Proliferation Of Hematopoietic Cells Research Articles

Structural Characterization of Polysaccharides Isolated from Panax notoginseng Medicinal Residue and Its Protective Effect on Myelosuppression Induced by Cyclophosphamide.

This study aims to establish the isolation and purification method of polysaccharides from medicinal residue of Panax notoginseng (PPN). The structure and protective effect of PPN on myelosuppression mice were investigated. One neutral polysaccharide (NPPN) and five acidic polysaccharides (APPN I, APPN II-A, APPN II-B, APPN III-A, and APPN III-B) were obtained. The results confirmed that NPPN, APPN I and APPN II-A are glycan with 1, 4 main chains. APPN III-A is a glycan. APPN II-B and APPN III-B are homogalacturonan pectin with 1, 4 main chains. This study demonstrated that NPPN played a bone marrow protective role in myelosuppression mice induced by cyclophosphamide. NPPN could relieve cell cycle arrest, reduce the apoptosis rate of marrow cells, and improve granulocyte-macrophage colony-stimulating (GM-CSF), thermoplastic polyolefin (TPO) and erythropoietin (EPO) serum level, which contributes to promoting the proliferation of hematopoietic cells.

Discover Novel Covalent Inhibitors Targeting FLT3 through Hybrid Virtual Screening Strategy

FMS-like tyrosine kinase 3 (FLT3) plays a very important role in regulating the proliferation, differentiation and survival of normal hematopoietic stem cells. Internal tandem duplications of the FLT3 gene (FLT3-ITD) mutations are present in 25% of all acute myeloid leukemia (AML) patients and are frequently associated with adverse clinical outcomes. Therefore, FLT3-ITD is a promising target for the treatment of AML. The use of covalent virtual screenings has shown that efficient rational approaches for the rapid discovery of new drugs scaffold. Herein, we report a hybrid virtual screening strategy that led to the discovery of FLT3 inhibitors. Using the combination of non-covalent docking and covalent docking, 8 compounds were found to inhibit FLT3, and G856-8335, S346-0154 are also effective against mutant FLT3. These two compounds also show selectivity to receptor tyrosine kinase (C-KIT), which has the potential for optimization. And this work can be extended to the screening of other covalent inhibitors.

Short-Term Reloading After Prolonged Unloading Ensures Restoration of Stromal but Not Hematopoietic Precursor Activity in Tibia Bone Marrow of C57Bl/6N Mice.

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sμg). To evaluate the effect of sμg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.

Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice.

Melanocortin receptor 1 (MC1-R) is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbs cholesterol homeostasis, increases arterial leukocyte accumulation and accelerates atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. Since various cell types besides leukocytes express MC1-R, we aimed at investigating the specific contribution of leukocyte MC1-R to the development of atherosclerosis. For this purpose, male Apoe-/- mice were irradiated, received bone marrow from either female Apoe-/- mice or MC1-R deficient Apoe-/- mice (Apoe-/- Mc1re/e) and were analyzed for tissue leukocyte profiles and atherosclerotic plaque phenotype. Hematopoietic MC1-R deficiency significantly elevated total leukocyte counts in the blood, bone marrow and spleen, an effect that was amplified by feeding mice a cholesterol-rich diet. The increased leukocyte counts were largely attributable to expanded lymphocyte populations, particularly CD4+ T cells. Furthermore, the number of monocytes was elevated in Apoe-/- Mc1re/e chimeric mice and it paralleled an increase in hematopoietic stem cell count in the bone marrow. Despite robust leukocytosis, atherosclerotic plaque size and composition as well as arterial leukocyte counts were unaffected by MC1-R deficiency. To address this discrepancy, we performed an in vivo homing assay and found that MC1-R deficient CD4+ T cells and monocytes were preferentially entering the spleen rather than homing in peri-aortic lymph nodes. This was mechanistically associated with compromised chemokine receptor 5 (CCR5)-dependent migration of CD4+ T cells and a defect in the recycling capacity of CCR5. Finally, our data demonstrate for the first time that CD4+ T cells also express MC1-R. In conclusion, MC1-R regulates hematopoietic stem cell proliferation and tissue leukocyte counts but its deficiency in leukocytes impairs cell migration via a CCR5-dependent mechanism.

Hypoxia preconditioned mesenchymal stem cell-derived exosomes induce ex vivo expansion of umbilical cord blood hematopoietic stem cells CD133+ by stimulation of Notch signaling pathway.

Mesenchymal stem cells (MSCs) are crucial cells that play an essential role in the maintenance, self-renewal, and proliferation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow niche. It has been proven that MSCs can be used as a feeder layer for the proliferation of HSCs to enhance the number of HPCs and HSCs. Recently, it has been demonstrated that MSC-derived exosome (MSC-DE) has critical roles in different biological processes in bone marrow (BM). In the current research, we examined the importance of hypoxia-preconditioned MSC-derived exosomes (HP-MSC-DE) and normoxia-preconditioned MSC-derived exosomes (NP-MSC-DE) in the self-renewal and long-term clonogenic potential of umbilical cord blood hematopoietic stem cells (UCB-HSCs). We showed that the secretion rate and component of the exosome (EXO) were changed in HP-MSC-DE compared to NP-MSC-DE. Notably, the Jagged-1 (Notch ligand) content of EXO was much more plentiful in HP-MSC-DE compared to NP-MSC-DE. The addition of HP-MSC-DE enriched by Jagged-1 to the co-culture system stimulates the Notch pathway on the membrane of UCB-HSCs CD133+ and enhances proliferation. HP-MSC-DE induction using an anti-Jagged-1 antibody suppresses all biological functions of the Jagged-1 protein. Importantly, HP-MSC-DE containing Jagged-1 could change the biology of HSCs CD133+ and increase the self-renewal capacity, quiescence, and clonogenic potential of CD133+ cells. Moreover, they support generating a large number of primitive cells. Our study signified the importance of HP-MSC-DE in the proliferation of UCB-HSCs CD133+, which manifested therapeutic applications of EXO in the enhanced number of HSCs and subsequently alleviated bone marrow transplantation.

Generating Large Numbers of Human Megakaryocytes from Pluripotent Stem Cells

The process of hemostasis and blood coagulation relies heavily on a sufficient supply of platelets (PLTs, also known as thrombocytes) within a person's bloodstream. Platelet transfusion is an effective treatment for thrombocytopenia-related diseases, yet paucity of supply and limited shelf-life (5 - 7 days) remain challenging. PLTs are generated by the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) into megakaryocytes (MKs), a rare subset of large polyploid bone marrow cells. Methods to produce MKs in vitro by inducing mesodermal specification and hematopoietic differentiation of human pluripotent stem cells (hPSCs) could provide a reliable and safe supply of PLTs for transfusion and would also be amenable to gene editing for generation of HLA-null universal PLTs. Culture methods to generate hPSC-derived MKs and PLTs have been described, yet published protocols lack standardization, are PSC line dependent and/or promote differentiation of other lineages, resulting in low MK cell yields and purity. The use of feeder cells and/or viral vectors also limits the clinical and scale-up applicability. Here, we describe an efficient feeder cell-free and serum-free culture system that promotes the selective differentiation of hPSCs from multiple ES and iPS lines into polyploid MKs with high purity and yields and ability to generate platelets.The 17-day protocol includes two stages: a 12-day stage to differentiate hPSCs into megakaryocytic-biased HSPCs through endothelial-to-hematopoietic transition (H-phase), and a 5-day stage to further differentiate HSPCs into mature MKs (MK-phase). at the start of the H-phase, hPSC aggregates were plated in mTeSR TM media on Matrigel ®-coated plates at 16 aggregates (100 - 200 µm in diameter, ~100 cells per aggregate) per cm 2 to allow attachment overnight (Day -1). The cells were then cultured in mesoderm-induction medium for 3 days (Day 0 - 3), and subsequently in hematopoietic specification medium for 9 days (Day 3 - 12). During this phase, PSC-derived HSPCs emerged from an adherent layer of endothelial cells and were released into suspension. On day 12 these nonadherent cells were harvested and seeded at 1 - 3.5 × 10 5 cells/mL in MK maturation medium containing thrombopoietin (TPO) and other hematopoietic growth factors and cultured for 5 additional days (MK-phase, Day 12 - 17). At the end of H-phase (day 12) and MK-phase (day 17) the cells were counted and assessed for HSPC markers (CD34/CD45), the erythroid marker glycophorin A (GlyA), MK markers (CD41a/CD42b), DNA ploidy profile and PLT production by flow cytometry and immunofluorescence microscopy. Two embryonic stem (ES) cell lines (H1 and H9), and two induced pluripotent stem (iPS) cell lines (WLS-1C and STiPS-R038) were used in this study.At the end of H-phase (Day 12), on average 48% (range: 34 - 72%) of cells released into suspension co-expressed CD41a and CD42b markers, with an average yield of 93 CD41a +CD42b + cells per seeded hPSC (range: 30 - 200, n = 4 for H9/1C, n = 3 for H1/R038). The cells also expressed CD34 (average of 78% CD34 + cells) and GlyA (average of 71% GlyA + cells), indicating that the H-phase may support differentiation of PSCs to megakaryocyte-erythroid progenitors. At the end of MK-phase (Day 17), on average 82% of the cells expressed CD41a (range: 70 - 99%), 62% of the cells co-expressed CD41a and CD42b (range: 40 - 85%), and an average of 253 CD41a +CD42b + cells were generated per seeded hPSC (range: 70 - 700 MKs, n = 11 for H1/H9/R038, n = 7 for 1C). Of note, less than 5% of cells expressed GlyA, showing that the culture system is specific for megakaryocytic differentiation. The DNA ploidy profile of the CD41a +CD42b + cells generated on Day 17 showed that on average 26% and 9% of cells had 4N and 8N+ DNA ploidy, respectively (n = 11). Multinucleated MKs could also be readily observed by immunofluorescence microscopy. These PSC-derived MKs produced an average of 3.5 PLTs (range: 1 - 10 PLTs, n = 11) based on viable CD41a +CD45 -GlyA - PLT-like particles with a similar size and CD41 expression as control PLTs prepared from fresh blood.In conclusion, we have developed a simple two-step, yet robust serum- and feeder-free culture system for generating high numbers of hPSC-MKs that are large, polyploid, and capable of shedding PLTs. This culture method provides a platform to study thrombopoiesis and is amenable to scale-up method development. DisclosuresNo relevant conflicts of interest to declare.

C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs.To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice vs. -MYC +/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin -Sca-1 +cKit + [LSK]), multipotent progenitors (MPPs; LSK CD48 +CD150 -), common myeloid progenitors (CMPs; Lin -Sca1 -cKit +), granulocyte-monocyte progenitors (GMPs; Lin -Sca-1 -cKit +CD34 +FCγR +), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease.We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit + and Cd11b + BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER T2+/-;Myc fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN. DisclosuresKuykendall: Pharmaessentia: Honoraria; Abbvie: Honoraria; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Blueprint: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria, Speakers Bureau. Komrokji: Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; Geron: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau.

Myeloid-Biased HSC Require Semaphorin4a from the Bone Marrow Niche for Self-Renewal Under Stress and Life-Long Persistence

A subset of hematopoietic stem cells with inherent myeloid and platelet bias (myHSC) is positioned at the top of hematopoietic hierarchy and considered the most primitive. Notably, lower proliferative output of myHSC correlates with a greater capacity for self-renewal indicating that quiescence is essential for their function. Hence, excessive myHSC expansion following inflammatory challenge is likely to put them at a higher risk of proliferation-induced damage. Given that inflammatory stress is unavoidable throughout live, we hypothesized that myHSC may uniquely depend on quiescence-inducing signals for their protection and long-term persistence. However, the nature of these signals remains largely unexplored.We have previously performed proximity-based analysis of the bone marrow niche to identify novel regulators of HSC quiescence (Silberstein et al, Cell Stem Cell 2016). Briefly, we defined the transcriptional profiles of osteolineage cells which were located in closer proximity to a transplanted HSC (proximal cells), and designated secreted factors with higher expression level in proximal cells as putative regulators of HSC quiescence. For the current study, we selected Semaphorin4a (Sema4a) - a known regulator of neural development, angiogenesis and immune response with no previously documented role in hematopoiesis - and examined its impact on myHSC function during inflammatory stress.We found that Sema4a was expressed in the niche cells (endothelium and osteoprogenitors) in mice and humans. Recombinant Sema4a reduced proliferation of mouse and human hematopoietic stem and progenitor cells ex vivo. Baseline analysis of young Sema4aKO mice revealed mild anemia, thrombocytosis, myeloid bias and a slight reduction in the proportion of HSC in the G0 phase suggesting that Sema4a regulates HSC quiescence and differentiation in vivo. Upon Poly(I:C) injection, Sema4aKO myHSC (Lin -Kit +Sca +CD48 -CD34 -CD150 high) displayed markedly increased cycling and upregulation of alpha-interferon and JAK-STAT signaling while “balanced” HSC (Lin -Kit +Sca +CD48 -CD34 -CD150 low) were unaffected. Similar exaggerated proliferative response in Sema4aKO myHSC was observed upon injection with IL-1β.Next, we investigated the long-term impact of inflammation-induced loss of myHSC quiescence. Aged Sema4aKO mice developed anemia, thrombocytosis, neutrophilia. Most significantly, we observed a two-fold expansion of phenotypic myHSC (but not balanced HSC) which displayed proliferative senescence, increased cellular stress and premature differentiation by scRNA-Seq, and a complete loss of reconstitution upon transplantation. In contrast, young Sema4aKO HSC showed a higher level of post-transplant chimerism consistent with their prior “pre-activated” state. Thus, loss of myHSC quiescence leads to increased sensitivity to inflammatory stressors and enhanced myHSC response but eventual collapse of regenerative function.In order to determine if the microenvironment served as a critical source of Sema4a for myHSC, WT myHSC were transplanted into lethally irradiated WT and Sema4aKO hosts. Strikingly, the majority of Sema4aKO recipients died while all WT recipients survived. Intra-vital imaging at 24 hours revealed a greater number of cells and clusters in Sema4aKO recipients suggesting that excessive early myHSC proliferation led to impaired self-renewal and engraftment failure. Finally, we found that Plexin D1 acts as a functional receptor for Sema4a on myHSC, since Plexin D1-deficient myHSC recapitulated the post-transplant phenotype of young Sema4aKO myHSC described above.Taken together, our data demonstrate that under the conditions of increased myeloid demand, protection from proliferative stress is critical for preserving myHSC function, and highlight a critical but previously unrecognized role for Sema4a-PlxnD1 axis in this process. Our study suggests that therapeutic augmentation of myHSC quiescence may alleviate the negative impact in inflammatory signaling, serve to improve marrow function in inflammatory diseases, and prevent development of myeloid malignancy. DisclosuresRadtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy. Kiem: Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy; VOR Biopharma: Consultancy. Scadden: Fate Therapeutics: Current holder of individual stocks in a privately-held company; Editas Medicines: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: sponsored research; FOG Pharma: Consultancy; Agios Pharmaceuticals: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Serum Level of IFN-a2 and Regulatory T-Cell Subsets Correlates with Disease Progression in De Novo Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a group of diseases with clonal proliferation of dysplastic bone marrow hematopoietic cells. Recent studies have established that the ‘immune signature’ is substantially different in low and high-risk MDS. Nevertheless, the exact mechanism by which the immune response of the disease progresses from an early stage to advanced MDS and acute myeloid leukemia (AML) remain poorly defined.In order to explore the key immune-related factors in the progression of MDS, a total of 56 patients with newly diagnosed MDS were enrolled in this prospective study, where patients were classified as 25 cases of low-risk group (the IPSS-R score, very good risk, good risk and intermediate risk) and 31 cases of high-risk group (the IPSS-R score, very poor risk, poor risk). Levels of 48 cytokines in patients' serum were determined using Luminex assays, and flow cytometry were used to measure the percentages and absolute counts of T regulatory cell (Treg) and its subsets: induced regulatory Treg (iTreg: CD45RO +CD3 +CD4 +CD25 +CD127low +) and naturally regulated Treg (nTreg: CD45RA +CD3 +CD4 +CD25 +CD127low +).Our results showed that there were significant differences in the expression of 19 cytokines between the high-risk group versus low-risk group (Figure 1A). MDS patients in high-risk group had lower serum levels of some interleukins with promoting or amplifying inflammation signals in directly or indirectly way, including IL-1a, IL-2, IL-4, IL-9, IL-12, IL-15 and IL-17ANGF; three growth factors, influencing the immune microenvironment by regulating the growth and differentiation of corresponding cells, including GM-CSF, PDGF-bb and b-NGF; three chemokines including GRO-a, IP-10 and MIP-1b,which regulates the migration of cells to their respective locations to play a pro-inflammatory role; two immunomodulatory cytokines including TNF-b and IFN-a2; leukemia inhibitory factor (LIF) as well as TNF-related apoptosis-inducing ligand (TRAIL) compared to the MDS patients in low-risk group(p<0.05). The higher serum levels of Eotaxin (p= 0.0493), facilitating proliferation and metastasis of tumor cells expressing the it's receptor, was identified in the higher-risk MDS patients. Although there is no significant difference of Treg cells in PB between the two groups, both the percentages and absolute counts of nTreg/iTreg was decreased in high-risk group of MDS patients (Figure 1B, p<0.05). After adjusting the Cox model according to the Revised International Prognostic Scoring System (IPSS-R), age and transfusion dependency, it was found that higher IFN-a2 level was correlated with longer progression-free survival (PFS) (Figure 1C, p=0.038, HR=0.89,95%CI: 0.797~0.994).These results suggested that the pro-inflammatory cytokines during the progression of MDS, the immune disorder of microenvironment and the transformation of nTreg to iTreg may be the key immunological mechanisms for the emergence of immune tolerance and transformation to AML. The serum level of IFN-a2, contributing to inhibit cancer cell proliferation and activate immune system, is an independent protective factor for disease progression or death in patients with MDS, which may be an effective indicator to predict the progression of MDS into higher risk category or transforming into AML. At the same time, it shows that some cytokines in serum are related to the progression and prognosis of MDS, which may provide a basis for early clinical intervention to improve the prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Real-World Evaluation of the Association between Elevated Blood Counts and Thrombotic Events in Polycythemia Vera (Analysis of Data from the REVEAL Study)

A Real-World Evaluation of the Association between Elevated Blood Counts and Thrombotic Events in Polycythemia Vera (Analysis of Data from the REVEAL Study)

Hemogen/BRG1 Cooperativity Is Required for Erythrocyte Maturation and Hemoglobin Production during Mammalian Erythropoiesis

Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unclear. We found that depletion of hemogen in human CD34 + erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to occupy chromatin at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization. DisclosuresNo relevant conflicts of interest to declare.

Megakaryocyte-specific knockout of the Mir-99b/let7e/125a cluster lowers platelet count without altering platelet function

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function.

Interleukin-1β suppression dampens inflammatory leucocyte production and uptake in atherosclerosis.

AimsTargeting vascular inflammation represents a novel therapeutic approach to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1β (IL-1β) using canakinumab, a monoclonal antibody, reduces the incidence of cardiovascular events in patients after myocardial infarction (MI). The biological basis for these beneficial effects remains incompletely understood. We sought to explore the mechanisms of IL-1β-targeted therapies.Methods and resultsIn mice with early atherosclerosis (ApoE–/– mice on a high-cholesterol diet for 6 weeks), we found that 3 weeks of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)-inflammasome inhibition or anti-IL-1β treatment (using either MCC950, an NLRP3-inflammasome inhibitor which blocks production and release of active IL-1β, or a murine analogue of canakinumab) dampened accumulation of leucocytes in atherosclerotic aortas, which consequently resulted in slower progression of atherosclerosis. Causally, we found that endothelial cells from atherosclerotic aortas lowered expression of leucocyte chemoattractants and adhesion molecules upon NLRP3-inflammasome inhibition, indicating that NLRP3-inflammasome- and IL-1β-targeted therapies reduced blood leucocyte recruitment to atherosclerotic aortas. In accord, adoptive transfer experiments revealed that anti-IL-1β treatment mitigated blood myeloid cell uptake to atherosclerotic aortas. We further report that anti-IL-1β treatment and NLRP3-inflammasome inhibition reduced inflammatory leucocyte supply by decreasing proliferation of bone marrow haematopoietic stem and progenitor cells, demonstrating that suppression of IL-1β and the NLRP3-inflammasome lowered production of disease-propagating leucocytes. Using bone marrow reconstitution experiments, we observed that haematopoietic cell-specific NLRP3-inflammasome activity contributed to both enhanced recruitment and increased supply of blood inflammatory leucocytes. Further experiments that queried whether anti-IL-1β treatment reduced vascular inflammation also in post-MI accelerated atherosclerosis documented the operation of convergent mechanisms (reduced supply and uptake of inflammatory leucocytes). In line with our pre-clinical findings, post-MI patients on canakinumab treatment showed reduced blood monocyte numbers.ConclusionsOur murine and human data reveal that anti-IL-1β treatment and NLRP3-inflammasome inhibition dampened vascular inflammation and progression of atherosclerosis through reduced blood inflammatory leucocyte (i) supply and (ii) uptake into atherosclerotic aortas providing additional mechanistic insights into links between haematopoiesis and atherogenesis, and into the beneficial effects of NLRP3-inflammasome- and IL-1β-targeted therapies.

Prevalence of the GFI1-36N SNP in Multiple Myeloma Patients and Its Impact on the Prognosis.

Transcription factor Growth Factor Independence 1 (GFI1) regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells. A single nucleotide polymorphism (SNP) variant of GFI1 (GFI1-36N: serine replaced by asparagine at position 36), has a prevalence of 5-7% among healthy Caucasians and 10-15% in patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) predisposing GFI-36N carriers to these diseases. Since GFI1 is implicated in B cell maturation and plasma cell (PC) development, we examined its prevalence in patients with multiple myeloma (MM), a haematological malignancy characterized by expansion of clonal PCs. Strikingly, as in MDS and AML, we found that the GFI1-36N had a higher prevalence among MM patients compared to the controls. In subgroup analyses, GFI1-36N correlates to a shorter overall survival of MM patients characterized by the presence of t(4;14) translocation and gain of 1q21 (≤3 copies). MM patients carrying gain of 1q21 (≥3 copies) demonstrated poor progression free survival. Furthermore, gene expression analysis implicated a role for GFI1-36N in epigenetic regulation and metabolism, potentially promoting the initiation and progression of MM.

High-volume endurance exercise training stimulates hematopoiesis by increasing ACE NH2-terminal activity.

One of the health benefits of endurance exercise training (ET) is the stimulation of hematopoiesis. However, the mechanisms underlying ET-induced hematopoietic adaptations are understudied. N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits proliferation of early hematopoietic progenitor cells. The angiotensin I-converting enzyme (ACE) NH2-terminal promotes hematopoiesis by inhibiting the anti-hematopoietic effect of Ac-SDKP. Here we demonstrate for the first time the role of ACE NH2-terminal in ET-induced hematopoietic adaptations. Wistar rats were subjected to 10 weeks of moderate-(T1) and high-(T2) volume swimming-training. Although both protocols induced classical ET-associated adaptations, only T2 increased plasma ACE NH2-domain activity (by 40%, P=0.0003) and reduced Ac-SDKP levels (by 50%, P<0.0001). T2 increased the number of hematopoietic stem cells (HSCs; ∼200%, P=0.0008), early erythroid progenitor colonies (∼300%, P<0.0001) and reticulocytes (∼500%, P=0.0007), and reduced erythrocyte lifespan (∼50%, P=0.022). Following, Wistar rats were subjected to T2 or T2 combined with ACE NH2-terminal inhibition (captopril (Cap) treatment: 10 mg.kg-1.day-1). T2 combined with ACE NH2-terminal inhibition prevented Ac-SDKP decrease and attenuated ET-induced hematopoietic adaptations. Altogether, our findings show that ET-induced hematopoiesis was at least partially associated with increased ACE NH2-terminal activity and reduction in the hematopoietic inhibitor Ac-SDKP.

Hematopoietic and Mesenchymal Stromal Cells: New Immunological Roles During Fungal Infections.

Adult stem cells are characterized not only by their regenerative and immunomodulatory capacity but also by their therapeutic potential in various pathologies that include hematological malignancies, cancer, and autoimmune and inflammatory diseases, among others. However, these cells seem to play a paradoxical role during the development of the immune response in some infectious diseases. As an example, Candida albicans can induce the proliferation and differentiation of hematopoietic stem cells (HSCs) and their progenitors, a process known as emergency hematopoiesis. Moreover, Aspergillus fumigatus and C. albicans, once recognized by mesenchymal stromal cells (MSCs), can induce an anti-inflammatory or proinflammatory profile, respectively, and, in turn, these cells can inhibit the growth of these fungal pathogens. Additionally, the transplantation of MSCs, in an experimental pulmonary model of paracoccidioidomycosis, has been shown to exacerbate the inflammatory response. More recently, in vitro studies have shown that MSCs recognize Paracoccidioides brasiliensis through a mechanism mediated by toll-like receptor (TLR)2, TLR4, and Dectin-1, which, in turn, induces a proinflammatory profile. This review describes the main mechanisms and immunomodulatory properties of HSCs and MSCs during infections caused by some medically important fungal pathogens described so far in literature.

Serum Leptin Level in Children with Acute Lymphoblastic Leukemia

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer .It is a disease of monoclonal proliferation of hematopoietic precursor cells of the lymphoid series within the bone marrow. Leptin is a peptide hormone which is predominantly produced by white adipose tissue. The main function of leptin in the human body is the regulation of energy expenditure and control of appetite .It is supposed to be involved in the pathogenesis of hematological malignancy through its proliferative, anti-apoptotic, and differentiating effects on hematopoietic neoplastic cells, in addition to its synergestic effect on vascular endothelial growth factor (VGEF) . Objectives To evaluate serum leptin level in children with acute lymphoblastic leukemia at diagnosis and at day 28 post induction chemotherapy,and to test the association between serum leptin levels and anthropometric measures of ALL patients and prognostic markers of ALL as age, gender, initial WBCS count, extramedullary infiltration to organs and CSF, cytogenetics, and response to treatment . Patients and Methods This is a case- control study performed at Clinical Pathology Department, Ain Shams University Hospitals. The study was conducted on 30 ALL pediatric patients admitted to Hematology &amp; Oncology Unit, Pediatric Department, Ain Shams University Hospitals.The patients were evaluated at diagnosis before intake of chemotherapy and at day 28 post-induction chemotherapy, and 25 healthy controls matched with patients in age, sex, BMI. Results Serum leptin level is elevated in ALL patients at diagnosis as compared to controls .It has no significant relation with patients age, sex, BMI. It is elevated in patients who received cranial radiotherapy and those who relapsed. .However, it has no significant relation with clinical data (fever, lymphadenopathy and organomegaly) and laboratory characteristics (CBC, percentage of blasts infiltrating bone marrow and peripheral blood, immunophenotyping and cytogenetics) of ALL patients. Conclusion Data from our study showed that elevated leptin level in ALL patients can be considered a risk factor involved in ALL pathogenesis.It can be used as a biomarker for ALL diagnosis, a prognostic factor for ALL patients on chemotherapy, and a predicting factor for relapse.

Gata2 haploinsufficiency promotes proliferation and functional decline of hematopoietic stem cells with myeloid bias during aging

AbstractDuring aging, hematopoietic stem cell (HSC) function wanes with important biological and clinical implications for benign and malignant hematology, and other comorbidities, such as cardiovascular disease. However, the molecular mechanisms regulating HSC aging remain incompletely defined. GATA2 haploinsufficiency driven clinical syndromes initially result in primary immunodeficiencies and routinely evolve into hematologic malignancies on acquisition of further epigenetic mutations in both young and older patients. Using a conditional mouse model of Gata2 haploinsufficiency, we discover that during aging Gata2 promotes HSC proliferation, monocytosis, and loss of the common lymphoid progenitor. Aging of Gata2 haploinsufficient mice also offsets enhanced HSC apoptosis and decreased granulocyte-macrophage progenitor number normally observed in young Gata2 haploinsufficient mice. Transplantation of elderly Gata2 haploinsufficient HSCs impairs HSC function with evidence of myeloid bias. Our data demonstrate that Gata2 regulates HSC aging and suggest the mechanisms by which Gata2 mediated HSC aging has an impact on the evolution of malignancies in GATA2 haploinsufficiency syndromes.

MPN-161: A Phase I Study of TP-3654, an Oral PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis (MF)

Context: MF is characterized by bone marrow fibrosis (BMF), ineffective hematopoiesis, splenomegaly, and debilitating symptoms. Expression of PIM-1 is significantly upregulated in MF hematopoietic cells, indicating that PIM-1 is a potential therapeutic target in MF. TP-3654, an oral small-molecule investigational PIM-1 kinase inhibitor with favorable selectivity against other kinases, has been shown to inhibit proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone and in combination with ruxolitinib normalizes leukocyte counts and reduces spleen size and BMF in JAK2V617F and MPLW515L murine MF models. Objective: To evaluate TP-3654 monotherapy in patients with MF. Design: Phase I, open-label, dose-escalation study (NCT04176198). Setting: Multicenter, US only. Patients or Other Participants: Key eligibility criteria include primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF); intermediate-2/high-risk MF per Dynamic International Prognostic Scoring System (DIPSS); previously failed or are ineligible to receive Janus kinase inhibitors (e.g., ruxolitinib, fedratinib); grade ≥2 BMF; platelet count ≥50x109/L; absolute neutrophil count ≥1x109/L; peripheral blood blast counts Interventions: Patients receive oral TP-3654. Dose escalation, performed using a Bayesian Logistic Regression Model with Escalation with Overdose Control, will continue until identification of the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) is achieved. Dose expansion will follow. Main Outcomes Measures: Primary objectives: to determine MTD and/or identify the RP2D of TP-3654 monotherapy. Secondary objectives: to assess preliminary activity (spleen volume reduction, symptom improvement, and BMF reduction), determine QT interval changes, and establish the pharmacokinetic profile. Exploratory objectives: to study pharmacodynamic markers in peripheral blood and BM biopsies. This study is currently recruiting.

Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability.

The therapeutic value of mesenchymal stromal cells (MSCs) for various regenerative medicine applications, including hematopoietic stem cell transplantations (HSCT), has been well-established. Owing to their small numbers in vivo, it becomes necessary to expand them in vitro, which leads to a gradual loss of their regenerative capacity. Stress-induced mitogen-activated protein kinase p38 (p38 MAPK) signaling has been shown to compromise the MSC functions. Therefore, we investigated whether pharmacological inhibition of p38 MAPK signaling rejuvenates the cultured MSCs and boosts their functionality. Indeed, we found that the ex vivo expanded MSCs show activated p38 MAPK signaling and exhibit increased oxidative stress. These MSCs show a decreased ability to secrete salutary niche factors, thereby compromising their ability to support hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation. We, therefore, attempted to rejuvenate the cultured MSCs by pharmacological inhibition of p38 MAPK - a strategy broadly known as "priming of MSCs". We demonstrate that priming of MSCs with a p-38 MAPK inhibitor, PD169316, boosts their niche-supportive functions via upregulation of various HSC-supportive transcription factors. These primed MSCs expand multipotent HSCs having superior homing and long-term reconstitution ability. These findings shed light on the significance of non-cell-autonomous mechanisms operative in the hematopoietic niche and point towards the possible use of pharmacological compounds for rejuvenation ofex vivocultured MSCs. Such approaches could improve the outcome of regenerative therapies involving in vitro cultured MSCs.