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The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81−/− mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection., Lactate is a by-product of glycolysis that can function via its G protein receptor GPR81. Here the authors show that LPS or Salmonella infection enhances glycolytic metabolism in bone marrow neutrophils, resulting in lactate production, which increases endothelial barrier permeability and mobilization of these neutrophils by targeting endothelial GPR81.

The mobilization efficiency of hematopoietic stem/progenitor cells from bone marrow (BM) to circulation by granulocyte colony-stimulating factor (G-CSF) is dramatically dispersed in humans and mice with no mechanistic lead for poor mobilizers. The regulatory mechanism for mobilization efficiency by dietary fat was assessed in mice. Fat-free diet (FFD) for 2 weeks greatly increased mobilization compared to normal diet (ND). The BM mRNA level of peroxisome proliferator-activated receptor δ (PPARδ), a receptor for lipid mediators, was markedly up-regulated by G-CSF in mice fed with ND and displayed strong positive correlation with widely scattered mobilization efficiency. It was hypothesized that BM fat ligand for PPARδ might inhibit mobilization. The PPARδ agonist inhibited mobilization in mice fed with ND and enhanced mobilization by FFD. Treatment with the PPARδ antagonist and chimeric mice with PPARδ+/- BM showed enhanced mobilization. Immunohistochemical staining and flow cytometry revealed that BM PPARδ expression was enhanced by G-CSF mainly in mature/immature neutrophils. BM lipid mediator analysis revealed that G-CSF treatment and FFD resulted in the exhaustion of ω3-polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). EPA induced the up-regulation of genes downstream of PPARδ, such as carnitine palmitoyltransferase-1α and angiopoietin-like protein 4 (Angptl4), in mature/immature neutrophils in vitro and inhibited enhanced mobilization in mice fed with FFD in vivo. Treatment of wild-type mice with the anti-Angptl4 antibody enhanced mobilization together with BM vascular permeability. Collectively, PPARδ signaling in BM mature/immature neutrophils induced by dietary fatty acids negatively regulates mobilization, at least partially, via Angptl4 production.

Hematopoietic stem and progenitor cells (HSPCs) are essential for daily mature blood cell production, host immunity, and osteoclast-mediated bone turnover. The timing at which stem cells give rise to mature blood and immune cells while maintaining the bone marrow (BM) reservoir of undifferentiated HSPCs and how these opposite tasks are synchronized are poorly understood. Previous studies revealed that daily light onset activates norepinephrine (NE)-induced BM CXCL12 downregulation, followed by CXCR4+ HSPC release to the circulation. Recently, we reported that daily light onset induces transient elevations of BM NE and tumor necrosis factor (TNF), which metabolically program BM HSPC differentiation and recruitment to replenish the blood. In contrast, darkness onset induces lower elevations of BM NE and TNF, activating melatonin production, which metabolically reprograms HSPCs, increasing their short- and long-term repopulation potential, and BM maintenance. How the functions of BM-retained HSPCs are influenced by daily light and darkness cycles and their clinical potential are further discussed.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of several cell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs), endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR), which is used as a marker to isolate them. EPCR/protease-activated receptor-1 (PAR1) signaling in endothelial cells has anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflammation. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR(+) LT-HSC BM retention and egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production, maintaining NO(low) LT-HSC BM retention with increased VLA4 expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generation and activate different PAR1 signaling that overcomes BM EPCR(+) LT-HSC retention, inducing their recruitment to the bloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulation of CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses new roles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1 signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production.

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor-positive (EPCR(+)) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α-converting enzyme (TACE), enhanced CXCL12-CXCR4-induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR(+) LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NO(low) EPCR(+) LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR(+) LT-HSCs, with potential clinical relevance for stem cell transplantation.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

MicroRNA-155 promotes G-CSF-induced mobilization of murine hematopoietic stem and progenitor cells via propagation of CXCL12 signaling

Migration of hematopoietic stem and progenitor cells (HSPC) migration from the blood, their transendothelial migration across the physical blood–bone marrow (BM) barrier into the BM is an active first step termed homing which is essential during embryo development and clinical BM transplantation. Steady-state HSPC egress from the bone marrow during homeostasis is dramatically accelerated during stress-induced recruitment as part of host defense mechanisms and is mimicked by clinical mobilization for HSPC transplantation. Cytokines (including G-CSF), chemotherapeutic agents, or other small molecule inhibitors (including the CXCR4 antagonist AMD3100) treatment disrupts signals retaining HSPC retention in the BM and enhances their massive recruitment to the circulation which is harnessed for clinical transplantation. Both these processes although unique, share some overlapping molecules, pathways, and cellular components. In this chapter, we will discuss the current understanding of key features and dynamic processes involved in HSPC mobilization and homing and the mechanistic insights including guidance cues and molecular pathways. In particular, we will discuss the involvement of microenvironmental interactions, SDF-1/CXCR4, VLA-4/VCAM-1, osteoblasts, osteoclasts, selectins, integrins, CD44/hyaluronic acid, matrix metalloproteinases, bioactive sphingolipids, complement proteins, and regulation by the nervous and immune systems. We will also discuss suggestive, experimental studies to improve homing and commonly used mobilizing agents.

Significance: Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. Recent Advances: ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROShigh short-term repopulating stem cells, and ROSlow quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. Critical Issues: Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. Future Directions: Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development. Antioxid. Redox Signal. 21, 1605–1619.

Innate immune neutrophils provide the first line of host defense against bacterial infections. Neutrophils under steady state rely almost entirely on glycolysis and exhibit very low levels of oxidative phosphorylation. The metabolite lactate has long been considered a "waste byproduct" of cell metabolism which accumulates during inflammation and sepsis. Increased plasma lactate levels in human patients is used as a marker for sepsis diagnosis. However, the direct effector actions of lactate, particularly in regulating neutrophil mobilization and function during inflammation has remained obscure. To better understand the metabolic consequences of BM neutrophil activation during the onset of inflammation, we tested how bacterial lipopolysaccharides (mimicking gram negative bacterial inflammation) introduced intraperitoneally (i.p.) affect neutrophil metabolism and mobilization. RNAseq of sorted BM neutrophils revealed that LPS-activated neutrophils upregulate enzymes catalyzing the first part of glycolysis (hexokinase and PFKL) and downregulate the expression of TCA cycle enzymatic genes. In addition, LPS enhanced neutrophil lactate production and release as indicated by higher levels of BM lactate and higher expression of LDHA and MCT4. In addition, LPS increased NADPH oxidase (NOX)-mediated reactive oxygen species and HIF-1α levels in BM neutrophils, which are up-stream of glycolytic enzymes and lactate production and release. Recently, we reported that i.p. lactate administration rapidly activated and mobilized neutrophils from BM to the circulation (ASH, 2017). To test if lactate acts preferentially on neutrophils, we also examined other types of hematopoietic cells. Interestingly, we found that lactate specifically and rapidly (i.e., within 4 hrs) mobilized neutrophils to the circulation whereas the levels of peripheral blood (PB) monocytes, lymphocytes, granulocyte monocyte progenitors (GMPs) and hematopoietic progenitor stem cells (LSK) were reduced following lactate administration. LPS treatment failed to mobilize activated ROShigh neutrophils to the PB in NOX-/- mice, while lactate administration partially rescued this defect following LPS treatment. Our data also reveal that the NOX/ROS axis operates upstream of lactate production in BM neutrophils since abnormal metabolic rates were found in NOX-/- neutrophils during the onset of the acute inflammatory responses. Moreover, we found that BM endothelial cells (BMEC) abundantly express the highly selective lactate receptor GPR81, and that neutrophil-released lactate increased BM vascular permeability via BMEC GPR81 signaling (ASH, 2017). Consistent with a role of the lactate/GPR81 axis in enhanced vascular permeability, we find that i.p. injected LPS reduced VE-Cadherin expression on highly permeable sBMECs in GPR81 dependent manner. Notably, neutralizing VE-Cadherin in GPR81-/- mice can rescue and elevate PB neutrophil levels, similarly to wild-type (WT) mice, suggesting that VE-Cadherin is downstream of GPR81 signaling and plays a role in neutrophil mobilization. Finally, to examine the potential clinical relevance of our findings, we infected WT, NOX-/- and GPR81-/- mice with Salmonella Typhimurium and found out that this pathogen drove high generation of ROS, elevated HIF-1αlevels, and triggered lactate production and release in WT BM neutrophils. In contrast, BM neutrophils of infected NOX-/- mice exhibited significantly lower HIF-1αand impaired lactate production and release. Consequently, WT mice infected with Salmonella had a higher levels of neutrophils in the blood, as compared to their NOX-/- or GPR81-/- mice counterparts. Altogether, our data reveal that the same regulatory mechanisms by which neutrophils respond to LPS challenges are used during bacterial infection with Salmonella. Our study highlights lactate released by BM neutrophils as a key pro-inflammatory stimulus of a novel immune-metabolic crosstalk which is triggered by infection and locally opens the BM vascular barrier to facilitate neutrophil mobilization and recruitment to sites of inflammation. Targeting this immune-metabolic crosstalk between lactate-producing neutrophils and the BM endothelium could be useful for the control of pathological neutrophil activation and mobilization during bacterial infections and help treatments of neutrophil related immune disorders. Disclosures No relevant conflicts of interest to declare.

The role of corticosterone (Cort), the immune system's major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment. © 2013 Macmillan Publishers Limited All rights reserved.

Cytokine-induced expansion of hematopoietic stem and progenitor cells (HSPCs) is not fully understood. In the present study, we show that whereas steady-state hematopoiesis is normal in basic fibroblast growth factor (FGF-2)–knockout mice, parathyroid hormone stimulation and myeloablative treatments failed to induce normal HSPC proliferation and recovery. In vivo FGF-2 treatment expanded stromal cells, including perivascular Nestin+ supportive stromal cells, which may facilitate HSPC expansion by increasing SCF and reducing CXCL12 via mir-31 up-regulation. FGF-2 predominantly expanded a heterogeneous population of undifferentiated HSPCs, preserving and increasing durable short- and long-term repopulation potential. Mechanistically, these effects were mediated by c-Kit receptor activation, STAT5 phosphorylation, and reduction of reactive oxygen species levels. Mice harboring defective c-Kit signaling exhibited abrogated HSPC expansion in response to FGF-2 treatment, which was accompanied by elevated reactive oxygen species levels. The results of the present study reveal a novel mechanism underlying FGF-2–mediated in vivo expansion of both HSPCs and their supportive stromal cells, which may be used to improve stem cell engraftment after clinical transplantation.

Adult hematopoietic stem cells (HSC) continuously replenish the blood with immune and blood cells with a finite life span, from the bone marrow (BM) reservoir of immature and maturing leukocytes. Regulation of HSC migration and development is essential for their function and blood cell production. These diverse and multiple states require a tight regulation to efficiently address host defense and repair requirements. Numerous recent studies disclose a central role for bone related cells in regulation of HSC and hematopoiesis. During ontogeny HSC home and seed the fetal BM in the last gestation period when the bone is already ossified. Ossification involves bone forming osteoblast- and bone degrading osteoclast activity and is considered essential for the formation of BM cavities and hematopoiesis. This synchronized association implies the need for active bone cells and bone turnover for HSC regulation. Osteoblastic cells and SDF- 1+/nestin+ reticular adventitial and CAR cells are crucial for regulation of HSC lodgment, self-renewal and function. Bone resorbing osteoclasts regulate bone turnover and progenitor cell detachment and release from the BM. Sympathetic signals from the nervous system activated by circadian rhythms or stress conditions control both bone turnover and HSC migration and development. In this review we discuss pathways and mechanisms involved in this orchestrated regulatory network. A special focus is made on the pivotal role of the SDF-1/CXCR4 axis as a determinant of HSC fate. Inflammation, DNA damage, cytokine mobilization, microgravity and aging are discussed as specific physiologic and pathologic events entailing dysregulation of the tightly balanced Bone-Brain-Blood triad.

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34 + progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells. © 2011 Nature America, Inc. All rights reserved.

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM. Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. Disclosures No relevant conflicts of interest to declare.

Therapeutic mobilization of vasculogenic progenitor cells is a novel strategy to enhance neovascularization for tissue repair. Prototypical mobilizing agents such as granulocyte colony-stimulating factor mobilize vasculogenic progenitor cells from the bone marrow concomitantly with inflammatory cells. In the bone marrow, mobilization is regulated in the stem cell niche, in which endosteal cells such as osteoblasts and osteoclasts play a key role. Because Wnt signaling regulates endosteal cells, we examined whether the Wnt signaling antagonist Dickkopf (Dkk)-1 is involved in the mobilization of vasculogenic progenitor cells. Using TOP-GAL transgenic mice to determine activation of β-catenin, we demonstrate that Dkk-1 regulates endosteal cells in the bone marrow stem cell niche and subsequently mobilizes vasculogenic and hematopoietic progenitors cells without concomitant mobilization of inflammatory neutrophils. The mobilization of vasculogenic progenitors required the presence of functionally active osteoclasts, as demonstrated in PTPε-deficient mice with defective osteoclast function. Mechanistically, Dkk-1 induced the osteoclast differentiation factor RANKL, which subsequently stimulated the release of the major bone-resorbing protease cathepsin K. Eventually, the Dkk-1–induced mobilization of bone marrow–derived vasculogenic progenitors enhanced neovascularization in Matrigel plugs. Thus, these data show that Dkk-1 is a mobilizer of vasculogenic progenitors but not of inflammatory cells, which could be of great clinical importance to enhance regenerative cell therapy.

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor κB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1+/c-Kit+/Lin− cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell–rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1+/c-Kit+/Lin− cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

Bone remodeling by bone-forming osteoblasts and bone-resorbing osteoclasts dynamically alters the bone inner wall and the endosteum region, which harbors osteoblastic niches for hematopoietic stem cells. Investigators have recently elucidated mechanisms of recruitment and mobilization; these mechanisms consist of stress signals that drive migration of leukocytes and progenitor cells from the bone marrow reservoir to the circulation and drive their homing to injured tissues as part of host defense and repair. The physical bone marrow vasculature barrier that is crossed by mobilized cells actively transmits chemotactic signals between the blood and the bone marrow, facilitating organ communication and cell trafficking. Osteoclasts play a dual role in regulation of bone resorption and homeostatic release or stress-induced mobilization of hematopoietic stem/progenitor cells. In this review, we discuss the orchestrated interplay between bone remodeling, the immune system, and the endosteal stem cell niches in the context of stem cell proliferation and migration during homeostasis, which are accelerated during alarm situations.

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

The mechanisms governing migration and extramedullary dissemination of leukemic cells remain obscure. In this study the migration and in vivo homing to the bone marrow of nonobese diabetic severe combined immunodeficient (NOD/SCID) mice injected with human precursor-B acute lymphoblastic leukemia (ALL) cells in comparison to normal CD34+ progenitors (both cord blood and mobilized peripheral blood) was investigated. Although migration and homing of both cell populations was dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions, major differences in receptor expression as well as the migratory capacity toward various concentrations of SDF-1 were found. Furthermore, unlike normal CD34+ progenitors, in vivo homing of the leukemic cells was superior when recipient NOD/SCID mice were not irradiated prior to transplantation. In addition, we report differences in the adhesion molecules activated following SDF-1 stimulation, documenting a major role for very late antigen 4 (VLA-4), but not VLA-5 and lymphocyte function-associated antigen-1 (LFA-1), in homing of precursor-B ALL cells. Interestingly, Toxin-B and pertussis toxin inhibited the homing of the leukemic cells but not that of normal CD34+ progenitors or normal CD10+/CD19+ precursor-B cells, revealing differences in CXCR4 signaling pathways that are based on changes that acquired by the leukemic cells. Altogether, our data provide new insights into different SDF-1–induced signaling, activation, and consequent motility between normal CD34+ and precursor-B ALL progenitors, which may lead to improved clinical protocols. (Blood. 2004;103: 2900-2907)

Successful blood and marrow transplant (BMT), both autologous and allogeneic, requires the infusion of a sufficient number of hematopoietic progenitor/stem cells (HPCs) capable of homing to the marrow cavity and regenerating a full array of hematopoietic cell lineages in a timely fashion. At present, the most commonly used surrogate marker for HPCs is the cell surface marker CD34, identified in the clinical laboratory by flow cytometry. Clinical studies have shown that infusion of at least 2 × 106 CD34+ cells/kg recipient body weight results in reliable engraftment as measured by recovery of adequate neutrophil and platelet counts approximately 14 days after transplant. Recruitment of HPCs from the marrow into the blood is termed mobilization, or, more commonly, stem cell mobilization. In Section I, Dr. Tsvee Lapidot and colleagues review the wide range of factors influencing stem cell mobilization. Our current understanding focuses on chemokines, proteolytic enzymes, adhesion molecules, cytokines and stromal cell-stem cell interactions. On the basis of this understanding, new approaches to mobilization have been designed and are now starting to undergo clinical testing. In Section II, Dr. Michele Cottler-Fox describes factors predicting the ability to mobilize the older patient with myeloma. In addition, clinical approaches to improving collection by individualizing the timing of apheresis and adjusting the volume of blood processed to achieve a desired product are discussed. Key to this process is the daily enumeration of blood CD34+ cells. Newer methods of enumerating and mobilizing autologous blood HPCs are discussed. In Section III, Dr. John DiPersio and colleagues provide data on clinical results of mobilizing allogeneic donors with G-CSF, GM-CSF and the combination of both as relates to the number and type of cells collected by apheresis. Newer methods of stem cell mobilization as well as the relationship of graft composition on immune reconstitution and GVHD are discussed.

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34(+) stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34(+)CD38(-)CXCR4(-) and CXCR4(+) cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34(+)CXCR4(+) (R4(+)) and CD34(+)CXCR4(-) (R4(-)) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34(+) cells. Coinjection of purified cells with 10 microg anti-CXCR4 mAb significantly reduced engraftment of all CD34(+) subsets, and 50 microg completely abrogated engraftment by R4(-) and CD34(+) cells. Importantly, R4(-) cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4(-) cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34(+) cells but yet CXCR4 dependent. In conclusion, R4(-) cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1-dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34(+) stem and progenitor cells, regulating their motility and repopulation capacities.

Chronic myelogenous leukemia (CML), a malignant myeloproliferative disorder originating from a pluripotent stem cell expressing the bcr-abl oncogene, is characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow (BM) into the circulation. Moreover, immature CD34+ CML cells have lower adhesion to stromal cells and fibronectin as well as lower engraftment potential in severe combined immunedeficient (SCID) and nonobese diabetic (NOD)/SCID mice than normal CD34+ cells. We report in this study that leukemic Philadelphia chromosome-positive (Ph+)CD34+ cells from newly diagnosed CML patients that express the chemokine receptor CXCR4 migrate in response to stromal-derived factor-1 (SDF-1). However, normal Ph-CD34+CXCR4+ cells derived from the same patient have significantly higher migration levels toward SDF-1. In contrast to their transwell migration potential, the SDF-1-mediated integrin-dependent polarization and migration of the Ph+CD34+CXCR4+ cells through extracellular matrix-like gels were significantly lower than for normal cells. Concomitantly, binding of these cells to vascular cell adhesion molecule-1 or fibronectin, in the presence of SDF-1, was also substantially lower. These findings suggest a major role for SDF-1-mediated, integrin-dependent BM retention of Ph+CD34+ cells.

The migration of immunocytes within the extracellular matrix (ECM) is influenced by the activation state of the incoming cell and its responses to the presence of chemokines and cytokines. We studied the regulatory role of TGF-beta1 on T cell homing to secondary lymphatic organs, such as the spleen, and chemotaxis within an ECM-like environment in using an ECM-like 3-dimensional gel system designed to follow the migration of individual leukocytes along chemokine gradients in real time. The numbers of migrating naive, but not memory T cells toward SDF-1alpha markedly increased after pre-incubating the cells with TGF-beta1 (0.25 ng/ml) for 24 h. The mechanisms underlying TGFbeta1-modulated migration involve the up-regulation of the expression of the SDF-1alpha receptor CXCR4, the enhancement of the SDF-1alpha-induced actin polymerization, and increased phosphorylation of Pyk2, a focal adhesion kinase involved in integrin-mediated lymphocyte migration, adhesion and interactions with ECM. Interestingly, priming of naive human T cells with TGF-beta1 increased homing of these cells to the spleen of NOD/SCID mice in a CXCR4-dependent manner. We propose that the effect of TGF-beta1 on the chemotaxis of naive T cells may be important in the locomotion of naive T cells toward SDF-1alpha-rich niches.

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34+CD38−/lowCXCR4+ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34−CD38−/lowLin− cells were not detected. Moreover, whereas freshly isolated CD34+CD38+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34+CD38+ cells. Homing of enriched human CD34+ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34+CD38−/lowCXCR4+cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34+ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gαiproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34+ cells. Homing of human CD34+ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34+CD38−/lowCXCR4+cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

Modulation of reactive oxygen species (ROS) levels in hematopoietic stem cells (HSC) is crucial to control HSC quiescence and blood formation. High ROS levels are required for leukocyte formation while low ROS levels are essential to maintain HSC quiescence. However, regulation of ROS content in HSC is poorly understood. Adhesion interactions between HSC and their bone marrow (BM) stromal cells (BMSC) via CXCL12/CXCR4 maintain HSC in a quiescence non-motile state, protecting them from 5-FU chemotherapy insult (Sugiyama, Immunity, 2006). Surface CXCL12 expression by BMSC is dependent on connexin-43 (Cx43) gap junctions mediated cell contact (Schajnovitz, Nat. Immunol., 2011) and BM hematopoietic stem and progenitor cells (HSPC) survive and eliminate excess ROS levels post 5-FU chemotherapy treatment, by transferring ROS to BMSC in a Cx43 dependent manner (Taniguchi, PNAS 2012). Here, we report that ROS content of BM HSPC inversely correlates with ROS levels in adjacent BMSC. Administration of the pro inflammatory cytokine G-CSF results in decreased HSPC Cx43 expression, elevated ROS levels and increased glucose uptake. Conversely, in the BM stromal microenvironment, G-CSF administration generated lower ROS level and reduced glucose uptake. Up-regulation of BM Sphingosine 1-Phosphate (S1P), a downstream target of G-CSF required for ROS production in HSPC, reduced stromal ROS content and proliferation. Accordingly, mice with reduced BM S1P levels (S1Plow) have lower BM content of HSPC, accompanied by reduced ROS, glucose uptake and lactate production in these cells. More importantly, BM from S1Plow mice has a 3 fold increased frequency of primitive ROSlow/ EPCR+ long-term repopulating cells, as evident by immunophenotypic analysis and long-term competitive repopulation assays. Concomitantly, S1Plow mice have increased content of BMSC with higher ROS levels and glucose uptake, leading to higher BM content of colony-forming unit fibroblasts. Our results reveal a dynamic and inverse metabolic relationship between BM HSC and the stroma microenvironment. We hypothesized that the opposite metabolic state of HSPC and BMSC is due to mitochondrial transfer between the two populations. Therefore, we created chimeric mice by transplanting mitochondria labeled GFP (mito-GFP) HSPC to wild type (WT) mice and detected 88% of the host BMSC to contain donor-derived mitochondria, indicating the existence of mitochondria transfer from hematopoietic cells to BMSC in vivo. This transfer is bidirectional, albeit at a lesser degree, as determined in reverse chimeric mice where up to 26% of the donor-derived HSPCs acquired recipient mitochondria. Mitochondrial transfer can be recapitulated also in vitro in an overnight co-culture system of mito-GFP HSPC and primary BMSC, resulting in mitochondrial transfer and increased ROS content in a subpopulation of osteogenic BM PDGFRα+/ Sca-1-/CD48dim stromal cells. Mitochondrial transfer is cell contact dependent and mediated by Cx43 gap junctions. In vitro co-culture of mito-GFPHSPC from Cx43 deficient (KO) mice with WT or Cx43 KO BMSC reduced 50% mitochondrial transfer to PDGFRα+/Sca-1-/CD48dim stromal cells. Contrarily, the mitochondrial transfer from WT HSPC to Cx43 KO stromal cells was not affected, revealing that Cx43 expression on HSPC, but not on BM stromal cells, is specifically required for mitochondrial transfer. Interestingly, in vitro inhibition of AMP-activated protein kinase (AMPK), a crucial metabolic regulator, dramatically increased mitochondrial transfer from HSPC to BMSC. Administration of the AMPK inhibitor BML in vivo increased ROS content of PDGFRα+/Sca-1- BMSC while decreasing it in HSPC, further suggesting that AMPK inhibition regulates mitochondrial transfer and ROS production. Our results imply that mitochondria are scavenged by the BM osteogenic microenvironment to prevent excessive ROS levels in the HSC pool and in parallel to activate bone formation. Altogether, we have discovered a dynamic, inverse metabolic state between BM HSPC and their supporting stromal microenvironment during quiescence, proliferationand differentiation of these two populations. Thus, blood cell production and bone generation take place at the expense of the other. This metabolic seesaw is mediated by mitochondrial transfer from HSPC to osteogenic BM stroma in a HSPC Cx43 gap-junction dependent manner and regulated through AMPK signaling. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions, a process utilized for BM harvest. Recently, we demonstrated that mouse long term repopulating hematopoietic stem cell (LT-HSC) BM retention and their rapid release to the blood circulation are governed by a switch in nitric oxide (NO) generation via distinct coagulation-related protease activated receptor 1 (PAR1) cascades (Gur-Cohen S. et al., NM, 2016). Herein we report that surface PAR1 expression can be exploited and serve as a positive predictive marker for the efficiency of human CD34+ HSPC mobilization among healthy donors in clinical G-CSF-induced mobilization for matched allogeneic transplantations. We found that PAR1 expression on circulating leukocytes before G-CSF administration was positively correlated with higher yields of mobilized leukocytes after stimulation (P0.5 x 109/L) from an average of 14.6 days to an average of 11 days post-transplant. In addition, a trend of accelerated platelet production was documented to be related with higher PAR1 expression by circulating leukocytes prior to G-CSF stimulation. Consequently, poor mobilizer donors were characterized by extremely low surface PAR1 expression on circulating CD34+ cells prior to G-CSF stimulations. Herein we present a case report of a thrombophilic donor carrying the MTHFR mutation, expressing exceedingly low PAR1 levels at baseline, with the outcome of inadequate numbers of mobilized CD34+ HSPC in the blood following G-CSF treatments. To further gain insight into the role played by PAR1 signaling in the regulation of G-CSF-induced HSPC mobilization, we used mice as a functional preclinical small animal model. We found that antagonizing PAR1 signaling attenuated both steady state release and G-CSF-induced HSPC mobilization. Furthermore, co-administration of G-CSF with PAR1 antagonist attenuated secretion of BM stromal CXCL12 and abrogated upregulation of surface CXCR4 and PAR1 expression by BM HSPCs, all leading to significantly reduced HSPC migration, differentiation and mobilization. In support, PAR1-/- mice failed to efficiently mobilize HSPCs in response to G-CSF compared to wild type counterparts. Enforced HSPC recruitment by G-CSF treatments dramatically accelerated PAR1-dependent NO production by eNOS, known to promote TACE-mediated EPCR shedding and rapid LT-HSC mobilization. Concomitantly, circulating steady state and G-CSF-mobilized stem cells lack surface EPCR expression. Intriguingly, while EPCR expression by primitive BM stem cells was transiently reduced after G-CSF treatments, antagonizing PAR1 signaling along with G-CSF stimuli blocked NO generation and synchronically expanded BM EPCR+ LT-HSC and their supportive stromal progenitor cells (MSPCs), as confirmed by increased repopulation in transplanted mice. Finally, we report an inverse PAR1 expression and regulation by BM HSPC and stromal MSPCs in mediating G-CSF-induced mobilization. G-CSF induced elevation of PAR1 expression on BM HSPCs, providing the driving force for their enhanced NO mediated migration, proliferation, differentiation and recruitment to the circulation. Contrary, the levels of PAR1 expression were reduced on MSPCs in response to G-CSF treatment, and following NO generation by eNOS activity and CXCL12 secretion, resulted in reduced mesenchymal differentiation leading to accumulated numbers of immature mesenchymal (CFU-F) and osteoblast (CFU-OB) progenitor cells. Taken together, our study identifies and highlights inverse PAR1 signaling and NO generation as essential regulator of G-CSF induced HSPC mobilization and MSPC development opening new avenues to advance therapeutics for enhancing clinical G-CSF induced stem cell mobilization and transplantation protocols. Disclosures Ruf: Iconic Therapeutics: Consultancy.

How bone marrow (BM) stem cells replenish the blood with mature cells while maintaining the reservoir of undifferentiated stem cells, is poorly understood. We report that murine leukocyte production and BM stem cell maintenance are regulated by light and darkness cues. We identified two daily peaks of BM stem and progenitor cell (HSPC) proliferation: the morning peak following light initiation (11 AM, ZT5) and the night peak following darkness (11 PM, ZT17). Both peaks are preceded by a transient elevation of tumor necrosis factor-alpha (TNFα) in the BM at 7 AM and at 7 PM, leading to increased reactive oxygen species (ROS) in HSPC and inducing their cycling. Reduced HSPC levels were observed either following ROS inhibition or in TNFα deficient mice. TNFα elevation augmented the levels of the TNFα converting enzyme (TACE) levels on HSPCs, promoting BM TNFα shedding. Interestingly, transient TNFα elevation was induced by switching light to darkness and vice versa, suggesting a role for TNFα as an internal mechanism of alert, preparing HSPC to cycle upon demand. While the morning HSPC peak was accompanied by increased egress and differentiation, the night peak was associated with retention and low differentiation. Norepinephrine (NE) generation has been shown to be driven by light-induced cues from the brain and to induce stem cell egress from the BM during the morning peak (Mendez-Ferrer et al, Nature 2008), while melatonin is an antioxidant that is mainly produced following the onset of darkness. We found that although NE and melatonin are continuously present in the BM, NE levels are predominantly augmented following initiation of light while melatonin is mostly elevated during the night. Administration of melatonin or inhibition of the sympathetic nervous system by β3-adrenergic receptor antagonist during the morning induced HSPC retention, decreasing their morning differentiation and egress. In accordance, injection of NE during the evening induced HSPC egress and differentiation at night. Taken together, these results reveal that TNFα via ROS generation regulates both light and darkness peaks of stem cell proliferation in the BM. However, the nervous system via NE secretion further drives their maturation and egress only during the morning peak. Looking for mechanisms of HSPC protection which are essential to avoid BM exhaustion, we found that melatonin prevented their differentiation and egress thus maintaining them in a primitive state during the darkness peak. Concomitant with the night peak, we also observed increased BM levels of rare activated αSMA/Mac-1 macrophage/monocyte cells. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). The high melatonin levels at night induced PGE2 signaling in the BM stem cell niche, regulating COX2high αSMA/Mac-1 macrophages, which restored low ROS levels, preventing stem cell differentiation and egress. Murine BM leukocytes differentiate predominantly during the light time and are therefore more responsive to inflammatory challenges during this time frame. Mimicking bacterial infections, endotoxin-induced mortality was shown to correlate with administration time, with very high mortality in mice treated at noon and very low mortality following midnight challenge (Halberg et al, Exp boil Med, 1960). We found that LPS administration in the afternoon resulted in a dramatic increase in BM neutrophils and monocytes production and recruitment which is lethal, in contrast to LPS injection at midnight with no immune activation. Reducing differentiation in the BM during the morning peak by administrating β3-adrenergic receptor antagonist, melatonin or ROS inhibition, all decreased the levels of myeloid cell production and recruitment following LPS challenge in the afternoon. Our results revealed that the morning peak involves HSPC proliferation, differentiation and egress, allowing HSPC to replenish the blood and the immune system with mature leukocytes on a daily basis. In contrast, the night peak induces HSPC proliferation with reduced differentiation and egress, allowing the renewal of the BM stem cell pool. In summary, we have identified two daily peaks in BM HSPC levels which are regulated via light and darkness cues that impact daily blood cell production, host immunity and renewal of the BM stem cell reservoir. Disclosures No relevant conflicts of interest to declare.

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) are active and essential first steps during embryonic development and in clinical stem cell transplantation. Rare, BM LT-HSCs endowed with the highest self-renewal and durable repopulation potential, functionally express the anticoagulant endothelial protein C receptor (EPCR) and PAR1. In addition to coagulation and inflammation, EPCR-PAR1 signaling independently controls a BM LT-HSC retention-release switch via regulation of nitric oxide (NO) production within LT-HSCs. EPCR+ LT-HSCs are maintained in thrombomodulin+ (TM) periarterial BM microenvironments via production of activated protein C (aPC), the major ligand for EPCR. Restriction of NO production by aPC-EPCR-PAR1 signaling, activates VLA4-mediated adhesion, anchoring EPCR+ LT-HSCs to the BM and protecting them from chemotherapy insult, sparing hematological failure and premature death (Gur-Cohen S. et al, Nat Med 2015). We report that transplanted EPCR+ LT-HSCs preferentially homed ‎to and were retained in the BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing treatment that block aPC binding and attenuate EPCR+ LT-HSC BM homing. Furthermore, short term aPC in vitro pretreatment dramatically augmented EPCR+ LT-HSC BM homing, lodgment and long-term repopulation. PAR1 deficient stem cells were irresponsive to treatment with aPC and displayed reduced BM homing efficiency, all pointing to the aPC-EPCR-PAR1 axis as a crucial mediator of BM LT-HSC homing. Additionally, aPC pretreated EPCR+ LT-HSCs had a striking advantage to competitively home to the BM. Consistently, BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with wild type stem cells in competitive repopulation assays. Importantly, the competitive homing results strongly imply that the BM available niches for newly arrived EPCR+ LT-HSCs are limited. Indeed, aPC pretreated EPCR+ LT-HSCs BM homing reached a plateau, as increasing the transplanted cell dose above 5x106 BM mononuclear cells, did not yield higher donor EPCR+ LT-HSC homing. These results reveal that there is a limited BM space for newly arrived transplanted EPCR+ stem cells to non-irradiated hosts. Importantly, we found that EPCR+ LT-HSCs can engraft the BM of non-conditioned mice with high efficiency, while remaining in a dormant, non-cycling state. Furthermore, the dormant homed EPCR+ LT-HSCs were later awakened and activated solely by treating the engrafted hosts with a low dose 5-FU chemotherapy, or with NO donor SNAP, revealing that preconditioning and clearance of occupied BM HSC niches are not required. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by unique BM arterioles, and not in the spleen. BM homed EPCR+ LT-HSCs were found adjacent to TM+ arterioles, imposing their retention. Homed BM EPCR+ LT-HSCs highly express full-length TM with intact lectin-like domain, and the BM TM+ endothelium was found to be enriched with a Glycocalyx layer, in particular with Heparan Sulfate Proteoglycan-2 (HSPG-2). HSGP-2 might specifically interact with the lectin-like domain of TM-expressingLT-HSCs, providing BM specific recognition and accelerated homing. Intriguingly, stabilizing TM function by in vitro pretreatment with platelet factor-4 (PF4) bypassed BM-derived cues and increased EPCR+/TM+ LT-HSC homing also to the spleen, suggesting a supportive role for PF4, highly secreted by BM megakaryocytes, in guiding EPCR+/TM+ LT-HSCs to the BM. Herein we define EPCR as a guidance molecule, navigating LT-HSC specifically to BM TM+ aPC-secreting blood vessels, allowing stem cell retention and protection from DNA damaging agents. The BM harbors a limited number of available stem cell niches for newly arrived transplanted EPCR+/TM+ LT-HSCs, and in vitro aPC pretreatment dramatically augments EPCR+/TM+ LT-HSC BM homing. Our findings provide new mechanistic insights and identify key players concerning LT-HSC homing specifically to the BM, leading to better repopulation following transplantation. This up-to-date approach and new knowledge may potentially lead to improved BM transplantation protocols and to prevent chemotherapy resistance of EPCR-expressing cancer stem cell mediated relapse. Disclosures Ruf: Iconic Therapeutics: Consultancy.

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 × 105mononuclear cells. We report that as few as 8 × 104 human cord blood mononuclear cells transplanted into NOD/SCID/B2mnull mice resulted in mutlilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 × 103 CD34+ cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2mnull mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2mnull mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 × 105mononuclear cells. We report that as few as 8 × 104 human cord blood mononuclear cells transplanted into NOD/SCID/B2mnull mice resulted in mutlilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 × 103 CD34+ cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2mnull mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2mnull mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.