ALBERT

Description: The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell cycle, DNA-damage checkpoint, differentiation and apoptosis. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. Positive as well as inhibitory signals are integrated into the phosphorylation of the RB protein to regulate the G1 to S-phase progression of the cell cycle. Despite the importance of RB in leukemia, the consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are still not clear and have been controversially discussed. Using Cre-enzyme expression driven by the hematopoietic specific Vav1-promotor, we generated mice that are constitutively deficient in RB (hemRb−/− animals) in HSPCs. HemRb−/− mice showed anemia with an increased number of reticulocytes in PB, consistent with a published role of RB in erythroid differentiation. In addition, the frequency of Mac-1 positive cells in BM was increased to 67% compared to 47% in control animals, whereas the frequency of B220 positive B-lymphoid cells was almost 10-fold reduced, without affecting the T-lymphoid compartment. HemRb−/− mice possessed a 3-fold enlarged spleen with a 5-fold increased number of colony-forming cells (CFCs) and severe extramedullary hematopoiesis, a phenotype also reported for animals transplanted with Rb−/− fetal liver cells. BM of hemRb−/− mice showed an almost 3-fold reduction of HSC frequency, measured by the cobblestone-area forming cell assay (CAFC) assay, but not a decrease in the number of HSCs determined by cell surface staining and flow cytometry. Upon transplantation into NOD/SCID animals or upon competitive transplantation into C57BL/6. CD45.1 animals, HSPCs from hemRb−/− mice contributed 4 to 6-fold less to hematopoiesis. HSPCs from hemRb−/− animals were neither impaired in their ability to home to the BM, nor did they show increased apoptosis. Finally, we detected a significant 4-fold decrease in stem cell function/numbers upon stress caused by 5-FU treatment in hemRB−/− mice compared to control animals. We conclude that upon transplantation/stress, HSPCs from hemRb−/− animals are impaired in their self-renewal function. HemRb−/− animals also showed a 2-fold increase in the frequency of CFCs in peripheral blood. As we detected no increased leukemia incidence in the hemRb−/− animals (now up to 1 year of age), loss of the tumor suppressor RB in hematopoietic cells might be regarded as necessary, but not sufficient for causing early onset leukemia. In summary, loss of RB results in context/localization dependent phenotypes in the hematopoietic hierarchy, influencing stem and progenitor cells in function, localization and differentiation ability.

Description: 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

Description: 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

Description: 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

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: The ability of healthy, older individuals to undergo stem cell mobilization in response to granulocyte colony-stimulating factor (G-CSF) has not been specifically investigated. Aging is usually associated with an overall decline in tissue and cellular function. Therefore it is anticipated that the ability to mobilize stem cells in response to G-CSF may be reduced in aged individuals, although whether or not a negative correlation exists between age and mobilization is still under debate. We analyzed the influence of aging on the mobilization proficiency in healthy mice. Young 2–3 month old and aged 20–26 month old, which corresponds to a human age of 70, were mobilized according to standard procedures. The frequency of hematopoietic progenitor cells in peripheral blood (PB) of aged mice was 3- fold elevated over the frequency found in PB of young mice. The aged animals possessed the ability to mobilize increased numbers of primitive hematopoietic cells despite a slight, but significant anemia. This 3-fold difference in stem cell frequency in PB was further confirmed by long-term transplantation experiments. To determine if the increased ability to mobilize is intrinsic to aged hematopoietic stem cells or driven by age-associated, extrinsic changes in the BM microenvironment, a competitive mobilization assay based on the Ly5.2/Ly5.1 mismatch system was used. The genetic origin of the CFCs was determined by flow cytometry. In this assay, as previously reported, aged BM HSCs contribute to only 30% chimerism in PB prior to mobilization, resulting in a 1:2 ratio in favor of young cells in PB chimerism 3 months post-transplant. In contrast, when we analyzed the CFC chimerism of the competitively transplanted animals post-mobilization, 85% of all CFC from PB were derived from cells from old mice, strongly arguing that increased mobilization proficiency is a) not a simple consequence of the increased CFC content in aged BM and b) to a large extent intrinsic to the aged primitive hematopoietic cell, and thus relatively independent from the aged BM microenvironment. These data also imply that the stroma stem cell/interaction might change with age.