Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

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Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro

Transfusion Medicine and Hemotherapy ◽

10.1159/000520932 ◽

2022 ◽

pp. 1-10

Author(s):

Patrick Wuchter ◽

Anke Diehlmann ◽

Harald Klüter

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Model Systems ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Hematopoietic Stem Cell Niche ◽

Cell Niche

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

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Normoxic Tumour Extracellular Vesicles Modulate the Response of Hypoxic Cancer and Stromal Cells to Doxorubicin In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21175951 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5951

Author(s):

Laura Patras ◽

Marcel H. A. M. Fens ◽

Pieter Vader ◽

Arjan Barendrecht ◽

Alina Sesarman ◽

...

Keyword(s):

Drug Resistance ◽

Extracellular Vesicles ◽

Hypoxia Inducible Factor ◽

B Cell Lymphoma ◽

Tumour Microenvironment ◽

Hypoxia Inducible Factor 1 ◽

Apoptotic Protein ◽

Donor Cells

Extracellular vesicles (EV) secreted in the tumour microenvironment (TME) are emerging as major antagonists of anticancer therapies by orchestrating the therapeutic outcome through altering the behaviour of recipient cells. Recent evidence suggested that chemotherapeutic drugs could be responsible for the EV-mediated tumour–stroma crosstalk associated with cancer cell drug resistance. Here, we investigated the capacity of tumour EV (TEV) secreted by normoxic and hypoxic (1% oxygen) C26 cancer cells after doxorubicin (DOX) treatment to alter the response of naïve C26 cells and RAW 264.7 macrophages to DOX. We observed that C26 cells were less responsive to DOX treatment under normoxia compared to hypoxia, and a minimally cytotoxic DOX concentration that mounted distinct effects on cell viability was selected for TEV harvesting. Homotypic and heterotypic pretreatment of naïve hypoxic cancer and macrophage-like cells with normoxic DOX-elicited TEV rendered these cells slightly less responsive to DOX treatment. The observed effects were associated with strong hypoxia-inducible factor 1-alpha (HIF-1α) induction and B-cell lymphoma–extra-large anti-apoptotic protein (Bcl-xL)-mediated anti-apoptotic response in normoxic DOX-treated TEV donor cells, being also tightly connected to the DOX-TEV-mediated HIF-1α induction, as well as Bcl-xL levels increasing in recipient cells. Altogether, our results could open new perspectives for investigating the role of chemotherapy-elicited TEV in the colorectal cancer TME and their modulatory actions on promoting drug resistance.

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Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽

10.1182/blood.v106.11.2190.2190 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2190-2190 ◽

Cited By ~ 1

Author(s):

Pieter K. Wierenga ◽

Ellen Weersing ◽

Bert Dontje ◽

Gerald de Haan ◽

Ronald P. van Os

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Late Antigen ◽

Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

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G-CSF Mediated Decrease in Bone-Marrow SDF-1 Level Is Neutrophil Dependent but CD26 Independent

Blood ◽

10.1182/blood.v112.11.3469.3469 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3469-3469

Author(s):

Pratibha Singh ◽

Seiji Fukuda ◽

Janardhan Sampath ◽

Louis M. Pelus

Keyword(s):

Bone Marrow ◽

Magnetic Beads ◽

Critical Role ◽

Alternative Mechanism ◽

Wild Type ◽

Hematopoietic Stem ◽

Osteoblast Activity

Abstract Interaction of CXCR4 expressed on hematopoietic stem and progenitor cells (HSPC) with bone-marrow stromal SDF-1 is believed to play a central role in retention or mobilization of HSPC. Recently, a mobilization regimen of G-CSF was shown to decrease osteoblast number resulting in reduced levels of bone-marrow SDF-1, however the detailed mechanism leading to this reduction is currently unknown. It is unlikely that G-CSF directly regulates osteoblast SDF-1 production since osteoblasts do not express G-CSF receptor. Proteolytic cleavage of SDF-1 by peptidase CD26 in the bone-marrow may be an alternative mechanism responsible for reduction of SDF-1 level. Although CD26 can cleave SDF-1 in vitro, direct evidence of SDF-1 cleavage by CD26 in vivo during G-CSF induced HSPC mobilization has not been demonstrated. We previously demonstrated that neutrophils are required for G-CSF induced HSPC mobilization and that CD26 expression on neutrophils, rather than HSPC, is critical for mobilization. To more fully understand the role of CD26 in altering SDF-1 protein/activity during G-CSF induced HSPC mobilization, we quantitated bone-marrow SDF-1 levels in CD26−/− and wild-type CD26+/+ mice by ELISA during G-CSF administration. A standard 4 day G-CSF mobilization regimen (100 μg/kg bid, sc × 4 days) decreased bone-marrow total SDF-1 from 4.55±0.3 to 0.52±0.06 ng/femur in wild-type CD26+/+ mice (8.7-fold) and from 4.51±0.3 to 0.53±0.05 ng/femur (8.5-fold) in CD26−/− mice. However, despite an equivalent decrease in SDF-1, total CFU mobilization and the absolute number of mobilized SKL cells were decreased (3.1 and 2.0 fold lower, respectively) in CD26−/− mice compared to wild-type CD26+/+ controls. These results suggest that the decrease in total SDF-1 level in marrow seen following G-CSF treatment is independent of CD26. Cytological examination of bone-marrow smears showed that the reduction in SDF-1 levels in bone-marrow of both wild-type CD26+/+ and CD26−/− mice following G-CSF administration correlated with an increase in total absolute bone-marrow neutrophil cell number, suggesting a role for neutrophils in modulation of SDF-1 protein. To determine if neutrophils affect osteoblast SDF-1 production, bone marrow Gr-1+ neutrophils from wild-type CD26+/+ and CD26−/− mice were purified using anti-Ly6G magnetic beads and co-cultured with MC3T3-E1 preosteoblasts in vitro. Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased pre-osteoblast SDF-1 production by similar amounts (15.4-fold vs 14.8-fold respectively), while Gr-1 neg cells from both wild-type CD26+/+ or CD26−/− were without effect on SDF-1 levels. Similarly, Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased SDF-1 produced by MC3T3-E1-derived osteoblasts from 1.85±0.3 to 0.52±0.06 ng/ml (3.5 fold) and 0.56±0.07 ng/ml (3.3 fold) respectively, with Gr-1neg cells having no effect. Gr-1+ neutrophils either from wild-type or CD26−/− mice, but not Gr-1neg cells, significantly induced apoptosis of MC3T3-E1 cells as measured by Annexin-V staining (70.5%±10.2 vs 71.2%±12.5 for wild-type CD26+/+ and CD26−/− neutrophils respectively) and significantly inhibited osteoblast activity (20-fold vs 20.6-fold for CD26+/+ and CD26−/− neutrophils respectively) as measured by osteocalcin expression. Furthermore, irrespective of G-CSF treatment, an inverse correlation between absolute neutrophil number and SDF-1 protein levels was observed, suggesting that G-CSF induces neutrophil expansion but does not directly affect SDF-1 production. Collectively, these results provide additional support for the critical role of neutrophils in G-CSF induced mobilization and strongly suggested that neutrophils directly regulate bone-marrow SDF-1 levels independent of CD26 activity.

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The Role of Microrna-196a-1 and Microrna-196b in Acute T-Lymphoblastic Leukemia and Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.1607.1607 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1607-1607

Author(s):

Ebru Coskun ◽

Eva Kristin von der Heide ◽

Cornelia Schlee ◽

Nicola Goekbuget ◽

Dieter Hoelzer ◽

...

Keyword(s):

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Mirna Precursor ◽

Acute Leukemias ◽

Aberrant Expression ◽

Gm Csf ◽

Fold Reduction ◽

Precursor Molecules

Abstract Abstract 1607 Poster Board I-633 INTRODUCTION Overexpression of the gene ERG (v-ets erythroblastosis virus E26 oncogene homolog) is an adverse prognostic factor in adult patients with acute T-lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). However, the underlying biology remains unknown. The aim of this study was to investigate the regulation of ERG expression by microRNAs (miRNAs) and to explore their potential role in acute leukemia and normal hematopoiesis. METHODS: A bioinformatic database search was carried out using the Targetscan, Pictar, and Human microRNA target tools to predict ERG regulating miRNAs. Verification of ERG as potential target of predicted miRNAs was performed by AMAXA transfection of miRNA precursor molecules in the myeloid leukemic cell line KG1a. After 24 hours (hrs) and 48 hrs total RNA was extracted using the Trizol reagent. Overexpression of the miRNAs was confirmed by TaqMan MicroRNA assays and ERG expression was determined by real-time RT-PCR. Moreover, specific binding of miRNAs to the 3'UTR of ERG was verified by luciferase reporter assays co-transfecting the ERG 3'UTR cloned into the psiCHECK-2 luciferase vector with miRNA precursor molecules. To investigate the expression of miRNAs during hematopoietic maturation, CD34 positive bone marrow cells from healthy individuals were in vitro cultured using the cytokines SCF and IL-3 (maintenance culture) with the addition of EPO or G-/GM-CSF. Cells were harvested after 3, 6, 9, 13, 16, and 20 days and miRNA expression levels were measured. The expression of miR-196a-1 and miR-196b was also studied in acute leukemias including bone marrow samples of adult patients with newly diagnosed T-ALL (n=105) and AML (n=34). RESULTS: By the database search, a total of 13 miRNAs were predicted to potentially regulate ERG and were further studied. Of these, only the miRNAs miR-196a-1 and miR-196b induced a significant reduction of ERG expression levels. After 24 hrs ERG was significantly down-regulated by 36% (after miR-196a-1 transfection) and by 42% (after miR-196b transfection) as well as after 48 hrs by 43% (after miR-196a-1 transfection) and by 47% (after miR-196b transfection) compared to the controls. The luciferase assays revealed a 30% and 40% luciferase activity reduction in miR-196a-1 and miR-196b transfected cells, respectively, compared to the miRNA-missense transfected cells. This confirmed the direct binding of these miRNAs to the ERG 3'UTR. During hematopoietic differentiation of normal CD34 positive progenitors, expression of miR-196a-1 was constant over time using the different cytokine conditions. In contrast, the expression of miR-196b decreased substantially during the in vitro differentiation (maintenance culture: 20-fold reduction; EPO: 18-fold reduction; G-/GM-CSF: 13-fold reduction - from day 0 to day 9). In acute leukemia, we found that miR-196a-1 was significantly higher expressed in AML compared to bone marrow samples of healthy donors (P=0.02). In T-ALL, miR-196a-1 was significantly up-regulated in patients with aberrant expression of myeloid markers (P=0.04), and miR-196b expression correlated with CD34 expression (P=0.003). In contrast to the reported adverse prognostic impact of ERG, expression of these miRNAs had no prognostic significance in T-ALL. CONCLUSION: This study identifies miR196a-1 and miR-196b as ERG regulators. We show that miR-196b is specifically down-regulated during hematopoietic differentiation, thus indicating a specific role of this miRNA in hematopoiesis. Moreover, the aberrant expression of miR-196a-1 and miR-196b in T-ALL and AML points to a potential role of these miRNAs in acute leukemias. Disclosures No relevant conflicts of interest to declare.

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Role of the Urokinase Receptor (uPAR) in the Cross-Talk of Hematopoietic Stem Cells with the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v112.11.324.324 ◽

2008 ◽

Vol 112 (11) ◽

pp. 324-324 ◽

Cited By ~ 2

Author(s):

Nunzia Montuori ◽

Patrizia Ricci ◽

Bianca Serio ◽

Valeria Visconte ◽

Claudio La Penna ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Mirror Image ◽

Cell Surface Receptor ◽

Hematopoietic Stem ◽

Stromal Microenvironment ◽

Surface Receptor ◽

Cell Adhesion And Migration

Abstract The urokinase-type plasminogen activator receptor (uPAR) is a cell-surface receptor involved in cell adhesion and migration. uPAR binds urokinase (uPA) and vitronectin (VN) and interacts with integrins and chemotaxis receptors. Soluble forms of uPAR (suPAR) have been detected in human plasma and urine. A cleaved form of suPAR (c-suPAR), lacking the N-terminal domain and exposing the sequence SRSRY (aa 88–92), stimulates cell migration by activating fMLP receptors. We recently demonstrated uPAR involvement in G-CSF-induced CD34+ hematopoietic stem cell (HSC) mobilization. We also demonstrated that c-suPAR could induce mobilization of hematopoietic stem/progenitor cells in mice. Since HSC mobilization and homing to bone marrow (BM) are mirror image processes which utilize the same mediators and similar signaling pathways, we investigated whether uPAR and its ligands could play a role in regulating CD34+ HSC interactions with the BM stroma, thus also contributing to HSC homing and engraftment to the BM. We found expression of uPA and VN in cultures of human BM stroma cells. Interestingly, stroma cells also produced suPAR and high amounts of c-suPAR, exposing the chemotactic SRSRY sequence. The role of the different soluble forms of uPAR produced by stroma cells in regulating HSC interactions with the BM microenvironment was analyzed by long term cultures (LTC) of BM and G-CSF mobilized CD34+ HSCs, in the presence of suPAR or the uPAR-derived uPAR84–95 peptide, corresponding to the active site of c-suPAR. Both suPAR and the uPAR84–95 peptide increased the number of adherent and released clonogenic progenitors from LTC of BM and G-CSF mobilized HSCs. To elucidate the mechanism of suPAR and c-suPAR effects on CD34+ HSC interactions with the stromal microenvironment, in vitro adhesion and proliferation assays were performed on CD34+ KG1 cells. suPAR treatment determined a significant increase in CD34+ KG1 cell adhesion whereas c-suPAR increased cell proliferation. Taken together, our results indicate that BM stroma produces soluble forms of uPAR that regulate CD34+ HSC interactions with BM microenvironment, their local proliferation and trafficking from and to BM.

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Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling

Blood ◽

10.1182/blood.v118.21.219.219 ◽

2011 ◽

Vol 118 (21) ◽

pp. 219-219

Author(s):

Daniela S. Krause ◽

Keertik Fulzele ◽

Kevin Barry ◽

Sutada Lotinun ◽

Roland Baron ◽

...

Keyword(s):

Bone Marrow ◽

Bone Remodeling ◽

Conflicts Of Interest ◽

Bone Matrix ◽

Myeloid Cells ◽

Hematopoietic Stem ◽

Inflammatory Protein ◽

G Protein Coupled

Abstract Abstract 219 Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated. We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected. In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls. In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling. Disclosures: No relevant conflicts of interest to declare.

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An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽

10.1182/blood.v118.21.2333.2333 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2333-2333

Author(s):

Brian D. Adams ◽

Shangqin Guo ◽

Haitao Bai ◽

Changchun Xiao ◽

E. Premkumar Reddy ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Hematopoietic Recovery ◽

Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

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Vav1, a Rho GTPase Guanine Exchange Factor (GEF) Is Required to Integrate gp130 Cytokine Signaling for Successful Engraftment of Murine Hematopoietic Stem Cells Following Irradiation

Blood ◽

10.1182/blood.v128.22.2149.2149 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2149-2149

Author(s):

Serena De Vita ◽

Yanhua Li ◽

Chad Everett Harris ◽

Meaghan McGuinness ◽

David A. Williams

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Rho Gtpase ◽

Macrocytic Anemia ◽

Cytokine Signaling ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Adult Mice

Abstract Successful engraftment of hematopoietic stem and progenitor cells (HSPCs) during bone marrow transplantation requires appropriate homing and retention of transplanted cells in the bone marrow (BM) and the activation of a proliferative program in response to signals from the hematopoietic microenvironment (HM). Molecular pathways regulating migration, homing and retention of HSPCs in the BM are integrated by RhoGTPasesincluding Rac and CDC42, however the complex cues that drive the proliferative response of these cells following transplantation are less clear. We have previously described the hematopoietic phenotype of adult mice lacking Vav1, a multi-domain, hematopoietic-specific GEF for Rac and CDC42. Deletion of Vav1 does not affect steady state hematopoiesis in adult mice, but severely compromises the engraftment potential of HSPCs. In the absence of Vav1, signal transduction from SDF1α is impaired in HSPCs, leading to abnormal localization and reduced retention of these cells in the HM, a phenotype similar to deficiency of Rac (Sanchez-Aguilera et al. PNAS, 2011). Here, we define an unexpected role for Vav1 in mediating post-irradiation proliferative responses of HSPCs. Surprisingly, we observed that deletion of Vav1 does not affect HSPC migration during ontogeny, a process largely mediated by SDF1α. The number of immunophenotypically and functionally defined HSPCs in Vav1-/- E13.5 fetal liver (FL) was comparable to WT (Table 1). Similarly, no difference was detected in HSPCs in the peripheral blood (PB) of E18.5 embryos or in the BM of newborn mice. However, similar to adult cells, Vav1-/- fetal HSCPs showed severely defective engraftment in lethally irradiated recipients (see Table 2). In marked contrast, both adult and fetal Vav1-/- HSCPs could engraft non-irradiated (Kit W/Wv Rag2- γc-) recipients, achieving successful correction of the macrocytic anemia and B cell leukopenia phenotype of recipient mice (Table 2, in red). Reduced proliferation of Vav1-/- HSPCs was also observed in vitro upon co-culture with primary irradiated stromal cells (Table 3). No differences among genotypes were detected when using non-irradiated stromal cells. These data suggest a distinct role for Vav1 in mediating responses of HSPCs to the HM after irradiation. To further clarify this phenotype, we investigated the role of individual soluble factors on proliferative responses of Vav1 HSPCs. Given their known role in expansion and proliferation of hematopoietic progenitors we focused on gp130 cytokines. We found that both IL-6 and IL-11, prominent members of this cytokine family, were increased in the BM of irradiated WT recipients, compared to both non-irradiated WT recipients (3x and 2x increase) and Kit W/Wv Rag2- γc- (2x and 3x increase) age- and sex-matched animals. To validate the potential role of IL-6 and IL-11 in Vav1 function, we stimulated HSPCs with both cytokines and observed that they induced phosphorylation of Vav1 and activation of Rac, but not CDC42. Using CFU assays, liquid culture experiments and BrdU analysis we confirmed that deletion of Vav1 abolishes the proliferative responses elicited by IL-6 and IL-11 on HSPCs in vitro. In summary, we show that Vav1 acts in HSPCs to mitigate responses to pro-inflammatory cytokines present in the HM during engraftment following irradiation. Manipulating the gp130-Vav-Rac axis in HSPCs could represent a strategy to enhance engraftment of normal cells in conditioned recipients. Disclosures Williams: bluebird bio: Research Funding; Novartis: Consultancy; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Pertussis toxin–sensitive G proteins regulate lymphoid lineage specification in multipotent hematopoietic progenitors

Blood ◽

10.1182/blood-2009-01-201939 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5757-5764 ◽

Cited By ~ 12

Author(s):

Anne Y. Lai ◽

Akiko Watanabe ◽

Tommy O'Brien ◽

Motonari Kondo

Keyword(s):

Bone Marrow ◽

Pertussis Toxin ◽

Distal Region ◽

Lineage Specification ◽

Differentiation Potential ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Lymphoid Lineage

Abstract Lymphoid and myeloid lineage segregation is a major developmental step during early hematopoiesis from hematopoietic stem cells. It is not clear, however, whether multipotent progenitors (MPPs) adopt a lymphoid or myeloid fate through stochastic mechanisms, or whether this process can be regulated by extracellular stimuli. In this study, we show that lymphoid lineage specification occurs in MPPs before lymphoid lineage priming, during which MPPs migrate from the proximal to the distal region relative to the endosteum of the bone marrow. Lymphoid-specified MPPs have low myeloid differentiation potential in vivo, but potently differentiate into myeloid cells in vitro. When treated with pertussis toxin, an inhibitor of G protein–coupled receptor signaling, lymphoid-specified MPPs regain in vivo myeloid potential, and their localization is dispersed in the bone marrow. These results clearly demonstrate that specific microenvironments that favorably support lymphoid or myeloid lineage development exist at structurally distinct regions in the bone marrow.

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