R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

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R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v128.22.5048.5048 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5048-5048

Author(s):

Kam Tong Leung ◽

Yorky Tsin Sik Wong ◽

Karen Li ◽

Kathy Yuen Yee Chan ◽

Xiao-Bing Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Rgs Proteins ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

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Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

Blood ◽

10.1182/blood-2002-10-3233 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3784-3793 ◽

Cited By ~ 166

Author(s):

Ryan Reca ◽

Dimitrios Mastellos ◽

Marcin Majka ◽

Leah Marquez ◽

Janina Ratajczak ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Calcium Flux ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Wide Range ◽

Normal Human ◽

Anaphylatoxin C3a

Abstract Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

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R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling

Blood Advances ◽

10.1182/bloodadvances.2020003307 ◽

2021 ◽

Author(s):

Kathy Chan ◽

Chi Zhang ◽

Yorky T. S. Wong ◽

Xiao-Bing Zhang ◽

Chi Chiu Wang ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

G Protein ◽

Ex Vivo ◽

Cell Trafficking ◽

Rgs Proteins ◽

Hematopoietic Stem ◽

Cell Functions ◽

Cd34 Cells

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine SDF-1 and its G-protein-coupled receptor CXCR4, which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13,and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cellsnot only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13 and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.

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Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient

Blood ◽

10.1182/blood-2004-04-1430 ◽

2005 ◽

Vol 105 (1) ◽

pp. 40-48 ◽

Cited By ~ 173

Author(s):

Marcin Wysoczynski ◽

Ryan Reca ◽

Janina Ratajczak ◽

Magda Kucia ◽

Neeta Shirvaikar ◽

...

Keyword(s):

Progenitor Cells ◽

Lipid Rafts ◽

Adhesion Molecule ◽

Actin Polymerization ◽

Membrane Lipid ◽

Small Gtpase ◽

Calcium Flux ◽

Urokinase Plasminogen Activator Receptor ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte–colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)– and bone marrow (BM)–derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.

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Protection of CML Progenitors from Bcr-Abl Tyrosine Kinase Inhibitor Mediated Apoptosis by the Bone Marrow Stromal Microenvironment.

Blood ◽

10.1182/blood.v110.11.3378.3378 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3378-3378

Author(s):

Bin Zhang ◽

Heiko Konig ◽

Tinisha Mcdonald ◽

Tessa L. Holyoake ◽

Dario Campana ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Direct Contact ◽

Hematopoietic Stem ◽

Transwell Insert ◽

Abl Tyrosine Kinase ◽

Stromal Microenvironment ◽

Cd34 Cells ◽

Tki Treatment

Abstract The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant hematopoietic stem and progenitor cells (HSPC). The bone marrow microenvironment regulates the self-renewal, proliferation and differentiation of HSPC. We investigated the role of microenvironmental interactions in resistance of CML HSPC to elimination by BCR-ABL tyrosine kinase inhibitors (TKI). CML CD34+CD38− primitive progenitor cells and CD34+CD38+ committed progenitor cells were cultured for 96 hours with IM (5μM), nilotinib (5μM) and dasatinib(150nM), in medium supplemented with low concentrations of growth factors, with and without irradiated primary human marrow stromal cells (immortalized by ectopic telomerase expression) followed by an assessment of apoptosis and proliferation. Culture with stroma did not result in significant alteration of apoptosis in the absence of TKI treatment (3.1±0.7% apoptosis for primitive progenitors with stroma and 2.7±0.9% without stroma, 3.7±0.2% for committed progenitors with stroma and 4.7±2.1% without stroma). Coculture with stroma completely protected CML primitive and committed progenitors from TKI-induced apoptosis. CML CD34+CD38− cells demonstrated 20±6% apoptosis following culture with IM in the absence of stroma, but only 3.8±1% apoptosis in the presence of stroma (p=0.04, n=4). Similarly, apoptosis with nilotinib decreased from 12.5±1.8% without stroma to 2.9±0.3% with stroma (p=0.033), and apoptosis with dasatinib decreased from 7.1±0.04% without stroma to 2.7±0.2% with stroma (p=0.001). Apoptosis of CML CD34+CD38+ cells also significantly decreased following TKI treatment with 12.9±4.0%, 10.6±3.2%, 8.4±2.3% apoptosis observed after IM, nilotinib and dasatinib treatment respectively without stroma and 7.1±1.2%, 4.8±1.0%, 3.7±0.4% with stroma, (p=0.04, p=0.03 and p=0.02 respectively, n=4). Culture with stroma resulted in mild reduction in CML progenitor proliferation in the absence of TKI treatment, but TKI treatment resulted in similar degrees of inhibition of proliferation regardless of the presence of stroma. Culture of CML CD34+ cells in a Transwell insert with 0.45μm pores, allowing free diffusion of stromal factors but preventing direct contact with stroma, was associated with reduction in the protective effect of stroma coculture (32.2% apoptosis without stroma, 14.7% with stroma, and 24.6% with Transwell insert). Addition of blocking antibodies to a4 integrin and N-cadherin did not affect survival of CML CD34+ cells in the absence of IM, but resulted in enhanced apoptosis of CML CD34+ cells cocultured with stroma after addition of IM (20.4% apoptosis without antibody, 28.9% with anti-N-cadherin, and 29.8% with anti-integrin antibody). We conclude that the bone marrow stromal microenvironment protects CML primitive and committed progenitors from pro-apoptotic effects of BCR-ABL TKI treatment. Direct contact-mediated interactions, likely through VLA-4 and N-Cadherin, play an important role in protecting CML CD34+ cells from TKI-mediated apoptosis. These observations indicate that measures aimed at interfering with the protective effects of stroma could be of benefit for the eradication of residual malignant progenitors in CML patients receiving BCR-ABL TKI treatment.

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Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.4040.4040 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4040-4040

Author(s):

Szabolcs Fatrai ◽

Simon M.G.J. Daenen ◽

Edo Vellenga ◽

Jan J. Schuringa

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Fold Increase ◽

Marrow Stromal Cells ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

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βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.3640.3640 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3640-3640

Author(s):

Sofieke E Klamer ◽

Paula B van Hennik ◽

Daphne C Thijssen-Timmer ◽

C. Ellen Van der Schoot ◽

Carlijn Voermans

Keyword(s):

Cell Surface ◽

Mrna Expression ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Cell Types ◽

Cell Surface Expression ◽

Type I ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins. The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Blood ◽

10.1182/blood.v122.21.217.217 ◽

2013 ◽

Vol 122 (21) ◽

pp. 217-217

Author(s):

Karin Golan ◽

Aya Ludin ◽

Tomer Itkin ◽

Shiri Cohen-Gur ◽

Orit Kollet ◽

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Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Surface Expression ◽

Daily Basis ◽

Hematopoietic Stem ◽

Activated Macrophage ◽

Sphingosine 1 Phosphate ◽

Stem And Progenitor Cells ◽

Primitive State

Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. 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). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures: No relevant conflicts of interest to declare.

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