Megakaryocyte-erythroid Progenitors Research Articles

Single-Cell RNA-Seq Reveals the Differentiation Hierarchy of Normal Human Bone Marrow and a Distinct Transcriptome Signature of Monosomy 7 Cells

Monosomy 7 is a frequent cytogenetic abnormality in hematopoietic malignancies and a general indicator of poor prognosis. Due to lack of distinct cell surface markers between monosomy 7 cells and normal cells, it is not feasible to physically separate aneuploid from diploid cells. We performed single-cell RNA-seq (scRNA-seq), which allows the entire transcriptome of large numbers of single cells to be assayed in an unbiased way, to investigate hematopoietic differentiation of normal and aneuploid human hematopoietic cells. Bone marrow samples were collected from four patients (P1-P4) with myelodysplastic syndrome, and four healthy volunteers. Conventional cytogenetics showed -7/7q- in bone marrow cells from P1, P3 and P4, and dup(1)(q21q32) in cells from P2; retrospectively, P2 was found positive for monosomy 7 as well as trisomy 8 by fluorescence in situ hybridization. Fresh CD34+CD38- and CD34+CD38+cells were sorted by flow cytometry and then subjected to Fluidigm C1 Single-Cell Auto Prep System for scRNA-seq. After excluding cells with low transcriptome coverage, 326 cells from P1 and P2 (analysis is in progress for P3 and P4), and 391 cells from healthy subjects were analyzed by comparison of transcriptomes from 17,071 genes. Nonlinear dimension reduction and visualization were achieved using t-distributed Stochastic Neighbor Embedding (tSNE). Cells from healthy controls clustered into seven subgroups based on their gene expression pattern, and each group could be associated with a previously reported hematopoietic cell type by known marker genes (Laurenti E, Nat Immunol, 2013). These cell types included hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP) (Fig 1a). Individual cells from healthy controls were ordered by Monocle software based on their expression profile similarity to uncover a differentiation hierarchy. A two-branch trajectory of development from HSC was revealed, with one branch progressing towards erythroid cell and the other to lymphoid/myeloid cells (Fig 1b). This pattern differs from the classic hematopoietic model, but is consistent with reports claiming existence of early-lymphoid-biased progenitors that retain myeloid but not erythroid potential (Doulatov S, Nat Immunol, 2010), and of dominance of multipotent and unipotent progenitors over scarce oligopotent progenitors in the adult marrow hematopoietic hierarchy (Notta F, Science, 2016). We compared single cells from patients and healthy controls for regional and chromosomal copy number differences in gene expression. We identified subclonal populations of cells from patients that showed decreased expression of chromosome 7 genes (60% in P1, and 55% in P2; Fig 1c and 1d), and increased expression of chromosome 8 (77% in P2) and chromosome 1 long arm genes (P2), at FDR=0.05 estimated with cells from health donors. Gene Ontology enrichment analysis using topGO indicated that cells with low global expression of chromosome 7 genes had dysregulated expression of immune related genes, including B cell receptor signaling pathway, T cell activation and differentiation, antigen receptor-mediated signaling pathway, as well as signal transduction and Fc-γ Receptor signaling pathway. ScRNA-seq analysis reveals a simple pattern of normal human hematopoietic development and the molecular signature of aneuploid cells from patients with developing "clonal evolution". This powerful method should improve characterization of functional changes in human cells with chromosome abnormalities. [Display omitted] DisclosuresDesierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

Synthetic miRNA Switch Technology Elucidates Heterogeneity in Regulation of Immortalized Megakaryocyte Cell Lines, Associated with Improvement of Platelet Generation Efficiency for Clinical Use

We have developed a robust in vitro platelet generation system using a self-renewing megakaryocyte (MK) cell line, imMKCL, which was established by introducing three doxycycline (DOX)-inducible transgenes, c-MYC, BMI1, and BCL-XL into human induced pluripotent stem cells (iPSCs)-derived megakaryocyte-erythroid progenitor (MEP) cells for immortalization (Nakamura et al. Cell Stem Cell 2014). This system is suitable for clinical application since imMKCLs proliferate from 106 to over 1010 cells in only 20 days in the presence of DOX (self-renewing stage), and when DOX is removed, imMKCLs enlarge, form proplatelets and release platelets (maturation stage). However, although imMKCLs display homogenous appearance at their self-renewing stage, only a minor population eventually produces platelets at a level comparable to MKs in vivo. Therefore, it is critical to resolve this heterogeneity in terms of maturation to improve the efficiency of platelet production for their practical use.In that regard, we herein apply microRNA (miRNA) switch technology to identify a subpopulation of imMKCLs with high capacity to produce platelets upon maturation. The synthetic miRNA responsive switches could be used for detecting and efficiently purifying cell populations based on endogenous miRNA activity (Miki et al. Cell Stem Cell 2015).First we performed a screening of miRNA switches to investigate the miRNA activities in imMKCLs at the self-renewing stage. Twenty-six miRNA candidates turned out to have some activities in imMKCLs in the presence of DOX. Among those miRNAs, we noticed that Let-7a-5p showed a clear distinguishable pattern with about 10% of the cells in the lower Let-7a-5p activity group (Group L), while other large majority in the higher activity group (Group H). When we sorted the two groups by flow cytometer even in the presence of DOX (self-renewing stage), Group L population in imMKCL revealed to produce significantly more platelets than Group H population (average of 9.9 vs 6.0 platelets per each MK, p<0.01) after DOX depletion. Transfecting Let-7a-5p inhibitor into Group H cells increased platelet production equivalent to the level of Group L, suggesting that Let-7a-5p suppresses the maturation of imMKCLs.Next, we found that the expression level of LIN28A but not LIN28B, a putative downstream target of c-MYC for self-renewal in imMKCL, in Group L was 8.3 times higher than that in Group H (p<0.01), where LIN28A is reported to downregulate the expression of Let-7a-5p by inhibiting the processing of Let-7 miRNA precursors by DICER (Piskounova et al. Cell 2011). This difference was not affected by Let-7a-5p inhibitor, in line with the role of LIN28A as the upstream regulator of Let-7.Finally, to address the targets of Let-7a-5p, we then performed RNA-seq analysis to compare gene expression profiles between Group L and Group H. We found that 691 genes were upregulated more than twice in Group L than in Group H. Among these, 48 genes were predicted to be the targets of Let-7a-5p according to three miRNA target prediction databases, miRSystem, miRDB, and miRSearch. Validation of 48 genes by qPCR confirmed the upregulation of several genes selectively in platelet-producing imMKCLs. Accordingly, transfecting Let-7a-5p inhibitor into Group H cells upregulated the expressions of these confirmed genes, strongly suggesting that they are the candidate targets of Let-7a-5p regulating maturation of imMKCLs.In conclusion, miRNA switch technology enabled us to purify an imMKCL population with high platelet production capability based on the activity of Let-7a-5p. Furthermore, this study revealed the heterogeneity of miRNA expressions in imMKCLs at their self-renewal stage, and suggests that miRNA-mediated gene regulation at this stage affect their maturation afterwards. DisclosuresNo relevant conflicts of interest to declare.

Architectural and Functional Heterogeneity of Hematopoietic Stem/Progenitor Cells in Non-Del(5q) Myelodysplastic Syndromes

IntroductionMyelodysplastic syndromes (MDS) are clinically diverse malignant disorders of aging with a propensity to evolve to acute myeloid leukemia (AML) or bone marrow failure. In early MDS, whole genome sequencing identified mutations distributed in few clones. Evidence have been provided for the existence of a MDS-initiating cell in cases harboring a 5q deletion. However, the clonal heterogeneity and its consequences on the phenotypic diversity of non-del(5q) MDS is little documented. Here we focused on studying the hierarchical organization and the functionality of clones defined by molecular profiling. The clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment was investigated and dominant clones were examined as MDS-initiating cells.Material and methodsBone marrow (BM) samples were obtained from 20 patients with non-del(5q) MDS enrolled in the national Programme Hospitalier de Recherche Clinique MDS-04 after informed consent in accordance with ethics committee guidelines. BM samples,cytaphereses from age-matched healthy individuals and cord bloods were used as controls. Targeted NGS of a selected panel of 39 genes was used to define the mutational landscape on BM mononuclear cells (MNC). To study the clonal architecture at the HSPC level, single CD34+CD38- cells were seeded in 96-well plates coated with MS-5 stromal cells and cultured in H5100 MyeloCult medium (StemCell Technologies, Vancouver, Canada) with cytokines for six weeks. For long-term culture-initiating cell (LTC-IC) assays, CD34+ progenitors were cultured for six weeks on MS-5-coated plates without cytokines and then tested for colony-forming cells. For clonogenic assays, CD34+CD38- cells were seeded in methylcellulose for two weeks. All animal experimentations were performed in NSG mice.ResultsIn the 20 cases of non-del(5q) MDS, genomic lesions were traced down to single CD34+CD38- HSPC-derived colonies. Clonal organization was mostly linear in 13/17 patients and branched in 4 cases with retention of a dominant subclone. The clone detected in LTC-IC compartment and that reconstituted short-term human hematopoiesis in xenotransplantation models was usually the dominant clone, which gave rise to the myeloid and to a lesser extent to the lymphoid lineage. Other mutations not detected in LTC-IC can appear in CD34+CD38- compartment or at the level of lineage-committed progenitors. The pattern of mutations may differ between common myeloid (CMP), granulo-monocytic (GMP) and megakaryocytic-erythroid (MEP) progenitors. For instance, a major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knockdown by shRNA in normal CD34+ progenitors impaired their granulocytic and erythroid differentiation. By contrast, a STAG2 gene mutation, not detected in CMP or MEP, amplified in a GMP, which drove the transformation to AML.ConclusionIn the present study, we characterized the first genetic hits that initiate disease in a dominant clone of the CD34+CD38- HSPC compartment, which exhibits LTC-IC activity and reconstitutes human short-term hematopoiesis in NSG mice. The genetic heterogeneity in non-del(5q) MDS arises within the HSPC compartment and in lineage-committed progenitors which ultimately support the transformation into AML. The clonal architecture of HSPC compartment and mutations selection along differentiation contribute to the phenotype of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation, and may guide the search for novel therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

An Autosomal-Recessive GFI1B Mutation Defines the Splice Isoform p37 As Essential for Biogenesis of Functional Human Platelets, but Dispensable for Erythropoiesis

Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner.We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected.Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9.Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. DisclosuresNo relevant conflicts of interest to declare.

MiR-125b modulates megakaryocyte maturation by targeting the cell-cycle inhibitor p19INK4D.

A better understanding of the mechanisms involved in megakaryocyte maturation will facilitate the generation of platelets in vitro and their clinical applications. A microRNA, miR-125b, has been suggested to have important roles in the self-renewal of megakaryocyte-erythroid progenitors and in platelet generation. However, miR-125b is also critical for hematopoietic stem cell self-renewal. Thus, the function of miR-125b and the complex signaling pathways regulating megakaryopoiesis remain to be elucidated. In this study, an attentive examination of the endogenous expression of miR-125b during megakaryocyte differentiation was performed. Accordingly, the differentiation of hematopoietic stem cells requires the downregulation of miR-125b, whereas megakaryocyte determination and maturation synchronize with miR-125b accumulation. The overexpression of miR-125b improves megakaryocytic differentiation of K562 and UT-7 cells. Furthermore, stage-specific overexpression of miR-125b in primary cells demonstrates that miR-125b mediates an enhancement of megakaryocytic differentiation after megakaryocyte determination, the stage at which megakaryocytes are negative for the expression of the hematopoietic progenitor marker CD34. The identification of miR-125b targets during megakaryopoiesis was focused on negative regulators of cell cycle because the transition of the G1/S phase has been associated with megakaryocyte polyploidization. Real-time PCR, western blot and luciferase reporter assay reveal that p19INK4D is a direct target of miR-125b. P19INK4D knockdown using small interfering RNA (siRNA) in megakaryocyte-induced K562 cells, UT-7 cells and CD61+ promegakaryocytes results in S-phase progression and increased polyploidy, as well as improved megakaryocyte differentiation, similarly to the effects of miR-125b overexpression. P19INK4D overexpression reverses these effects, as indicated by reduced expression of megakaryocyte markers, G1-phase arrest and polyploidy decrease. P19INK4D knockdown in miR-125b downregulated cells or p19INK4D overexpression in miR-125b upregulated cells rescued the effect of miR-125b. Taken together, these findings suggest that miR-125b expression positively regulates megakaryocyte development since the initial phases of megakaryocyte determination, and p19INK4D is one of the key mediators of miR-125b activity during the onset of megakaryocyte polyploidization.

Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia.

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34− AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34− myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34− precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34− SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34− AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

Myeloproliferative neoplasms as a model of a stem cell disease

Myeloproliferative neoplasms (MPN) are clonal disorders of the hematopoietic stem cell. Somatic gain of function mutations in the genes for JAK2, CALR or MPL can be found in about 90% of patients with MPN and are thought to be the disease driving mutations. In addition, accumulation of mutations in genes encoding epigenetic regulators, including TET2, ASXL1, DNMT3A and EZH2, transcription factors and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. We have studied the effects of the JAK2-V617F mutation on the hematopoietic stem cell (HSC) compartment in mouse models of MPN and using limiting-dilution and single-cell transplantations we could show that that MPN disease can be initiated clonally from a purified single HSC carrying JAK2-V617F as the sole genetic alteration. Expression of JAK2-V617F in HSCs promoted cell division and expanded long-term repopulating capacity to a compartment with intermediate expression levels of lineage markers. Mice double mutant for JAK2-V617F and loss of the epigenetic regulator gene Ezh2 in hematopoieisis showed acceleration of disease and a more severe MPN phenotype with early progression to myelofibrosis. RNA sequencing in Ezh2 deficient hematopoietic stem cells and megakaryocytic erythroid progenitors identified highly upregulated genes, including Lin28b, and Hmga2, and Chip-qPCR analysis of their promoters revealed decreased H3K27me3 deposition. Expression of JAK2-V617F together with homozygous loss of Ezh2 resulted in greatly increased MPN disease initiation in limiting dilution transplantation and added repopulating capacity to stem and progenitor compartments that normally do not show long-term engraftment. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and demonstrate synergistic effects with JAK2-V617F on the HSC compartment.

Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction

AbstractBipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor–mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter–sorted cell populations, we found that the Lin−CD34+CD38midCD45RA−FLT3−MPL+CD36−CD41− population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB. We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.

Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis.

Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2 In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2(-/-) mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2(+/-) mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F-expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN.

Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways.

BackgroundRecent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.ResultsTo clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: “Pre-MEP,” enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; “E-MEP,” strongly biased towards erythroid differentiation; and “MK-MEP,” a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output.ConclusionsOur study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0939-7) contains supplementary material, which is available to authorized users.

Leukemia cell infiltration causes defective erythropoiesis partially through MIP-1α/CCL3.

Leukemia often results in severe anemia, which may significantly contribute to patient mortality and morbidity. However, the mechanisms underlying defective erythropoiesis in leukemia have not been fully elucidated. In this study, we demonstrated that insufficient erythropoiesis in an immunocompetent acute myeloid leukemia (AML) murine model was due to reduced proliferation of megakaryocyte erythroid progenitors and increased apoptosis of erythroblasts. Colony-forming cell assays indicated that the leukemic bone marrow (BM) plasma inhibited erythroid colony formation, whereas they had no inhibitory effect on other types of colonies. Cytokine array analysis demonstrated that the chemokine CCL3 was elevated in the plasma of AML mice and patients. CCL3 inhibited erythroid differentiation of hematopoietic stem cells, common myeloid progenitors and especially megakaryocytic-erythroid progenitors. Administration of the CCR1 antagonist partially recovered the yield of erythroid colonies in the presence of CCL3 or leukemic BM plasma. Mechanistically, we observed an increase of p38 phosphorylation and subsequent downregulation of GATA1 after CCL3 treatment. Furthermore, knockdown of CCL3 attenuated leukemic progression and alleviated anemia. Therefore, our results demonstrate that elevated CCL3 in the leukemic environment suppresses erythropoiesis via CCR1-p38 activation, suggesting a novel mechanism for the erythroid defects observed in leukemia.

Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

Simultaneous measurement of human hematopoietic stem and progenitor cells in blood using multicolor flow cytometry.

Hematopoietic stem cells are the source of all inflammatory cell types. Discovery of specific cell surface markers unique to human hematopoietic stem (HSC) and progenitor (HSPC) cell populations has facilitated studies of their development from stem cells to mature cells. The specific marker profiles of HSCs and HSPCs can be used to understand their role in human inflammatory diseases. The goal of this study is to simultaneously measure HSCs and HSPCs in normal human venous blood using multicolor flow cytometry. Our secondary aim is to determine how G-CSF mobilization alters the quantity of each HSC and HSPC population. Here we show that cells within the CD34+ fraction of human venous blood contains cells with the same cell surface markers found in human bone marrow samples. Mobilization with G-CSF significantly increases the quantity of total CD34+ cells, blood borne HSCs, multipotent progenitors, common myeloid progenitors, and megakaryocyte erythroid progenitors as a percentage of total MNCs analyzed. The increase in blood borne common lymphoid and granulocyte macrophage progenitors with G-CSF treatment did not reach significance. G-CSF treatment predominantly increased the numbers of HSCs and multipotent progenitors in the total CD34+ cell population; common myeloid progenitors and megakaryocyte erythroid progenitors were enriched relative to total MNCs analyzed, but not relative to total CD34+ cells. Our findings illustrate the utility of multicolor flow cytometry to quantify circulating HSCs and HSPCs in venous blood samples from human subjects. © 2016 International Clinical Cytometry Society.

Leukemia Cell Infiltration Causes Defective Erythropoiesis Partially through MIP-1alpha

Leukemia often results in severe anemia, which may significantly contributes to mortality and morbidity of the patients. However, the mechanisms underlying the insufficient erythropoiesis in leukemia have been poorly understood. In this study, with our recently established non-irradiated MLL-AF9 acute myeloid leukemia (AML) murine model (Cheng H et al, Blood 2015), we observed a significant decrease in hemoglobin and red blood cells (RBCs) of Peripheral blood (PB) in the leukemic mice (n=6 per group, p=0.0122 vs p=0.0003). The absolute numbers of the erythroblasts at different stages (Pro Es, Ery.A, Ery.B, Ery.C) in bone marrow (BM) were also reduced. Consistently, by gene set enrichment analysis (GSEA) of microarray data of LKS+ cells (GSE52506 ) from leukemic mice, we found significant down-regulation of erythroid differentiation related genes such as GATA1, FOG-1, LMO2 and KLF1. These genes were more significantly inhibited in megakaryocytic-erythroid progenitors (MEPs) and Pro Es from the leukemic mice. Notably, the MEPs were the most reduced subset among all the committed hematopoietic progenitor cells (HPCs) during leukemia progression (90% decrease compared to control, p = 0.0007). MEPs were gradually accumulated in the G0 phase (from 22% to 70%, p<0.001). In contrast, erythroblasts (Pro Es, Ery.A, Ery.B) were more cycling (G1/S/G2/M) in leukemic mice and the proportions of Annexin V+ cells in erythroblasts but not in MEPs were also increased during leukemia development. Colony-forming cell (CFC) assays revealed that BM plasma of leukemia mice exerted an inhibitory effect on both BFU-Es and CFU-Es of BM mononuclear cells (BMMNCs) but not on other types of colonies (40% decrease for CFU-Es, 60% decrease for BFU-Es, p<0.001). Consistently, BM plasma of AML patients could also reduce the yield of BFU-Es and CFU-Es from CD34+ cord blood cells (n=7, p=0.006).To determine which cytokines may be responsible for the inhibitory effect, we collected serum and BM plasma from control and leukemic mice for cytokine array analysis. Among the elevated cytokines, MIP-1alpha was previously reported to be up-regulated in leukemic stem cells and its higher expression was found in the majority of patients with leukemia and a subset of patients with lymphoma and myeloma according to the Oncomine data set. We also confirmed it in a cohort of untreated AML patients (n=32). Importantly, AML patients with higher expression of MIP-1alpha showed reduced survival time (median=13.08 months) compared with the patients with lower expression (median=25.86 months) based on the leukemia-gene-atlas (LGA) analysis (n=72). By the CFC assay and single cell culture with different subsets of hematopoietic stem cells (HSCs) and HPCs, MIP-1alpha was able to largely mimic the inhibitory effect on the erythroid differentiation at both stem cell and progenitor cell levels. Mechanistically, we observed higher expression of MIP-1alpha receptor CCR1 in HSCs, MEPs and erythroblasts than CCR5. Administration of CCR1 antagonist, BX471 could partially recover the yield of erythroid colonies after treatment of MIP-1alpha or leukemia BM plasma. An increase of phosphorylation of p38 (phos p38) and resulted down-regulation of GATA1 after MIP-1alpha treatment were documented by Western blots and immunostaining. In summary, our results demonstrate that leukemic cell infiltration causes severe inhibition of erythropoiesis largely at different erythroid precursor levels and this inhibitory effect is at least partially medicated by elevated MIP-1alpha level via CCR1-p38 activation in the leukemic microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Enhancer Accessibility during Erythropoiesis and Megakaryopoiesis Correlates with Lineage-Specific Gene Expression

Enhancers are epigenetic regulatory modules critical to lineage-specific transcript expression. Understanding the development and maintenance of enhancers will help clarify lineage commitment decisions. The ENCODE Project Consortium used a variety of cell lines to define enhancer regulatory maps. Our goal is to build ENCODE-like enhancer maps for primary mouse hematopoietic cells. To accomplish this we have performed genome-wide open chromatin surveys (Assay for Transposable-Accessible Chromatin [ATACSeq]) and transcriptome analysis (RNASeq) in enriched populations of hematopoietic stem and progenitor cells (LSK), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), and erythroid and megakaryocytic progenitors (CFUE and CFUMeg, respectively). In addition to ATACSeq and RNASeq we performed ChIPSeq in erythroblasts (EB) and megakaryocytes (MEG). Together these data provide a high-quality map of essential enhancers and correlated transcription profiles at specific stages of hematopoiesis.Enhancers are associated with DNase I hypersensitivity and monomethylation at lysine 4 of histone H3 (H3K4me1), while active enhancer regions (AERs) are also acetylated at lysine 27 of histone H3 (H3K27ac). We identified several thousand candidate enhancer regions (cERs) in EB and MEG (Table). More than 70% of cERs also contained the H3K27ac mark and thus were candidate AERs (cAERs). In 90% of cases cAERs were closer to an active transcriptional start site (TSS) than to any other TSS. 23 of these erythroid cAERs were shown to be active in a luciferase assay.We next evaluated the major trends in acquisition and maintenance of cERs during hematopoietic differentiation and their correlation with altered gene expression. We focused on cAERs and examined their appearance and retention in progenitor cells (Table). Almost all cell-specific (97% of EB and 93% of MEG) cAERs are in accessible chromatin as monitored by ATACSeq (HC cAERs). This high degree of correlation allows us to use overlaps with progenitor ATACSeq data to estimate the presence of the cAERs identified in the mature cells. Our initial results show that the mature cell HC cAERs overlap with progenitor cell ATACSeq data (MEP and CMP) is greater for EB cAERs than MEG, suggesting that chromatin in these cells is more similar to that of EB. However, in LSK, 50% of both EB and MEG cAERs overlap with ATACSeq peaks, suggesting that half of cell-specific cAERs are present in LSK while the rest are established during differentiation. In contrast, 82% of shared EB and MEG cAERs overlapped with LSK ATACSeq peaks and were maintained throughout differentiation.Candidate super enhancers (cSEs) are the cAERs with the highest levels of H3K27ac as measured by ChIPSeq. Within the top 2% of AERs we identified 101 EB and 98 MEG HC cSEs (i.e., overlap ATACSeq peaks). There was no overlap between the EB and MEG cSEs, indicating that cSEs are more cell-specific than all cAERs (Χ2 ≤ 0.04). RNASeq data confirmed that 92% of cSEs were closest to an active TSS. These data indicate that cSEs are more cell-specific than cAERs and are associated with increased gene expression.Approximately 96% of EB-specific cSEs correlated with ATACSeq peaks in CFUE chromatin, 94% in MEP, 82% in CMP, and 65% in LSK. In contrast, 95% of MEG-specific cSEs correlated with ATACSeq peaks in CFUMeg chromatin, 81% in MEP and CMP, and 75% in LSKs. The higher percentage of MEG cSEs in LSK is significantly different from all MEG cAERs (Χ2 ≤ 0.003), indicating that, unlike cAERs, MEG-specific cSEs are established early and maintained throughout differentiation.We have shown that cAERs, especially cSEs, are highly cell-specific in primary murine erythroid and megakaryocytic cells and correlate with gene expression. Examining changes in cAERs, cSEs, and gene expression allows us to map the specific epigenetic changes in chromatin that define erythroid and megakaryocytic differentiation. These results will enable us to test hypotheses about the mechanism of erythroid and megakaryocytic lineage commitment.TableEB-specificMEG-specificCommonNo.PercentNo.PercentNo.PercentcER7153n/a4196n/a619n/acAER6097100%3064100%468100%HC cAER593497%284893%43493%HC cAER + CFUE ATACSeq581898%n/an/an/an/aHC cAER + CFUMeg ATACSeqn/an/a260692%n/an/aHC cAER + MEP ATACSeq559194%150953%39491%HC cAER + CMP ATACSeq475480%147352%38589%HC cAER + LSK ATACSeq371963%136848%35181% DisclosuresNo relevant conflicts of interest to declare.

Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP), which resides downstream of common myeloid progenitor (CMP). However, the definition of unipotent Meg progenitor (MegP) is still controversial.To shed light on the heterogeneity of hematopoietic stem/progenitor cells (HSPCs), we analyzed the expression level of 96 lineage-specific genes in a single cell. While most of MEPs exclusively expressed erythroid- but not Meg-specific genes, a fraction of CMP cells (6.25%) showed distinct and exclusive expression pattern of platelet or Meg-specific genes. Among them were the cell surface molecules including CD41, the contents of alpha-granules of platelets and Meg-lineage specific transcription factors. Based on these data, we hypothesized that MegP might exist within the CD41+ CMP fraction.In fact, a fraction (7.9 ± 3.2 %) of CMPs expressed significant level of CD41 in FACS analysis, but none of the other CD34+ cells including hematopoietic stem cells (HSCs), MEPs and granulocyte-macrophage progenitors (GMPs) expressed CD41. Purified CD41+CMPs exhibited myeloblast-like immature morphological characteristics. Interestingly, cells with 4N-nuclei were exclusively found in CD41+CMPs (1%), indicating the initiation of endomitosis and polyploidization process has already taken place at this early stage of hematopoiesis.A conventional in vitro culture condition revealed that CD41+CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41-CMPs generated all types of myeloid colonies. To analyze Meg-lineage potential, we cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. The majority of single CD41+CMPs gave rise to pure megakaryocyte colonies.In addition, the novel immunodeficient mouse strain, which was recently developed by us and could support human Meg-lineage hematopoiesis much more than any strains available today, enabled us to evaluate in vivo differentiation potential of lineage-restricted progenitors. As a result, CD41+CMPs showed unipotent and robust Meg-lineage differentiation potential compared to MEPs.In order to elucidate gene expression profiling of CD41+CMPs more precisely, we performed cDNA microarray experiment. GSEA (Gene Set Enrichment Analysis) revealed that CD41+CMPs up-regulated Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulated erythroid and GM lineage-affiliated genes, clearly reflecting their lineage potentials.We also evaluated lineal relationship of these populations in vitro. While MEP did not give rise to CD41+CMPs, CD41-CMPs successfully generated MegPs, suggesting that CD41+CMPs could be the progeny of CD41-CMPs.Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs on per-cell-basis, indicating that this population is the major source of Megs rather than MEP.Finally, to evaluate the pathophysiological significance of MegPs in human diseases, we analyzed population frequencies of each stem/progenitor population in essential thrombocythemia (ET) patients' bone marrow. The MegP pool dramatically expanded in ET BM as compared to that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We also evaluated JAK2 mutation (V617F) burden in each stem/progenitor population by utilizing the digital PCR method with allele specific probes and the amplicon sequencing-based detection. As a result, JAK2 mutation is highly concentrated to MegP population in accordance with expansion of MegP, suggesting that this population should play an important role in pathogenesis of ET.In summary, we have prospectively identified the unipotent MegP population, which was endowed with robust Meg potential and emerged directly from CMPs bypassing MEPs. The newly-identified MegPs robustly contribute to physiological and pathological human megakaryopoiesis including ET. DisclosuresNo relevant conflicts of interest to declare.

Blvrb Mutation Induces Thrombocytosis Via Redox Dysregulation in the Heme Degradation Pathway

Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin (Tpo)/c-MPL liganding is dispensable for definitive thrombopoiesis establishing that fundamentally important modifier loci remain unelucidated. We completed RNASeq of highly-purified platelets from 7 essential thrombocythemia (ET) subjects [4 harbored the JAK2V617F mutation and 3 were genotypically normal] and 5 healthy controls, and developed an iterative algorithm to identify 33 non-synonymous SNVs that are putatively linked to the ET phenotype. Transcripts possessing these candidate SNVs were enriched on average in early-stage megakaryocyte/erythroid progenitors (MEPs), and became more restricted in terminally-differentiating megakaryocytes and erythroblasts. Genotypic studies using an expanded ET cohort (N = 36), followed by statistical association analyses using controls from the 1000 Human Genomes Project and an independently-genotyped cohort of healthy controls (N = 208), established that 5 SNVs (excluding JAK2V617F) were associated with ET. A single mutation (BLVRBS111L; p = 0.0006) retained its significance as a thrombocytosis risk allele using genotypic data from a secondary cohort with reactive (non-clonal) thrombocytosis (RT, N = 53), suggesting a function as an independent driver mutation of enhanced thrombopoiesis. TheBLVRB (biliverdin IXβ reductase) gene functions downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) within the heme degradation pathway to catalyze reduction of biliverdin (BV) tetrapyrrole(s) as an intermediary redox substrate in Bilirubin (BR) generation. Bacterially-expressed and purified recombinant BLVRBS111L showed defective enzymatic activity [compared to BLVRBWT (wild-type)] using flavin mononucleotide [flavin reductase (FR) activity; p <0.0001] and BV IXβ dimethyl esters (biliverdin reductase (BVR) activity; p <0.0001), the latter specifically generated by coupled heme oxidation as verdin-restricted BLVRB activity probes. The loss-of-mutation BLVRBS111L NAD(P)H-dependent redox coupling caused higher baseline ROS (reactive oxygen species) accumulation in lentivirus-transduced CD34+-derived induced pluripotent stem cells (iPSC/BLVRBS111L), results sharply contrasting with ROS neutralization in iPSC/BLVRBWT exposed to tert-butyl hydroperoxide (TBHP) as the oxidant stress source (p < 0.00001). Disparate redox coupling/ROS handling in genetically-modified primary CD34+ multipotential progenitor cultures established disproportionate expansion of primitive CFU-GEMMs in CD34+/BLVRBS111L (p = 0.001), and an absolute increase of BFU-E in CD34+/BLVRBWT (p = 0.001). Collagen-based progenitor cultures demonstrated a statistically-significant increase of CD41+ CFU-MKs in CD34+/BLVRBS111L cells (p <0.01) with no increased CFU-MKs in CD34+/BLVRBWT cells. Cumulative distribution plots of parallel Tpo-suspension cultures confirmed divergent ROS accumulation between MK/BLVRBWT and MK/BLVRBS111L, differences that were identifiable pre-terminal differentiation (Day 0, p = 0.0007), most pronounced at Day 5 corresponding to peak MK BLVRB expression across genotypes (p = 8 x 10-7), and persistent at terminal differentiation (Day 10; p = 0.05). Maximally disparate ROS accumulation (Day 5) corresponded to greatest size disparity and temporally-earlier (and sustained at Day 10) CD41 expression in MK/BLVRBS111L (p = 0.03). A bilineage (Tpo/Epo) differentiation model, designed to characterize erythroid/megakaryocyte (E/Meg) progenitor balance arising from common MEPs demonstrated no evidence for differential Mk (CD41+/Glycophorin A-) lineage balance, suggesting that BLVRBS111L ROS-promoting effects accelerate post-commitment expansion downstream of MEP lineage fate decisions. These data provide the first evidence linking redox coupling to MK lineage fate and expansion in humans, either via activity as a flavin reductase, in partnered electron exchange with an unidentified protein, or as a verdin-regulated redox coupler regulated by heme degradation and isomer-restricted BV IXβ (or IXδ, IXγ) generation; in principle, development of BLVRB-specific redox inhibitors represent innovative approaches to selectively alter a regulatory pathway controlling MK lineage expansion and human platelet counts. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor Bach1 and Bach2 Control Common Myeloid Progenitor Cell Differentiation Under Infectious Stimuli

Background: Erythrocyte and granulocyte/macrophage develop from common myeloid progenitor (CMP) (Akashi et al., 2000). Differentiation of hematopoietic progenitor cells is precisely controlled by multiple transcription factors, among which GATA1, C/EBPα, C/EBPβ and Spi-C play pivotal roles in erythrocyte and granulocyte/macrophage differentiation (Mancini et al., 2012; Pongubala et al., 2008; Hirai et al., 2006; Haldar et al., 2014). However, the mechanism by which the differentiation of CMP controlled under infectious condition has been unclear. Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements (Oyake et al., 1996). They promote B cell development by repressing the myeloid genes such as Cebpb and Spic in common lymphoid progenitor cells (Itoh-Nakadai et al., 2014). In addition, Bach1 regulates several target genes related to iron/heme homeostasis such as globin genes and hemeoxygenase-1, and Bach2 may similarly regulate these genes (Igarashi, 2014). Therefore, it is expected that both Bach1 and Bach2 play redundant roles in erythropoiesis. To figure out their roles in erythroid and myeloid cell differentiation, we performed hematological and transcriptomics analyses using Bach1-/- Bach2-/- (double-deficient; DD) mice.Methods: The generation of DD mice on the C57BL/6J background and Bach2 reporter mice with red fluorescent protein coding cDNA inserted in the Bach2 locus were described previously (Itoh-Nakadai et al., 2014). Mice between 8-12 weeks old were analyzed in the present study. Bone marrow (BM) cells were stained with specific combinations of antibodies to identify erythroid/myeloid progenitor and mature cells (Sheila et al., 2008; Cornelis et al., 2007; Socolovsky et al., 2001). Flow cytometry analysis and cell sorting were performed by using FACSAriaⅡ(BD) and FlowJo software (TreeStar). For infectious simulation of CMP, sorted CMPs were incubated with 1μg/ml LPS (Sigma) for 48h and RNA was purified with RNeasy micro kit (Qiagen). Quantitative PCR by using SuperscriptⅢ reverse transcriptase (Invitrogen) and Light Cycler system (Roche) was performed according to manufacturer's instructions. Microarray analysis by using Sure-Print G3 mouse GE microarray slide (Agilent) was performed as previously described (Itoh-Nakadai et al., 2014) and the results were analyzed by using GeneSpring software (Agilent). We used Gene Set Enrichment Analysis (GSEA) to interpret gene expression data (Subramanian et al., 2005; Mootha et al., 2003). LPS stimulation (50 μg/body) of mice was performed as previously described (Ryan et al., 2008). Data were analyzed by the two-sided Student's t-test and p - values of <0.05 were considered statistically significant.Results: DD mice show mild normocytic anemia compered to wild-type (WT), Bach1-/-, and Bach2-/- mice (hemoglobin; 14.4±0.2, 14.0±0.3, 13.5±0.3 and 11.9±0.7 g/dl, for WT, Bach1-/-, Bach2-/- and DD, respectively, p<0.05 for comparison between DD and other genotypes, n=7). Immature and mature erythroblast populations were significantly decreased in BM of DD (immature; 25.8±1.78, 15.6±1.4, mature; 27.6±3.3, 17.4±2.3×106/body for WT and DD, respectively, p<0.05, n=6). Megakaryocyte-erythroid progenitor (MEP)/granulocyte-monocyte progenitor (GMP) ratio was significantly decreased in BM of DD (MEP/GMP: 0.13±0.01, 0.07±0.01 for WT and DD, respectively, p<0.05, n=5). Bach2 expression was detected in CMP, MEP and even GMP by using Bach2-RFP mice. LPS stimulation of WT CMP significantly decreased mRNA levels of Bach1, Bach2 and Gata1. On the other hand, Cebpb and Spic mRNA levels were significantly increased. LPS stimulation of WT mice induced significant increase of granulocyte and decrease of erythrocyte and B lymphocyte in BM, which was consistent with previous reports. It was also shown that LPS stimulation significantly decreased MEP/ GMP ratio. According to the clustering analysis of the microarray data of CMP sorted from WT and DD mice, they showed clearly different expression profiles. GSEA showed that CMP of DD skewed to myeloid cell lineage and lost the erythroid gene expression compared to WT.Conclusions: Bach1 and Bach2 control the differentiation of CMP to erythroid cell or myeloid cell by repressing myeloid genes such as Cebpb and Spic. Infectious stimuli may promote myeloid cell differentiation by reducing the expression of Bach1 and Bach2 in CMP. DisclosuresFujiwara:Chugai Pharmaceutical CO., LTD: Research Funding. Harigae:Chugai Pharmaceutical CO., LTD: Research Funding.

Megakaryocytic Fate Specification and Maturation

Megakaryocyte maturation and polyploidization are essential for effective platelet formation. In a tightly controlled process, bipotent megakaryocyte-erythroid progenitors (MEP) differentiate into diploid megakaryoblasts; undergo a progressive increase in ploidy, resulting in very large cells with multilobated polyploid nuclei; and undergo extensive cytoplasmic maturation in preparation for proplatelet release. Recent studies have begun to unravel the complex genetic and epigenetic changes that promote megakaryocyte specification from hematopoietic stem and progenitor cells, and that control subsequent megakaryocyte polyploidization and maturation. The transcriptional regulatory network underlying specification from MEP is emerging, with data revealing that many of the same factors that regulate specification also play critical roles in the final stages of megakaryocyte maturation, whereas other factors are critical for specification, but must be then inactivated in order for cell maturation to occur. Megakaryopoiesis is disrupted in several malignant and non-malignant disorders that result in significant morbidity and mortality. Mutations are inherited or acquired, and affect megakaryocyte specification, maturation and subsequent platelet formation, or both. Novel, findings will be covered in the context of normal transcriptional regulation, potential mechanisms of megakaryocyte dysplasia in MDS, and transformation to acute megakaryoblastic leukemia. These studies build the basis for our understanding of inherited and acquired megakaryocytic abnormalities and thrombocytopenias. Disclosures No relevant conflicts of interest to declare.

Members of the Mir-99/100~125 Tricistrons Cooperatively Induce a Pre-Leukemic Myeloproliferative Disorder

MicroRNAs (miRNAs) reflect the best studied class of regulatory non-coding RNAs (ncRNAs), which control genetic networks with key cellular functions. In vertebrate genomes, a significant number of miRNA genes are located closely adjacent to each other in miRNA polycistrons. The mature miRNAs of the three human miR-99/100~125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are processed from one single transcript and are highly expressed in acute promyelocytic leukemia (APL). Expression profiling by qPCR in sorted murine hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), megakaryocytic erythroid progenitors (MEPs) and granulocytic monocytic progenitors (GMPs) revealed high expression levels of miR-99/100 and miR-125 family members in HSCs and CMPs. However, the consequences of the coordinated expression of the miRNAs belonging to different seed families on self-renewal and proliferation of HSCs and myeloid progenitors and their contribution to the pathogenesis of APL are not well understood.To elucidate the genetic interactive network of miR-99/100~125 miRNAs and the role of each individual miRNA within this network, we generated a set of eight different constructs covering any permutation of miRNA family members from the two miR-99/100~125 clusters on hsa11 and hsa21 (miR-99a, miR-125b-2, let-7c, miR-99a/let-7c, miR-100/miR-125b-1, let-7a-2/miR-125b-1, miR-100/let-7a-2/miR-125b-1 and miR-99a/let-7c/miR-125b-2). Lentiviral overexpression of these constructs in human hematopoietic stem and progenitor cells (HSPCs) resulted in a significant reduction of monocytic/macrophage colony-forming units (CFU-M; 2.2-2.8-fold, p≤0.05) and granulocytic CFU-Gs (2-4.3-fold, p≤0.05) in methylcellulose-based CFU assays exclusively for miR-125-containing bi-/tricistronic constructs (miR-100/miR-125b-1, let-7a-2/miR-125b-1, miR-100/let-7a-2/miR-125b-1 and miR-99a/let-7c/miR-125b-2), but not for the single miRNA expression constructs. Accordingly, during myelomonocytic differentiation HSPCs transduced with those miR-125-containing bi-/tricistronic constructs gave rise to a major population of monomorphic, non-adherent cells devoid of granulocytic and monocytic markers, which was not present in single miRNA-transduced cells. In murine isogenic transplantation experiments (N=105), only the combined miRNA expression of miR-125b with let-7 and/or miR-99/100 led to the expansion and retention of immature Gr-1(low)/Mac-1(+)/B220(-) cells in the bone marrow (1.6-1.8fold; p≤0.01). Accordingly, either the CMP or GMP compartment of transplanted mice was expanded in miR-125-containing bi-/tricistronic constructs (CMPs 1.6-fold in let-7a-2/miR-125b-1, GMPs 1.8-1.9-fold in miR-100/miR-125b-1 and tricistrons; p≤0.01;), but not upon single miRNA overexpression (1.1-1.3-fold; p≥0.1).Global gene expression profiling of human HSPCs transduced with the eight miRNA constructs revealed a core expression signature commonly regulated by the four miR-125b-containing bi-/ tricistronic constructs (367 genes upregulated [>1.5-fold]; 417 genes downregulated). Strikingly, this core signature is enriched for genes with concordant expression in leukemic stem cells (LSCs) and HSCs (FDR q≤1.8x10-14). The genes of the core signature were not or only modestly affected in the context of the single miRNAs.Thus, the miR-99/100~125 tricistron miRNAs form an interaction network, wherein the combined activity of miR-125b with let-7 and/or miR-99/100 family members converged to induce a stem cell signature creating a synthetic phenotype. The synthetic phenotype can only be observed in the combination of two or all three miRNAs but not for each miRNA alone, and is generated by miR-99/100 and/or let-7 altering the hematologically dominant miR-125 phenotype. This interactive network might explain the genomic miR-99/100~125 clustering and reveals a novel cooperative mode to induce self-renewal and a differentiation block in myeloid progenitor cells, predisposing them to leukemic transformation in APL. DisclosuresNo relevant conflicts of interest to declare.