Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell

Hematopoietic stem cells (HSCs) are at the apex of lifelong blood cell production. Recent clonal analysis studies suggest that HSCs are heterogeneous in function and those that contribute to homeostatic production may be distinct from those that engraft during transplant.

We developed a rapid mobilization regimen utilizing a unique CXCR2 agonist (an N-terminal truncated MIP-2a) and the CXCR4 antagonist AMD3100. A single subcutaneous injection of both agents together resulted in rapid mobilization in mice with a peak progenitor cell content in blood reached within 15 minutes. This mobilization was equivalent to a 5-day regimen of G-CSF. This rapid mobilization is the result of synergistic signaling, and was blocked in CXCR4 or CXCR2 knockout mice, confirming receptor and mechanism specificity. Mobilization is caused by synergistic release of MMP-9 from neutrophils and mobilization was blocked in MMP-9 knockout mice, mice treated with an anti-MMP-9 antibody, TIMP-1 transgenic mice, or mice where neutrophils were depleted in vivo using anti-GR-1 antibody. In vivo confocal imaging of mice demonstrated that the mobilization regimen causes a rapid and transient increase in bone marrow vascular permeability, "opening the doorway" for hematopoietic egress to the peripheral blood.

Transplantation of 2x10⁶ peripheral blood mononuclear cells (PBMC) from the rapid regimen resulted in a 4 or 6 day quicker recovery of neutrophils and platelets, respectively, compared to a G-CSF mobilized graft (n=12 mice per group, P<0.01). In limiting dilution competitive transplants, the rapid regimen demonstrated a greater than 2-fold enhancement in competitiveness (n=30 mice/treatment group, 2 individual experiments, P<0.001). Additionally, in secondarily transplanted mice, competitiveness of the rapidly mobilized graft increased as measured by contribution to chimerism, while G-CSF mobilized grafts remained static (n=16 mice/group, P<0.01). Surprisingly, despite robust enhancement in both short and long-term engraftment by the rapidly mobilized graft, phenotypic analysis of the blood of mobilized mice for CD150+ CD48- Sca-1+ c-kit+ Lineage ^neg (SLAM SKL) cells, a highly purified HSC population, showed lower numbers of phenotypically defined HSCs than in the G-CSF group.

These data suggested that a unique subset of "highly engraftable" HSCs (heHSCs) are mobilized by the rapid regimen compared to G-CSF. However, as our earlier studies were performed using grafts that contained the total PBMC fraction (similar to the clinical apheresis product) we could not rule out the potential contribution of accessory cells to the enhanced engrafting ability of the heHSCs. Therefore, in 3 independent experiments, we mobilized large cohorts of mice with the rapid regimen or G-CSF and sorted SLAM SKL cells from the PBMC fraction and competitively transplanted equal numbers of SLAM SKL cells from either the rapid regimen or G-CSF and tracked contribution to chimerism over 36 weeks. Remarkably, the heHSCs from the rapid regimen demonstrated a 2-fold enhancement in competitiveness compared to SLAM SKL cells from the G-CSF group (n=17 mice/group, P<0.0004).

While appreciation for HSC heterogeneity has grown, methods are lacking for prospectively isolating differing HSC populations with known biologic function, to study molecular heterogeneity. Like panning for gold, we sought to use the differential mobilization properties of our rapid regimen and G-CSF as a "biologic sieve" to isolate the heterogeneous HSC populations from the blood. We again flow sorted SLAM SKL cells from mice mobilized with the rapid regimen or G-CSF and performed RNA-Seq analysis of the purified populations. The heHSCs mobilized by the rapid regimen had a unique transcriptomic signature compared to G-CSF mobilized or random HSCs acquired from bone marrow (P<0.000001). Strikingly, gene set enrichment analysis (GSEA) demonstrated that the heHSCs had a gene signature highly significantly clustered to that of fetal liver HSCs, further demonstrating the selective harvesting of a subset of highly engraftable stem cells.

Our results mechanistically define a new mobilization strategy, that in a single day can mobilize a graft with superior engraftment properties compared to G-CSF, and selectively mobilize a novel population of heHSCs with an immature molecular phenotype capable of robust long-term engraftment.