Adhesion Of Hematopoietic Stem Cells Research Articles

Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of hematopoietic stem cells.

Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF-VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.

Cysteine Cathepsins and Their Prognostic and Therapeutic Relevance in Leukemia

AbstractCysteine cathepsins are lysosomal proteases that require Cys-His ion pair in their catalytic site for enzymatic activity. While their aberrant expression and oncogenic functions have been widely reported in solid tumors, recent findings suggest that these proteases also play an important role in the pathogenesis of hematological malignancies. In this review, we summarize the potential clinical implications of cysteine cathepsins as diagnostic and prognostic markers in leukemia, and present evidences which supports the utility of these proteases as potential therapeutic targets in hematological malignancies. We also highlight the available information on the expression patterns, regulation, and potential functions of cysteine cathepsins in normal hematopoiesis and hematological malignancies. In hematopoiesis, cysteine cathepsins play a variety of physiological roles including regulation of hematopoietic stem cell adhesion in the bone marrow, trafficking, and maturation. They are also involved in several functions of immune cells which include the selection of lymphocytes in the thymus, antigen processing, and presentation. However, the expression of cysteine cathepsins is dysregulated in hematological malignancies where they have been shown to play diverse functions. Interestingly, several pieces of evidence over the past few years have demonstrated overexpression of cathepsins in leukemia and their association with worst survival outcomes in patients. Strategies aimed at altering the expression, activity, and subcellular localization of these cathepsins are emerging as potential therapeutic modalaties in the management of hematological malignancies. Recent findings also suggest the involvement of these proteases in modulating the immune response in leukemia and lymphomas.

Retraction: Role of Microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

This Retraction follows an Expression of Concern relating to this article previously published by Portland Press.This article is being retracted from Bioscience Reports at the request of the authors following receipt of a notification from a reader, alerting the authors to the duplications in Figure 4A as well as duplicated figures from another published article by the same authors in Zhongguo Shi Yan Xue Ye Xue Za Zhi (10.19746/j.cnki.issn1009-2137.2019.02.046).The authors wish to retract the article. The Editor-in-Chief and Editorial Board agree with the retraction.

Expression of Concern: Role of Microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates.

Role of microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells.

Background: The present study investigated the effects of microvascular endothelial cells (MECs) on the chemotaxis, adhesion and proliferation of bone marrow hematopoietic stem cells (HSCs) ex vivo. Methods and Results: MECs were collected from the lung tissue of C57BL/6 mice, and HSCs were isolated with immunomagnetic beads from bone marrow of GFP mice. MECs and HSCs were co-cultured with or without having direct cell–cell contact in Transwell device for the measurement of chemotaxis and adhesion of MECs to HSCs. Experimental results indicate that the penetration rate of HSCs from the Transwell upper chamber to lower chamber in ‘co-culture’ group was significantly higher than that of ‘HSC single culture’ group. Also, the HSCs in co-culture group were all adherent at 24 h, and the co-culture group with direct cell–cell contact had highest proliferation rate. The HSC number was positively correlated with vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) levels in supernatants of the culture. Conclusions: Our study reports that MECs enhance the chemotaxis, adhesion and proliferation of HSCs, which might be related to cytokines SDF-1 and VEGF secreted by MECs, and thus MECs enhance the HSC proliferation through cell–cell contact. The present study revealed the effect of MECs on HSCs, and provided a basis and direction for effective expansion of HSCs ex vivo.

EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential.

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

Deconvolution of Hematopoietic Commitment Decisions By Genome-Wide Analysis of Progressive DNA Methylation Changes

Recent advances in single cell transcriptome analyses have resulted in the derivation of new models to describe the hierarchical organization of the mammalian hematopoietic system. While such an approach appears to be effective at discerning the trajectory of differentiation from hematopoietic stem cells (HSCs) to a given mature lineage, it remains a challenge to identify definitive points where specific lineage fates become restricted. The characterization of molecular events that correspond to such commitment decisions is critical to our interrogation of the existence and nature of serial bifurcation steps that are hypothesized to underlie the process of hematopoiesis. 5-Methylcytosine is a stable epigenetic modification, whose remodeling at specific CpG residues appears to be integral to the process of enforcing lineage-restricted gene expression programs. We have previously observed that the remodeling of the DNA methylome appears to be both progressive and irreversible during the process of hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. In order to pursue this concept further, we used tagmentation-based whole-genome bisulfite sequencing to generate a genome-wide DNA methylation map of murine hematopoiesis. This map encompasses 26 different FACS-purified populations, ranging from LT-HSCs through to terminally differentiated blood cell lineages. Across all cell populations studied, we identified 147,232 differentially methylated regions (DMRs). In line with our previous data, hierarchical clustering of these DMRs revealed coordinately regulated regions that show progressive and unidirectional lineage-specific DNA methylation dynamics during hematopoietic differentiation that would be indicative of a molecular mechanism of cell fate restriction. Single cell DNA methylome analysis indicated that methylation programming may be exclusive for a specific lineage within each cell analyzed, supporting the use of this data to establish the discreet points at which lineage commitment occurs. Along these lines, lineage-specific DMRs could already be identified within the early hematopoietic stem and multipotent progenitor compartments, supporting the concept that lineage restriction occurs early during differentiation and providing a potential molecular basis for so called lineage-priming/bias. Indeed, a phylogenetic tree of the entire hematopoietic system could be constructed purely based on methylation remodeling events that took place in the Lin-, Sca-1+, c-Kit+ compartment. To gain further insight into how the DNA methylation programming relates to regulation of gene expression, we generated a comprehensive single cell transcriptome map encompassing the entire hematopoietic component of the bone marrow. Integration of DNA methylation dynamics with single cell gene expression dynamics provided evidence for anti-correlation between the transcriptional program and DNA methylation. However, loss of DNA methylation was not invariably associated with an increase in gene expression, suggesting that DNA methylation has more a permissive rather than an instructive role in regulating gene expression programs. We next applied our data set to the exploration of the phenomenon of myeloid lineage bias that has been described in aged mice, by investigating the methylomes of young and aged HSCs. Compared to young HSCs, we identified 3,275 DMRs in aged HSCs, which were predominantly associated with loss of DNA methylation and affected genes involved in HSC adhesion and migration, such as Vwf and ITGB3. Remarkably, 46 % of aging DMRs overlapped with the methylome programs identified in hematopoietic differentiation and were enriched for genes involved in integrin signaling, platelet activation and aggregation. This data suggests that HSC aging results in remodeling of the DNA methylome in vivo, in a manner that is associated with a differentiation bias towards the megakaryocytic lineage. Together, our work provides a rich resource to investigate DNA methylation changes in normal or diseased hematopoiesis, across a broad range of conditions such as aging. Disclosures Lipka: InfectoPharm GmbH: Employment.

Diverging impact of cell fate determinants Scrib and Llgl1 on adhesion and migration of hematopoietic stem cells.

Cell fate determinants Scrib and Llgl1 influence self-renewal capacity of hematopoietic stem cells (HSCs). Scrib-deficient HSCs are functionally impaired and lack sufficient repopulation capacity during serial transplantation and stress. In contrast, loss of Llgl1 leads to increased HSC fitness, gain of self-renewal capacity and expansion of the stem cell pool. Here, we sought to assess for shared and unique molecular functions of Llgl1 and Scrib by analyzing their interactome in hematopoietic cells. Interactome analysis was performed by affinity purification followed by mass spectrometry. Motility, migration and adhesion were assessed on primary murine HSCs, which were isolated by FACS sorting following conditional deletion of Scrib or Llgl1, respectively. Imaging of Scrib-deficient HSCs was performed by intravital 2-photon microscopy. Comparison of Scrib and Llgl1 interactome analyses revealed involvement in common and unique cellular functions. Migration and adhesion were among the cellular functions connected to Scrib but not to Llgl1. Functional validation of these findings confirmed alterations in cell adhesion and migration of Scrib-deficient HSCs in vitro and in vivo. In contrast, genetic inactivation of Llgl1 did not affect adhesion or migratory capacity of hematopoietic stem cells. Our data provide first evidence for an evolutionarily conserved role of the cell fate determinant Scrib in HSC adhesion and migration in vitro and in vivo, a unique function that is not shared with its putative complex partner Llgl1.

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.

Methylation of HAI-2/SPINT2 in Bone Marrow Mesenchymal Stromal Cells of MDS and AML Patients Affects Hematopoietic Stem Cell Survival and Adhesion

In recent years, the role of tumor microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stem/stromal cell (BMMSC) contribution to disease progression remains poorly explored. We had previously performed a microarray analysis of myelodysplastic syndrome (MDS) patient-derived BMMSC (MDS-BMMSC) and found an underexpression of HAI-2/SPINT2, an endogenous inhibitor of the hepatocyte growth factor (HGF) activator. This gene has been described as methylated in various cancer types and has been associated with disease progression. Despite of being related to the pathogenesis of several neoplasms, the role of HAI-2/SPINT2 has not yet been fully elucidated in hematological diseases, such as MDS and acute myeloid leukemia (AML). Thus, the aim of this study was to evaluate HAI-2/SPINT2 expression in derived BMMSC and total bone marrow (BM) of healthy donors (HD), MDS and AML patients as well as in BMMSC treated with 5-Azacitidine (Aza), a DNA methyltransferase (DNMT) inhibitor. To achieve this, we collected BM hematopoietic cells and plastic-adherent BMMSC from aspirates of HD, MDS and AML patients. BMMSC were expanded to passage 4 and defined as CD73+/CD90+/CD105+/CD45-/CD34-/CD31-/HLA-DR-. A total of 29 HD and 121 patients at diagnosis (MDS=72 [low-risk=46, high-risk=26], AML with myelodysplastic related changes (AML-MRC)=17 and de novo AML=32) were included. HAI-2/SPINT2 mRNA was significantly decreased in MDS- (0.34[0.01-2.06];P <.01) compared to HD-BMMSC (0.89[0.46-1.59]). When patients were stratified according to WHO classification, HAI-2/SPINT2 expression was lower in both low-risk (0.31[0.01-1.33]) and high-risk (0.43[0.01-2.06]) MDS-BMMSC. Similar results were found in total BM: HAI-2/SPINT2 transcripts were significantly decreased in MDS (0.41[0.01-2.53];P <.01), AML-MRC (0.38[0.014-0.84];P <.01) and AML patients (0.33[0.01-2.07];P <.001) compared to HD (0.91[0.19-4.79]). To investigate whether this loss of expression was due to HAI-2/SPINT2 methylation, BMMSC were treated with Aza (1µM or IC50 value) for 48h. In MDS- and AML-BMMSC, Aza treatment resulted in a pronounced upregulation of HAI-2/SPINT2 mRNA and protein levels. Moreover, Aza treatment of HD-BMMSC did not improve the HAI-2/SPINT2 mRNA and protein levels as much as the observed in MDS- and AML-BMMSC. To better understand the role of HAI-2/SPINT2 downregulation in BMMSC physiology, its expression was inhibited in a BM stromal cell line (HS5). As previously reported, HAI-2/SPINT2 silencing resulted in an increased secretion of HGF, known to be overexpressed in plasma of MDS patients and considered a prognostic factor in MDS and AML patients (Matsuda et al., Leukemia, 2004). Moreover, after co-culture, HAI-2/SPINT2 knockdown improved survival of blasts isolated from AML-MRC and AML patients. We also observed an increased adhesion of CD34+ hematopoietic stem cells (HSC) to HAI-2/SPINT2 silenced HS5 cells. This prompted us to analyze the expression of cell adhesion molecules in MDS- and AML-BMMSC. We observed a significant augment in the expression of CD49b and CD49d integrins in MDS- and AML-compared to HD-BMMSC. Taken together, SPINT2 inhibition improves HGF secretion, consequently with alteration in molecule receptor adhesion, resulting in an increased expression of integrins (CD49b and CD49d) responsible for cell-to-cell adhesion. Thus, reactivation of HAI-2/SPINT2 levels after Aza treatment indicates that this gene is probably epigenetically silenced by methylation in MDS and AML, and is possibly a tumor suppressor gene. Interestingly, nowadays, epigenetic therapy by Aza is the first-line treatment for MDS patients, and induces prolonged survival and delayed AML evolution. Likewise, our results suggest that HAI-2/SPINT2 may play a role in deregulation of HGF cytokine secretion with consequently alteration in HSC adhesion and growth/survival. Tumor microenvironment niche is currently known to play a critical role in cancer initiation and progression, thus HAI-2/SPINT2 may contributes to functional and morphological abnormalities of microenvironment niche and with the stem/progenitor cancer cell progression. Hence, downregulation in HAI-2/SPINT2 gene expression, due to methylation in MDS- and AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Emerging Strategies to Enhance Homing and Engraftment of Hematopoietic Stem Cells

Successful clinical outcomes from transplantation of hematopoietic stem cells (HSCs) depend upon efficient HSC homing to bone marrow (BM), subsequent engraftment, and, finally, BM repopulation. Homing of intravenously administered HSCs from peripheral blood (PB) through the circulation to the BM stem cell niches, which is the first critical step that precedes their engraftment, is enforced by chemotactic factors released in the BM microenvironment that chemoattract HSCs. These chemotactic factors include α-chemokine stromal-derived factor 1 (SDF-1), the bioactive phosphosphingolipids sphingosine-1-phosphate (S1P) and ceramid-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. Stem cells may also respond to a Ca2+ or H+ gradient by employing calcium- or proton-sensing receptors, respectively. In this review, we will present emerging strategies based on ex vivo manipulation of graft HSCs that are aimed at enhancing the responsiveness of HSCs to BM-secreted chemoattractants and/or promoting HSC adhesion and seeding efficiency in the BM microenvironment.

Maintenance of leukemic and normal hematopoietic stem cells in bone marrow niches by EVI1-regulated GPR56

Acute myeloid leukemia (AML) with high ecotropic viral integration site-1 (EVI1) expression (EVI1high AML) is classified as a refractory leukemia with a poor prognosis. We identified G protein-coupled receptor 56 (GPR56) as a novel marker for EVI1high AML, which is an orphan adhesion G protein-coupled receptor (GPCR). GPR56 was found to be associated with high cell adhesion and anti-apoptotic activities in EVI1high AML through activation of RhoA signaling. Moreover, in Gpr56-/- mice, the number of hematopoietic stem cells (HSCs) in bone marrow was significantly decreased with proportional increases in the spleen and peripheral blood, reflecting extramedullary hematopoiesis. The number of Gpr56-/- HSC progenitor cells in the G0 phase was significantly reduced with impaired adhesion and the loss of GPR56 function, which diminished the in vivo repopulating ability of HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of quiescence and cellular adhesion of EVI1high AML and HSCs in the bone marrow niche. Moreover, given that GPR56 expression in leukemia stem cells is much higher than that in HSCs, GPR56 is a candidate therapeutic target for leukemia stem cells in EVI1high AML.

Mimicking the Bone Marrow Niche in Vitro: Establishment of a 3D Perfusion Culture System for Mesenchymal Stromal Cells

IntroductionHematopoiesis takes place in the bone marrow niche. The niche is created by a specific interplay of different cell types and signalling molecules such as growth factors and cytokines. The self-renewal and survival of hematopoietic stem cells (HSCs) as well as their proliferation and differentiation are stimulated by these microenvironmental factors. Hematological disorders such as leukemia are associated with disruptions of the microenvironment within the bone marrow niche. To get further insights into the interplay of cells and molecules creating the niche and potential alterations due to hematological disorders, it is of importance to develop a model which mimics the niche in vitro. Thus, the aim of this study was to establish a 3D perfusion culture system for human mesenchymal stromal cells (MSCs), which constitute an important supportive cellular component of the bone marrow niche.MethodsWe used a modular perfusion culture system (Will W. Minuth, Regensburg, Germany). The system was already applied for culturing of a variety of different tissues, but not yet for culturing human MSCs.MSCs were cultured on porous membrane filters consisting of mixed cellulose esters (pore size: 0.45 µm). Membranes were placed into a container, which was permanently perfused with fresh culture medium. Thus, the cells were constantly provided with nutrition while the accumulation of toxic metabolic products was prevented.To establish the system, several parameters were varied in order to find out optimal conditions for the MSCs in perfusion culture. The influence of the following parameters was analyzed: the material of the membranes, the cell seeding volume, the position of the membranes in the perfusion culture container, the concentration of HEPES buffer in the medium and its flow rate. The viability of the MSCs in different culture conditions was tested by applying an MTS assay. Additionally, morphology of MSCs and the expression of exemplary selected genes important for the bone marrow niche (CXCL12 and JAG1) were analyzed. MSCs cultured under conventional cell culture conditions served as controls. Statistical analysis was performed by using the Student’s t-test (GraphPad Prism 6).ResultsWe could show that the amount of viable MSCs cultured under ideal conditions in perfusion culture was considerably higher than in conventional cell cuture (OD450: 0.37 ± 0.03 vs. 0.2 ± 0.01, P-value < 0.01 %). A perfusion rate of 20.8 µL per minute and a HEPES concentration of 50 mM were observed to be optimal for the viability and growth of the MSCs. The cells showed no differences in gene expression levels due to the different culture conditions.Table 1:Relative quantification of CXCL12 and JAG1 mRNA levels in MSCsDeltaCTCXCL12DeltaCTJAG1Perfusion culture8.4025.967Conventional cell culture8.0086.750Additionally, the MSCs cultured on membranes formed 3D-like networks. In perfusion culture, the MSCs seemed to grow in a more orderly manner compared to conventional cell culture conditions. No differences in morphology were observed due to the different culture conditions.DiscussionThe 3D perfusion culture system is sufficient to increase the viability and the growth of the MSCs without changing the gene expression profile of exemplary chosen genes relevant for homing and adhesion of HSCs in the bone marrow niche. The morphology of MSCs also did not change due to the different culture conditions.ConclusionThis system can be used for further experiments including co-culturing experiments with MSCs and HSCs and/ or leukemic blasts and might be an important option to mimic the hematopoietic stem cell niche in vitro. DisclosuresNo relevant conflicts of interest to declare.

Macrophages and Bone Marrow Stem Cell Niches: The Roles of PGE2 and Coagulation

Blood-forming hematopoietic stem cells (HSC) functionally express the chemokine receptor CXCR4, which is required to control their directional motility, cell cycle, adhesion, and bone marrow (BM) repopulation. HSC are mostly BM-retained in a quiescent, non-motile mode via CXCR4-induced adhesion interactions with BM niche cells, which functionally express surface CXCL12. Surface CXCL12-mediated adhesion interactions with BM stromal cells protect quiescent CXCR4+ stem cells from DNA damaging agents while preserving their developmental potential. On the other hand, CXCL12 secretion by BM stromal cells and its release to the blood increase CXCR4 expression and signaling in hematopoietic stem and progenitor cells (HSPC), inducing their egress and clinical mobilization. Finally, CXCR4+ HSC also follow CXCL12 gradients to the BM when they home back from the blood. Intriguingly, dynamic CXCR4/CXCL12 signaling cascades control not only HSC retention in the BM, but also their homing to the BM and their release and mobilization to the blood. In addition to their central role in host defense, myeloid cells participate in organ homeostasis, including HSC localization. Monocyte-derived bone resorbing osteoclasts cleave and release endosteal membrane-bound CXCL12, stem cell factor, and osteopontin, factors needed for HSC adhesion and BM retention, leading to CXCL12/CXCR4-mediated HSPC mobilization. Recently, we identified a rare population of BM αSMA+ macrophages that highly express, which protects HSC from inflammatory insults via COX-2-mediated PGE2 secretion and reactive oxygen species inhibition. In vitro PGE2 upregulates CXCR4 on enriched human cord blood CD34+ and murine HSC via cAMP activation, leading to enhanced CXCL12-induced migration, homing and BM repopulation. However, preliminary results reveal that in vivo PGE2 treatment reduced CXCR4 expression on BM HSC by increasing surface CXCL12 expression by stromal cells. Surface CXCL12 upregulation is due to PGE2-mediated secretion and autocrine signaling of lactate, which is followed by cAMP inhibition in BM stromal cells. Indeed, antagonizing COX-2 or activating cAMP induced HSPC mobilization via BM CXCL12 secretion and increased CXCR4 expression and signaling in HSPC. Coagulation cascades also navigate HSC localization. We revealed that anticoagulant microenvironments in the BM mediate HSC adhesion and retention via inhibition of nitric oxide (NO) production and HSC migration. Physiologic stress induced extensive production of the pro-coagulant factor thrombin, which activates NO generation, CXCL12 secretion and enhanced CXCR4+ stem cell motility and mobilization. We found multinucleated pre-osteoclast cell clusters in femoral metaphysis expressing Tissue Factor (TF) a potent initiator of coagulation leading to thrombin generation. Osteoclast maturation and stress signals also activate TF induced pro-coagulation cascades, mediating HSPC egress and recruitment to the circulation. Daily light and darkness cues regulate many physiological processes, including osteoclast/osteoblast bone remodeling, BM CXCL12 production and secretion and CXCR4+ HSC egress. In addition to the previously identified morning peak of HSC egress to the blood, we identified two peaks of BM HSC proliferation: a morning peak in conjunction with stem cell egress and an evening peak of HSC expansion without egress. Higher BM levels of the HSC-protecting aSMA+ monocyte/macrophages documented in the evening and their associate “low-CXCR4/high CXCL12” BM microenvironment provide the mechanism for these fluctuating patterns. Finally, we found higher PGD2 levels and reduced PGE2 levels in monocyte/macrophages in the morning, whereas at night we documented the opposite patterns. PGD2 induced CXCL12 secretion and HSC egress, while PGE2 induced their retention. Our studies attribute central roles for BM myeloid cells in HSC regulation. Disclosures No relevant conflicts of interest to declare.

PGE2 Promotes BM Hematopoietic Stem Cell Retention Via Stromal Lactate Production­­­­, cAMP and CXCL12/CXCR4 Regulation

The CXCR4/CXCL12 axis is essential for retention and protection from DNA damage of quiescent hematopoietic stem cells (HSC) in their bone marrow (BM) niches. Murine CXCR4+ HSC tightly adhere to BM stromal cells which functionally express cell surface CXCL12. Stress induces secretion of CXCL12 by BM stromal cells and its release to the circulation, mediating hematopoietic stem and progenitor cell (HSPC) egress, recruitment and clinical mobilization. Previously, we reported that Prostaglandin E2 (PGE2), highly produced by COX-2+ BM αSMA+ monocyte/macrophages, upregulates surface CXCR4 expression on enriched human CD34+ HSPC and their CXCL12 induced motility via cAMP activation in vitro. PGE2 inhibits intracellular reactive oxygen species (ROS) generation in HSPC and also increases membrane bound CXCL12 expression by BM stromal cells leading to HSC adhesion to their niche supporting cells in vivo, overall contributing to BM stem cell retention. We also found that elevation in cAMP activation promotes CXCL12 secretion from BM stromal cells, and another report has recently shown that lactate signaling via its major receptor HCA-1 inhibits cAMP.Thus, we hypothesized that the major metabolite lactate, cAMP and PGE2 cross-regulate BM stem cell retention by modulating the CXCR4/CXCL12 axis. We found that both hematopoietic stem cells and BM stromal cells functionally express the lactate receptor HCA-1. Stimulation with PGE2 elevated lactate production by BM stromal cells and stimulation with a HCA-1 receptor agonist, or with lactate, both elevated membrane bound expression of CXCL12 on BM stromal cells. Moreover, since cAMP is elevated by PGE2 signaling whereas lactate signaling was shown to inhibit cAMP, we tested the role of cAMP in CXCL12 expression and secretion by BM stromal cells. We found that in vitro the cAMP enhancer forskolin increased CXCR4 expression by HSPC and in vivo forskolin administration reduced membrane bound CXCL12 levels and elevated CXCL12 secretion as expected. Conversely, in vivo forskolin co-administered with lactate, elevated membrane bound CXCL12 levels and reduced CXCL12 secretion, indicating that lactate limits cAMP elevation and promotes surface CXCL12 expression by BM stromal cells. In accordance,inhibition of cAMP under PGE2 stimulation both in vitro and in vivo, augmented membrane bound CXCL12 expression and inhibited CXCR4 upregulation, mimicking the effects of lactate. We found that PGE2 administration in vivo resulted in reduced CXCR4 expression on primitive BM HSPCs however in vitro PGE2 elevated CXCR4 expression on enriched HSPC. Our results suggest that PGE2 signaling in vivo induces secretion of the metabolite lactate by BM stromal cells, increasing membrane bound CXCL12 expression and reducing expression of CXCR4 on HSPC via cAMP inhibition.Importantly, repeated in vivo administration of PGE2, lactate or its receptor HCA-1 agonist (once daily for 2 days), all reduced CXCR4 expression and steady state egress of HSPC to the bloodcirculation. Thus, PGE2 via downstream lactate secretion acts as a BM stem cell retaining factor. In accordance, we found that in vivo inhibition of PGE2 production by repeated (once daily for five days) injections of COX-2 inhibitors, such as Meloxicam led to HSPC mobilization. This mobilization was abrogated by co-administration of lactate, suggesting that in vivo inhibition of meloxicam induced CXCL12 secretion and release by lactate prevents HSPC mobilization. We found that in vivo COX-2 inhibition reduced membrane expression of CXCL12 by BM stromal cells and elevated surface CXCR4 expression by BM HSPC in a ROS dependent manner. Moreover, neutralization of CXCR4 or CXCL12 by specific antibodies, or ROS by its scavenger NAC, all blocked meloxicam induced stem and progenitor cell mobilization. These results reveal that COX-2 inhibition increased BM CXCL12 secretion and its release to the blood, upregulated CXCR4 leading to HSPC mobilization in a ROS and CXCL12 dependent manner.In conclusion, our results reveal that PGE2 enhances both cAMP elevation and lactate secretion by BM stromal cells in the vicinity of hematopoietic stem cells. Lactate acts in an autocrine manner modulating surface CXCL12 expression by BM niche cells and reduced CXCR4 expression by hematopoietic stem cells via inhibition of cAMP, promoting retention and preservation of hematopoietic stem cells in their BM niches. DisclosuresNo relevant conflicts of interest to declare.

CD44 standard and CD44v10 isoform expression on leukemia cells distinctly influences niche embedding of hematopoietic stem cells

BackgroundA blockade of CD44 is considered a therapeutic option for the elimination of leukemia initiating cells. However, anti-panCD44 can interfere with hematopoiesis. Therefore we explored, whether a CD44 variant isoform (CD44v)-specific antibody can inhibit leukemia growth without attacking hematopoiesis. As a model we used CD44v10 transfected EL4 thymoma cells (EL4-v10).MethodsThe therapeutic efficacy of anti-panCD44 and anti-CD44v10 was evaluated after intravenous application of EL4/EL4-v10. Ex vivo and in vitro studies evaluated the impact of anti-panCD44 and anti-CD44v10 as well as of EL4 and EL4-v10 on hematopoietic stem cells (HSC) in cocultures with bone marrow stroma cells with a focus on adhesion, migration, cell cycle progression and apoptosis resistance.ResultsIntravenously injected EL4-v10 grow in bone marrow and spleen. Anti-panCD44 and, more pronounced anti-CD44v10 prolong the survival time. The higher efficacy of anti-CD44v10 compared to anti-panCD44 does not rely on stronger antibody-dependent cellular cytotoxicity or on promoting EL4-v10 apoptosis. Instead, EL4 compete with HSC niche embedding. This has consequences on quiescence and apoptosis-protecting signals provided by the stroma. Anti-panCD44, too, more efficiently affected embedding of HSC than of EL4 in the bone marrow stroma. EL4-v10, by catching osteopontin, migrated on bone marrow stroma and did not or weakly interfere with HSC adhesion. Anti-CD44v10, too, did not affect the HSC – bone marrow stroma crosstalk.ConclusionThe therapeutic effect of anti-panCD44 and anti-CD44v10 is based on stimulation of antibody-dependent cellular cytotoxicity. The superiority of anti-CD44v10 is partly due to blocking CD44v10-stimulated osteopontin expression that could drive HSC out of the niche. However, the main reason for the superiority of anti-CD44v10 relies on neither EL4-v10 nor anti-CD44v10 severely interfering with HSC – stroma cell interactions that, on the other hand, are affected by EL4 and anti-panCD44. Anti-panCD44 disturbing HSC embedding in the osteogenic niche weakens its therapeutic effect towards EL4. Thus, as far as leukemic cells express CD44v isoforms, the therapeutic use of anti-panCD44 should be avoided in favor of CD44v-specific antibodies.

Serine Protease Inhibitor Kunitz-Type 2 Is Downregulated in Myelodysplastic Syndromes and Modulates Cell–Cell Adhesion

Myelodysplastic syndromes (MDS) are clonal disorders involving hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, a defective hematopoiesis supporting capacity of mesenchymal stromal cells (MSCs) in the microenvironment niche has been implicated in MDS pathophysiology. The interaction between the dysfunctional MSCs MDS and HSC regulates diverse adhesion-related processes, such as progenitor cell survival, proliferation, differentiation, and self-renewal. As previously reported, a microarray analysis identified serine protease inhibitor kunitz-type 2 (SPINT2), an inhibitor of hepatocyte growth factor (HGF) activation, to be downregulated in MSCs from MDS patients. To define the role of SPINT2 in MDS hematopoietic microenvironment, an analysis of the effect of SPINT2 silencing in MSCs was carried out. We herein reported significantly lower levels of SPINT2 whereas HGF was expressed at higher levels in MSCs from MDS patients compared with healthy controls. SPINT2 underexpression results in an increased expression, production, and secretion of HGF and stromal cell-derived factor 1 (SDF-1) by MSCs. An increased adhesion of normal HSC or malignant cells onto MSCs silenced for SPINT2 was also observed. The altered MSCs adhesion in SPINT2-knockdown cells was correlated with increased CD49b and CD49d expression and with a decrease in CD49e expression. Our results suggest that the SPINT2 underexpression in the MSC from MDS patients is probably involved in the adhesion of progenitors to the bone marrow niche, through an increased HGF and SDF-1 signaling pathway.

A novel mechanism of abnormal hematological indices in liver cirrhosis: Bone marrow endothelial cell dysfunction caused by humoral inhibitor affects the hematopoietic function of bone marrow

Abnormal hematological indices (HIs), a complication of liver cirrhosis (LC), present difficulties in the treatment of LC and pose a serious threat to the survival of patients. LC is a dynamic wound-healing process that occurs in response to repeated liver injury and is a chronic disorder associated with changes in various organs and tissues. It has been reported that humoral inhibitor in the formation of LC could affect the hematogenic functions of bone marrow (BM) by acting on erythroid differentiation. This indicates that the BM microenvironment is affected by humoral inhibitor in LC. Bone marrow endothelial cells (BMECs) are very important components of the BM microenvironment that function as the cytoskeleton to support the adhesion of hematopoietic stem cells (HSCs). In addition, they can secrete cytokines, which have important functions in regulating positioning, homing, proliferation, differentiation and other functions of HSCs on the BM microenvironment. These functions of BMECs may be affected due to direct contact with blood and long-term exposure to an environment with humoral inhibitor in the presence of LC. Multiple studies have shown that during the formation of LC, hepatic sinusoid endothelial cells were damaged and secreted cytokines and matrix proteins. Moreover, these cytokines and matrix proteins were involved in the formation and development of LC. Similar in function to mature-stage BM, liver at the embryonic stage also functions as a type of hematogenic organ. With similar anatomical position and functions to that of hepatic sinusoid endothelial cells, BMECs may undergo similar changes and impair hematogenic function of BM. More importantly, we found even more convincing evidence in that the humoral inhibitor in LC could lead to the ultrastructural damage of BMECs that were positively related to the degree of severity of LC. Therefore, we hypothesise the existence of a novel mechanism for abnormal HIs in LC: the continuous humoral inhibitor may lead to abnormal cytokine secretion of BMECs and attenuate their supporting functions, and such alterations of BMECs may lead to BM microenvironment disorder and dysfunction of HSCs, finally causing abnormal HIs.

Periostin Acts As An Important Cell Cycle Regulator Of Adult Hematopoietic Stem Cells Via Binding To Integrin-αvβ3

Osteoblasts are one of the important cellular components of the niche for hematopoietic stem cells (HSCs) in mammalian bone marrow (BM). Integrin receptors not only play a key role in HSC adhesion within the BM niche but also transfer regulatory signals from the microenvironment to HSCs. Periostin (Postn or osteoblast specific factor-1; OSF-1) is expressed in osteoblasts in addition to many other tissues, and acts as a ligand for Integrin-αvβ3 (ITGAV-B3). We identified POSTN as an important regulator of the cell cycle in adult murine HSCs. POSTN inhibited culture induced proliferation of HSCs thereby decreasing the total number of cells following 2-5 day culture of primitive HSCs, identified as CD150+CD48-Lin-Sca-1+c-kit+ (CD150 KLS) cells with SCF and TPO, while increasing the proportion of long-term (LT-) HSCs. Culture for 5 days with POSTN decreased the short-term (ST-) engraftment of progeny of 200 CD150 KLS cells, while significantly increasing LT- engraftment of the donor derived cells. A significant fraction of CD150 KLS cells expressed ITGAV as well as ITGB3. POSTN did not affect proliferation of HSCs in vitro following blocking of ITGAV with neutralizing antibodies. Among the important cell cycle regulators, we found an increase in p27kip1 expression in HSCs. Preliminary studies on possible signaling mechanisms involved, showed that POSTN inhibits Akt phosphorylation, known to mediate inhibition of both expression and activation of p27Kip1. Intravenous infusion of recombinant POSTN protein significantly decreased proliferation of hematopoietic progenitors as shown by Brdu incorporation and Hoechst/Pyronin staining. Interestingly, POSTN infusion also led to an increase in the number of KLS as well as CD150 KLS cells in the BM. Studies on characterization of the hematopoietic system of Postn-/- mice are underway. To further determine the role of ITGAV in HSCs, we used blocking antibodies against ITGAV and performed homing and engraftment studies. No effect on either homing potential or engraftment of ST- and LT- engraftment was seen. However, the competitive repopulation of ITGAV- CD150 KLS cells was significantly lower that that of ITGAV+ CD150 KLS cells (isolated using non-blocking antibodies). Therefore, our studies confirm the importance of ITGAV expression on primitive HSCs as well as presents POSTN as an important cell cycle regulator in the hematopoietic system. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem/Progenitor Cell Retention in the Bone Marrow Depends On Tissue Specific Heparan Sulfate Proteoglycans

AbstractAbstract 637The hematopoietic stem cell niche is known to govern the function of hematopoietic and leukemic stem cells. One constituent of the niche is the extracellular matrix comprised of several elements including a group of molecules called heparan sulfate proteoglycans (HSPGs). These molecules are present in the microenvironment of all tissue stem cells and are critical in the dynamic interactions between stem cells and niche elements.Heparan sulfate proteoglycan synthesis depends upon the glycosyltransferase, Ext1, a known tumor suppressor with a skeletal phenotype (exostoses) when mutated in affected humans. In order to investigate the role of HSPGs in the hematopoietic stem cell niche we conditionally deleted the Ext1 gene in the recently described Mx1+ osteolineage stem/progenitor cell population (Park et al., 2012). Our studies show that Ext1 and its biological product, HSPGs, play a major role in the maintenance of hematopoietic stem cells (HSCs) by modulating the bone marrow microenvironment.Ext1 deletion in osteolineage cells results in a marked loss of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow and an increase in circulating colony forming units (CFU-Cs) and HSCs (designated as lineage -, c-Kit+, Sca1+, CD 150 + and CD48-) both in the spleen and peripheral blood, suggesting a key role of heparan sulfate proteoglycans in the retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. Importantly, histomorphometry analysis showed no defect in bone remodeling upon Ext1 deletion.In order to investigate whether the above phenotype could be therapeutically exploited in the context of bone marrow stem/progenitor cell mobilization, we administered the heparan sulfate proteoglycan inhibitors, heparin and protamine sulfate to mice in combination with conventional mobilization regimes such as G-CSF. Pharmacological inhibition of HSPG induced a significant improvement of hematopoietic stem/progenitor cell mobilization over G-CSF treatment alone, an effect that was completely abrogated in the absence of endogenous HSPGs. Furthermore, by combining HSPG inhibition with G-CSF, we could mobilize a population of HSPCs with superior in-vivo self-renewing ability.Mechanistically, we observed that Ext1 deletion in stromal cells leads to a constitutive activation of the AKT pathway through the PDK1-mediated phosphorylation of AKT-Thr-308, which in turn phosphorylates and inactivate FOXO1 transcription factor. Expression profiling of genes transcriptionally regulated by FOXO1 revealed that VCAM1, a key molecule driving HSCs adhesion to their niche, was highly down regulated in Ext1 deficient cells. VCAM1 protein levels were also decreased accompanied by a profound reduction in HSC adhesion to Ext1 deficient cells in vitro.Taken together our results suggest that Ext1/HSPGs control hematopoietic stem/progenitor cells retention in the bone marrow through an AKT/FOXO1/VCAM1 dependent pathway and that targeting heparan sulfate proteoglycans is a strategy for mobilizing stem cells with superior self renewing capability to the blood.We are grateful to Lexicon Genetics and the MMRRC for kindly providing the Ext1 mouse model. Disclosures:Wagers:BD Biosciences: Consultancy; iPierian, Inc.: Consultancy; MPM Capital: Consultancy; Novartis: Honoraria. Scadden:Fate Therapeutics: Consultancy, Equity Ownership; Genzyme: Consultancy.