Bone and region-specific targeting of SCF and CXCL12 in stromal niche cells reveals local and lineage-specific regulation of terminal hematopoiesis Free

LepR⁺ stromal cells in the bone marrow microenvironment produce essential regulatory factors like SCF-which promotes hematopoietic stem and progenitor cells (HSPC) maintenance- and CXCL12, which orchestrates HSPC retention and quiescence. Current models using global niche-specific Cre drivers (e.g., LepR-Cre) do not allow interrogation of whether stromal cells regulate HSPCs locally or at a distance.

To address this, we utilized the Zic1-Cre mouse model, which was recently developed and proved to selectively target vertebral skeletal stem cells and their derivatives, sparing other bones (Sun J, Nature, 2023). We first confirmed that Zic-cre targets LepR⁺ stromal cells with Zic1-Cre;mT/mG reporter mice, which express membrane GFP (mGFP) in cells that have undergone Cre-recombination and membrane Tomato in other cells. Confocal images showed selective mGFP expression in reticular stromal cells in the dorsal vertebrae but no mGFP in ventral vertebrae or other bones. Flow cytometry and scRNA seq analyses of mGFP⁺ and mGFP^- stromal cells showed that mGFP⁺ cells expressed key niche markers including LepR, Cxcl12, and Kitl (SCF), and lacked endothelial and hematopoietic markers, confirming their identity as LepR⁺ perivascular stromal niche cells. These findings establish Zic1-Cre as a precise tool for regional niche interrogation.

Using Zic1-Cre and LepR-Cre to conditionally delete Scf, we found a near-complete loss of bone marrow SCF level in LepR-Cre mice, while Zic1-Cre mice showed a significant ~2.9-fold reduction in vertebrae SCF (1.9 pg vs 0.66 pg/vertebrae, p=0.011). Deletion of SCF with LepR-Cre caused widespread hematopoietic defects, including reduced long-term HSC (2.9x10³ vs 1.8x10³ per femur, p=0.017; 2.1x10³ vs 1.4x10³ per lumbar vertebra, p=0.3) and erythroid progenitors (CFU-E) (18.3x10⁴ vs 4.9x10⁴ per femur, p<0.0001; 11.8x10⁴ vs 3.3x10⁴ per lumbar vertebra, p<0.0001). In contrast, Zic1-Cre-mediated SCF deletion led to a localized ~ 60% reduction in CFU-E (8.3x10⁴ vs 3.4x10⁴ per lumbar vertebra, p=0.007) in the vertebrae, with minimal impact on HSCs. Surprisingly, both Cre models showed ~40% fewer mature neutrophils, despite unchanged granulocyte progenitor (GP) counts. The numbers and functions of other myeloid progenitors were unaffected, but colony assay revealed impaired GP function in Scf-deleted mice (Ctrl=252 vs LepR-cre=130 vs Zic1-cre=158/1000 cells, p=0.017, p=0.047), indicating differential requirements of SCF levels in HSC and committed progenitors and identifying SCF as a novel regulator of terminal granulopoiesis. Confocal imaging showed that although SCF was exclusively depleted in the Zic1-Cre dorsal vertebrae, CFU-E and neutrophils remained evenly distributed across vertebral segments. This spatial uncoupling between SCF source and cellular localization demonstrates that LepR⁺-cell-derived SCF acts over long distances within the marrow to support erythroid and granulocytic differentiation.

We then conditionally deleted CXCL12 in dorsal vertebral stromal cells by generating Zic1-CreΔCxcl12 mice. In agreement with previous studies showing that most of the BM CXCL12 is produced by the endothelium we found minimal reductions in CXCL12 in the BM extracellular fluid. However, Zic1-CreΔCxcl12 mice displayed a 1.4-fold increase in late erythroblasts in the vertebra (6.7x10⁶ vs 9.6x10⁶ per lumbar vertebra, p=0.0314), while early erythroblasts showed a non-significant rise. CFU-Es, reticulocytes and red blood cells (RBC) remained unchanged. In LepR-CreΔCxcl12 mice, we found a 40% increase in erythroid cells from CFU-Es to reticulocytes. Confocal analyses of Zic1-CreΔCxcl12 mice showed that late erythroblasts increased -exclusively- in the dorsal vertebrae. These results indicate that stromal-derived CXCL12 inhibits terminal erythropoiesis by acting on proximal -but not distal- cells.

Together, these results reveal that LepR⁺ stromal cells regulate hematopoiesis by acting on both proximal and distal cells depending on the cytokine and cell lineage examined. They also reveal exquisite sensitivity of different lineages to SCF and CXCL12; and demonstrate that LepR⁺-cell-derived SCF is a novel positive regulator of granulopoiesis while LepR⁺-cell-derived CXCL12 negatively regulates erythropoiesis. The Zic1-Cre model provides a powerful platform for dissecting spatially restricted niche functions and reveals unexpected complexity in the regulation of hematopoiesis by bone marrow stromal cells.