CXCL12 Production by Early Mesenchymal Progenitors Is Required for Hematopoietic Stem Cell Maintenance

Abstract 510

Hematopoietic stem cells (HSCs) reside in a specialized microenvironment in the bone marrow that provides key signals required for HSC maintenance. Neither the stromal cell population(s) that comprise this stem cell niche nor their HSC-regulatory signals are well defined. In particular, there is controversy about the relative importance of the endosteal niche, which is comprised of osteoblasts and their progenitors, and the perivascular niche, which is comprised of endothelial cells, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem and progenitor cells (including Nestin-GFP+ cells). CXCL12 is a key component of the stem cell niche that regulates HSC trafficking and function. CXCL12 is constitutively expressed by several stromal cell populations, including endothelial cells, osteoblasts, and perivascular stromal cells. Here we generated a floxed allele of Cxcl12 to conditionally delete Cxcl12 from candidate niche cells in the bone marrow and assess the effect on HSCs. Deletion of Cxcl12 in endothelial cells and mature osteoblasts was mediated by the Tie2-Cre recombinase (Cre) and osteocalcin (Oc)-Cre transgenes, respectively. To target Cxcl12 deletion in CAR cells and osteoprogenitors, we used the osterix (Osx)-Cre transgene. Finally, to target multipotent mesenchymal progenitors, we used the Prx1-Cre transgene. Prx1 is a transcription factor expressed early during limb bud mesoderm development, and Prx1-Cre targets all cells derived from limb bud mesoderm.

We first performed lineage mapping studies using transgenic mice carrying a knock-in of the green fluorescent protein (GFP) gene into the Cxcl12 locus. As reported previously, the highest CXCL12 expression was observed in CAR cells, which is a heterogeneous perivascular stromal cell population that contains osteoprogenitors. Lineage mapping with Osx- and Prx1-Cre show that both transgenes mediate efficient and equivalent recombination in nearly all mature osteoblasts, osteoblast progenitors, and CAR cells. Prx1-Cre, but not Osx-Cre, also targets a novel subset of PDGFRα⁺ Sca⁺ mesenchymal progenitors. We show that deletion of Cxcl12 from mature osteoblasts has no effect on HSC number or function. Deletion of Cxcl12 from Osx-Cre-targeted stromal cells, which includes osteoprogenitors and CAR cells, results in constitutive mobilization of hematopoietic progenitors; however, HSC number and function are normal. Cxcl12 deletion in endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 in mesenchymal progenitors is associated with a marked loss of HSCs and HSC quiescence. In addition to endothelial cells, we thus identify a novel Prx1-Cre targeted subset of mesenchymal progenitors that appears to be necessary to support HSCs. These data show that expression of CXCL12 from stromal cells in the perivascular region is required for HSC maintenance.