The percentage of murine hematopoietic stem and progenitor cells, which present with a loss of function upon treatment with the genotoxic agent hydroxyurea, is inversely correlated to the mean lifespan of inbred mice, including the long-lived C57BL/6 and short-lived DBA/2 strains. Quantitative trait locus mapping in BXD recombinant inbred strains identified a region spanning 12.5 cM on the proximal part of chromosome 11 linked to both the percentage of dysfunctional hematopoietic stem and progenitor cells as well as regulation of lifespan. By generating and analyzing reciprocal congenic mice for this locus, we demonstrate that this region indeed determines the sensitivity of hematopoietic stem and progenitor cells to hydroxyurea. These cells do not present, as previously anticipated, with differences in cell cycle distribution, and also not with changes in the frequency of cells undergoing apoptosis, senescence, replication stalling and re-initiation activity, excluding that variations in proliferation, replication or viability underlie the distinct response of these cells from the congenic and parental strains. An epigenetic aging clock in blood cells was accelerated in C57BL/6 mice congenic for the DBA/2 version of the locus. We verified pituitary tumor-transforming gene-1 (pttg1)/securin as the quantitative trait gene regulating the differential response of hematopoietic stem and progenitor cells to hydroxyurea treatment and which might thus likely be linked to the regulation of lifespan.

The G protein-coupled receptor 56 (GPR56) was identified as part of the molecular signature of functionally validated leukemic stem cells isolated from patients with acute myeloid leukemia (AML). This report now demonstrates particularly high expression of GPR56 in patients with mutant NPM1 and FLT3-length mutation and association of high GPR56 expression with inferior prognosis in a large patient cohort treated in two independent multicenter phase III trials. Functional relevance of GPR56 expression was validated in mice, in which co-expression of Gpr56 significantly accelerated HOXA9-induced leukemogenesis and vice versa knockdown of Gpr56 delayed onset of HOXA9/MEIS1-induced AML. Overexpression of Gpr56 grossly changed the molecular phenotype of Hoxa9-transduced cells affecting pathways involved in G protein-coupled receptors (GPRCs) and associated intracellular signaling. Blockage of surface GPR56 by an anti-GPR56 antibody successfully impaired engraftment of primary human AML cells. In summary, these data demonstrate that high expression of GPR56 is able to contribute to AML development and characterize the GPR56 as a potential novel target for antibody-mediated antileukemic strategies.

Interactions between leukemic cells and the bone marrow microenvironment are essential for the maintenance and progression of myeloid leukemias. G-protein coupled receptor 56 (Gpr56) is an adhesion molecule which collaborates with the extracellular matrix through interaction with collagen III binding and transglutaminase 2, and by this activating the Rho A response pathway. In the hematopoietic hierarchy, GPR56 expression is highest in stem cells and decreasing in expression with differentiation. It is a poor prognostic factor in cytogenetically normal acute myeloid leukemia (AML), and was identified as a member of a signature expressed in functionally validated human AML stem cells (Eppert et al., Nature Medicine 2011). To test its functional relevance for AML, we first analyzed the expression of GPR56 in primary patient samples (n= 74) with different genotypes from the AML dataset available in the TCGA database. Normal karyotype patients showed significantly higher expression of GPR56 (3.1 fold p<0.05), when compared to patient samples with translocations. Analysis within the normal karyotype dataset showed high GPR56 expression independent of NPM1 or FLT3-ITD mutations. Quantification of GPR56 expression by TaqMan PCR confirmed high expression both in the CD34-positive and CD34-negative leukemic subpopulations in normal karyotype AML, indicating high expression of this protein in the compartment enriched for leukemic stem cells. As GPR56 expression was high in normal karyotype AML, known to be characterized by aberrant homeobox expression, we tested the functional relevance of Gpr56 expression in a murine Hoxa9 bone marrow (BM) transplantation model. Aberrant expression of Hoxa9 in 5-FU mobilized stem and progenitor cells increased GPR56 expression 1.3 fold (p<0.05), but failed to induce short latency AML up to an observation time of over 160 days post transplant. Retrovirally engineered aberrant overexpression of Gpr56 alone also did not induce any perturbations with regard to engraftment and cell differentiation over 90 days post transplant in mice. However, overexpression of Hoxa9 together with Gpr56 induced a significant and 1.8 fold increase in colony forming cell activity (CFC) (p<0.05), followed by a 1.6 fold (p<0.05) increase in secondary CFCs when compared to HoxA9 alone. Mice transplanted with BM cells co-transduced with HoxA9 and Gpr56 rapidly developed AML after a median latency of 139 days post transplant (HoxA9 MIEG3 N=8, HoxA9 Gpr56 N=10, p<0.05). The mice displayed splenomegaly (mean 0.47g ± 0.07), and a high expression of myeloid markers on leukemic cells (Mac-1: 31%-57%; Mac-1/Gr-1: 24%-53%; Gr-1: 0.5%-16%). Cells co-transduced with Gpr56 and HoxA9 showed a decrease in Cxcr4 expression, when compared to HoxA9 alone and a 6-fold reduction in CFCs (p<0.05) when cells were introduced to an in-vitro SDF-1 gradient migration assay. However, BM cells co-transduced with Hoxa9 and Gpr56 did not show any significant drop in their homing capacity in vivo. Of note, leukemogenicity of Hoxa9 BM cells co-transduced with Meis1 was dependent on Gpr56 expression, as knockdown of Gpr56 expression via shRNA doubled the latency time in transplanted mice (median latency 55 days compared to 95 days, respectively p<0.05). Knockdown of Gpr56 induced a decrease in cells capable of attaching to the stroma in the adhesion cobble-stone area forming cell assay (CAFC) (6.5 fold reduction). However, cells able to home to the BM in vivoincreased 2.3 fold (p<0.05), suggesting that the delay in disease progression was due to a decrease in attaching to the niche.

The decline in hematopoietic function seen during aging involves a progressive reduction in the immune response and an increased incidence of myeloid malignancy, and has been linked to aging of hematopoietic stem cells (HSCs). The molecular mechanisms underlying HSC aging remain unclear. Here we demonstrate that elevated activity of the small RhoGTPase Cdc42 in aged HSCs is causally linked to HSC aging and correlates with a loss of polarity in aged HSCs. Pharmacological inhibition of Cdc42 activity functionally rejuvenates aged HSCs, increases the percentage of polarized cells in an aged HSC population, and restores the level and spatial distribution of histone H4 lysine 16 acetylation to a status similar to that seen in young HSCs. Our data therefore suggest a mechanistic role for Cdc42 activity in HSC biology and epigenetic regulation, and identify Cdc42 activity as a pharmacological target for ameliorating stem cell aging.

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that whereas Rac1 activity is up-regulated upon stem cell factor, integrin, or CXCL12 stimulation, R-Ras activity is inversely up-regulated. Expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1-activity whereas deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras(-/-) HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras(-/-) HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras(-/-) mice showed enhanced responsiveness to G-CSF for HPC mobilization and exhibited decreased bone marrow homing. Transplantation experiments indicate that the R-Ras deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.

BackgroundThe small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated.Design and MethodsCre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry.ResultsErythropoiesis in Rac1(-/-);Rac2(-/-) mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen.ConclusionsThese data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

Aged hematopoietic stem cells (HSCs) are impaired in supporting hematopoiesis. The molecular and cellular mechanisms of stem cell aging are not well defined. HSCs interact with nonhematopoietic stroma cells in the bone marrow forming the niche. Interactions of hematopoietic cells with the stroma/microenvironment inside bone cavities are central to hematopoiesis as they regulate cell proliferation, self-renewal, and differentiation. We recently hypothesized that one underlying cause of altered hematopoiesis in aging might be due to altered interactions of aged stem cells with the microenvironment/niche. We developed time-lapse 2-photon microscopy and novel image analysis algorithms to quantify the dynamics of young and aged hematopoietic cells inside the marrow of long bones of mice in vivo. We report in this study that aged early hematopoietic progenitor cells (eHPCs) present with increased cell protrusion movement in vivo and localize more distantly to the endosteum compared with young eHPCs. This correlated with reduced adhesion to stroma cells as well as reduced cell polarity upon adhesion of aged eHPCs. These data support a role of altered eHPC dynamics and altered cell polarity, and thus altered niche biology in mechanisms of mammalian aging.

Hematopoiesis, the process in which blood cells are generated from hematopoietic stem and progenitor cells (HSPCs) is primarily confined to the bone cavities. The interactions of hematopoietic cells with stroma cells forming niches inside the bone cavities are central to hematopoiesis, as these regulate cell proliferation, self-renewal and differentiation. Hematopoietic cell/stroma interactions have thus been, in analogy to the immunological synapse, named stem/progenitor cell synapses. So far, visualization of the behavior of somatic stem and progenitor cells in an undisturbed in vivo environment has not been reported for the mammalian system and consequently, the cellular dynamics of stem, progenitor and differentiated cells in vivo are only poorly defined. We developed and performed intravital time-lapse 2-photon microscopy in the marrow of the long bones (tibia) of mice to study the behavior and dynamics of differentiated hematopoietic cells as well as HPCs and HSCs in close vicinity to the endosteum in vivo over time. We demonstrate that HPCs as well as HSCs reside in close vicinity to the endosteum, further supporting the notion of an endosteal stem cell niche, and that they are, in contrast to differentiated macrophages and dendritic cells, solitary and immobile. Both HPCs and HSCs occupy distinct positions relative to the endosteum and show cell protrusion movement consistent with an active stem/progenitor cell synapse. Lastly, we report that aged HSCs show increased protrusion movement and localize more distantly to the endosteum compared to young HSCs. In addition, aged HSCs present with reduced adhesion to stroma as well as reduced polarity upon adhesion in vitro, implying a connection between altered stem cell dynamics in vivo and stem cell aging. The intravital imaging technology developed might establish a basis for further delineating additional important questions in stem cell biology like cellular mechanisms of hematopoietic stem cell self-renewal and differentiation in the context of the stroma/niche in vivo.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine G-CSF has become the preferred source of HSPCs for clinical stem cell transplants. However, up to 10% of donors fail to mobilize sufficient numbers of stem cells impeding autologous transplants or significantly delaying transplant recovery time. Consequently, novel regimens are warranted to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in the mouse, we map the epidermal growth factor receptor (EGFR) to a genetic region on murine chromosome 11 modifying G-CSF-mediated HSPC mobilization. Expression levels of EGFR in HSPCs were inversely correlated with HSPC mobilization, implying a negative role for EGFR signaling in mobilization. Genetic reduction of EGFR activity (waved2 mice) or treatment with the EGFR inhibitor erlotinib increased stem cell mobilization up to 5-fold in combination with G-CSF. Increased mobilization due do alteration of EGFR activity correlated with reduced activity of Cdc42 and consequently, inhibition of Cdc42 activity in vivo by a specific Cdc42 inhibitor similarly enhanced mobilization. Our findings reveal a novel signaling pathway regulating stem cell mobilization and thus provide new rationale for targeted pharmacological approaches to further improve HSPC mobilization and thus transplantation outcomes.

Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. p190-B GTPase Activating Protein (GAP) which regulates RhoA activity has been implicated in IGF-1 signaling via cAmp-response element binding proteins (CREB) activity regulating cell size during fetal development (Sordella, Dev Cell, 2002). Since IGF-1 and CREB play important roles in hematopoiesis, we hypothesize that p190-B is critical for HSC functions. Mice lacking p190-B die before birth. p190-B−/− embryos at 14.5 day post coitum exhibited fetal liver (FL) hypocellularity associated with profound anemia. Surprisingly, p190-B−/− FL showed normal frequency of myeloid colonies and a two-fold increase in day 28 and 35 CAFC frequency compared to WT. Overall, the number of progenitors per p190-B−/− liver was decreased while the number of HSC remained unchanged compared to WT FL, suggesting maintenance of the HSC pool in p190-B−/− FL despite the anemia. The repopulating capacity of p190-B−/− FL was assessed by serial competitive repopulation assay. Short-term repopulating ability of p190-B−/− FL was modestly increased compared to WT as assessed by peripheral blood (PB) chimerism 3 months post-transplant (58.3 ± 3.5% vs 45.5±5.4%, p<0.05, respectively). Remarkably, in secondary and tertiary recipients, p190-B−/− FL exhibited dramatically enhanced engraftment compared to WT (65.9±6.4% vs 29.3±6%, p<0.01; 21.7±7.9% vs 1.5±0.5%, p<0.05, respectively). The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. BM analysis of secondary recipients revealed loss of the HSC pool in WT engrafted animals, while HSC frequency was near normal in p190-B−/− engrafted mice (LinnegScaposKitpos frequency: 0.034±0.019% vs 0.219±0.110%, p<0.05, respectively). Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSKMac1low) demonstrated similar enhanced engraftment in the BM 4 months post transplant. p190-B-mediated enhanced HSC engraftment was associated with normal proliferation and differentiation of p190-B−/− progenitors. Interestingly, a pool of quiescent HSC was maintained in serially transplanted p190-B−/− animals, since Pyronin Yneg LSK cells were still present in the BM of p190-B−/− engrafted mice while this population disappeared in WT controls. p190-B−/− FL cells also exhibited increased colony-forming unit homing to BM compared to WT controls. Together, these results suggest that loss of p190-B is associated with HSC quiescence and more efficient homing of HSCs that may contribute to long term regeneration of the hematopoietic stem cell pool after transplantation.

Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell division cycle. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. The consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are incompletely understood. Here, we report that mice genetically deficient in Rb in all hematopoietic cells (Vav-Cre Rb knockout [KO] animals) showed altered contribution of distinct hematopoietic cell lineages to peripheral blood, bone marrow, and spleen; significantly increased extramedullary hematopoiesis in the spleen; and a 2-fold increase in the frequency of hematopoietic progenitor cells in peripheral blood. Upon competitive transplantation, HSPCs from Vav-Cre Rb KO mice contributed with an at least 4- to 6-fold less efficiency to hematopoiesis compared with control cells. HSPCs deficient in Rb presented with impaired cell-cycle exit upon stress-induced proliferation, which correlated with impaired function. In summary, Rb is critical for hematopoietic stem and progenitor cell function, localization, and differentiation.

Rac GTPases (i.e. Rac1, Rac2 and Rac3), a subfamily of Rho GTPases, control actin organization and have overlapping as well as distinct roles in cell survival, proliferation, and differentiation in various hematopoietic cell lineages (Gu et al, Science 2003, Cancelas et al, Nature Med 2005). Using conditional gene-targeting in mice, we have previously demonstrated that Rac1 and Rac2 deficiency causes anemia with abnormal erythrocyte cytoskeleton and decreased deformability (Kalfa et al, Blood 2006). In the present studies, we found by colony assays that although bone marrow (BM) BFU-E activity was unaltered from that of the wild type (WT) mice, Rac1−/−;Rac2−/− erythroid bursts had a strikingly different morphology appearing as round, small, dense colonies, likely a manifestation of motility defects associated with Rac GTPase deficiency. Total CFU-Es recovered from Rac1−/−;Rac2−/− BM were as low as 25% of that in WT mice (p<0.05). To further assess erythroblast differentiation, BM cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood 2001). Flow cytometry analysis revealed that proerythroblasts and basophilic erythroblasts in the BM were significantly decreased in Rac1−/−;Rac2−/− (∼30–50% of WT content) while the terminal differentiation to orthochromatic erythroblasts was comparable. In vivo BrdU labeling and flow cytometry with 7-AAD and annexin-V in combination with staining for CD71 and Ter119 revealed no difference in proliferation or survival between WT and Rac1−/−;Rac2−/− erythroid cells after the proerythroblast stage. These data suggest that deficiency of Rac1 and Rac2 GTPases affect erythropoiesis mainly at the early stages of BFU-E and CFU-E formation but not during terminal differentiation to orthochromatic erythroblasts. Given the prominent role of Rac GTPases in regulating actin structure, we next evaluated the possible involvement of Rac GTPases in enucleation, the terminal step of erythropoiesis that likely requires significant actin remodeling. We performed quantitative analysis in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable nucleic acid-staining dye. The frequency of enucleated red cells (SYTO16-low, Ter119-positive population) was similar in the WT and the Rac1−/−;Rac2−/− erythroid cultures. However, application of a Rac GTPase inhibitor, NSC23766, to the WT or the Rac1−/−;Rac2−/− erythroid cultures during the enucleation phase resulted in an inhibition of enucleation up to 80% dose-dependently (figure 1). Rac1 and Rac2 deficiency led to a compensatory elevation of Rac3 activity that was effectively suppressed by NSC23766, as demonstrated by immunoblotting in the Rac1−/−;Rac2−/− erythroblasts and effector-domain pull-down studies. Moreover, NSC23766 inhibited Rac1, Rac2, and Rac3 activities as well as actin polymerization of the erythroblasts. Thus, Rac1, Rac2, and Rac3 have redundant but essential roles in supporting actin dynamics necessary for the nucleus extrusion during the enucleation process.

There is significant individual variation in humans in their ability to mobilize hematopoietic stem and progenitor cells (HSPC) from bone marrow (BM) to peripheral blood upon G-CSF stimulation. Understanding the molecular mechanism underlying this variation may provide clinically significant targets to improve HSPC mobilization. Using forward genetics, we have previously demonstrated that a locus on chromosome 11 is responsible for murine inter-strain variation in G-CSF mediated HSPC mobilization (Geiger et. al., Exp. Hematol., 2004). Novel subcongenic animals were generated that further confined the locus to a 5 Mbp interval. Of the 12 genes in this interval, epidermal growth factor receptor (EGFR) was identified as a candidate gene for regulating inter-strain differences in mobilization. Using real-time PCR, we demonstrated that EGFR is expressed in both murine and human HSPC and that the level of EGFR expression is inversely correlated with GCSF-induced mobilization proficiency of murine HSPC. To further demonstrate a role of EGFR signaling in mobilization, C57BL/6 mice were treated with G-CSF and increasing concentrations of murine recombinant epidermal growth factor (EGF). Our results demonstrated that a single dose of EGF (0.8 ug/g) significantly inhibited mobilization efficiency (approx. 4-fold) (46.7±13 CFC/37.5ul PB with G-CSF treated mice vs. 12.67±1.5 CFC/37.5ul PB with G-CSF + EGF treated mice). To determine whether the reduction in mobilization efficiency is dependent upon EGFR activity, EGFR mutant mice (waved-2+/−) were treated with G-CSF and EGF. In contrast to control mice, EGF had no significant inhibitory effect on G-CSF-mediated mobilization in waved-2+/− mice indicating that EGFR signaling is necessary for inhibition of mobilization by EGF (46.7±13.0 vs. 12.67±1.5 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control mice compared to 14.2±3.7 vs. 12.1±3.6 CFC/37.5ul PB with waved-2+/− mice). To determine if EGFR-signaling regulated mobilization is HSC intrinsic, we performed “competitive mobilization experiments” in which equal numbers of BM cells (4x106) from either control or waved-2−/− mice and competitor Ly5.1 cells were transplanted into lethally irradiated BoyJ mice and subsequently treated with G-CSF and EGF. Animals transplanted with control cells demonstrated a significant reduction in mobilization efficiency in response to EGF compared to animals transplanted with waved-2−/− cells (20.2±7.2 vs. 7.89±1.2 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control transplanted mice compared to 20.3±4.5 vs. 18.67±1.2 CFC/37.5ul PB with waved-2−/− transplanted mice) indicating that EGFR activity in hematopoietic cells is necessary to confer inhibition of mobilization efficiency by EGF. Taken together, these findings identify EGFR signaling as a negative regulator and a genetic modifier of G-CSF induced mobilization proficiency and a potential novel target to enhance HSPC mobilization efficiency.

Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in close association with a highly organized 3-dimensional structure formed by stroma cells, referred to as the niche. Mobilization of HSPCs from bone marrow to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF) requires de-adhesion of HSPCs from the niche. The influence of aging of HSPCs on cell-stroma interactions has not been determined in detail. Using a mouse model of G-CSF-induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately 5-fold greater in aged mice. Competitive mobilization experiments confirmed that enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic cells from aged mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma and with elevated levels of GTP-bound Cdc42. These results might indicate that stroma-stem cell interactions are dynamic over a lifetime and result in physiologically relevant changes in the biology of primitive hematopoietic cells with age.