophenotypic HSCs (pHSC) and MPPs compared to wild-type controls (blue). Values indicate the percentage of mCherry-positive cells ± S.D. in each fraction for n=3 mice.

Figure 2. Hoxb5 distinguishes between LT-HSC and non-LT-HSC

a, Experimental schematic for long-term haematopoietic reconstitution assays. CD45.1⁺ recipient mice were lethally irradiated and competitively transplanted with 10 or 3 Hoxb5-tri-mCherry HSCs and 2×10⁵ CD45.1⁺/CD45.2⁺ supporting cells. For secondary transplants, 1×10⁷ whole bone marrow (WBM) cells or 100 sorted LSK cells were transferred from primary recipient mice. b, 16 week chimerism in primary recipients receiving 10 Hoxb5^neg (n=9 mice), Hoxb5^lo (n=13 mice), or Hoxb5^hi (n=18 mice) pHSCs. Each column represents an individual mouse. c, 16 week chimerism following WBM secondary transplant. d, Average donor lineage contribution in 10-cell primary transplants. Error bars denote S.D. e, Individual donor chimerism by lineage in WBM secondary recipients. Each line represents an individual mouse (n=6 mice for Hoxb5^neg, n=5 mice for Hoxb5^lo, and n=8 mice for Hoxb5^hi).

Figure 3. Hoxb5-negative cells contaminate previous HSC definitions

Flow cytometry plots of 12-week-old C57BL/6 bone marrow depicting Hoxb5-tri-mCherry reporter activity in previously reported HSC immunophenotypes. a, pHSC (LSK CD150⁺CD34^–/loFlk2⁻) Hoxb5⁺(red) and Hoxb5⁻ (blue). b, CD11a⁻ (LSK CD150⁺CD34^–/loCD11a⁻). c, HSC-1 (LSK CD150⁺CD48^–/loCD229^–/loCD244⁻). d, Fraction I (LSK CD150⁺CD34^–/loCD41⁻). e, CD150⁺CD48⁻CD41⁻ cells, currently used to identify HSCs in situ. Wild type FMO used to define Hoxb5 negativity for each panel. f, Summary percentage of Hoxb5-positive and negative cells in characterized HSC subfractions. Error bars denote S.D. (n=5 mice). Refer to Extended Data Fig. 7a–d for gating scheme.

Figure 4. LT-HSC exhibit near homogenous attachment to VE-cadherin positive cells

a, Tissue preparation and representative images of tibial bone marrow plug after paraformaldehyde fixation (Day 0), treatment with Reagent-1 (Day 7, Day 14) . b, Localization of Hoxb5-positive cells (red and arrows) and VE-cadherin-positive cells (green) in 3D-reconstructed images. Scale bar 30µm c, Representative 2D images of direct (top panel) and non-direct (bottom panel) association of Hoxb5-positive cells (red) with VE-cadherin-positive cells (green). Scale bar 10µm. d, Frequency of Hoxb5-positive cells (n=287 cells, from n=3 mice) and random spots (n=600 spots, from n=3 mice) plotted against proximity to VE-cadherin-positive cells. unpaired student’s t-test (p<0.0001). e, Average number of Hoxb5-positive cells in proximal, medial, and distal regions of tibia (n=3 mice). unpaired student’s t-test.