Pathological Hematopoiesis Research Articles

Abstract P128: Endothelial Nitric Oxide Determines Hematopoietic Cell Fate and Osteoclastogenesis By Modulating CSF1 During Hypertension

Introduction: Loss of endothelial nitric oxide (NO) is common in hypertension (HTN). The bone marrow niche contains endothelial cells; however, how endothelial NO affects hematopoiesis and bone in HTN remains undefined. Hypothesis: We hypothesized that NO loss in HTN drives hematopoiesis in the bone marrow, later favoring osteoclastogenesis and bone resorption. Methods and Results: By microcomputed tomography, eNOS deficient mice (eNOS -/- ) had reduced femoral cortical area (Figure 1A) and trabecular bone volume fraction (Figure 1B) compared to wildtype mice (WT). Flow cytometry showed that granulocyte-monocyte (Figure 1C) and common lymphoid progenitors (Figure 1D) were markedly higher in eNOS -/- than in WT. mRNA analysis revealed loss of endothelial NO increased expression of cathepsin K (Ctsk), colony stimulating factor-1 receptor (CSF1R), and tartrate resistant acid phosphatase (TRAP) in the bone marrow. We exposed murine bone-marrow endothelial cells (BMECs) to 5% (normotension), 5% with L-NAME, or 10% (HTN) cyclic strain. Compared to 5% strain, BMECs exposed to L-NAME or 10% strain exhibited markedly increased CSF1 production, an essential cytokine for myelopoiesis and osteoclastogenesis (Figure 1E and F). In coculture experiments, we also found that macrophages cultured with BMECs undergoing 5% strain with L-NAME or 10% strain exhibited higher expression of Ctsk, CSF1R, and TRAP, indicating osteoclast formation. Conclusion: Endothelial NO likely inhibits osteoclastogenesis and bone resorption. The loss of NO through mechanical stretch or inhibiting eNOS increases CSF1 production and hematopoiesis. This shows a potential mechanism by which NO loss leads to pathological hematopoiesis and bone resorption during HTN.

Expression of microRNA-155 in thalassemic erythropoiesis.

Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in β-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with β-thalassemia/HbE. However, the expression and function of miR-155, especially in β-thalassemia, have not yet been explored. To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from β IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in β-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in β-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in β-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. miR-155 regulates IE in β-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Analysis of 7 cases of pediatric acute myeloid leukemia with DEK-NUP214 fusion gene

Objective: To investigate the clinical features, treatment regime, and outcome of pediatric acute myeloid leukemia (AML) with DEK-NUP214 fusion gene. Methods: The clinical data, genetic and molecular results, treatment process and survival status of 7 cases of DEK-NUP214 fusion gene positive AML children admitted to the Pediatric Blood Diseases Center of Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from May 2015 to February 2022 were analyzed retrospectively. Results: DEK-NUP214 fusion gene positive AML accounted for 1.02% (7/683) of pediatric AML diagnosed in the same period, with 4 males and 3 females. The age of disease onset was 8.2 (7.5, 9.5) years. The blast percentage in bone marrow was 0.275 (0.225, 0.480), and 6 cases were M5 by FAB classification. Pathological hematopoiesis was observed in all cases except for one whose bone marrow morphology was unknown. Three cases carried FLT3-ITD mutations, 4 cases carried NRAS mutations, and 2 cases carried KRAS mutations. After diagnosis, 4 cases received IAE induction regimen (idarubicin, cytarabine and etoposide), 1 case received MAE induction regimen (mitoxantrone, cytarabine and etoposide), 1 case received DAH induction regimen (daunorubicin, cytarabine and homoharringtonine) and 1 case received DAE induction regimen (daunorubicin, cytarabine and etoposide). Complete remission was achieved in 3 cases after one course of induction. Four cases who did not achieved complete remission received CAG (aclarubicin, cytarabine and granulocyte colony-stimulating factor), IAH (idarubicin, cytarabine and homoharringtonine), CAG combined with cladribine, and HAG (homoharringtonine, cytarabine and granulocyte colony-stimulating factor) combined with cladribine reinduction therapy, respectively, all 4 cases reached complete remission. Six patients received hematopoietic stem cell transplantation (HSCT) after 1-2 sessions of intensive consolidation treatment, except that one case was lost to follow-up after complete remission. The time from diagnosis to HSCT was 143 (121, 174) days. Before HSCT, one case was positive for flow cytometry minimal residual disease and 3 cases were positive for DEK-NUP214 fusion gene. Three cases accepted haploid donors, 2 cases accepted unrelated cord blood donors, and 1 case accepted matched sibling donor. The follow-up time was 20.4 (12.9, 53.1) months, the overall survival and event free survival rates were all 100%. Conclusions: Pediatric AML with DEK-NUP214 fusion gene is a unique and rare subtype, often diagnosed in relatively older children. The disease is characterized with a low blast percentage in bone marrow, significant pathological hematopoiesis and a high mutation rate in FLT3-ITD and RAS genes. Low remission rate by chemotherapy only and very high recurrence rate indicate its high malignancy and poor prognosis. Early HSCT after the first complete remission can improve its prognosis.

ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis.

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.

The immunological role of mesenchymal stromal cells in patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a common hematological malignant disease, characterized by malignant hematopoietic stem cell proliferation in the bone marrow (BM); clinically, it mainly manifests clinically mainly by as pathological hematopoiesis, hemocytopenia, and high-risk transformation to acute leukemia. Several studies have shown that the BM microenvironment plays a critical role in the progression of MDS. In this study, we specifically evaluated mesenchymal stromal cells (MSCs) that exert immunomodulatory effects in the BM microenvironment. This immunomodulatory effect occurs through direct cell-cell contact and the secretion of soluble cytokines or micro vesicles. Several researchers have compared MSCs derived from healthy donors to low-risk MDS-associated bone mesenchymal stem cells (BM-MSCs) and have found no significant abnormalities in the MDS-MSC phenotype; however, these cells have been observed to exhibit altered function, including a decline in osteoblastic function. This altered function may promote MDS progression. In patients with MDS, especially high-risk patients, MSCs in the BM microenvironment regulate immune cell function, such as that of T cells, B cells, natural killer cells, dendritic cells, neutrophils, myeloid-derived suppressor cells (MDSCs), macrophages, and Treg cells, thereby enabling MDS-associated malignant cells to evade immune cell surveillance. Alterations in MDS-MSC function include genomic instability, microRNA production, histone modification, DNA methylation, and abnormal signal transduction and cytokine secretion.

G-CSF/GM-CSF-induced hematopoietic dysregulation in the progression of solid tumors.

There are two types of abnormal hematopoiesis in solid tumor occurrence and treatment: pathological hematopoiesis, and myelosuppression induced by radiotherapy and chemotherapy. In this review we primarily focus on the abnormal pathological hematopoietic differentiation in cancer induced by tumor‐released granulocyte colony‐stimulating factor (G‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). As key factors in hematopoietic development, G‐CSF/GM‐CSF are well‐known facilitators of myelopoiesis and mobilization of hematopoietic stem cells (HSCs). In addition, these two cytokines can also promote or inhibit tumors, dependent on tumor type. In multiple cancer types, hematopoiesis is greatly enhanced and abnormal lineage differentiation is induced by these two cytokines. Here, dysregulated hematopoiesis induced by G‐CSF/GM‐CSF in solid tumors and its mechanism are summarized, and the prognostic value of G‐CSF/GM‐CSF‐associated dysregulated hematopoiesis for tumor metastasis is also briefly highlighted.

Progress in diagnostic staging of myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are a group of acquired heterogeneous clonal disorders that originate from hematopoietic stem cells. It is characterized by a reduction in one or more blood lines in the myeloid system with abnormal development, pathological hematopoiesis and a high risk of transformation to acute myeloid leukemia (AML). In 2016, the World Health Organization (WHO) revised the diagnostic staging criteria for MDS, but with the development of next-generation sequencing (NGS) technology, the diagnostic staging of MDS has opened a new era of accurate diagnosis and individualized treatment. In this paper, we review the diagnostic typing of MDS.

Multistage hematopoietic stem cell regulation in the mouse: A combined biological and mathematical approach

SummaryWe have reconciled steady-state and stress hematopoiesis in a single mathematical model based on murine in vivo experiments and with a focus on hematopoietic stem and progenitor cells. A phenylhydrazine stress was first applied to mice. A reduced cell number in each progenitor compartment was evidenced during the next 7 days through a drastic level of differentiation without proliferation, followed by a huge proliferative response in all compartments including long-term hematopoietic stem cells, before a return to normal levels. Data analysis led to the addition to the 6-compartment model, of time-dependent regulation that depended indirectly on the compartment sizes. The resulting model was finely calibrated using a stochastic optimization algorithm and could reproduce biological data in silico when applied to different stress conditions (bleeding, chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependent model of immature hematopoiesis provides new avenues to a better understanding of both normal and pathological hematopoiesis.

Single-Cell Methylome and Transcriptome High-Resolution Maps Define the Effects of Epigenetic Modifiers on the Landscape of Hematopoietic Differentiation

Epigenetic modifications are critical determinants of cell identity and play a central role in normal hematopoietic differentiation. Moreover, mutations in epigenetic modifiers including TET2 and IDH2 mutations, which result in a decreased ability to de-methylate DNA, are frequent in myeloid leukemias and clonal hematopoiesis of indeterminate potential. This raises a central question in normal and pathologic hematopoiesis - how do epigenetic modifiers shape the differentiation landscape?To address this question, we applied high-throughput single-cell RNA-seq (scRNAseq) to reconstruct high-resolution maps of hematopoietic differentiation, with and without disruption of central epigenetic modifiers (Tet2 and Idh2). Briefly, FACS-sorted lineage-negative bone marrow cells were isolated from sixteen-week old Mx1 -cre wildtype (Wt, n=6), Mx1 -cre Tet2fl/fl (n= 6) or Mx1 -cre Idh2R140Q/Wt (n=3) mice, 4 weeks following recombination. In total, 45,100 cells underwent droplet-based scRNAseq. Clustering of Wt cells identified 25 distinct progenitor populations, recapitulating previous reports and providing a map of normal mouse hematopoietic differentiation.Tet2knockout (KO) resulted in a 1.5-fold increase in uncommitted cluster HSC4 (Flt3hi, c-Kithi, Cd34low) (Figure A-B), consistent with a potential differentiation block resulting from Tet2-KO. Notably, the HSC differentiation block did not uniformly affect differentiation into committed progenitors, but resulted in a 2-fold decrease in erythroid precursors and a 1.5-fold increase in monocyte precursors. These findings were evident by scRNA-seq four months before this phenotype was observed by flow cytometry. While the expression profiles of these committed erythroid and monocytic progenitors were not different from Wt progenitors, the degree of priming to these fates within uncommitted Tet2-KO HSCs (HSC clusters 1-4) showed reduced priming towards the erythroid fate and increased priming towards the monocytic fate (Figure C). In contrast, mutant Idh2 resulted in different skews of hematopoiesis than Tet2-KO, including an increase in neutrophil precursors, while there was no significant change in the frequency of erythroid precursors (Figure D). These data demonstrate that Tet2 and Idh2 mutations, both of which disrupt de-methylation, have distinct effects on hematopoietic differentiation.To explore the link between aberrant DNA methylation upon Tet2 - KO and the observed differentiation skews, we performed joint single-cell methylation and RNAseq, as well as ATAC-bisulfite sequencing. As expected, Tet2-KO resulted in higher global methylation. However, the increase in methylation was stochastically distributed across the genome, resulting in higher methylation heterogeneity (Figure E), and associated with higher cell-to-cell variation in transcriptional profiles. Importantly, we found that stochastic methylation changes result in deterministic differentiation skews due to differential sensitivity to methylation of binding motifs of lineage-defining transcription factors (TFs). For instance, motifs of erythroid-related TFs (including Nrf1, E2f4 and E2f1), but not monocytic-related TFs, showed a marked enrichment of CpGs and methylation-sensitive binding (Figure F).These data demonstrate the ability of high-resolution single-cell mapping of hematopoiesis to identify the exact point of disruption of HSC differentiation due to alterations in epigenetic modifiers. Specifically, these data show that Tet2-KO results in reduced erythroid but increased monocytic priming of HSCs, leading to skewed differentiation. Moreover, single-cell epigenomic analyses identified that while the hypermethylation effect of Tet2-KO is distributed widely across the genome, the skews in lineage priming result from biases in the sensitivity of lineage-defining TFs to CpG methylation at their motifs. The skews may therefore arise not from a change in methylation of a particular locus, but rather through broad lack of demethylation in targets of erythroid priming TFs, resulting from inherent enrichment of these motifs in CpGs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood.Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time.CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors.To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Combined Biological and Modeling Approach of Hematopoiesis: From Native to Stressed Erythropoiesis

We developed here a new mathematical modelling approach which reconciliates native and stress hematopoiesis, focused on hematopoietic stem and progenitor compartments. We first proposed an erythropoiesis model leaning on a minimum of 6 cell-amplification compartments, able to reproduce native erythropoiesis. A phenylhydrazine-induced hemolytic stress was next applied, and in vivo data were used to estimate model parameters through an optimized algorithm and integrated regulatory processes. A reduction of all stem cell compartments was observed, due to a drastic differentiation without proliferation during 7 days, followed by a huge proliferation in all compartments including long-term hematopoietic stem cells, before returning to normal values. The 6-compartment model applied to stress erythropoiesis was complexified by integrating regulatory processes. Thus, regulation of hematopoiesis appears dispensable during native hematopoiesis but mandatory for stress hematopoiesis. In conclusion this multi-step and time-dependent model of immature hematopoiesis opens new opportunities to understand development of normal or pathological hematopoiesis.

Single-cell transcriptomic landscape of human blood cells.

High throughput single-cell RNA-seq has been successfully implemented to dissect the cellular and molecular features underlying hematopoiesis. However, an elaborate and comprehensive transcriptome reference of the whole blood system is lacking. Here, we profiled the transcriptomes of 7551 human blood cells representing 32 immunophenotypic cell types, including hematopoietic stem cells, progenitors and mature blood cells derived from 21 healthy donors. With high sequencing depth and coverage, we constructed a single-cell transcriptional atlas of blood cells (ABC) on the basis of both protein-coding genes and long noncoding RNAs (lncRNAs), and showed a high consistence between them. Notably, putative lncRNAs and transcription factors regulating hematopoietic cell differentiation were identified. While common transcription factor regulatory networks were activated in neutrophils and monocytes, lymphoid cells dramatically changed their regulatory networks during differentiation. Furthermore, we showed a subset of nucleated erythrocytes actively expressing immune signals, suggesting the existence of erythroid precursors with immune functions. Finally, a web portal offering transcriptome browsing and blood cell type prediction has been established. Thus, our work provides a transcriptional map of human blood cells at single-cell resolution, thereby offering a comprehensive reference for the exploration of physiological and pathological hematopoiesis.

Leukemia risk and rate of radiation dose accumulation. Part 2: Comparative analysis of leukocytic indices and dynamics of peripheral blood values in relation to external gamma-exposure dose

The results of a retrospective analysis of leukocytic indices and dynamics of peripheral blood values in relation to external gamma-exposure dose among the personnel of the first atomic production facility in Russia were presented. The study was performed on the basis of the database “Leukemia in the cohort of workers of the Mayak Production Association employed in 1948-1958”. The database contains hematological, clinical and dosimetry information on the two groups of workers: the study group includes individuals with leukemia as the cause of death (n=84); control group includes personnel without hematological cancer pathology (n=300). The control group was composed by selecting 3-4 internal control cases for each leukemia case taking into account gender, age of the start of the exposure and the same accumulated absorbed dose of gamma-exposure to red bone marrow. Based on 19592 analysis of peripheral blood we had described hematopoiesis shifts along cell lines, performed correlation analysis of interrelation between hemogram values and radiation dose, analyzed particular characteristics of blast cells’ appearance in peripheral blood flow in the period prior to leukemias. Comparative analysis of various leukocytic indices was performed among Mayak PA personnel for the first time. The diverse nature of the response of bone marrow hematopoiesis was noted; more pronounced hematological shifts were indicated regarding more intensive production exposure. A number of hematopoietic features were detected among people exposed to production radiation: 1) cytopenia in relation to minimum accumulated doses of radiation exposure in both studied groups; 2) more pronounced lability of hemograms in relation to increased accumulation dose of external gamma-exposure and period of radiation exposure among individuals who had developed leukemia in future in comparison to controls; 3) correlation between peripheral blood cells and accumulated doses of external gamma-exposure was characterized by the highest values of coefficients among individuals with future leukemias; 4) myeloblasts were most often registered among all the blast cells; proerythroblasts were characterized by the shortest average period from the start of the exposure and to the moment of their appearance in the peripheral blood flow (178 days in the study group); polychromatophile erythroblasts in peripheral blood were registered only among the workers with future leukemia; 5) when comparing leukocytic indices in proportion to the categories of accumulated doses of external gamma-exposure, a pronounced variability in the ratio of blood cells was observed among exposed individuals who were subsequently diagnosed with leukemia, especially in the range of 2.0-2.5 Gy. Thus, a comprehensive analysis of hematological parameters, including the assessment of leukocytic indices, is more informative than studying a standard hemogram. Particular features of the dynamics of the cellular composition of peripheral blood can be used as indicators of pathological hematopoiesis in exposed workers prior to clinical manifestation of leukemia.

Deregulated NKL Homeobox Genes in B-Cell Lymphoma.

Recently, we have described physiological expression patterns of NKL homeobox genes in early hematopoiesis and in subsequent lymphopoiesis. We identified nine genes which constitute the so-called NKL-code. Aberrant overexpression of code-members or ectopically activated non-code NKL homeobox genes are described in T-cell leukemia and in T- and B-cell lymphoma, highlighting their oncogenic role in lymphoid malignancies. Here, we introduce the NKL-code in normal hematopoiesis and focus on deregulated NKL homeobox genes in B-cell lymphoma, including HLX, MSX1 and NKX2-2 in Hodgkin lymphoma; HLX, NKX2-1 and NKX6-3 in diffuse large B-cell lymphoma; and NKX2-3 in splenic marginal zone lymphoma. Thus, the roles of various members of the NKL homeobox gene subclass are considered in normal and pathological hematopoiesis in detail.

An R-Derived FlowSOM Process to Analyze Unsupervised Clustering of Normal and Malignant Human Bone Marrow Classical Flow Cytometry Data.

Multiparameter flow cytometry (MFC) is a powerful and versatile tool to accurately analyze cell subsets, notably to explore normal and pathological hematopoiesis. Yet, mostly supervised subjective strategies are used to identify cell subsets in this complex tissue. In the past few years, the implementation of mass cytometry and the big data generated have led to a blossoming of new software solutions. Their application to classical MFC in hematology is however still seldom reported. Here, we show how one of these new tools, the FlowSOM R solution, can be applied, together with the Kaluza® software, to a new delineation of hematopoietic subsets in normal human bone marrow (BM). We thus combined the unsupervised discrimination of cell subsets provided by FlowSOM and their expert-driven node-by-node assignment to known or new hematopoietic subsets. We also show how this new tool could modify the MFC exploration of hematological malignancies both at diagnosis (Dg) and follow-up (FU). This can be achieved by direct comparison of merged listmodes of reference normal BM, Dg, and FU samples of a representative acute myeloblastic case tested with the same immunophenotyping panel. This provides an immediate unsupervised evaluation of minimal residual disease. © 2019 International Society for Advancement of Cytometry.

A20 deficiency in hematopoietic stem cells causes lymphopenia and myeloproliferation due to elevated Interferon-\u03b3 signals

Inflammation and inflammatory cytokines have been shown to exert both positive and negative effects on hematopoietic stem cells (HSCs) and hematopoiesis. While the significance of inflammation driven hematopoiesis has begun to unfold, molecular players that regulate this phenomenon remain largely unknown. In the present study, we identified A20 as a critical regulator of inflammation controlled hematopoietic cell fate decisions of HSCs. A20 deficiency in HSCs leads to increased differentiation of myeloid cells and myeloproliferation. Analysis of erythroid lineage cells of A20 deficient mice indicated a striking reduction of erythrocytes in the bone marrow (BM), but elevated numbers in the spleen. Loss of A20 in HSCs causes a severe blockade of B cell differentiation in the BM and absence of peripheral B cells in the spleen, liver and blood. T cell differentiation studies revealed a reduction of both T cell progenitors and differentiated T cells in the thymus and altered T cell numbers in the spleens of A20 mutant mice. Analysis of lineage committed progenitors of the myeloid, erythroid and lymphoid lineages specified an altered composition in the A20 deficient BM. Genetic studies identified that specific loss of A20 in the myeloid lineage cells results in myeloproliferation. Bone marrow transplantation studies and mixed bone marrow chimera studies suggested an involvement of inflammatory cytokines, particularly interferon (IFN)- γ, in the onset of myeloproliferation and lymphopenia of A20 deficient mice. Finally, ablation of IFNγ signals in A20 deficient mice rescued the hematopoietic defects. In essence, these studies highlight a previously unknown role for A20 in the restriction of inflammation driven pathologic hematopoiesis. We believe that our studies based on A20 mutant mice will be helpful in understanding the pathophysiology and in the treatment of patients with A20 (TNFAIP3) mutations.

Pericytes in Bone Marrow.

Bone marrow environments are composed of multiple cell types, most of which are thought to be derived from mesenchymal stem cells. In mouse bone marrow, stromal cells with CD45- Tie2- CD90- CD51+ CD105+ phenotype, Nestin-GFP+, CXCL12-abundant reticular (CAR) cells, PDGFRα+ Sca-1+ or CD51+ PDGFRα+, and Prx-1-derived CD45- Ter119- PDGFRα+ Sca-1+ populations select for MSC activity. There is evidence that these stromal cell populations display some significant overlap with each other and comprise important cellular constituents of the hematopoietic stem cell niche. Moreover, these mesenchymal cell populations share characteristics in their location as they all are found around bone marrow vessels (can be called "pericytes"). In this chapter, with reviewing the recent literatures, how the pericytes relate to physiological and pathological hematopoiesis is argued.

Clinical Charcteristics and Prognostic Analysis of 28 cases of Pediatric Myelodysplastic Syndrome

To analyze the clinical features and prognosis of 28 children with myelodysplastic syndrome (MDS) and to screen the high risk factors affecting the prognosis so as to provide the new ideas for standard of clinical diagnosis and therapy. The clinical data of 28 children with newly diagnosed MDS treated in our hospital from March 1994 to July 2016 were analyzed retrospectively, the features of disease onset and the results of laboratory examination were summarized, all MDS children were followed up, the prognosis and the high risk factors affecting the prognosis were evaluated. In all 28 MDS children, the ratio of male to female was 1.8∶1, the incidence of MDS was observed in boys, while the low incidence of MDS was found in older children. The clinical manifestations were mainly the decrease of three series blood cells in 16 cases (57.14%), other cases presented simple anemia (7.1%), simple thrombocytopenia (7.1%), neutropenia with anemia (14.29%), and anemia with thrombocytopenia (14.28%).The bone marrow image showed mainly hyperplasia (82.14%), and the pathological hematopoiesis, moreover the manifistation of pathological hematopoiesis was different in forma and degree; the bone marrow biopsy showed the typical abnormal localization of immature precursor(ALIP) accepted for 33.33%; the chromosome karyotype detection showed the detected rate of chronosome abnormality was 41.18%. The median follow-up time was 1.75 years. 5 children with MDS received the hematopoietic stem cell transplantation (HSCT), among them 1 dead and 4 maintained CCR; Out of other 23 patients no-received HSCT, 7 cases given up treatment after confirmed diagnosis, 16 cases received the chemotherapy (2 cases given up treatment after CR, 5 cases transformed into AML, 3 cases relapsed, 3 cases maintained CCR), 11 cases dead, 9 cases failed to be followed up. The 5-years OS rate and EFS rate in all patients were predicted as (38.2±11.3)% and (35.3±11.3)%,respectively, among them, the OS and EFS rates of patients received the HSCT allo superior to those of patients did not received HSCT ［(80.0±17.9）% vs.(22.8±11.5）%］ (P<0.05) and ［(80.0±17.9)% vs (17.5±11.1)%］(P<0.05). Analysis showed that in addition to receiving the HSCT(P<0.05), platelet decrease in peripheral blood(P<0.01), the age, sex, existance of micromegakaryocytes in bone marrow and progressive MDS or no influenced not on the prognosis(P>0.05). The children MDS is rare and easy to be misdiagnosis, moreover displays more high heterogeneity and poor prognosis, thereby the early diagnosis is crucial, in addition, the system of prognosis evaluation is imperative to be perfected. The HSCT may be the effective method for curative treatment of childhood MDS.

The master regulator FUBP1: its emerging role in normal cell function and malignant development.

The human Far Upstream Element(FUSE)Binding Protein 1 (FUBP1) is a multifunctional DNA- and RNA-binding protein involved in diverse cellular processes. FUBP1 is a master regulator of transcription, translation, and RNA splicing. FUBP1 has been identified as a potent pro-proliferative and anti-apoptotic factor by modulation of complex networks. FUBP1 is also described either as an oncoprotein or a tumor suppressor. Especially, FUBP1 overexpression is observed in a growing number of cancer and leads to a deregulation of targets that includes the fine-tuned MYC oncogene. Moreover, recent loss-of-function analyses of FUBP1 establish its essential functions in hematopoietic stemcell maintenance and survival. Therefore, FUBP1 appears as an emerging suspect in hematologic disorders in addition to solid tumors. The scope of the present review is to describe the advances in our understanding of the molecular basis of FUBP1 functions in normal cells and carcinogenesis. We also delineate the recent progresses in the understanding of the master role of FUBP1 in normal and pathological hematopoiesis. We conclude that FUBP1 is not only worth studying biologically but is also of clinical relevance through its pivotal role in regulating multiple cellular processes and its involvement in oncogenesis.

Bloody Zebrafish: Novel Methods in Normal and Malignant Hematopoiesis.

Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebrafish is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identified in genetic and chemical screens using the zebrafish model. Moreover, zebrafish enables in vivo imaging of hematopoietic stem cell generation and differentiation during embryogenesis, and adulthood. Finally, zebrafish has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebrafish model has proven beneficial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the field.