Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches

Abstract

Although haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche, most published experimental manipulations of the HSC niche have affected the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance. Cxcl12(DsRed) knock-in mice (DsRed-Express2 recombined into the Cxcl12 locus) showed that Cxcl12 was primarily expressed by perivascular stromal cells and, at lower levels, by endothelial cells, osteoblasts and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation. Different stem and progenitor cells thus reside in distinct cellular niches in bone marrow: HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.

Figure 1. Endothelial cells and perivascular stromal cells are the major sources of Cxcl12 in bone marrow

a–c, In Cxcl12^DsRed/+ mice, DsRed was primarily expressed by perivascular cells throughout the bone marrow. d–f, Cxcl12-DsRed and Scf-GFP expression strongly overlapped around sinusoids throughout the bone marrow of Scf^gfp/+; Cxcl12^DsRed/+ mice. g–i, Col2.3-GFP⁺ bone-lining osteoblasts expressed Cxcl12-DsRed in the bone marrow of Col2.3-GFP⁺; Cxcl12^DsRed/+ mice. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, blue) in c, f and i. j, CD45/Ter119⁻Scf-GFP⁺ perivascular stromal cells expressed Cxcl12-DsRed in Scf^gfp/+; Cxcl12^DsRed/+ (blue histogram) but not in Scf^gfp/+ control marrow (red histogram). k, CD45/Ter119⁻Cxcl12-DsRed^high cells expressed Scf-GFP in Scf^gfp/+; Cxcl12^DsRed/+ (blue) but not in Cxcl12^DsRed/+ control marrow (red). l, CD45/Ter119⁻PDGFRα⁺ perivascular stromal cells expressed Cxcl12-DsRed in Cxcl12^DsRed/+ (blue) but not control (+/+; red) marrow. m, VE-cadherin⁺ endothelial cells expressed Cxcl12-DsRed in Cxcl12^DsRed/+ (blue) but not control (+/+; red) marrow. n, 0.5% of CD45/Ter119⁺ haematopoietic cells expressed Cxcl12-DsRed in Cxcl12^DsRed/+ but not in control marrow (Supplementary Fig. 1g). o, CD45/Ter119⁻Col2.3-GFP⁺ osteoblasts from enzymatically dissociated bone expressed Cxcl12-DsRed in Col2.3-GFP; Cxcl12^DsRed/+ (blue) but not Col2.3-GFP control (red) mice. p, Cxcl12 transcript levels by qRT-PCR in different subpopulations of bone marrow cells (n=3–6). BM, whole bone marrow cells. Peri, EYFP⁺ perivascular stromal cells from Lepr-cre; loxpEYFP mice. Endo, VE-cadherin⁺ bone marrow endothelial cells. Ob, Col2.3-GFP⁺ osteoblasts. Hema, CD45/Ter119⁺Cxcl12-DsRed⁺ haematopoietic cells. All data represent mean±s.d. from at least three independent experiments. Two-tailed student’s t-tests were always used to assess statistical significance (*P<0.05, **P<0.01, ***P<0.001). Scale bars are 20 um.

Figure 2. CXCL12 produced by endothelial cells promotes HSC maintenance

a, Bone marrow and spleen cellularity (n=8–9) and (b) HSC frequency in Tie2-cre; Cxcl12^fl/fl mice versus littermate controls (n=10–11). c, 3×10⁵ donor bone marrow cells from Tie2-cre; Cxcl12^fl/fl mice gave significantly lower levels of donor myeloid, B, and T cell reconstitution in irradiated mice (three experiments with a total of 12–14 recipients per genotype). d–f, Tie2-cre; Cxcl12^fl/fl mice had normal frequencies of MPPs, LMPPs, CMPs, MEPs, GMPs (d), CLPs (e), and committed B lineage progenitors in bone marrow (f) (n=3–4). g–i, Tie2-cre; Cxcl12^fl/fl mice had normal frequencies of myeloerythroid colony-forming progenitors in bone marrow (g), spleen (h), and blood (i) (n=3–6). Δ, recombined Cxcl12^fl allele; con, negative control mice with +/+ or fl/+ or fl/fl Cxcl12 genotypes (without cre). Data are mean±s.d. (*P<0.05, **P<0.01, ***P<0.001, #=0.057).

Figure 3. CXCL12 produced by Lepr-expressing perivascular stromal cells retains HSCs and colony-forming progenitors in the bone marrow

a, b, Cellularity (a, n=6) and HSC frequency (b, n=6–7) in the bone marrow and spleen of Lepr-cre; Cxcl12^fl/− mice and littermate controls. c, 3×10⁵ bone marrow cells from Lepr-cre; Cxcl12^fl/− mice gave normal levels of donor myeloid, B, and T cell reconstitution in irradiated mice (three experiments with a total of 15 recipient mice per genotype). d–f, Lepr-cre; Cxcl12^fl/− mice had normal frequencies of MPPs, LMPPs, CMPs, MEPs, GMPs (d), CLPs (e), and committed B lineage progenitors in bone marrow (f) (n=3). g–i, Lepr-cre; Cxcl12^fl/− mice had normal frequencies of myeloerythroid colony-forming progenitors in bone marrow (g) but significantly increased frequencies in spleen (h), and blood (i) (n=3–5). j, 6×10⁵ mononucleated blood cells from Lepr-cre; Cxcl12^fl/− mice gave long-term multilineage reconstitution in irradiated mice, while blood cells from littermate controls did not. Δ, recombined Cxcl12^fl allele; -, germline deleted Cxcl12 allele or Cxcl12^DsRed allele; con, control mice. Data represent mean±s.d. (*P<0.05, **P<0.01, ***P<0.001).

Figure 4. CXCL12 produced by osteoblasts promotes the maintenance of early lymphoid progenitors but not HSCs

a, b, Cellularity (a, n=4) and HSC frequency (b, n=4) in the bone marrow and spleen of Col2.3-cre; Cxcl12^fl/fl mice and littermate controls. c, 3×10⁵ bone marrow cells from Col2.3-cre; Cxcl12^fl/fl mice gave significantly lower levels of donor cell reconstitution in the T and B cell lineages but not in the myeloid lineage relative to control bone marrow cells (three experiments with a total of 13–14 recipients per genotype). d, 20 CD150⁺CD48⁻Lineage⁻Sca1⁺cKit⁺ HSCs from Col2.3-cre; Cxcl12^fl/fl mice gave normal donor cell reconstitution (three experiments with a total of 14–15 recipients per genotype), including normal levels of myeloid, B, and T cells (Supplementary Fig. 8c). e–h, Col2.3-cre; Cxcl12^fl/fl bone marrow had normal frequencies of MPPs, CMPs, MEPs, GMPs (e), and committed B lineage progenitors (h) but significantly reduced frequencies of CLPs (f) and IL7Rα⁺LMPPs (g) (n=3–5). i, Col2.3-cre; Cxcl12^fl/fl mice had normal frequencies of myeloerythroid colony-forming progenitors in the bone marrow, spleen, and blood (n=3–6). j, Some Lin⁻IL7Rα⁺ early lymphoid progenitors were adjacent to the endosteum. k–m, Prx1-cre; Cxcl12^fl/fl mice exhibited significant reductions in bone marrow cellularity (k, n=3–4) and the frequencies of HSCs (l), CLPs, and IL7Rα⁺LMPPs (m, n=4–5).Δ, recombined Cxcl12^fl allele; con, control mice. *P<0.05, **P<0.01, ***P<0.001.