. 2021 Mar;591(7850):438-444.

doi: 10.1038/s41586-021-03298-5. Epub 2021 Feb 24.

A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis

Bo Shen¹, Alpaslan Tasdogan¹, Jessalyn M Ubellacker¹, Jingzhu Zhang¹, Elena D Nosyreva², Liming Du¹, Malea M Murphy¹, Shuiqing Hu³, Yating Yi⁴, Nergis Kara¹, Xin Liu¹, Shay Guela¹, Yuemeng Jia¹, Vijayashree Ramesh¹, Claire Embree¹, Evann C Mitchell¹, Yunduo C Zhao⁵, Lining A Ju⁵, Zhao Hu⁵, Genevieve M Crane⁶, Zhiyu Zhao¹, Ruhma Syeda³, Sean J Morrison^{7

8}

Affiliations

¹ Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
² Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA.
³ Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁴ Department of Restorative Sciences, School of Dentistry, Texas A&M University, Dallas, TX, USA.
⁵ School of Biomedical Engineering, Heart Research Institute and Charles Perkins Center, The University of Sydney, Darlington, New South Wales, Australia.
⁶ Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA.
⁷ Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA. Sean.Morrison@UTSouthwestern.edu.
⁸ Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA. Sean.Morrison@UTSouthwestern.edu.

PMID: 33627868
PMCID: PMC7979521
DOI: 10.1038/s41586-021-03298-5

A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis

Bo Shen et al. Nature. 2021 Mar.

. 2021 Mar;591(7850):438-444.

doi: 10.1038/s41586-021-03298-5. Epub 2021 Feb 24.

Authors

Affiliations

¹ Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
² Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA.
³ Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁴ Department of Restorative Sciences, School of Dentistry, Texas A&M University, Dallas, TX, USA.
⁵ School of Biomedical Engineering, Heart Research Institute and Charles Perkins Center, The University of Sydney, Darlington, New South Wales, Australia.
⁶ Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA.
⁷ Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA. Sean.Morrison@UTSouthwestern.edu.
⁸ Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA. Sean.Morrison@UTSouthwestern.edu.

PMID: 33627868
PMCID: PMC7979521
DOI: 10.1038/s41586-021-03298-5

Abstract

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors^1-6. LEPR⁺ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors^7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin^13,14, distinguishes peri-arteriolar LEPR⁺ cells poised to undergo osteogenesis from peri-sinusoidal LEPR⁺ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR⁺osteolectin⁺ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin⁺ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin⁺ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin⁺ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin⁺ cells depleted osteolectin⁺ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.

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Conflict of interest statement

COMPETING INTERESTS

The authors declare no competing interests.

Figures

**Extended Data Figure 1.. *Oln*^mT reporter mice showed that Osteolectin is expressed by osteoblasts, osteocytes, and chondrocytes in addition to peri-arteriolar LepR⁺ stromal cells.**
(**a-c**) The mouse *Osteolectin* gene was modified to insert an mTomato-WPRE-pA cassette between the 5’ untranslated region and exon 3 to generate the targeting vector. These sites were selected to avoid disrupting conserved intronic sequences. Open boxes indicate untranslated regions and black boxes indicate translated regions of *Osteolectin*. The targeted founder mouse (F0) was identified by southern blotting (b) using 5’ and WPRE probes (black bars). (c) PCR genotyping of genomic DNA confirmed germline transmission of the *Oln*^mT allele. Mice were backcrossed at least three times onto a C57BL/Ka background before analysis. (d) *Osteolectin* transcript levels by qRT-PCR in *Oln*-Tomato⁺ and *Oln*-Tomato⁻ bone marrow cells (3 mice analyzed in 3 independent experiments). (e) Flow cytometry gates to distinguish *Oln*-mTomato⁺LepR⁺ from *Oln*-mTomato⁻LepR⁺ stromal cells in enzymatically dissociated bone marrow. (f) *Osteolectin* transcript levels by qRT-PCR in *Oln*-Tomato⁺LepR⁺ and *Oln*-Tomato⁻LepR⁺ bone marrow cells (4 mice analyzed in 4 independent experiments). (g) We did not detect *Oln*-Tomato in VE-cadherin-expressing bone marrow endothelial cells (4 mice per genotype analyzed in 4 independent experiments). (h and i) Femur epiphysis (h) and diaphysis (i) from *Oln*^mT/+; *Col1a1*2.3*-EGFP mice (images are representative of 3 independent experiments from 8–10-week-old mice). In panel h, the arrowhead points to *Oln*-Tomato⁺*Col1a1***2.3*-EGFP⁺ osteoblasts in trabecular bone and the arrow points to *Oln*-Tomato⁺*Col1a1***2.3*-EGFP⁻ hypertrophic chondrocytes in the growth plate (scale bar = 100μm). Panel i shows *Oln*-Tomato⁺*Col1a1*2.3*-EGFP⁺ osteoblasts at the endosteum (arrow), *Oln*-Tomato⁺ osteocytes (arrowhead), and *Oln*-Tomato⁺ periosteal cells (asterisk) (scale bars = 40μm). All data represent mean ± SD.

**Extended Data Figure 2.. Bone marrow *Oln*-mTomato⁺ cells localized mainly around arterioles in the diaphysis.**
(a) A low magnification view of the femur diaphysis showed *Oln*-Tomato⁺ stromal cells associated with arterioles enriched in the center of the marrow. In this image, arteriolar and sinusoidal blood vessels were distinguished based on size, morphology, and continuity of the basal lamina, visualized using anti-laminin antibody staining as previously described (images are representative of 3 independent experiments; scale bar = 200μm). (b) *Oln*^mT/+ femur bone marrow showing that, in contrast to the diaphysis (Fig. 1h), most Sca-1⁺ arterioles were not surrounded by *Oln*-Tomato⁺ stromal cells in the metaphysis. The *Oln*-Tomato⁺ cells in this panel were osteoblasts and osteocytes associated with trabecular bone (scale bar = 500μm). **(c, d**) Gene set enrichment analysis showing significant enrichment of genes associated with osteogenesis in CD45⁻Ter119⁻CD31⁻*Scf*-GFP⁺*Oln*-mTomato⁺ stromal cells and adipogenesis in CD45⁻Ter119⁻CD31⁻*Scf*-GFP⁺*Oln*-mTomato⁻ stromal cells (FDR, false discovery rate; NES, normalized enrichment score; 4 mice analyzed in 4 independent experiments). (**e-f**) The mouse *Osteolectin* gene was modified by inserting an iCreER-WPRE-pA cassette between the 5’ untranslated region and exon 3 to generate the targeting vector. These sites were selected to avoid disrupting conserved intronic sequences. Open boxes indicate untranslated regions and black boxes indicate translated regions of *Osteolectin*. The targeted founder mouse (F0) was identified by Southern blotting (f) using 5’ and WPRE probes (black bars). (g) PCR genotyping of genomic DNA confirmed germline transmission of the *Oln*^iCreER allele. Mice were backcrossed at least three times onto a C57BL/Ka background before analysis. (h) Deep imaging of *Oln*^iCreER/+; *Rosa26*^{loxp-tdTomato/+} femur bone marrow 3 days after tamoxifen administration at 2 months of age, showing that *Oln*-Tomato⁺ cells were exclusively peri-arteriolar in the diaphysis (images are representative of 3 independent experiments; scale bar = 200μm). (i and j) Deep imaging of the femur epiphysis (i) and diaphysis (j) one month after tamoxifen administration at 2 months of age (images are representative of 3 independent experiments). Panel i shows *Oln*-Tomato⁺ hypertrophic chondrocytes in the growth plate (arrow) and *Oln*-Tomato⁺*Col1a1***2.3*-EGFP⁺ osteoblasts in trabecular bone (arrowhead; scale bar = 60mm). Panel j shows *Oln*-Tomato⁺*Col1a1***2.3*-EGFP⁺ osteoblasts at the endosteum (arrow), an *Oln*-Tomato⁺ osteocyte (arrowhead), and *Oln*-Tomato⁺ periosteal cells (asterisk; scale bars = 30μm). (k) *Col1a1*-CreER; *Rosa26*^{loxp-tdTomato/+}; *Col1a1*2.3*-EGFP mice were treated with tamoxifen at 2 months of age and the percentage of *Col1a1*2.3*-EGFP⁺ osteoblasts that were Tomato⁺ was assessed 3 days to 1 month later (3 mice per time point analyzed in 3 independent experiments). All data represent mean ± SD.

**Extended Data Figure 3.. Osteolectin⁺ cells create a niche for CLPs but not other hematopoietic stem/progenitor cells.**
(a) 2-month-old *Oln*^iCreER/+; *Rosa26*^{loxp-tdTomato/+} mice were sublethally irradiated 3 days after tamoxifen administration. Two weeks later, none of the adipocytes in the bone marrow were Tomato⁺ (image representative of 3 independent experiments; scale bar = 60μm). (b) Representative H&E stained sections from ossicles formed by *Oln*-Tomato⁺ (left) or *Oln*-Tomato⁻ (right) stromal cells sorted from *Oln*^mT/+ mice showing bone (arrowheads), hematopoietic cells (arrow), and adipocytes (asterisk; images are representative of 5 independent experiments; scale bar = 100μm). (c-e) *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl littermate controls were treated with tamoxifen at 2-months of age. One month later, blood from *Oln*^iCreER/+; *Scf*^fl/fl mice showed normal white blood cell (c), red blood cell (d) and platelet (e) counts (6 mice per genotype analyzed in 3 independent experiments). (f-k) *Oln*^iCreER/+; *Scf*^fl/fl mice and littermate controls exhibited no significant differences in the frequencies of B220⁺ B cells (f), CD3⁺ T cells (g), Gr-1⁺Mac-1⁺ myeloid cells (h), CD41⁺ megakaryocyte lineage cells (i), CD71⁺/Ter119⁺ erythroid lineage cells (j), HSCs or MPPs in the spleen (k; 6 mice per genotype analyzed in 3 independent experiments). (l) Bone marrow cells from *Oln*^iCreER/+; *Scf*^fl/fl mice and littermate controls gave similar levels of donor cell reconstitution upon competitive transplantation into irradiated mice (5 donor mice per genotype analyzed in 3 independent experiments). The differences between *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl mice were not statistically significant in c-l. (m) *Oln*^mT/+; *Scf*^GFP/+ femur bone marrow showing *Oln*-Tomato⁺ osteoblasts at the endosteum were negative for *Scf*-GFP, while peri-arteriolar *Oln*-Tomato⁺ stromal cells were positive for *Scf*-GFP (representative of 3 independent experiments; scale bar = 80μm). (n) *Oln*^mT/+; *Col1a1***2.3*-EGFP femur bone marrow showing *Col1a1***2.3*-EGFP⁺ osteoblasts at the endosteum were *Oln*-Tomato⁺ (representative of 3 independent experiments; scale bar = 100μm). All data represent mean ± SD. Statistical significance was assessed using matched samples two-way ANOVAs followed by Sidak’s (c-e, and k-l) or Tukey’s (f-j) multiple comparisons tests.

**Extended Data Figure 4.. Flow cytometry gating strategy for the isolation of hematopoietic stem and progenitor cell populations.**
(a-d) Representative flow cytometry gates used to identify the hematopoietic stem and progenitor cell populations in the bone marrow (a, b), and T lineage progenitors in the thymus (c, d). The markers used to identify each of the cell populations characterized in this study are listed in Supplementary Table 1. (e) Representative flow cytometry gates showing that more than 50% of IL7Rα⁺Lineage⁻ cells were Lin⁻Sca1^lowc-kit^lowIL7Ra⁺Flt3⁺ CLPs and most of the non-CLP IL7Rα⁺Lineage⁻ (Flt3⁻) cells were CD19⁺, likely to be other early B lineage progenitors (4 mice analyzed in 3 independent experiments). All data represent mean ± SD.

**Extended Data Figure 5.. *Oln*-mTomato⁺ osteoblasts and osteocytes in the metaphysis do not express *Scf*-GFP and *Oln*-mTomato⁺ cells in the diaphysis expand during fracture healing.**
The differences among treatments in cell frequencies were also evident in absolute numbers. (a-c) Epiphysis (a) and metaphysis (**b, c**) of *Oln*^mT/+; *Scf*^GFP/+ femur bone marrow showing that hypertrophic chondrocytes (arrowhead in a; scale bars = 30μm) as well as osteoblasts and osteocytes associated with trabecular bone (arrowhead in c) were negative for *Scf-*GFP (images are representative of 3 independent experiments; scale bars = 400μm (b) and 100μm (c)). Most of the *Oln-*Tomato staining in the metaphysis reflects *Col1a1*2.3-GFP*⁺ osteoblasts and osteocytes associated with trabecular bone, as shown in Fig. 1d and Extended Data Fig. 2b. The boxed area in panel b is magnified in panel c. (d) Image of the metaphysis of *Scf*^GFP/+ femur bone marrow showing limited non-specific staining by anti-tdTomato antibody (scale bar = 100mm). (e-g) *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl littermate control mice were fed tamoxifen chow from 2–4 months of age. They exhibited no significant differences in the frequencies of HSCs, MPPs, GMPs, MEPs, or CMPs in the bone marrow (e), or B220⁺ B cells (f), or CD3⁺ T cells (g) in the bone marrow or spleen. The differences between *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl mice were not statistically significant in e-g (5 mice per genotype analyzed in 3 independent experiments). (**h, i**) Three days after tamoxifen, femurs were fractured in 2-month-old *Oln*^mT/iCreER; *Scf*^fl/fl mice and *Oln*^mT/+; *Scf*^fl/fl littermate controls then the bone marrow was analyzed two weeks later. HSC and MPP frequencies did not significantly change during fracture healing. In contrast, CLP frequencies significantly increased in *Oln*^mT/+; *Scf*^fl/fl control but not in *Oln*^mT/iCreER; *Scf*^fl/fl mice (h). The frequency of Osteolectin⁺ cells significantly increased in both *Oln*^mT/iCreER; *Scf*^fl/fl mice and *Oln*^mT/+; *Scf*^fl/fl controls (I; 5 mice per genotype analyzed in 3 independent experiments). (j) Localization of IL7Rα⁺Lineage⁻ lymphoid progenitors in the marrow of the fractured as compared to control (CON) femur (4 mice per treatment analyzed in 4 independent experiments). All data represent mean ± SD. Statistical significance was assessed using matched samples two-way ANOVAs followed by Sidak’s (e-i) or Tukey’s multiple comparisons tests (h), or Cochran-Mantel-Haenszel test (j).

**Extended Data Figure 6.. CLPs and peri-arteriolar Osteolectin⁺ cells are depleted during aging but most other hematopoietic stem and progenitor cell populations are not.**
The differences among treatments in cell frequencies were also evident in absolute numbers. (a-d) *Oln*^mT/+ mice received daily subcutaneous injections with PBS or 40 μg/kg human PTH for 28 days. PTH treated mice exhibited significantly thicker cortical bone (**a, b**) and significant reductions in the frequencies of Osteolectin⁺ cells (c) and CLPs (d; 8 mice per treatment analyzed in 4 independent experiments). Micro CT images (b, scale bar = 800μm) of cortical bone are representative of 8 independent experiments. (e, f) The frequency of LepR⁺ cells (e) and the percentage of LepR⁺ cells that were *Oln*-mTomato⁺ (f) in *Oln*^mT/+ bone marrow at 2 to 18 months of age (8 mice per time point analyzed in 4 independent experiments). (g) Femur bone marrow from an 18-month-old *Oln*^mT/+ mouse showing an arteriole surrounded by *Oln*-mTomato⁺ cells (arrowhead) and an arteriole lacking *Oln*-mTomato⁺ cells (arrow) in the diaphysis (image is representative of 4 independent experiments; scale bar = 100μm). (h) The frequency of CLPs in *Oln*^mT/+ mice at 2 to 18 months of age. (i-m) During aging, the frequencies of HSCs (i) and MPPs (j) in the bone marrow increased while the frequencies of GMPs (k), CMPs (l), and MEPs (m) did not significantly change (8 mice per time point analyzed in 4 independent experiments). (n) Localization of IL7Rα⁺Lineage⁻ cells in the bone marrow of 2- and 18-month old mice (7 mice per time point analyzed in 3 independent experiments). (o) Aging significantly depleted Osteolectin⁺ cells associated with arterioles in both endosteal and non-endosteal regions of diaphysis bone marrow (8 mice per time point analyzed in 4 independent experiments). (p) Number of IL7Rα⁺Lineage⁻ cells per 100 μm of Osteolectin⁺ or Osteolectin⁻ arteriole at 2 and 18 months of age (7 mice per time point analyzed in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using paired t-tests (a, c, d, o, p), one-way ANOVA followed by Dunnett’s multiple comparisons test (e), matched samples two-way ANOVAs followed by Dunnett’s multiple comparisons tests (f, h-m), Cochran-Mantel-Haenszel test (n), or Mann–Whitney tests followed by Holm-Sidak’s multiple comparisons adjustments (o).

**Extended Data Figure 7.. *Oln*^iCreER/+; *Scf*^fl/fl mice exhibited reduced lymphopoiesis and impaired bacterial clearance after *Listeria* infection.**
The same differences that were evident among treatments in the frequencies of cell populations were also evident in absolute numbers. (**a-c**) *Oln*^iCreER/+; *Scf*^fl/fl mice and littermate controls were treated with tamoxifen at 2 months of age and then 3 days later were administered *Listeria* orally. Relative to *Scf*^fl/fl littermate controls, *Oln*^iCreER/+; *Scf*^fl/fl mice had decreased B (a) and T (b) cell counts in the spleen 5 days after *Listeria* infection and increased bacterial CFUs in the spleen 10 days after infection (c; 11–16 mice per genotype analyzed in 3 independent experiments). (**d-k**) *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl littermate controls were fed tamoxifen from 2 to 4-months of age then 3 days later were administered *Listeria* intraperitoneally. Relative to *Scf*^fl/fl littermate controls, *Oln*^iCreER/+; *Scf*^fl/fl mice exhibited reduced frequencies of Pre-proB cells, but not ProB or PreB cells, in the bone marrow (d) as well as ETPs and DN1 cells in the thymus (e) at 5 and/or 10 days after infection. *Oln*^iCreER/+; *Scf*^fl/fl mice also exhibited reduced numbers of NK cells in the spleen (f) as well as B cells (g), T cells (h), and NK cells (i) in mesenteric lymph nodes (mLN) at 5 and 10 days after infection. (j) *Oln*^iCreER/+; *Scf*^fl/fl mice exhibited increased bacterial CFUs in mLN at 5 and 10 days after infection. (k) The percentage of CD3⁺ T cells that were IFN-γ⁺ did not significantly differ between the spleens of *Oln*^iCreER/+; *Scf*^fl/fl mice and littermate controls (18–27 mice per genotype analyzed in 3 independent experiments). (l-n) Compared to *Scf*^fl/fl controls, *Oln*^iCreER/+; *Scf*^fl/fl mice had decreased spleen B (l) and T (m) cell counts, and increased spleen bacterial CFUs (n) at 5 and 10 days after infection with *Listeria* (21–27 mice per genotype analyzed in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using matched samples two-way ANOVAs followed by Sidak’s multiple comparisons tests (a, b, d-n) or Wilcoxon matched-pairs test (c).

**Extended Data Figure 8.. Mechanical stimulation is required for the maintenance of peri-arteriolar, but not peri-sinusoidal, niches for lymphoid progenitors in the bone marrow.**
(a) The effect of voluntary running for 4 weeks on femoral cortical bone mineral density (6 mice per treatment analyzed in 3 independent experiments). (b) Voluntary running for 4 weeks did not significantly affect the frequencies of HSCs, MPPs, GMPs, MEPs, CMPs, or CLPs in calvarium bone marrow (5 mice per treatment analyzed in 3 independent experiments). (c-h) Hindlimb unloading (c) for 2 weeks did not significantly affect forelimb cortical bone mineral density (d), cortical thickness (e), the percentage of LepR⁺ cells that were *Oln*-mTomato⁺ (f), or the frequencies of HSCs, MPPs, GMPs, MEPs, CMPs, or CLPs in humerus bone marrow (g). (h) Hindlimb unloading for 2 weeks did significantly reduce hindlimb (femur) cortical bone mineral density (5 mice per treatment analyzed in 3 independent experiments). (i) All point current amplitude histograms of the electrical recordings in Fig. 4h. The single channel conductance was 15 ± 1 pS from the Gaussian fits to the amplitude histograms. The y-axis shows the number of events. (j, k) Single channel current recordings and corresponding all point current histograms of *Piezo1* deficient *Oln*-mTomato⁺ cells isolated from *Oln*^mT/iCreER; *Piezo1*^fl/fl mice. The data were collected at −60 mm Hg applied pressure and at the holding potential of +60 mV, without (j) or with (k) 40 μM Yoda1 in the pipette. (l) *Piezo1* transcript levels by qRT-PCR in *Oln*-Tomato⁺ cells from *Oln*^mT/iCreER; *Piezo1*^fl/fl and *Oln*^mT/+; *Piezo1*^fl/fl control mice (6 mice per genotype analyzed in 3 independent experiments). (m and n) The effect of *Piezo1* deletion in Osteolectin⁺ cells on femoral cortical bone mineral density (m) and the percentage of Osteolectin⁺ cells that incorporated a 48 hour pulse of BrdU (n; 6 mice per genotype analyzed in 3 independent experiments). (o) Location of IL7Rα⁺Lineage⁻ cells in the bone marrow of *Oln*^mT/iCreER; *Piezo1*^fl/fl and *Oln*^mT/+; *Piezo1*^fl/fl control mice (6 mice per genotype analyzed in 3 independent experiments). (p-s) We treated *Col1a1*-creER; *Piezo1*^fl/fl; *Oln*^mT/+ mice and littermate controls with tamoxifen at 2 months of age and analyzed them 1 month later. *Piezo1* deletion in osteoblasts significantly reduced femur bone mineral density (p) and cortical thickness (q) but not the frequencies of Osteolectin⁺ cells (r) or HSCs, MPPs, GMPs, MEPs, or CLPs in the bone marrow (s; 5–6 mice per genotype analyzed in 3 independent experiments). (t-w) We analyzed the femurs of *Lepr*^cre/+; *Piezo1*^fl/fl; *Oln*^mT/+ mice and littermate controls at 4 months of age and found that *Piezo1* deletion in LepR⁺ cells did not significantly reduce bone mineral density (t) but did reduce cortical thickness (u). *Piezo1* deletion in LepR⁺ cells also significantly reduced the frequencies of Osteolectin⁺ cells (v) and CLPs (w) without affecting the frequencies of HSCs, MPPs, GMPs, MEPs, or CMPs (w) in the bone marrow (5 mice per genotype analyzed in 3 independent experiments). (x-z) Compared to *Scf*^fl/fl littermate controls, *Oln*^iCreER/+; *Piezo1*^fl/fl mice had decreased B (x) and T (y) cell counts in the spleen 5 days after oral *Listeria* infection and increased bacterial CFUs in the spleen 10 days after infection (z; 11–16 mice per genotype analyzed in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using paired t-tests (a, l, m, n, p-r, t-v, z), matched samples two-way ANOVAs followed by Sidak’s (b, d-f, h, s, w-y) or Holm-Sidak’s multiple comparisons adjustment (g), or Cochran-Mantel-Haenszel test (o).

**Figure 1.. Osteolectin is expressed by peri-arteriolar LepR⁺ cells.**
Images are representative of 3–5 experiments with 8–10-week-old mice. (**a-c**) Flow cytometric analysis of enzymatically dissociated bone marrow from *Oln*^mT/+ mice (4 mice per genotype in 4 independent experiments). (b) Most *Oln*-Tomato⁺ cells were LepR⁺. (c) Only a small minority of LepR⁺ cells were *Oln*-Tomato⁺. (d and e) Representative femur sections showing the epiphysis (d) and diaphysis (e) from 2–4-month-old *Oln*^mT/+; *Col1a1*2.3*-EGFP mice. Panel d shows *Oln*-Tomato⁺ hypertrophic chondrocytes (arrow) and *Oln*-Tomato⁺*Col1a1***2.3*-EGFP⁺ osteoblasts in trabecular bone (arrowhead; scale bar = 30μm). Panel e shows *Oln*-Tomato⁺*Col1a1*2.3*-EGFP⁺ osteoblasts in the endosteum (arrow) and *Oln*-Tomato⁺ osteocytes (arrowhead; scale bars = 25μm). (f and g) Deep imaging of *Oln*^mT/+ femur bone marrow. The boxed area in panel f is magnified in panel g. Panel f shows peri-arteriolar *Oln*-Tomato⁺ cells (arrowhead) as well as *Oln*-Tomato⁺ cells on the endosteal surface (arrow; scale bar = 100μm). Panel g shows *Oln*-Tomato⁺LepR⁺ peri-arteriolar stromal cells (arrowhead) and *Oln*-Tomato⁻LepR⁺ peri-sinusoidal stromal cells (arrow; scale bar = 40μm). (h) *Oln*-Tomato⁺ cells around Sca-1⁺ arterioles (arrow) but not Endomucin^high sinusoids (scale bar = 200μm). All data represent mean ± SD.

**Figure 2.. Osteolectin⁺ cells are short-lived osteogenic progenitors.**
(a) Percentage of CFU-F colonies that were Tomato⁺ when formed by bone marrow cells from 2 month-old *Lepr*^cre/+; *Rosa26*^{loxp-tdTomato/+} or *Oln*^iCreER/+; *Rosa26*^{loxp-tdTomato/+} mice, 3 days after tamoxifen administration (4 independent experiments). (b) Percentage of LepR⁺, LepR⁺*Oln*-mTomato⁺, and LepR⁺*Oln*-mTomato⁻ bone marrow stromal cells that formed CFU-F colonies. (c and d) Osteogenic (c) and adipogenic (d) differentiation of adherent cells cultured from LepR⁺*Oln*-mTomato⁺ or LepR⁺*Oln*-mTomato⁻ stromal cells (6 independent experiments in b-d). (e) The average numbers of perilipin⁺ adipocytes or alkaline phosphatase⁺ (ALP⁺) osteogenic cells that spontaneously differentiated per CFU-F colony after 1 week of culture (4 independent experiments). (f) Percentage of LepR⁺, LepR⁺*Oln*-mTomato⁺ and LepR⁺*Oln*-mTomato⁻ bone marrow stromal cells that incorporated a 24-hour pulse of BrdU in vivo (4 independent experiments). (g) *Oln*^iCreER/mT; *Rosa26*^loxp-EGFP/+ mice were treated with tamoxifen at 2 months of age and the percentage of *Oln*-Tomato⁺ cells that were EGFP⁺ was assessed (3 independent experiments). (h and i) *Oln*^iCreER/+; *Rosa26*^{loxp-tdTomato/+}; *Col1a1*2.3*-EGFP mice were treated with tamoxifen at 2 months of age and the percentages of LepR⁺ stromal cells (h) and *Col1a1*2.3*-EGFP⁺ osteoblasts (i) that were Tomato⁺ was assessed (5 independent experiments). (j) Bone, adipocytes, and hematopoiesis in ossicles formed by CFU-F cultured from LepR⁺*Oln*-mTomato⁺ or LepR⁺*Oln*-mTomato⁻ stromal cells (80 ossicles per cell population derived from 20 mice in 5 independent experiments). (k-l) Femurs were fractured 3 days after tamoxifen administration to *Oln*^iCreER/+; *Rosa26*^{loxp-tdTomato/+}; *Col1a1*2.3*-EGFP mice. Two weeks later the number of LepR⁺ cells per femur (k) and the percentage of LepR⁺ cells that were Tomato⁺ (l) were assessed (5 independent experiments). All data represent mean ± SD. Statistical significance was assessed using an unpaired t-test (a), paired t-tests (**c, k**, l), matched samples one-way ANOVAs followed by Tukey’s multiple comparisons tests (b, f), Wilcoxon test (d), Wilcoxon test followed by Holm-Sidak’s multiple comparisons adjustment (e), or Cochran-Mantel-Haenszel test (j).

**Figure 3.. Osteolectin⁺ peri-arteriolar stromal cells maintain early lymphoid progenitors by synthesizing SCF.**
Images are representative of 3 independent experiments. The same differences that were evident among the frequencies of cell populations were also evident in absolute numbers. (a, b) In femur bone marrow from 2-month-old *Scf*^GFP/+; *Oln*^mT/+ mice, *Scf*-GFP was expressed by both *Oln*-mTomato⁺ peri-arteriolar cells (arrowhead in a) and *Oln*-mTomato⁻ peri-sinusoidal cells (arrow in a; scale bar = 20μm). (c-d) *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl littermate control mice were treated with tamoxifen at 2-months of age then bone marrow and spleen cellularity (c), and hematopoietic stem and progenitor cell frequencies in the bone marrow (d) were analyzed one month later (3 independent experiments). (**e, f**) Localization of IL7Rα⁺Lineage⁻ lymphoid progenitors (arrows) adjacent to peri-arteriolar Osteolectin⁺ cells (scale bar = 15μm; 9 mice in 3 independent experiments). (g) Localization of IL7Rα⁺Lineage⁻ cells in the bone marrow of *Oln*^iCreER/+; *Scf*^fl/fl and *Scf*^fl/fl littermate control mice (6 mice per genotype in 3 independent experiments). (h-j) *Oln*^iCreER/+; *Scf*^fl/fl mice and littermate controls were fed tamoxifen from 2–4 months of age and the bone marrow (h) and thymus (**i, j**) were analyzed (3 independent experiments). (k and l) Compared to *Scf*^fl/fl controls, *Oln*^iCreER/+; *Scf*^fl/fl mice had decreased CLP frequency in the bone marrow (k) and worse survival (l) after *Listeria* infection (17–27 mice per genotype in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using matched samples two-way ANOVAs followed by Sidak’s multiple comparisons tests (c, d, h-k), Cochran-Mantel-Haenszel tests (f, g), or Mantel-Cox test (l).

**Figure 4.. Mechanical stimulation is required for the maintenance of peri-arteriolar niches for lymphoid progenitors in the bone marrow.**
The same differences that were evident among the frequencies of cell populations were also evident in absolute numbers. (a-c) The effect of voluntary running for 4 weeks on femoral cortical thickness (a), the percentage of LepR⁺ cells that were *Oln*-mTomato⁺ (b), and the frequencies of hematopoietic stem and progenitor cells in the bone marrow (c; 6–10 mice per treatment in 3–5 independent experiments). (d-g) The effect of hindlimb unloading for 2 weeks on femur cortical thickness (d), the percentage of LepR⁺ cells that were *Oln*-mTomato⁺ (e), the frequencies of stem and progenitors cells in the bone marrow (f), and the percentage of Osteolectin⁺ cells that incorporated a 48 hour pulse of BrdU (g; 5 mice per treatment in 3 independent experiments). (h) Single channel current recordings in *Oln*-mTomato⁺ cells at the indicated applied pressures. O and C denote the open and closed states of the channels. (i) Normalized open probability–pressure relationship from *Oln*-mTomato⁺ cells recorded with (blue) or without (white) 40 μM Yoda1 in the patch pipette (6 mice in 6 independent experiments). (j) Yoda1 mediated Ca⁺⁺ response in *Oln*-mTomato⁺ cells isolated from *Oln*^mT/iCreER; *Piezo1*^fl/fl or *Oln*^mT/+; *Piezo1*^fl/fl littermate control mice (5 mice per genotype in 3 independent experiments). (k) Western blot of Osteolectin⁺ cells from *Oln*^mT/iCreER; *Piezo1*^fl/fl or *Oln*^mT/+; *Piezo1*^fl/fl mice (representative of three independent experiments). (l-n) The effect of *Piezo1* deletion in Osteolectin⁺ cells on cortical thickness (l), the percentage of LepR⁺ cells that were *Oln*-mTomato⁺ (m), and the frequencies of stem and progenitor cells in bone marrow (n; 6 mice per genotype in 3 independent experiments). All data represent mean ± SD. Statistical significance was assessed using paired t-tests (a, b, g, l, m) and matched samples two-way ANOVAs followed by Sidak’s (c-e, j, n) or Holm-Sidak’s (f) multiple comparisons adjustments.

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Exercise generates immune cells in bone.
Saçma M, Geiger H. Saçma M, et al. Nature. 2021 Mar;591(7850):371-372. doi: 10.1038/d41586-021-00419-y. Nature. 2021. PMID: 33627859 No abstract available.
Osteolectin⁺ stromal cells: Mechanical stimulation improves bone regeneration and supports bacterial clearance after fracture.
Ehnert S, Histing T, Nüssler AK. Ehnert S, et al. Signal Transduct Target Ther. 2021 Jul 7;6(1):257. doi: 10.1038/s41392-021-00680-7. Signal Transduct Target Ther. 2021. PMID: 34234107 Free PMC article. No abstract available.
Runner's niche: multipurpose stromal cells maintained by exercise.
Dietlein N, Rodewald HR. Dietlein N, et al. Trends Immunol. 2021 Oct;42(10):841-843. doi: 10.1016/j.it.2021.08.007. Epub 2021 Aug 31. Trends Immunol. 2021. PMID: 34479798

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A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis

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A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis

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