scholarly journals Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5522-5522
Author(s):  
Yu Han ◽  
Huaquan Wang ◽  
Zonghong Shao
Keyword(s):  
Myelodysplastic Syndrome ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Increased Risk ◽  
Macrophage Colony

Abstract The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, inhibiting tumor cell apoptosis and anti-tumor immune reactions. In this study, we analyzed the role of macrophages from MDS patients in vitro. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Compared with that in the normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and signal regulatory protein alpha (SIRPα) were lower. Levels of inducible nitric oxide synthase(iNOS) secreted by macrophages in MDS were increased. In conclusion, monocyte-derived macrophages are impaired in myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Yu Han ◽  
Huaquan Wang ◽  
Zonghong Shao
Keyword(s):  
Myelodysplastic Syndrome ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Control Group ◽  
Phagocytic Capacity ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Increased Risk ◽  
Macrophage Colony

Background. The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. We analyzed the role of macrophages from MDS patients in vitro. Methods. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Phagocytic capacity of macrophages was measured with carboxyfluorescein succinimidyl ester and fluorescent microspheres. CD206 and signal regulatory protein alpha (SIRPα) on macrophages were detected by flow cytometry. Inducible nitric oxide synthase (iNOS) was measured by ELISA method. Results. Compared with normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and SIRPα were lower. Levels of iNOS secreted by macrophages in MDS were increased. Conclusions. Monocyte-derived macrophages are impaired in myelodysplastic syndromes.

Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1068-1068
Author(s):  
Naoko Takebe ◽  
Thomas MacVittie ◽  
Xiangfei Cheng ◽  
Ann M. Farese ◽  
Emily Welty ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4101-4101
Author(s):  
Wen-ming Chen ◽  
Zi-xing Chen ◽  
Jian-nong Cen ◽  
Jun He ◽  
Xiao-li Jiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Biological Characteristics ◽  
Bone Marrow Mononuclear Cell ◽  
Feeder Layer ◽  
Rt Pcr ◽  
Osteoblastic Cell Line ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1460-1460
Author(s):  
Laura A Paganessi ◽  
Lydia Luy Tan ◽  
Sucheta Jagan ◽  
Robin Frank ◽  
Antonio M. Jimenez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Gm Csf ◽  
Cytokine Treatment ◽  
Ex Vivo Culture ◽  
Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 143-143
Author(s):  
Jarrod A Dudakov ◽  
Alan M Hanash ◽  
Lauren F. Young ◽  
Natalie V Singer ◽  
Mallory L West ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Thymic Epithelial Cells ◽  
Immune Competence ◽  
Strong Negative Correlation ◽  
Hematopoietic Stem ◽  
Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

Role of PDGF/PDGFR in Regulation of Thrombopoiesis: A Possible Explanation for Imatinib Mesylate-Induced Thrombocytopenia in the Treatment of CML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3348-3348
Author(s):  
Mo Yang ◽  
Fanyi Meng ◽  
Jie yu Ye ◽  
Yue Xu ◽  
Bin Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Imatinib Mesylate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Potential Effect ◽  
Mice Model ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Bone Marrow Histology

Abstract Abstract 3348 Platelet-derived growth factor (PDGF), a platelet alpha-granule molecule, imply their potential effect in the regulation of megakaryocytopoiesis and thrombopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on human megakaryocytes and platelets (Yang et al, Thromb Haemastasis, 1997) and CD34+ cells, and their ability to mediate a mitogenic response. PDGF promoted the ex vivo expansion of human hematopoietic stem (CD34+) and progenitor (CD41+ CD61+) cells. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. PDGF stimulated in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also showed a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF activated the PI3k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors with subsequent activation of the PI3k/Akt pathway. The study provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia in the treatment of CML. Disclosures: No relevant conflicts of interest to declare.

Two Distinct Mechanisms Contribute to Granulomonocytic Hyperplasia in Chronic Myelomonocytic Leukemias (CMML)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 309-309
Author(s):  
Raphael Itzykson ◽  
Olivier Kosmider ◽  
Aline Renneville ◽  
Margot Morabito ◽  
Dorothee Buet ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Single Mutation ◽  
Monocyte Count ◽  
Kras Mutations ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Recurrent Mutations

Abstract Abstract 309 Background: The granulomonocytic (GM) hyperplasia of CMML has been attributed to GM-CSF hypersensitivity triggered by mutations in the CBL/RAS pathway according to the prevailing model in juvenile myelomonocytic leukemias (Kotecha Cancer Cell 2008). Recurrent mutations affecting epigenetic (eg TET2 and ASXL1) and splicing (eg SRSF2) machineries, or cytokine signaling (N/KRAS, CBL, JAK2) are present in most CMML cases, but none is specific of CMML. In 224 CMML patients (pts), we found TET2 (58%), SRSF2 (47%) and ASXL1 (38%) to be the most frequently mutated genes; only 66 (35%) cases had mutations in cytokine signaling genes (CBL, N/KRAS, JAK2, FLT3, KIT) (abstract submitted). We analyzed the differentiation of CD34+populations from genetically annotated CMML pts to address the mechanisms of GM hyperplasia in CMML. Methods: CD34+ populations (hematopoietic stem cells [HSC]; multipotent [MPP]; common myeloid [CMP] and granulomonocytic progenitors [GMP] defined by the CD34/CD38/CD90/CD123/CD45RA panel; Majeti Cell Stem Cell 2007) from 28 genetically annotated CMML and TET2 mutated MPN (n=8) or MDS (n=5) cases were cloned and genotyped for each mutation identified in mature CD14+ cells, and differentiated in vitro. Results: Early clonal dominance, with at least one mutation in > 75% of HSC/MPP clones, was found in all cases. In 18/19 pts with ≥2 mutations, a linear succession of mutations was found, with signaling mutations often following TET2 or ASXL1 mutations. Contrasting with the dominance of first events in HSC/MPP, second events reached clonal dominance in GMP, suggesting that they provide a selective advantage during the early steps of myeloid differentiation. We next analyzed the clonogenicity of peripheral blood (PB) CD34+ cells in the presence of GM-CSF (10 ng/mL) in 20 CMML cases and 4 controls. GM-CSF hypersensitivity (clonogenicity > mean+2SD of controls) was found in 7 (35%) cases. A mutation in a signaling gene was found in 6/7 pts (1 homozygous JAK2, 1 homozygous CBL, 4 heterozygous N/KRAS mutations), compared to 3/13 in pts without GM-CSF hypersensitivity (2 JAK2, 1 CBL, all heterozygous; P=.02) Median WBC was 29.2 and 11.4 G/L in pts with and without GM-CSF hypersensitivity, respectively (P=.08). The proportion of GMP in bone marrow (BM) CD34+cells was not significantly different in 33 CMML pts compared to 15 age-matched controls. Clonogenicity of GMP was similar in CMML and controls, except for a trend toward increased clonogenicity in pts with mutations in signaling genes. In contrast, the proportion of MPP and CMP was higher in CMML than in controls (P<.01 and P <.05, resp.). In erythromyeloid conditions (SCF, IL-3, G-CSF & EPO), both CMP and to a lesser extent MPP had an increased ability to form GM colonies at the expense of erythroid colonies (P <.001 and P<.01, resp.). Compared to healthy CMP, CMML CMP had and increased ability to mature into GMP in short-term culture, and increased PU.1 mRNA expression (P<.05), without significant changes in the levels of GATA1, CEBPA and CEBPB. Finally, in 16 pts, the proportion of GM colonies differentiating from CMP at the expense of erythroid colonies was inversely correlated to patient hemoglobin level (P=.002). Thus, premature GM differentiation of CMP, and to a lesser extent MPP, appears as the dominant mechanism of GM hyperplasia in CMML, whereas GM-CSF hypersensitivity and GMP expansion contribute only in the minority of patients with mutations in signaling genes. We next explored a possible link between early clonal dominance of TET2 mutations and premature GM differentiation. In TET2 mutated MPN (n=8) or MDS (n=5), the PB monocyte count was significantly correlated to the size of the TET2-mutated clone in the CD34+/CD38− (P=.006) rather than in the CD34+/CD38+ population (P=.08). Finally, functional invalidation by shRNA of TET2 in CD34+/CD38− followed by culture in the presence of SCF, IL-3, G-CSF & EPO caused a GM expansion that was not observed in CD34+/CD38+ cells. Similar analyses are underway for ASXL1. Conclusion: Our results suggest that early clonal dominance of mutations affecting the epigenetic machinery leading to premature GM differentiation of multipotent progenitors, rather than GM-CSF hypersensitivity, is the main mechanism of GM hyperplasia in CMML. This suggests a model whereby a single mutation can lead to different phenotypes, depending on the stage of differentiation at which the mutation has gained clonal dominance. Disclosures: No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

Autophagy Regulation and Dysfunction in Bone Marrow Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4623-4623
Author(s):  
Fernando V Pericole ◽  
Mariana Lazarini ◽  
Adriana S. S. Duarte ◽  
João Machado-Neto ◽  
Sara T. Olalla Saad
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Molecular Machinery ◽  
Total Bone Marrow

Abstract Abstract 4623 Introduction: Autophagy is a catabolic pathway by which cytoplasmic materials are degraded into the lysosome and it is also a quality control system for proteins and organelles. Autophagy plays an important role in cell adaptation to starvation, hypoxia, cell survival and cancer. Its core molecular machinery is tightly linked to metabolic pathways, such as LKB1/AMPK and mTORC1. Autophagy has been shown to play several important roles in cancer. Indeed, multiple autophagy genes have been characterized as tumor suppressor genes. In hematopoietic system, autophagy is required during myeloid and lymphoid differentiation, terminal erythroid mitochondrial clearance, production of proplatelets and also differentiation of monocytes into macrophages. Interestingly, autophagy seems disturbed in most bone marrow malignancies. Evidence in mice suggests that autophagy suppression (ATG7 or ATG5 knockdown models) in hematopoietic stem cells may be implicated in Acute Myeloid Leukemia (AML) pathogenesis. In Multiple Myeloma (MM), in vitro studies using cell lines showed autophagy activation and lysosome inhibitors (such as chloroquine) are currently been used in various combination treatments in clinical trials. Aim: The aim was to characterize the expression of autophagy machinery key genes (BECN1, MAP1LC3A, SQSTM1), as well as hypoxia master regulator (HIF1A) in total bone marrow cells from bone marrow malignancies: myelodysplasia (MDS), MM and AML patients, excluding acute promyelocytic leukemia. Methods: BECN1, MAP1LC3A, SQSTM1 and HIF1A levels were verified, by q-PCR, in diagnostic (or without any treatment) BM aspirates from 22 normal donors, 30 MDS (17 low-risk and 13 high-risk, according 2008 WHO classification), 43 AML and 11 MM patients. Results: BECN1 gene expression was increased in MM, compared with control group. All other groups did not differ from the control group. Comparing diseases amongst each other, AML had a lower BECN1 expression, compared with low-risk MDS and with MM (Figure 1A). Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2203-2210 ◽  
Author(s):  
GP Bagnara ◽  
G Zauli ◽  
L Vitale ◽  
P Rosito ◽  
V Vecchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Diamond Blackfan Anemia ◽  
Clear Explanation ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte- conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.

Sign in / Sign up

Export Citation Format

Share Document