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We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

The erythroblastic island (EBI) is a multicellular structure forming an erythropoietic niche consisting of a central macrophage surrounded by a rosette of maturing erythroblasts. Since their discovery more than 60 years ago, simultaneous quantification and visualization of EBIs remain difficult. Although flow cytometry enables high-throughput quantification of cell aggregates co-expressing macrophage and erythroblast markers, it cannot visually confirm whether the aggregates are genuine EBIs. While immunofluorescence microscopy allows visualization of EBIs, its low throughput limits its use for quantification. In the current study we employed nine-channel imaging flow cytometry (IFC) to develop a method to directly visualize and quantify EBIs in the mouse bone marrow. We found that EBI central macrophages do express F4/80, VCAM-1, and CD169, but not CD11b or Ly6G, and that CD11b+Ly6G+F4/80– granulocytes are found associated at the periphery of 40%–60% EBIs. Furthermore, we show for the first time using IFC that in vivo treatment with the hematopoietic stem cell-mobilizing cytokine granulocyte colony-stimulating factor (G-CSF) reduced EBI frequency in the bone marrow by more than 100-fold. These results indicate that mobilizing doses of G-CSF cause a collapse of EBIs in the bone marrow.

It has recently emerged that tissue-resident macrophages are key regulators of several stem cell niches orchestrating tissue formation during development, as well as postnatally, when they also organize the repair and regeneration of many tissues including the hemopoietic tissue. The fact that macrophages are also master regulators and effectors of innate immunity and inflammation allows them to coordinate hematopoietic response to infections, injuries, and inflammation. After recently reviewing the roles of phagocytes and macrophages in regulating normal and pathologic hematopoietic stem cell niches, we now focus on the key roles of macrophages in regulating erythropoiesis and iron homeostasis. We review herein the recent advances in understanding how macrophages at the center of erythroblastic islands form an erythropoietic niche that controls the terminal differentiation and maturation of erythroblasts into reticulocytes; how red pulp macrophages in the spleen control iron recycling and homeostasis; how these macrophages coordinate emergency erythropoiesis in response to blood loss, infections, and inflammation; and how persistent infections or inflammation can lead to anemia of inflammation via macrophages. Finally, we discuss the technical challenges associated with the molecular characterization of erythroid island macrophages and red pulp macrophages.

In normoxia, hypoxia-inducible transcription factors (HIFs) are rapidly degraded within the cytoplasm as a consequence of their prolyl hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes. We have previously shown that hematopoietic stem and progenitor cells (HSPCs) require HIF-1 for effective mobilization in response to granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist AMD3100/plerixafor. Conversely, HIF PHD inhibitors that stabilize HIF-1 protein in vivo enhance HSPC mobilization in response to G-CSF or AMD3100 in a cell-intrinsic manner. We now show that extrinsic mechanisms involving vascular endothelial growth factor receptor-2 (VEGFR2), via bone marrow (BM) endothelial cells, are also at play. PTK787/vatalanib, a tyrosine kinase inhibitor selective for VEGFR1 and VEGFR2, and neutralizing anti-VEGFR2 monoclonal antibody DC101 blocked enhancement of HSPC mobilization by FG-4497. VEGFR2 was absent on mesenchymal and hematopoietic cells and was detected only in Sca1+ endothelial cells in the BM. We propose that HIF PHD inhibitor FG-4497 enhances HSPC mobilization by stabilizing HIF-1α in HSPCs as previously demonstrated, as well as by activating VEGFR2 signaling in BM endothelial cells, which facilitates HSPC egress from the BM into the circulation.

SummaryMouse hematopoietic tissues contain abundant and heterogeneous populations of tissue-resident macrophages attributed trophic functions in control of immunity, hematopoiesis and bone homeostasis. A systematic strategy to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node, unexpectedly revealed macrophage surface marker staining typically emanated from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters and mRNA were all detected in non-macrophage cell populations including isolated stem and progenitor cells. The profile of macrophage remnant association reflects adhesive interactions between macrophages and other cell types in vivo. Applying this knowledge, reduced macrophage remnant attachment to BM granulocytes in Siglec1 deficient mice was associated with compromised emergency granulocytosis, revealing a function for Siglec1-dependent granulocyte-macrophage interactions. Analysis of published RNA-seq data for purified macrophage and non-macrophage populations indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single cell analysis of disaggregated tissues.

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.

International audience; Hematopoiesis and bone interact in various developmental and pathological processes. Neurogenic heterotopic ossifications (NHO) are the formation of ectopic hematopoietic bones in peri-articular muscles that develop following severe lesions of the central nervous system such as traumatic cerebral or spinal injuries or strokes. This review will focus on the hematopoietic facet of NHO. The characterization of NHO demonstrates the presence of hematopoietic marrow in which quiescent hematopoietic stem cells (HSC) are maintained by a functional stromal microenvironment, thus documenting that NHOs are neo-formed ectopic HSC niches. Similarly to adult bone marrow, the NHO permissive environment supports HSC maintenance, proliferation and differentiation through bidirectional signaling with mesenchymal stromal cells and endothelial cells, involving cell adhesion molecules, membrane-bound growth factors, hormones, and secreted matrix proteins. The participation of the nervous system, macrophages and inflammatory cytokines including oncostatin M and transforming growth factor (TGF)-β in this process, reveals how neural circuitry fine-tunes the inflammatory response to generate hematopoietic bones in injured muscles. The localization of NHOs in the peri-articular muscle environment also suggests a role of muscle mesenchymal cells and bone metabolism in development of hematopoiesis in adults. Little is known about the establishment of bone marrow niches and the regulation of HSC cycling during fetal development. Similarities between NHO and development of fetal bones make NHOs an interesting model to study the establishment of bone marrow hematopoiesis during development. Conversely, identification of stage-specific factors that specify HSC developmental state during fetal bone development will give more mechanistic insights into NHO.

The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.

Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor (G-CSF)-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: We discovered prostacyclin as a novel HSC regulator enriched in endosteal bone marrow niches. Ex vivo and in vivo treatment with clinical prostacyclin analogs enhanced HSC reconstitution potential and protect HSC from stress-mediated exhaustion. These findings open new therapeutic avenues to improve HSC engraftment after autologous transplantations by short ex vivo pulse treatment with prostacyclin analogs; and limit loss of HSC and acquired bone marrow failure by protecting HSC in patients undergoing radiation or chemotherapy treatment for non-hematologic malignancies. Prostacyclin analogs are safe, used clinically for vascular diseases, and thus have potential for swift repurposing for the therapies mentioned above.

The bone marrow (BM) contains a mosaic of niches specialized in supporting different maturity stages of hematopoietic stem and progenitor cells such as hematopoietic stem cells and myeloid, lymphoid, and erythroid progenitors. Recent advances in BM imaging and conditional gene knockout mice have revealed that niches are a complex network of cells of mesenchymal, endothelial, neuronal, and hematopoietic origins, together with local physicochemical parameters. Within these complex structures, phagocytes, such as neutrophils, macrophages, and dendritic cells, all of which are of hematopoietic origin, have been found to be important in regulating several niches in the BM, including hematopoietic stem cell niches, erythropoietic niches, and niches involved in endosteal bone formation. There is also increasing evidence that these macrophages have an important role in adapting hematopoiesis, erythropoiesis, and bone formation in response to inflammatory stressors and play a key part in maintaining the integrity and function of these. Likewise, there is also accumulating evidence that subsets of monocytes, macrophages, and other phagocytes contribute to the progression and response to treatment of several lymphoid malignancies such as multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma, as well as lymphoblastic leukemia, and may also play a role in myelodysplastic syndrome and myeloproliferative neoplasms associated with Noonan syndrome and aplastic anemia. In this review, the potential functions of macrophages and other phagocytes in normal and pathologic niches are discussed, as are the challenges in studying BM and other tissue-resident macrophages at the molecular level.

Hematopoietic stem cells (HSC) reside in perivascular regions of the bone marrow (BM) embedded within a complex regulatory unit called the niche. Cellular components of HSC niches include vascular endothelial cells, mesenchymal stromal progenitor cells and a variety of mature hematopoietic cells such as macrophages, neutrophils, and megakaryocytes-further regulated by sympathetic nerves and complement components as described in this review. Three decades ago the discovery that cytokines induce a large number of HSC to mobilize from the BM into the blood where they are easily harvested, revolutionised the field of HSC transplantation-curative for immune-deficiencies and some malignancies. However, despite now routine use of granulocyte-colony stimulating factor (G-CSF) to mobilise HSC for transplant, only in last 15 years has research on the mechanisms behind why and how HSC can be induced to move into the blood began. These studies have revealed the complexity of the niche that retains HSC in the BM. This review describes how BM niches and HSC themselves change during administration of G-CSF-or in the recovery phase of chemotherapy-to facilitate movement of HSC into the blood, and research now leading to development of novel therapeutics to further boost HSC mobilization and transplant success.

Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilization of hematopoietic stem cells (HSC) for autologous transplantation in cancer patients. Increasing the number of available HSC prior to mobilization is a potential strategy to overcome this deficiency. Bone marrow (BM) macrophages regulate HSC niches and promote engraftment of donor HSC post-transplant. In this study, we examined if manipulating macrophages using a modified long-acting Fc fusion protein of CSF1 could influence HSC niches to expand the HSC pool. Acute CSF1-Fc treatment (4 x daily injection) in C57Bl/6 mice induced a robust systemic myeloproliferative response that peaked 1 week post the initial injection. BM and spleen homeostasis were disrupted during peak myelopoiesis, including supressed lymphopoiesis and temporary engraftment of BM hematopoietic stem and progenitor cell (HSPC) in liver. CSF1-Fc effects were transient as by 2 weeks post first CSF1-Fc injection, overall haematopoiesis had normalized, but the number of HSC in BM (1.6 fold), and particularly in spleen (5.9 fold), were significantly elevated compared to controls. This was not due to direct CSF1-Fc actions on HSC as multiple experimental approaches confirmed that HSC and multipotent progenitors do not express CSF1-receptor. The increase in splenic HSC corresponded with a sustained phenotype shift in red pulp macrophages characterized by expression of the BM HSC niche macrophage marker CD169. We observed minimal evidence of in situ macrophage proliferation, indicating specialised adaptation of monocyte-derived macrophages to the CSF1-Fc altered splenic environment. Importantly, CSF1-Fc pre-treatment enhanced HSC mobilization (2.5 fold) into blood after G-CSF treatment when compared to controls. Competitive transplantation demonstrated the CSF1-Fc + G-CSF mobilized blood graft had improved reconstitution potential with no leukocyte lineage bias. Overall, the data suggest that CSF1-Fc treatment resulted in sustained adaptation of resident macrophages with improved capability to support extramedullary HSC niches resulting in a systemic increase in the absolute number of HSC. Therefore, conditioning patients with CSF1 could be a potential therapeutic strategy to overcome poor mobilization and subsequently improve autologous transplantation outcomes.

The unique interactions of leukemia cells with the bone marrow (BM) microenvironment (niche) are critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-selectin is a key niche component mediating acute myeloid leukemia (AML) chemoresistance, highlighting E-selectin as a promising therapeutic target. In this study, we found canonical E-selectin receptors CD44 and CD162 to be crucial for E-selectin adhesion, as mouse AML cells lacking both receptors failed to bind to E-selectin. We then developed an in vitro model to assess the chemo-sensitivity of mouse AML blasts adhering to various vascular adhesion molecules; this showed that E-selectin uniquely boosts AML cell survival to chemotherapy, but only when CD44/CD162 are present. Likewise when transplanted into recipient mice, CD44/CD162-/- AML cells were significantly more sensitive to chemotherapy compared to wildtype AML. Together these results suggest that CD44 and/or CD162 are key E-selectin receptors involved in AML chemoresistance. To validate these findings in human, we used CRISPR-Cas9 gene editing to selectively suppress CD44 or CD162 from the human AML cell line KG1a. Using our in vitro chemo-sensitivity assay, we showed that E-selectin could not promote AML survival in the absence of either CD44 or CD162, confirming our findings in mice. However interestingly, KG1a cells could still bind to E-selectin in the absence of CD44 or CD162, suggesting the involvement of several E-selectin receptors that can play different roles – either binding or signalling. To conclude, we described a novel form of niche-mediated chemoresistance that can be modelled in vitro, and identified CD44 and CD162 as key AML cell surface receptors involved. These findings highlight blockade of E-selectin or its receptors as a novel strategy to improve the treatment of AML.

Despite positive testing in animal studies, more than 80% of novel drug candidates fail to proof their efficacy when tested in humans. This is primarily due to the use of preclinical models that are not able to recapitulate the physiological or pathological processes in humans. Hence, one of the key challenges in the field of translational medicine is to “make the model organism mouse more human.” To get answers to questions that would be prognostic of outcomes in human medicine, the mouse's genome can be altered in order to create a more permissive host that allows the engraftment of human cell systems. It has been shown in the past that these strategies can improve our understanding of tumor immunology. However, the translational benefits of these platforms have still to be proven. In the 21st century, several research groups and consortia around the world take up the challenge to improve our understanding of how to humanize the animal's genetic code, its cells and, based on tissue engineering principles, its extracellular microenvironment, its tissues, or entire organs with the ultimate goal to foster the translation of new therapeutic strategies from bench to bedside. This article provides an overview of the state of the art of humanized models of tumor immunology and highlights future developments in the field such as the application of tissue engineering and regenerative medicine strategies to further enhance humanized murine model systems. Stem Cells 2015;33:1696–1704

PURPOSE OF REVIEW The nature and function of macrophages at the center of erythroblastic islands is not fully understood. This review discusses novel findings on the phenotypic and molecular characterization of erythroblastic island macrophages, and their role in regulating normal and pathological erythropoiesis. RECENT FINDINGS The phenotype to prospectively isolate erythroblastic island macrophages from mouse bone marrow has been identified. In-vivo depletion of erythroblastic island macrophages causes blockade of erythroblast maturation and delays erythropoietic recovery following chemical insults. The cytokine granulocyte colony-stimulating factor arrests medullary erythropoiesis by depleting erythroblastic island macrophages from the bone marrow. In-vivo ablation of macrophages improves anemia associated with β-thalassemia and reduces red blood cell counts in the mouse model of polycythemia vera. The role of cell adhesion molecules regulating interactions between erythroblastic island macrophages and erythroblasts has been clarified, and mechanisms of pyrenocyte engulfment by erythroblastic island macrophages have been demonstrated to involve Mer tyrosine kinase receptor. SUMMARY Prospective isolation of mouse erythroblastic island macrophages together with new genetic mouse models to specifically target erythroblastic island macrophages will enable molecular studies to better define their role in controlling erythroblast maturation. These studies have revealed the key role of erythroblastic island macrophages in regulating normal erythropoiesis and could be interesting targets to treat β-thalassemia or polycythemia vera.

Osteal macrophages (osteomacs) contribute to bone homeostasis and regeneration. To further distinguish their functions from osteoclasts, which share many markers and growth factor requirements, we developed a rapid, enzyme-free osteomac enrichment protocol that permitted characterization of minimally manipulated osteomacs by flow cytometry. Osteomacs differ from osteoclasts in expression of Siglec1 (CD169). This distinction was confirmed using the CD169-diphtheria toxin (DT) receptor (DTR) knock-in model. DT treatment of naïve CD169-DTR mice resulted in selective and striking loss of osteomacs, whilst osteoclasts and trabecular bone area were unaffected. Consistent with a previously-reported trophic interaction, osteomac loss was accompanied by a concomitant and proportionately striking reduction in osteoblasts. The impact of CD169(+) macrophage depletion was assessed in two models of bone injury that heal via either intramembranous (tibial injury) or endochondral (internally-plated femoral fracture model) ossification. In both models, CD169(+) macrophage, including osteomac depletion compromised bone repair. Importantly, DT treatment in CD169-DTR mice did not affect osteoclast frequency in either model. In the femoral fracture model, the magnitude of callus formation correlated with the number of F4/80(+) macrophages that persisted within the callus. Overall these observations provide compelling support that CD169(+) osteomacs, independent of osteoclasts, provide vital pro-anabolic support to osteoblasts during both bone homeostasis and repair.

Introduction/Background We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signalling pathways. Material and method Twenty-one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (bone volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Results Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 ± 0.5) compared to contralateral sides (0.56 mm3 ± 0.4), P = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. qPCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice. Conclusion Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro-inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interactionof c-MybwiththecoactivatorCBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore themolecularmechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction inmyeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia. Refereed/Peer-reviewed

We have previously shown vascular E (endothelial)-selectin to play a key role in niche-mediated chemo-resistance in Acute Myeloid Leukaemia (AML). Now we report that the cell surface glycosylation of AML blasts -and thus their E-selectin-binding potential- alters during therapy and queried whether these variations influence treatment outcome. Using preclinical mouse models of 11q23-rearranged (MLL-AF9) monomyelocytic AML, we found that although AML blasts in untreated mice display a range of E-selectin-binding potentials, the blasts with highest E-selectin-binding potential dominate in bone marrow (BM) after 24hr cytarabine therapy. Indeed, the highest 10% of AML blasts for E-selectin-binding were >12-fold (p=0.0014) more likely to survive post-chemotherapy. These data raise the question whether E-selectin binding potential itself may prospectively identify AML blasts with heightened regenerative potential. An alternative explanation could be that high-binding AML blasts predominate after chemotherapy simply due to survival advantages mediated by vascular niche E-selectin interaction. To investigate this, BM AML blasts were FACs sorted from donor C57BL/6 mice based on E-selectin-binding potential (highest and lowest 10%) and transplanted into recipient mice (1,500 AML blasts/recipient, 8/gp). No significant differences in duration of disease-free survival was observed. Thus E-selectin-binding potential itself does not prognostically identify the most potent Leukemia Reconstituting Cells (LRC) that initiate relapse. To determine instead whether the AML blasts that bind E-selectin can dominate during stress because of the survival advantage of interacting with E-selectin at the niche, an identical parallel experiment was performed, the only difference being that donor mice had E-selectin blocked (GMI-1271 100mg/kg BiD) for the last 48h prior to BM harvest, FACs sort of AML blasts and recipient mouse transplant. This time we observed a significant (2-fold) extension in disease-free survival in the recipients of high-binding AMLs (from donors treated with GMI-1271) compared to all other groups (p=0.012, median disease-free survival 33 vs. 63 days). Together these data indicate that administration of E-selectin antagonists, even as a single agent, may potentially improve patient outcomes - in cases where heightened E-selectin binding potential is observed. Next we investigated the intracellular AML signaling pathways potentially dampened by E-selectin absence/blockade. Two common pathways used by malignant cells for survival/regeneration are the PI3K/AKT/mTOR/ NF-kB and RAS/MAPK/ERK pathways. We have already shown the absence (in Sele-/- mice), or therapeutic blockade of E-selectin (with GMI-1271) in mice significantly dampens PI3K/AKT/NF-kB signaling in BM AML blasts in vivo. However, AML blasts can utilize alternative pathways such as RAS/MAPK/ERK for survival signaling as well, especially in the 30% of AMLs with NRAS mutations. So we determined whether MAPK/ERK signaling in AML could be similarly altered by E-selectin absence/blockade. Indeed MAPK/ERK phosphorylation was significantly reduced (2-fold) in BM AML blasts from host mice treated 24hr with GMI-1271 and in Sele-/- hosts. Thus contact with vascular E-selectin induces a range of survival/regenerative signaling pathways within BM AML blasts that would be highly advantageous for the blast in times of stress. In summary we show, (1) vascular niche E-selectin blockade by GMI-1271 dampens malignant AML reconstitution/survival potential in vivo when administered as sole agent alone, (2) That E-selectin blockade mediates these effects via dampening a range of intracellular survival/regeneration signalling pathways in the malignant cell, and finally (3) these data suggest E-selectin blockade may synergise with other specific pathway inhibitors to improve treatment outcomes - but only for malignant cells that are appropriately glycosylated to interact with E-selectin. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership.

We have recently found that the vascular adhesion molecule E(endothelial)-selectin is a critical bone marrow niche component mediating acute myeloid leukemia (AML) chemo-resistance. Clinical trials involving the use of E-selectin mimetics to improve efficacy of conventional AML therapy are in progress. In this study we investigate the identity of the AML cell surface receptors mediating vascular E-selectin-induced chemo-resistance. E-selectin has two well-characterised receptors, CD162 also known as P-Selectin Glycoprotein Ligand-1 (PSGL-1), and CD44 isoform HCELL (Hematopoietic Cell E-selectin/L-selectin Ligand) with other cell surface glycoproteins (such as ESL-1) and glycolipids also binding E-selectin after Sialyl Lewisx/a glycosylation. To investigate which of these AML cell surface receptors are responsible for mediating vascular E-selectin survival signalling, we first investigated if each co-localized with E-selectin on AML cell surface by confocal imaging. Human CD34+ AML KG1a cells were labelled ± adhesion to fluorescently E-selectin-IgM. Confocal imaging revealed that although both canonical CD44 and CD162 receptors co-localized with E-selectin at the site of cell contact, only CD162 became strongly polarized at E-selectin binding site, while CD44 remained widely distributed across the cell surface. To dissect a functional role for each of these canonical receptors in human AML, CRISPR-Cas9 gene editing was used to selectively delete CD44 and/or CD162 from human AML KG1a cells. We found that although deletion of both CD44 and CD162 receptors reduced KG1a E-selectin-IgM binding potential (3-fold), deletion of either receptor alone did not. Next, we investigated whether deletion of either receptor reversed E-selectin-mediated chemo-resistance in an in vitro chemosensitivity assay. KG1a cells were seeded in wells pre-coated with a range of vascular adhesion molecules commonly expressed in the bone marrow niche, then monitored for cell survival after 48hr treatment ± cytarabine. In this in vitro assay, we found that adhesion to E-selectin significantly increased parental KG1a survival to chemotherapy (p=0.0035). No similar increase in survival was observed following adhesion to P-selectin, or with integrin ligands ICAM-1 and PE-CAM-1. When we repeated the assay using Crispr CD44 deleted KG1a AML we found significant E-selectin-mediated chemo-resistance was still observed (p=0.027) even in the absence of CD44. These results suggest CD44 is not the receptor mediating AML chemo-resistance. In contrast E-selectin-mediated chemo-resistance was abrogated in the CD162 Crispr deleted human KG1a AMLs. Together these data suggest CD162/PSGL-1 expressed on the surface of human AML KG1a appears to be the receptor mediating vascular E-selectin chemo-resistance. This would be a completely novel role described for CD162 which is conventionally known as a homing molecule. To confirm this new role for CD162 in mediating AML chemo-resistance can be replicated in pre-clinical models in vivo, we next generated (11q23-rearranged) AML from CD44-/- and/or CD162-/- gene-deleted mice by retroviral transduction of murine hematopoietic stem cells with MLL-AF9 which then were transplanted into wildtype mice. Cohorts of leukemic mice (n=8/gp) were administered induction therapy (cytarabine/doxorubicin) to monitor impact on disease-free survival. In contrast to AMLs from wildtype, we found absence of CD162 in murine AMLs lead to a pronounced chemo-sensitisation in vivo resulting in a significant (6-fold, p=0.0004) extension in overall disease-free survival duration, compared to either no chemotherapy gene-deleted AML controls, or to treated wildtype AML controls. These in vivo murine data confirm the identification of an exciting new role for CD162 as an important cell surface receptor mediating therapy resistance in AML. In conclusion, we describe a novel form of niche-mediated chemo-resistance and identify CD162 as a key AML cell surface receptor involved in both human and mouse AML therapy resistance. CD162/PSGL-1 expression has not previously been implicated in direct therapy resistance. Together these findings help extend our knowledge on the potential mechanisms by which therapeutic blockade of vascular E-selectin can significantly improves therapy outcomes. Disclosures Levesque: GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Winkler:GlycoMimetics: Patents & Royalties.

Granulocyte colony-stimulating factor (G-CSF) and the CXCR4 chemokine receptor antagonist Plerixafor are used to mobilize hematopoietic stem and progenitor cells (HSPC). However, in the autologous setting, prior chemotherapy and radiotherapy impair HSPC mobilizing response to G-CSF and Plerixafor thereby precluding autologous HSC transplantation. We now demonstrate in mice that oncostatin M (OSM) production in the bone marrow (BM) is dramatically increased in response to G-CSF and that OSM counteracts the mobilizing effect of G-CSF and Plerixafor indirectly through the BM stroma. Indeed, by immunohistochemistry, a 4-day G-CSF treatment strongly increased OSM protein expression in cell clusters containing F4/80+ macrophages in the endosteal and central region of the mobilized BM. OSM concentration was 7-fold higher in BM fluids of G-CSF mobilized mice compared to control mice as measured by ELISA. To investigate OSM effects on HSPC mobilization, C57BL/6 mice (WT) and mice lacking the OSM receptor gene (OSMR-/-) were treated with 125 μg/kg human G-CSF bidaily for 2-6 days and/or Plerixafor (10 mg/kg) one hour before harvest. Absence of OSMR increased HSPC mobilization with a 4-fold increase in colony forming cells (CFC), phenotypic Lin-Kit+Sca1+ HSPC (LKS+ cells) and LKS+Flt3-CD48-CD150+ HSC into the blood at days 2 and 4 of G-CSF treatment and increased accumulation of HSPC and HSC in the spleen of OSMR-/- mice at day 6 of G-CSF treatment. In a competitive repopulation assay with serial dilutions of mobilized blood, deletion of OSMR gene enhanced ≈4-fold mobilization of functional reconstituting HSC in response to G-CSF as shown by Poisson's distribution analysis. HSPC mobilization in response to Plerixafor was also significantly enhanced with a doubling of CFC, HSPC and HSC into the blood of OSMR-/- mice compared to WT. OSMR gene deletion also significantly enhanced HSPC mobilization (3-fold) in response to G-CSF plus Plerixafor combination, the most effective mobilization regimen currently approved. To further demonstrate that endogenous OSM restrains HSPC mobilization in response to G-CSF, we produced a recombinant mouse OSM-trap, a dimeric protein chimera made of the extracellular domain of mouse OSMR and mouse gp130 fused with a mutant Fc fragment of mouse IgG2a. Co-administration of this OSM-trap together with G-CSF also increased HSPC and HSC mobilization into the blood in response to G-CSF compared to mice treated with G-CSF and control trap construct. This negative effect of endogenous OSM on HSPC mobilization was indirectly mediated by BM stromal cells. qRT-PCR on BM myeloid and stromal cells sorted from steady-state or mobilized mice showed that Osm mRNA is mostly expressed by CD11b+F4/80+CD169+VCAM1+ macrophages and CD11b+F4/80-Ly6G+ neutrophils. In sharp contrast, Osmr mRNA was undetectable in any sorted BM leukocyte population, but abundantly expressed by CD45-Lin-CD31+Sca1+CD105+ endothelial and CD45-Lin-CD31-CD51+PDGFRα+/-Sca-1+/- mesenchymal progenitor cells. OSMR protein was expressed by vascular beds in the BM as detected by IHC. Confirming the indirect effect of OSM, we found that recombinant OSM had no effect on HSPC chemotaxis in response to CXCL12, or HSPC adhesion to immobilized VCAM-1 or selectins in vitro. However, HSPC freshly isolated from the BM of OSMR-/- mice had enhanced chemotaxis in response to CXCL12 compared to HSPC isolated from WT mice. In conclusion, G-CSF increases OSM expression in the BM, and this appears to restrain HSPC mobilization in response to G-CSF via OSMR-expressing endothelial and mesenchymal cells that decreases HSPC responsiveness to CXCL12 gradient. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership.

Potent functional HSCs are enriched at the endosteal bone marrow (BM), which comprises ∼10% of total BM. To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes overexpressed in the endosteal relative to central BM. Prostacyclin/prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteal versus central BM (27-fold). PTGIS is the sole enzyme for biosynthesis of PGI2. PGI2 has no reported roles in HSC regulation in the BM and was chosen for further investigation. We found PGI2 analogue iloprost treatment of sorted BM lineage- Kit+ Sca1- (LKS+) cells in vitro for 7 days potently reduced proliferation and differentiation compared to vehicle control. BM cells pulse treated ex vivo with iloprost for 1 hour also resulted in 14-fold increased multilineage reconstitution potential compared to vehicle controls in competitive transplant assay. Iloprost administration in vivo partially rescued BM HSC reconstitution potential in mice sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. These data suggest that PGI2 protects HSC from stress. Mechanistically, we found iloprost pulse treatment in vitro was associated with increased pSTAT3 and cell cycle progression in HSCs. Additionally, we found ∼4-fold greater proportion of untreated LKS+ HSPC homing to the BM of recipients co-administered iloprost + G-CSF compared to vehicle + G-CSF at 4 hours post-transplant. We found that G-CSF administration also decreased Scf and Cxcl12 mRNA expression in BM, which were partially rescued with iloprost co-administration. These data suggest that PGI2 protects HSC during stress by acting directly on HSCs and indirectly to preserve BM niche functions. In summary, we identified PGI2 as a novel HSC niche factor abundant in the endosteal BM.

Neurological heterotopic ossifications (NHO) are frequent complications of traumatic brain and spinal cord injuries (SCI). NHO are abnormal ossifications within periarticular muscles resulting in joint ankylosis, vessel and nerve compression. To elucidate NHO pathogenesis, we have developed the first murine model of SCI-NHO, in which NHO is induced in cardiotoxin-injured muscle only following a SCI. However, what stem cell populations within muscle differentiate into osteoblasts and directly contribute to NHO is unclear. We hypothesized that injured muscles do not repair normally after SCI, and instead osteogenic differentiation of skeletal muscle-resident stem cells takes place. We focused on the two progenitor populations known to reside in muscles: satellite cells (SCs) and fibro- adipogenic progenitors (FAPs). SCs, which specifically express the transcription factor Pax7, differentiate into myoblasts, whereas FAPs, which express are of mesenchymal origin and express Prrx1+ instead, can differentiate into fibroblasts and adipocytes in vitro. We had shown that both populations can deposit calcium under osteogenic condition in vitro. We have now generated Pax7CreER x R26-ZsGreen (Pax7ZsG) and Prrx1Cre x R26-ZsGreen (Prrx1ZsG) to specifically trace the fate of SCs and FAPs respectively in our NHO model in vivo. We demonstrate that after SCI, Pax7+ SCs fail to proliferate and regenerate myofibers with instead persistent proliferation of Prrx1+ FAPs differentiating into osteocalcin+ osteoblasts in SCI-NHO at 21 days post-surgery. The above suggest that SCI causes muscle repair failure with instead proliferation and osteogenic differentiation of FAPs residing in the muscle. In addition, SCI significantly upregulated platelet-derived growth factor receptor (PDGFR) expression in FAPs indicating dysregulation of PDGFR signalling may be involved in the NHO development.

The central macrophage (MΦ) in erythroblastic islands (EI) is critical to erythropoiesis by providing iron and growth factors, and mediating enucleation of the maturing erythroblasts. As MΦ are key effectors of inflammation, we investigated the effect of lipopolysaccharides (LPS) on erythropoiesis in vivo. LPS (2.5mg/kg/d for 2 days) in C57BL/6 mice caused the whitening of the bone marrow (BM) with dramatically decreased erythroblast and reticulocyte numbers. Imaging flow cytometry was used to visualize structural changes and quantify numbers of EI. LPS treatment in vivo caused a marked loss of EI in the BM. This suppressive effect of LPS on BM erythropoiesis was TLR4-dependent as it was absent in TLR4 KO mice. By qRT-PCR and flow cytometry, TLR4 is not expressed by BM erythroblasts but by myeloid cells including EI MΦ. Together with the fact that addition of 100ng/mL LPS into BFU-E assays does not inhibit BFU-E colony growth, these suggest that the suppressive effect of LPS on erythroblasts is indirectly mediated. It is known that LPS mobilizes HSC in a G-CSF-dependent manner. As MΦ express the G-CSF receptor (GCSFR), we explored LPS effects in GCSFR KO mice. Surprisingly, although HSC did not mobilize in response to LPS in GCSFR KO mice, BM erythropoiesis was still suppressed with reduced numbers of erythroblasts. Unexpectedly however, the BM from LPS-treated GCSFR KO mice was still red with high numbers of erythrocytes. The explanation of this paradox is that LPS treatment causes BM vascular leakage in GCSFR KO mice with blood plasma volume in the BM 2.9-fold higher compared to LPS-treated WT mice (measured by i.v. injection of Evans Blue). In conclusion our data show that LPS suppresses medullar erythropoiesis indirectly in a G-CSF-independent manner but GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS.

Summary Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly heterogeneous in terms of microvasculature and thus oxygen concentration. The importance of hypoxia and HIFs in the hematopoietic environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoiesis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in hemodialysis patients. Pharmacological stabilization of HIF-1α protein with PHD inhibitors is also emerging as an important regulator of HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the bone marrow in vivo, thereby protecting them from high doses of irradiation and accelerating hematological recovery. Recent findings also show that stabilization of HIF-1α increases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerixafor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation. These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology

The dynamics of the hematopoietic system in steady state requires precise tuning to maintain the pool of deeply dormant hematopoietic stem cells (HSCs), while allowing the more proliferative HSCs to actively regenerate hematopoietic tissue. This chapter reviews some recent advances in dissecting the interactions between mesenchymal stem cells (MSCs) and HSCs firstly in mice and then in humans. More refined flow cytometry and 3D confocal fluorescent microscopy analyses of Nes-green fluorescent protein (GFP) transgenic mice revealed GFP positivity did not define a single population of bone marrow MSCs. Despite the complexity of HSC niches in the mouse bone marrow and the fact that exquisitely sensitive genetic studies with conditional gene induction or ablation in vivo in mice cannot be repeated in humans, progress has been made to better characterize genuine clonal multipotent MSCs and their HSC-supportive role in human bone marrow.

Macrophages, named for their phagocytic ability, participate in homeostasis, tissue regeneration and inflammatory responses. Bone and adjacent marrow contain multiple functionally unique resident tissue macrophage subsets which maintain and regulate anatomically distinct niche environments within these interconnected tissues. Three subsets of bone-bone marrow resident tissue macrophages have been characterised; erythroblastic island macrophages, haematopoietic stem cell niche macrophages and osteal macrophages. The role of these macrophages in controlling homeostasis and repair in bone and bone marrow niches is reviewed in detail.

Purpose of Review: Steady-state hematopoiesis in adult bone marrow requires the maintenance of a small pool of hematopoietic stem cells (HSCs) by self-renewing symmetric division. HSCs can be divided into potent rarely dividing HSCs which function as long-term reserve and more proliferative HSCs which contribute to maintaining the blood and immune cell pool. Extrinsic instructions provided by unique microenvironments (niches) regulate the fate of individual HSCs and progenitors. This review discusses the latest findings in respect to the organization and function of these niches. Recent Findings: It has recently emerged that mesenchymal stem cells, various osteoblastic progenitors and sinusoidal endothelial cells all critically regulate HSCs within niches. Each of these niche cells expresses different arrays of signaling proteins which differentially regulate HSCs and progenitors. HSCs have been reported in two types of niches. However, as osteoblastic/mesenchymal niches and perivascular niches overlap anatomically, this makes the dichotomy between osteoblastic niches for quiescent HSCs and endothelial niches for more proliferative HSCs a too simplistic model. Indeed local blood perfusion in a niche alone can functionally separate HSC populations. SUMMARY: The fate of each individual HSC is likely to be the result of the unique balance between signals elicited by proteins expressed by mesenchymal/osteoblastic progenitors, sinusoidal endothelial cells and physicochemical cues such as local blood perfusion and hypoxia in each individual niche. More sophisticated three-dimensional fluorescence microscopy techniques on whole mount bone fragments should provide new insights in the spatial organization of niches relative to bone and microcirculation in the bone marrow.

Hematopoietic stem cells (HSC) reside in specific peri-vascular niches in the bone marrow (BM). We have shown interactions with the inflammatory vascular adhesion molecule E-(endothelial)-selectin awakens HSC (Nat Med 2012). We now report that BM vascular cell-surface E-selectin expression is strongly upregulated during HSC mobilization regimens raising the question of a role for endothelial E-selectin in HSC transplant outcome. G-CSF was administered to cohorts of E gene-deleted or wildtype C57BL/6 mice together with E-selectin antagonist Uproleselan (Upro, GMI-1271). We found absence (Sele-/- mice) or therapeutic blockade (Upro) of E-selectin alone in steady-state hosts did not alter levels of circulating peripheral blood (PB) HSC. In contrast absence or therapeutic blockade of E-selectin strongly synergized with mobilizing regimens such as G-CSF or cyclophosphamide+G-CSF by boosting long-term engraftment and reconstitution potential of mobilized blood. The effect was pronounced boosting reconstitution potential over G-CSF-alone-mobilized blood 24-fold ( Two hypotheses may explain how therapeutic E-selectin-blockade promotes potency of mobilized HSC;Dampening of inflammatory activation at the BM niche induced by G-CSF that drive HSC exhaustion,Directly preventing adhesion-mediated HSC commitment during trans-endothelial transmigration from BM into blood. To investigate inflammatory mediator profiles in the BM, cohorts of mice were administered saline control or G-CSF ± Upro (125ug/kg G-CSF, 40mg/kg Upro BiD for 3 days) then femoral BM fluids flushed for cytokine profiling (LegendPlex). As anticipated, G-CSF administration significantly increased concentration of classic pro-inflammatory cytokines (TNF-α, IL-1β, IL-23, IFN-β) in BM associated with HSC activation and loss of quiescence in the BM. No similar boost in inflammatory mediators was observed in BM from mobilized mice co-administered Upro. Similarly pronounced metabolic changes could be observed in BM HSC following in vivo G-CSF administration (such as a significant doubling in HSC Mitochondrial Membrane potential [MOMP] suggesting increased energy demands with HSC activation) was similarly reversed in vivo by Upro co-administration. Together these results suggest blockade of E-selectin on activated vasculature significantly dampens BM HSC activation following G-CSF administration - potentially shielding HSC self-renewal potential. Next we investigated whether the transient interactions with endothelial E-selectin during trans-endothelial transmigration from BM into blood also directly affects HSC potency. BM HSPC harvested from G-CSF plus Upro-treated mice were loaded in transwells pre-coated with recombinant adhesion molecules (P-selectin, E-selectin and CD14-Fc as control) and HSC induced to transmigrate through coated pores towards CXCL12 gradient. After 3hr cell numbers were enumerated and exactly 100 transmigrated HSC from each well transplanted/recipient in a LT-CR transplant assay. Analysis of % CD45.2+ donor HSC reconstitution confirmed a pronounced 10-fold drop in reconstitution potential of HSC transmigrated through E-selectin compared to P-selectin or control coated transwells (p=0.0027) confirming that transient adhesive interactions with E-selectin, such as would occur on activated vasculature during HSC transmigration from BM into the blood, strongly impact subsequent HSC reconstitution upon transplantation. In summary, transient interactions between intravasating HSC with E-selectin on BM vasculature inadvertently compromises reconstitution potential of up to 96% of conventionally harvested HSC, indicating an unexpected disadvantage with current HSC harvesting procedures. These studies also point the way forward to a simple remedy, administration of E-selectin antagonist (Upro) together with G-CSF during HSC mobilization, to improve HSC transplant outcomes. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Marlton:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Perkins:Novartis Oncology: Honoraria. Levesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

Haematopoietic stem cells (HSCs) are regulated by their immediate microenvironment or niche. The most potent functional HSCs are enriched at the endosteum near the bone, which comprises ~10% of total bone marrow (BM). To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes that were >2-fold overexpressed in the endosteal BM relative to the central BM. In this screen, we uncovered known essential HSC niche factors overexpressed in the endosteal BM such as Scf, Cxcl12, and Angpt1, which validated our approach. Among the genes overexpressed in the endosteal BM, prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteum (>10-fold by qRT-PCR, p In initial experiments where we cultured fluorescence activated cell sorted (FACS) BM lineage- Kit+ Sca-1- (LKS+) haematopoietic stem and progenitor cells (HSPCs) for 7 days in serum free conditions, we found iloprost treatment potently reduced proliferation and differentiation compared to vehicle controls, assessed by flow cytometry phenotyping (p We next sought to determine whether iloprost affects HSC function in vivo. In steady state, low dose 0.1mg/kg iloprost administration to mice for 15 days did not alter BM HSPC phenotypes by flow cytometry nor HSC reconstitution potential in competitive transplants compared to vehicle controls suggesting PGI2 does not alter HSC function in homeostasis. To further test the effect of PGI2 following stress, mice were sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. We found low dose iloprost administration partially rescued BM HSC reconstitution potential 21 days following irradiation (p To understand the extrinsic mechanisms through which PGI2 regulates HSC in the BM following stress, we performed a HSC homing assay using naïve donors transplanted into 2-day G-CSF administered recipients. Analysis of HSPC homing to the BM at 4 hours post-transplantation revealed ~4-fold greater proportion of LKS+ HSPC homing to the BM of iloprost co-administered recipients compared to vehicle controls (p In summary, we have identified PGI2 as a novel HSC niche factor abundant in the endosteal BM. PGI2 analogues like Iloprost are well-tolerated and used clinically to treat vascular diseases such as pulmonary arterial hypertension and Raynaud's phenomenon. Our research suggests that PGI2 analogues can be rapidly repurposed in the clinic to improve HSC transplant outcomes and protect against BM failure following acute stressors such as accidental irradiation or inflammation. Disclosures Levesque: GlycoMimetics: Equity Ownership, Patents & Royalties.

In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.

The concept of hematopoietic stem cell (HSC) niche was formulated in 1978, but HSC niches remained unidentified for the following two decades largely owing to technical limitations. Sophisticated live microscopy techniques and genetic manipulations have identified the endosteal region of the bone marrow (BM) as a preferential site of residence for the most potent HSC - able to reconstitute in serial transplants - with osteoblasts and their progenitors as critical cellular elements of these endosteal niches. This article reviews the path to the discovery of these endosteal niches (often called 'osteoblastic' niches) for HSC, what cell types contribute to these niches with their known physical and biochemical features. In the past decade, a first wave of research uncovered many mechanisms responsible for HSC homing to, and mobilization from, the whole BM tissue. However, the recent discovery of endosteal HSC niches has initiated a second wave of research focusing on the mechanisms by which most primitive HSC lodge into and migrate out of their endosteal niches. The second part of this article reviews the current knowledge of the mechanisms of HSC lodgment into, retention in and mobilization from osteoblastic niches.

We compared potential trafficking mechanisms used by human (h) multipotent mesenchymal stem cells (MSC) derived from bone marrow (bm) or placenta (p). Both hbmMSC and hpMSC expressed a broad range of cell surface adhesion molecules including beta1-integrins (CD29) and CD44. Array data showed that both hbmMSC and hpMSC expressed mRNA for the cell adhesion molecules CD54 (ICAM-1), E-cadherin, CD166 (ALCAM), CD56 (NCAM), CD106 (VCAM-1), CD49a, b, c, e and f (integrins alpha1, 2, 3, 4 and 6), integrin alpha11, CD51 (integrin alphaV), and CD29 (integrins beta1). Functional binding of hpMSC, but not hbmMSC to VCAM-1 was demonstrated using recombinant chimeric constructs. Neither bone marrow nor placental MSC expressed ligands to endothelial selectins such as PSGL-1 or sialyl Lewis X (sLe(x)) carbohydrates and neither were able to bind functionally to chimeric constructs of the endothelial selectins CD62E (E-selectin) and CD62P (P-selectin). Furthermore, MSC expressed a restricted range of transferases necessary for expression of sLe(x), with no detectable expression of fucosyl transferases IV or VII. Placental MSC, but not hbmMSC, expressed mRNA for the chemokine receptors CCR1 and CCR3, and both hbmMSC and hpMSC expressed mRNA for CCR7, CCR8, CCR10, CCR11, CXCR4 and CXCR6. Intracellular chemokine receptor protein expression of CCR1, CCR3, CXCR3, CXCR4 and CXCR6 was detected in both hbmMSC and hpMSC. Cell surface expression of chemokine receptors was much more restricted with only CXCR6 displaying a strong signal on hbmMSC and hpMSC. Although cell surface expression of CXCR4 was not detected, MSC migrated in response to its ligand, CXCL12 (SDF-1). Thus, hbmMSC and hpMSC have an almost identical profile for cell surface adhesion and chemokine receptor molecules at the mRNA and protein levels. However, at the functional level, hpMSC likely utilise VLA-4-mediated binding in a superior manner to hbmMSC and thus may have superior engraftment properties to hbmMSC in vivo.

Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1α (HIF-1α) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1α protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF-1α transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability.Disclosure of potential conflicts of interest is found at the end of this article.

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)–induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF–induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

Objective. The twoendothelial selectins, P- and E-selectin, are critically importantfor adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. Materials and Methods. Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed6monthsposttransplant.Bonemarrowlineage-negative(Lin)Sca-1c-kitcellswere then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoieticpotentialwasassessedusinginvitrophenotypingandcolony-formingassaysand in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. Results. Long-term competitive repopulating HSCs were Linc-kit bright and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of LinSca-1ckit cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S12 compared to the other cultures. In contrast, E-selectin enhanced the differentiation of LinSca-1c-kit cells into cells that expressed the granulocytematurationmarker,Gr-1,accompaniedbylossofCFCpotentialfromthesecultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. Conclusion. These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S12 in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of LinSca-1c-kit cells, allowing enhanced ex vivo expansion of CFC and CFU-S12 but not HSCs. 2005 International Society for Experimental Hematology. Published by Elsevier Inc.

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9–deficient mice, NE × CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE × CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12α protein expression in the bone marrow of Ne × CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BM-Macs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-Lin-Kit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP+ HSC. Depletion of recipient CD169+ Macs abated engraftment of donor phenotypic HSC by 70% at 5 wk post-transplant and reduced BM reconstitution potential in competitive secondary transplants. In conclusion BM contains a myeloablation-resistant self-repopulating Mac subset that is necessary for efficient and/or sustained HSC BM engraftment following transplantation. Therefore interpretation of transplantation chimera experiments must take into account the persistence of this subset of host macrophages that support donor HSC engraftment.

Objective. Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic progenitor and stem cells from the bone marrow into the peripheral blood. Although the mechanisms leading to the mobilization of primitive hematopoietic cells is not fully understood, it has been noted that the yield of mobilization in humans is correlated to the down-regulation of c-KIT/CD117 expression on mobilized cells. We sought to determine the mechanisms responsible for the reduced expression of c-KIT on mobilized hematopoietic progenitor cells. Materials and Methods. Mice were mobilized with G-CSF and primitive hematopoietic cells were collected from bone marrow and blood to analyze c-KIT expression. Using cell lines expressing mouse and human c-KIT and a recombinant protein comprising the entire extracellular domain of human c-KIT, we analyzed by flow cytometry and immunoblotting the proteolytic cleavage of c-KIT by proteases released in bone marrow extracellular fluids extracted from mobilized mice. Results. Administration of G-CSF into mice results in the reduction of c-KIT expression on primitive hematopoietic cells in bone marrow and peripheral blood. Bone marrow extracellular fluids isolated from G-CSF-mobilized mice contain serine proteases that cleave c-KIT into discrete fragments. Proteases capable of cleaving c-KIT include neutrophil elastase, cathepsin G, proteinase-3 and matrix metalloproteinase-9. Conclusions. In addition to transcriptional controls, exocytosis, and ligand-induced internalization, the direct proteolytic cleavage of c-KIT by neutrophil and macrophage proteases represents a novel pathway to regulate the levels of c-KIT expression at the surface of hematopoietic cells and may be responsible in part for the down-regulation of c-KIT expression on mobilized hematopoietic progenitors in vivo.

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

Objective. Hematopoietic stem and progenitor cells normally reside in the bone marrow but can be mobilized into the peripheral blood following treatment with granulocyte colony-stimulating factor (G-CSF) or myelosuppressive chemotherapy. Although the number of transplants performed with mobilized blood currently exceeds those performed with bone marrow, little is known of the molecular mechanisms responsible for this phenomenon. We sought to determine whether mobilization induced by G-CSF or chemotherapy was triggered by common or distinct mechanisms. Methods. Balb/c mice were mobilized with either G-CSF alone, cyclophosphamide alone, or the combination of both agents. Spleens, peripheral blood, bone marrow extracellular fluids, and cells were taken at different time points and analyzed for the expression of VCAM-1, the number of peripheral blood progenitor cells, concentration of neutrophil proteases, and number of granulocytes. Results. Administration of either G-CSF or the myelosuppressive agent cyclophosphamide results in a sharp reduction of VCAM-1/CD106 expression in the bone marrow that coincides with the accumulation of granulocytic precursors and release of active neutrophil proteases neutrophil elastase and cathepsin G that directly cleave VCAM-1/CD106 in vitro. These events follow precisely the kinetics of hematopoietic progenitor cell mobilization into the peripheral blood. Conclusion. We have identified a commonality of events during mobilization induced by either G-CSF or chemotherapy, which include the accumulation in the bone marrow of active neutrophil proteases that directly cleave VCAM-1 and lead to the sharp reduction of VCAM-1 expression in this tissue.

The cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34+ HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF–overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF–induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF–induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF–induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.© 1998 by The American Society of Hematology.

The interaction between p145(o-KIT) and p210(bcr-abl), transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34(+) cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34(+) cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34(+) cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) from CML CD34(+)CD38(+) cells and the more primitive CML CD34(+)CD38(-) cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34(+) cells showed exclusive growth of Phi cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of beta(1)-integrin-mediated adhesion of CML CD34(+) cells to fibronectin was not increased when compared with the effect on normal CD34(+) cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML, (C) 1998 by The American Society of Hematology.