Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.     

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.     

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca²⁺ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K⁺ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.     

Regulation of hematopoietic stem cells in the niche

Hematopoiesis provides a suitable model for understanding adult stem cells and their niche. Hematopoietic stem cells(HSCs) continuously produce blood cells through orchestrated proliferation, self-renewal, and differentiation in the bone marrow(BM). Within the BM exists a highly organized microenvironment termed "niche" where stem cells reside and are maintained. HSC niche is the first evidence that a microenvironment contributes to protecting stem cell integrity and functionality in mammals. Although multiple models exist, recent progress has principally elucidated the cellular complexity of the HSC niche that maintains and regulates HSCs in BM. Here we introduce the development and summarize the achievements of HSC niche studies.     

Influence of a dual-injection regimen,plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

Background aimsInadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis.MethodsWe used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4⁺ fraction of CD34⁺ HSCs, we could improve engraftment. Human cord blood-derived CD34⁺ cells and human bone marrow-derived MSCs were used for these studies.ResultsWhen MSCs were transplanted 1 week before CD34⁺ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34⁺ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4⁺ cells in the CD34⁺ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages.ConclusionsPrior MSC and HSC cotransplantation followed by manipulation of the CXCR4–SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.     

Coordinate Regulation of Stem Cell Competition by Slit-Robo and JAK-STAT Signaling in the Drosophila Testis

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.     

Mesenchymal stem cells in hematopoietic stem cell transplantation

Mesenchymal stromal/stem cells (MSC) of bone marrow (BM) origin not only provide the supportive microenvironmental niche for hematopoietic stem cells (HSC) but are capable of differentiating into various cell types of mesenchymal origin, such as bone, fat and cartilage. In vitro and in vivo data suggest that MSC have low inherent immunogenicity, modulate/suppress immunologic responses through interactions with immune cells, and home to damaged tissues to participate in regeneration processes through their diverse biologic properties. MSC derived from BM are being evaluated for a wide range of clinical applications, including disorders as diverse as myocardial infarction and newly diagnosed diabetes mellitus type 1. However, their use in HSC transplantation, either for enhancement of hematopoietic engraftment or for treatment/prevention of graft-versus-host disease, is far ahead of other indications. Ease of isolation and ex vivo expansion of MSC, combined with their intriguing immunomodulatory properties and their impressive record of safety in a wide variety of clinical trials, make these cells promising candidates for further investigation.     

Circulating CXCR4-positive stem/progenitor cells compete for SDF-1-positive niches in bone marrow,muscle and neural tissues: an alternative hypothesis to stem cell plasticity

The trans-differentiation hypothesis of adult tissue-specific stem cells has been recently questioned because of insufficient proof that the so-called plasticity experiments were performed on pure populations of tissue-specific stem cells. It was shown recently, for example, that the formation of haematopoietic colonies by muscle cells depended on the presence of haematopoietic stem/progenitor cells residing within the muscle tissue and hence was not related to the plasticity of the muscle stem cells. The explanation for the presence in, or homing into, muscles of haematopoietic stem cells is, however, not clear. In our study, we hypothesised that muscle tissues secrete stromal-derived factor (SDF)- 1, an alpha-chemokine for haematopoietic stem cells (HSC), which could attract HSC circulating in peripheral blood into muscle tissue. We found, using RT-PCR and immunocytochemistry, that SDF-1 was expressed in human heart and skeletal muscles. Moreover, muscle satellite cells, which are pivotal for regeneration of muscle, highly expressed on their surface CXCR4, a G-protein-coupled receptor that binds SDF-1. To determine whether the CXCR4 receptor is functional on muscle satellite/progenitor cells, we stimulated murine satellite cells (the C2C12 cell line) with SDF-1 and demonstrated the phosphorylation of p42/44 MAPK and AKT serine-threonine kinase in these cells. Moreover, we showed that SDF-1 gradient chemoattracts these cells. We postulate that the CXCR4-positive muscle satellite and CXCR4-positive HSC circulating in the peripheral blood compete for occupancy of SDF-1-positive stem cell niches that are present in bone marrow and muscle tissues. Thus, we suggest that competition for common niches by various circulating CXCR4-positive stem cells and their ability to home to the SDF-1-positive niches in various organs, is a better explanation than stem cell plasticity of why (i) haematopoietic colonies can be cultured from muscles and (ii) early muscle progenitors could be cultured from bone marrow.     

Heterogeneous populations of bone marrow stem cells--are we spotting on the same cells from the different angles?

Accumulated evidence suggests that in addition to hematopoietic stem cells (HSC), bone marrow (BM) also harbors endothelial stem cells (ESC), mesenchymal stem cells (MSC), multipotential adult progenitor cells (MAPC), pluripotent stem cells (PCS) as well as tissue committed stem cells (TCSC) recently identified by us. In this review we discuss the similarities and differences between these cell populations. Furthermore, we will present the hypothesis that all of these versatile BM derived stem cells are in fact different subpopulations of TCSC. These cells accumulate in bone marrow during ontogenesis and being a mobile population of cells are released from BM into peripheral blood after tissue injury to regenerate damaged organs. Furthermore, since BM is a "hideout" for TCSC, their presence in preparations of bone marrow derived mononuclear cells should be considered before experimental evidence is interpreted simply as trans-differentiation or plasticity of HSC. Finally, our observation that the number of TCSC accumulate in the bone marrow of young animals and their numbers decrease during senescence provides a new insight into aging and may explain why the regeneration processes becomes less effective in older individuals.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.     

Cytokines and hematopoietic stem cell mobilization

Hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of many hematologic malignancies, chemotherapy sensitive relapsed acute leukemias or lymphomas, multiple myeloma; and for some non-malignant diseases such as aplastic anemia and immunodeficient states. The hematopoietic stem cell (HSC) resides in the bone marrow (BM). A number of chemokines and cytokines have been shown in vivo and in clinical trials to enhance trafficking of HSC into the peripheral blood. This process, termed stem cell mobilization, results in the collection of HSC via apheresis for both autologous and allogeneic transplantation. Enhanced understanding of HSC biology, processes involved in HSC microenvironmental interactions and the critical ligands, receptors and cellular proteases involved in HSC homing and mobilization, with an emphasis on G-CSF induced HSC mobilization, form the basis of this review. We will describe the key features and dynamic processes involved in HSC mobilization and focus on the key ligand-receptor pairs including CXCR4/SDF1, VLA4/VCAM1, CD62L/PSGL, CD44/HA, and Kit/KL. In addition we will describe food and drug administration (FDA) approved and agents currently in clinical development for enhancing HSC mobilization and transplantation outcomes.     

Hematopoietic stem cell niche: An interplay among a repertoire of multiple functional niches

Background

Hematopoietic stem cell (HSC) niche of the BM provides a specialized microenvironment for the regulation of HSCs. The strict control of HSCs by the niche coordinates the balance between the proliferation and the differentiation of HSCs for the homeostasis of the blood system in steady states and during stress hematopoiesis. The osteoblastic and vascular niches are the classically identified constituents of the BM niche.

Scope of review

Recent research broadens our understanding of the BM niche as an assembly of multiple niche cells within the BM. We provide an overview of the HSC niche aiming to delineate the defined and possible niche cell interactions which collectively modulate the HSC integrity.

Major conclusions

Multiple cells in the BM, including osteoblasts, vascular endothelia, perivascular mesenchymal cells and HSC progeny cells, function conjunctively as niche cells to regulate HSCs.

General significance

The study of HSC niche cells and their functions provides insights into stem cell biology and also may be extrapolated into the study of cancer stem cells. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit+ stem/progenitors. In MMP-9-/- mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.     

microRNA-150 regulates mobilization and migration of bone marrow-derived mononuclear cells by targeting Cxcr4

The interaction between chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor (SDF)-1, plays an important role in stem cell mobilization and migration in ischemic tissues. MicroRNAs (miRs) are key regulators of stem cell function and are involved in regulation of stem cell survival and differentiation to adopt different cell lineages. In this study, we show that ischemia inhibits the expression of miR-150 in BM-derived mononuclear cells (MNC) and activates its target Cxcr4 gene. Our results show that miR-150/CXCR4 cascade enhances MNC mobilization and migration. By using mouse acute myocardial infarction (MI) model, we found that MNCs in peripheral blood (PB) were increased significantly at day 5 after AMI as compared to control group and the number of CXCR4 positive MNCs both in bone marrow (BM) and PB was also markedly increased after MI. Analysis by microarray-based miRNA profiling and real-time PCR revealed that the expression of miR-150 which targets Cxcr4 gene as predicted was significantly downregulated in BM-MNCs after MI. Abrogation of miR-150 markedly increased CXCR4 protein expression suggesting its target gene. To show that miR-150 regulates MNC mobilization, knockdown of miR-150 in BM-MNCs by specific antisense inhibitor resulted in their higher migration ability in vitro as compared to scramble-transfected MNCs. Furthermore, in vivo BM transplantation of MNCs lacking miR-150 expression by lentiviral vector into the irradiated wild type mice resulted in the increased number of MNCs in PB after AMI as compared to control. In conclusion, this study demonstrates that ischemia mobilizes BM stem cells via miR-150/CXCR4 dependent mechanism and miR-150 may be a novel therapeutic target for stem cell migration to the ischemic tissue for neovascularization and repair.     

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 ([Formula: see text]-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.     

Niche-to-niche migration of bone-marrow-derived cells

During ontogenesis, haematopoietic stem cells (HSCs) relocate between extra-embryonic and embryonic compartments. Similarly, site-specific homing of HSCs is ongoing during adulthood. With the expanding knowledge of HSC physiology, a new paradigm emerges in which HSCs and haematopoietic progenitor cells (HPCs) migrate to defined microenvironments within the bone marrow (BM) and to 'activated' or 'inducible' niches elsewhere. Here, we summarize current understanding of HSC niche characteristics, and the physiological and pathological mechanisms that guide HSC homing both within the BM and to distant niches in the periphery, promoting new vessel growth in tumours and ischaemia. Recent observations suggest that features of the HSC niche might also be recapitulated in pre-metastatic sites. Clusters of BM-derived HPCs promote invasion of disseminating cancer cells. Clear clinical benefits can be foreseen by modulating HSCs and their microenvironments, in promoting tissue regeneration, and inhibiting tumourigenesis and cancer metastasis.     

Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.     

TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G₀ state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)–induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.