CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance

Abstract

Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts and perivascular stromal cells including endothelial cells, CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin-green fluorescent protein (GFP)-positive mesenchymal progenitors have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C-X-C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations. Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1-cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Figure 1. Targeting Cxcl12 deletion in bone marrow stromal cell populations

Lineage mapping was by performed by generating Osx-Cre ROSA26^Ai9/+ Cxcl12^gfp/+ mice (a) or Prx1-Cre ROSA26^Ai9/+ Cxcl12^gfp/+ mice (b). Cells that had undergone Cre-mediated recombination express tdTomato (red). Cells that express CXCL12 also express GFP (green). Counterstaining with DAPI highlights nuclei (blue). Shown are representative photomicrographs of femur sections. The left panel shows tdTomato fluorescence, the middle panel GFP fluorescence, and the right panel both. Original magnification 40X. (c) Representative dot plots showing GFP and tdTomato expression in CD45⁻ lineage⁻ stromal cells harvested from Osx-Cre ROSA^Ai9/+ Cxcl12^gfp/+ mice (left) or Prx1-Cre ROSA26^Ai9/+ Cxcl12^gfp/+ mice (right). GFP^bright CAR cells are boxed. (d) Shown is the percentage of CAR cells that express tdTomato (n=5). (e) CXCL12 mRNA expression relative to β-actin mRNA is shown on total bone marrow RNA (n=6–13). (f) CXCL12 protein in bone marrow extracellular fluid as measured by ELISA (n=3–5). (g) CAR cells were sorted from Osx-Cre Cxcl12^gfp/fl (Osx-GFP), or Prx1-Cre Cxcl12^gfp/fl (Prx1-GFP) mice and RNA prepared. Shown is CXCL12 mRNA expression relative to β-actin mRNA (n=5–7). (h) CD31⁺ lineage⁻ CD45⁻ endothelial cells were sorted from Tie2-Cre-targeted and control mice, and RNA was prepared. Shown is CXCL12 mRNA expression relative to β-actin mRNA (n=3). *P < 0.05, **P < 0.01; ***P < 0.001.

Figure 2. Deletion of Cxcl12 in defined stromal cell population results in the selective loss of HSCs and lymphoid progenitors

(a). Representative dot plots showing the gating strategy to identify HPC populations. Data are gated on lineage⁻ cells. (b) The number of CD150⁺ CD48⁻ CD41⁻ c-Kit⁺ Sca⁺ Lineage⁻ (KSL) cells, (c) CD150⁺ CD41⁻ CD48⁻ CD34⁻ Flk2⁻ KSL (dormant HSCs), (d) CD34⁻ Flk2⁻ KSL cells, (e) common lymphoid progenitors (CLP), (f) B lymphoid progenitors (BLP), and (g) pre-pro B cells per femur is shown. (h) The number of earliest thymic progenitors (ETP) per thymus is shown. Data represent the mean ± SEM of 6–13 mice. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Deletion of Cxcl12 in defined stromal cell populations results in HPC mobilization and a selective loss of repopulating activity and HSC quiescence

(a) Competitive repopulation assays were performed with a one to one ratio of donor and wild-type competitor bone marrow. Shown is the percentage of donor peripheral leukocytes over time. Data represent the mean ± SEM of 10–16 mice. (b) The percentage of donor cells in the bone marrow of the indicated lineage 14 weeks after transplantation is shown. Data represent the mean ± SEM of 5–6 mice. (c & d) The percentage of cells in the indicated stage of the cell cycle is shown for KSL (c) and CD150⁺ CD48⁻ CD41⁻ KSL cells (d). The number of colony-forming cells (CFU-C) in the blood (e) or spleen (f) is shown. Data represent the mean ± SEM of 5–15 mice. *P < 0.05; **P < 0.01; ***P < 0.001. na = not available.

Figure 4. Prx1-Cre differentially targets a PDGFRα⁺ Sca⁺ CXCL12 expressing mesenchymal progenitor cell population

(a) Representative dot plots showing tdTomato and CD31 expression in bone marrow cells from the indicated mice. Data are gated on CD45⁻ lineage⁻ cells. (b) The percentage of CD31⁺ CD45⁻ lineage⁻ cells that express tdTomato is shown. (c) Representative dot plot showing the gating strategy to identify CD45⁻ lineage⁻ PDGFRα⁺ Sca⁺ cells (boxed). Data are gated on CD45⁻ lineage⁻ cells. (d) Representative dot plots showing GFP and tdTomato expression in CD45⁻ lineage⁻ PDGFRα⁺ Sca⁺ (PαS) cells from Osx-Cre ROSA^Ai9/+ Cxcl12^gfp/+ (left panel) or Prx1-Cre ROSA26^Ai9/+ Cxcl12^gfp/+ mice (right panel). (e) Shown is the percentage of lineage⁻ PDGFRα⁺ Sca⁺ cells that express tdTomato (n=3). *P < 0.001; **P < 0.01. (f) TdTomato⁺ and tdTomato⁻ PαS cells were sorted from Prx1-Cre ROSA26^Ai9/+ Cxcl12^gfp/+ mice and colony-forming fibroblast (CFU-F) assays performed. Shown are representative photomicrographs of the cultures on day 14. (g) Number of CFU-F per 100 sorted PαS cells. Data represent the mean ± SEM of three independent experiments. Cells were harvested from the CFU-F cultures on day 14 and replated under osteogenic (h) or adipogenic (i) culture conditions. Shown are representative photomicrographs of cells stained for alkaline phosphatase or oil red O, respectively. Scale bar = 50 µM.