Erythroblast Differentiation Research Articles

Impaired Terminal Erythroid Maturation in β0-Thalassemia/HbE Patients with Different Clinical Severity.

Anemia in β-thalassemia is associated with ineffective erythropoiesis and a shortened lifespan of erythroid cells. The limited differentiation of β-thalassemic erythroblasts has been documented, but the characteristic feature of terminal erythroid maturation and its physiological relevance are not clearly described in β-thalassemias. Here, the red blood cell and reticulocyte cellular characteristics were determined in patients with β0-thalassemia/HbE in comparison to patients with iron deficiency anemia and healthy normal subjects. Severely affected β0-thalassemia/HbE patients showed the highest increase in immature reticulocytes, but the number of total erythrocytes was the lowest. Despite similar ranges of hemoglobin levels, β0-thalassemia/HbE patients had a higher number of reticulocytes and a greater proportion of immature fraction than patients with iron deficiency anemia did. In vitro CD34+ hematopoietic progenitor cells’ culture and flow cytometry analysis were conducted to investigate the erythroid maturation and mitochondrial clearance in β0-thalassemia/HbE erythroid cells as compared to normal cells. The delayed erythroid maturation and evidence of impaired mitochondria clearance were observed in β0-thalassemia/HbE cells at the terminal stage of differentiation. Additionally, increased transcript levels of genes related to erythroid mitophagy, BNIP3L and PINK1, were revealed in β0-thalassemia/HbE erythroblasts. The findings indicate that the erythroid maturation is physiologically relevant, and that the restoration of terminal maturation represents a potential therapeutic target for β-thalassemias.

Pan-Cancer Analysis Based on EPOR Expression With Potential Value in Prognosis and Tumor Immunity in 33 Tumors.

BackgroundErythropoietin receptor (EPOR), a member of the cytokine class I receptor family, mediates erythropoietin (EPO)-induced erythroblast proliferation and differentiation, but its significance goes beyond that. The expression and prognosis of EPOR in cancer remain unclear.MethodsThis study intended to perform a pan-cancer analysis of EPOR by bioinformatics methods. Several databases such as GTEx, TCGA, CCLE, and others were used to explore the overall situation of EPOR expression, and the correlation of EPOR expression with prognosis, microRNAs (miRNAs), immune infiltration, tumor microenvironment, immune checkpoint genes, chemokines, tumor mutation burden (TMB), microsatellite instability (MSI), methyltransferases, and DNA mismatch repair (MMR) genes in 33 tumors was analyzed. In addition, we compared the promoter methylation levels of EPOR in cancer tissues with those in normal tissues and performed protein–protein interaction network, gene–disease network, and genetic alteration analyses of EPOR, and finally enrichment analysis of EPOR-interacting proteins, co-expressed genes, and differentially expressed genes.ResultsThe TCGA database showed that EPOR expression was upregulated in BLCA, CHOL, HNSC, KIRC, LIHC, STAD, and THCA and downregulated in LUAD and LUSC. After combining the GTEx database, EPOR expression was found to be downregulated in 18 cancer tissues and upregulated in 6 cancer tissues. The CCLE database showed that EPOR expression was highest in LAML cell lines and lowest in HNSC cell lines. Survival analysis showed that high EPOR expression was positively correlated with OS in LUAD and PAAD and negatively correlated with OS in COAD, KIRC, and MESO. Moreover, EPOR had a good prognostic ability for COAD, LUAD, MESO, and PAAD and also influenced progression-free survival, disease-specific survival, disease-free survival, and progression-free interval in specific tumors. Further, EPOR was found to play a non-negligible role in tumor immunity, and a correlation of EPOR with miRNAs, TMB, MSI, and MMR genes and methyltransferases was confirmed to some extent. In addition, the enrichment analysis revealed that EPOR is involved in multiple cancer-related pathways.ConclusionThe general situation of EPOR expression in cancer provided a valuable clinical reference. EPOR may be target gene of hsa-miR-575, etc. A pan-cancer analysis of panoramic schema revealed that EPOR not only may play an important role in mediating EPO-induced erythroblast proliferation and differentiation but also has potential value in tumor immunity and is expected to be a prognostic marker for specific cancers.

Transmission Electron Microscopy to Follow Ultrastructural Modifications of Erythroblasts Upon ex vivo Human Erythropoiesis

Throughout mammal erythroid differentiation, erythroblasts undergo enucleation and organelle clearance becoming mature red blood cell. Organelles are cleared by autophagic pathways non-specifically targeting organelles and cytosolic content or by specific mitophagy targeting mitochondria. Mitochondrial functions are essential to coordinate metabolism reprogramming, cell death, and differentiation balance, and also synthesis of heme, the prosthetic group needed in hemoglobin assembly. In mammals, mitochondria subcellular localization and mitochondria interaction with other structures as endoplasmic reticulum and nucleus might be of importance for the removal of the nucleus, that is, the enucleation. Here, we aim to characterize by electron microscopy the changes in ultrastructure of cells over successive stages of human erythroblast differentiation. We focus on mitochondria to gain insights into intracellular localization, ultrastructure, and contact with other organelles. We found that mitochondria are progressively cleared with a significant switch between PolyE and OrthoE stages, acquiring a rounded shape and losing contact sites with both ER (MAM) and nucleus (NAM). We studied intracellular vesicle trafficking and found that endosomes and MVBs, known to be involved in iron traffic and heme synthesis, are increased during BasoE to PolyE transition; autophagic structures such as autophagosomes increase from ProE to OrthoE stages. Finally, consistent with metabolic switch, glycogen accumulation was observed in OrthoE stage.

Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythroblasts.

The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell derived erythrocytes and enhanced erythroid terminal differentiation. RNA sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 accelerated F-actin remodeling in terminally differentiated erythrocytes, favoring their maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation were associated with F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature human pluripotent stem cell-derived erythrocytes for use in translational applications.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+\u2009cells and causes decreased levels of hemoglobin

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

PDE9 Inhibition By IMR-687 Improves Markers of Beta-Thalassemia in the Hbb th1/th1 Experimental Mouse Model

IntroductionHbb th1/th1 mice lack functional beta-globin, leading to decreased hemoglobin (Hb) production, decreased mature red blood cells (RBCs), and ineffective erythropoiesis. The model mimics several of the pathological changes seen in beta-thalassemia. IMR-687 (tovinontrine) is a highly selective phosphodiesterase-9 (PDE9) inhibitor which increases intracellular cGMP. The aim of this study was to assess the effect of IMR-687 on markers of beta-thalassemia in the Hbb th1/th1 mouse model.MethodsEighteen Hbb th1/th1 mice were divided into three groups of six animals each and dosed once daily, by gavage, for 30 days with IMR-687 at 30 or 60 mg/kg/day or vehicle control. On day 30, blood was collected for routine hematology including measurement of Hb, reticulocytes and RBCs, and spleen tissue was dissociated to assess erythrocyte differentiation using flow cytometry analysis of immature (Ery.B: Ter119 highCD71 highFSC low) and mature (Ery.C: Ter119 highCD71 lowFSC low) erythroblast populations. Animals were observed daily for mortality and measured for total body weight (Day 0 and Day 30) and spleen to body weight ratio at necropsy (Day 30).ResultsAfter 30 days of treatment, the groups of mice treated with IMR-687 at 30 or 60 mg/kg/day showed a mean increase in Hb of 1.0 g/dL (p<0.05) and 1.5 g/dL (p<0.01), respectively, relative to vehicle controls. RBCs showed a statistically significant increase (p<0.05) following treatment with IMR-687 at 30 or 60 mg/kg/day (6.9 and 7.2 x10 6 cells/ml, respectively) relative to vehicle (6.2 x10 6 cells/ml). The number of reticulocytes decreased after treatment with IMR-687 (only in the 30 mg/kg/day group, 1.8 x10 6 cells/ml, p<0.05) relative to vehicle (2.2 x10 6 cells/ml). Flow cytometry analysis of spleens showed that both doses of IMR-687 increased (p<0.05) the percentage of Ery.C cells (24.2% and 28.5% in 30 and 60 mg/kg/day, respectively) relative to vehicle (16.6%,) and decreased (p<0.05) the percentage of Ery.B cells (38.4% and 36.9% in 30 and 60 mg/kg/day, respectively) relative to vehicle (43.5%), resulting in a maturation ratio (Ery.B/Ery.C) that favored mature RBCs. There were no deaths nor any significant differences in total body weight or spleen to body weight ratio in animals treated with IMR-687 relative to vehicle controls.ConclusionsAdministration of IMR-687 at 30 or 60 mg/kg/day for 30 days improved markers of disease progression in a mouse model of beta-thalassemia, as shown by a statistically significant increase in Hb and RBCs and decrease in reticulocytes as well as improved differentiation of splenic erythroblasts. Both dose levels of IMR-687 were well tolerated with no treatment-related deaths or abnormal clinical signs. These results support a role for IMR-687 in beta-thalassemia by enabling RBC maturation and improving ineffective erythropoiesis, key components in ameliorating disease pathology. Clinical testing of IMR-687 (up to 400 mg) as a once daily, oral tablet is currently ongoing in a Phase 2 study of patients with beta-thalassemia (NCT04411082). [Display omitted] DisclosuresMaciel: Imara Inc.: Research Funding. Carvalho: Imara Inc.: Research Funding. Rignault: Imara Inc.: Research Funding. Allali: Imara Inc.: Research Funding. OCain: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of β0-Thalassemia/Hemoglobin E Patients

Introduction: β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. Methods: To elucidate the correlation between ineffective erythropoiesis and β⁰-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. Results: The β⁰-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β⁰-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. Discussion/Conclusion: The degree of erythroid expansion and maturation arrest contributes to the severity of β⁰-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

A Phase 1 Trial of MEC (Mitoxantrone, Etoposide, Cytarabine) in Combination with Ixazomib (MLN9708) for Relapsed/ Refractory Acute Myeloid Leukemia (AML): Final Results

A Phase 1 Trial of MEC (Mitoxantrone, Etoposide, Cytarabine) in Combination with Ixazomib (MLN9708) for Relapsed/ Refractory Acute Myeloid Leukemia (AML): Final Results

In Vitro Erythropoietic Response to Enzyme Replacement Therapy in Gaucher Type 1 Patients

Introduction. Gaucher disease type 1 (GD, OMIM #230800) is the most common inherited lysosomal storage disorder resulting from lysosomal glucocerebrosidase (GBA, acid-β-glucosidase) deficiency and subsequent accumulation of glucosylceramide in macrophages. This leads to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). Anemia is attributed to bone marrow infiltration by Gaucher cells, to inflammation and to the presence of splenomegaly; however the underlying pathophysiological mechanism hasn't been fully elucidated.Aim. The aim of this study is to evaluate the in vitro erythroid status at baseline (T0) and the response to Enzyme Replacement Therapy (ERT) after 6 months (T1) and 1 year (T2). We evaluated the expansion and differentiation in erythroid cultures of CD34+ cells obtained from peripheral blood of GD type 1 patients at diagnosis before starting ERT (T0), at 6 months (T1) and at 1 year (T2) of ERT.Methods. CD34+ cells were obtained by immunomagnetic separation from peripheral blood mononuclear cells of GD patients (n=3) and controls (n=5). Isolated CD34+ were evaluated by in vitro clonogenic capacity assays using the methylcellulose semisolid culture medium after 14-16 days of culture. Hematopoietic colonies (burst-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage (CFU-M)) were scored with an inverted microscope. CD34+ cells were expanded in StemSpan medium, 1% StemSpan CC100 cytokine cocktail, 2 U/ml Erythropoietin (EPO) and 1 μM dexamethasone. Differentiation was induced in StemSpan medium with 10 ng/ml SCF and 10 U/ml EPO. Proliferation and surface marker expression (CD34, CD45, Glycophorine A, CD71) were examined by flow cytometry. Cell morphology was analyzed on cytocentrifuged smears stained with May-Grunwald-Giemsa.Results. At T0 we observed a higher number of BFU-E in GD samples (126±23 BFU-E/105 cells) compared to controls (49±12 BFU-E/105 cells) (Tab.1). Similarly, higher macrophage CFU-M were observed in GD samples (29±12 CFU-M/105 cells) compared to controls (10±1 CFU-M/105 cells) (Tab.1). The BFU-E and CFU-M morphology showed no difference between GD patients and controls. The increased proliferation of erythroid precursors of GD patients was associated to impaired ability to differentiate in liquid cultures after 14 days (45,9% proerythroblasts and only 4% polichromatophilic erythroblasts) compared to controls (27% and 19%, respectively) (Tab.1). Flow cytometry confirmed an impaired differentiation of GD erythroblasts compared to controls at day 14 of the erythroid liquid cultures, as shown by high percentage of CD71high/GlyAlow early erythroid precursor cells and low percentage of CD71high/GlyAhigh mature erythroblasts (GD: 73,9 and 4,9%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1). In samples from GD patients, after 1-year treatment with ERT (T2), we observed a reduction in the number of BFU-E compared to T0, reaching values similar to controls (GD T0: 126±23 BFU-E/105 cells, T2: 41±11 BFU-E/105 cells; controls: 49±12 BFU-E/105 cells) and an increased number of mature erythroid cells generated in GD liquid cultures (Tab. 1). Also CFU-M were reduced at T2 compared to T0 (GD T0: 29±12 CFU-M/105 cells, T2: 8±5 CFU-M/105 cells; controls: 10±1 CFU-M/105 cells) (Tab.1). An increased percentage of more differentiated erythroid cells at T2 compared to T0 was confirmed by flow cytometry in cultures from GD patients, as shown by low percentage of the CD71high/GlyAlow early erythroid precursor cells and high percentage of CD71high/GlyAhigh mature erythroblasts on day 14 (GD: 44,7 and 45,6%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1).Conclusions. These preliminary data suggest that anemia in GD might partly be related to a delay in erythroid differentiation. ERT treatment seems to correct the dyserythropoiesis by improving erythroid differentiation. Since glucosylceramide accumulates in macrophages, that are key elements in the erythroid niche, impaired erythroid differentiation might be related to the pathological micro-environment, rather than intrinsic hematopoietic stem cell defect. More extensive studies are needed to better understand the pathophysiology of anemia and pancytopenia of this rare disease.This study was partially supported by Sanofi Genzyme. [Display omitted] DisclosuresCappellini:Celgene: Honoraria; Novartis: Speakers Bureau; Vifor: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau.

High Mobility Group Box 1 Protein (HMGB1) Mediates Anemia of Inflammation through Lineage Skewing and Direct Inhibition of EPO Signaling

Erythropoiesis is sensitive to inflammation and its associated mediators. We previously identified the damage-associated molecular pattern, high mobility group box 1 protein (HMGB1), as a causative factor involved in the development of anemia using a murine model of chronic inflammation, and recently, showed that HMGB1 negatively affects human erythropoiesis, in part, through altered erythropoietin (EPO) signaling and hematopoietic stem and progenitor cell (HSPC) lineage commitment. Despite these findings, a mechanistic understanding of erythroid dysfunction in the presence of HMGB1 is lacking.We first documented a dose-dependent logarithmic difference in erythroid cell growth at Days 7 and 11 of culture of CD34+ HSPCs during erythroid differentiation as well as a relatively smaller loss of cells at Day 14 between HMGB1-treated and vehicle-treated cultures. These results suggest that HMGB1 exerts its effects at distinct stages of erythroid differentiation: at both the progenitor and the precursor stages. Indeed, flow analysis of erythroblast differentiation showed that HMGB1 caused an accumulation of precursors at the basophilic erythroblast stage. The loss of poly- and orthochromatic erythroblasts resulted from increased apoptosis at these stages of erythroid differentiation as assessed by annexin-V staining. Induction of apoptosis was confirmed by a dose-dependent increase in apoptosis and 3-fold increase in cleaved caspase-3 in HUDEP-2 cells, an erythroid cell line. Furthermore, a mutant form of HMGB1 (3S-HMGB1) in which critical cysteine residues were mutated to abolish the activity of the protein failed to alter erythroid proliferation and survival.HMGB1 appears to deprive differentiating erythroid cells of growth and survival signals through attenuated phosphorylation of downstream EPO effectors including pJAK2 and pSTAT5, independent of SHP1/2 activity. As a consequence of perturbed EPO signaling, erythroid cells contained reduced levels of nuclear HSP70 and GATA1, which ultimately led to apoptosis. Importantly, EPO co-administrated with either tumor necrosis factor (TNF), interleukin-1β (IL-1β), or IL-6 led to phosphorylation levels of JAK2-STAT5 comparable to controls, suggesting that direct inhibition of EPO signaling is specific to HMGB1.To document the effect of HMGB1 on EPO-dependent and independent progenitors, erythroid progenitor growth and differentiation were monitored at 24hr intervals in culture. CFU-E but not BFU-E numbers were decreased following treatment with HMGB1. Moreover, HMGB1-treated cells expressed high levels of GATA2 indicating deregulation of GATA transcription factor switching. Myelo-erythroid culture systems demonstrated that HMGB1 strongly inhibited erythroid commitment of CD34+ HSPCs while it favored the generation of increased numbers of CD11b+ and CD13+ expressing myeloid cells. The induced switch in lineage commitment is consistent with increased GATA2 expression and decreased GATA1 expression. Further, we observed that erythroid and hematopoietic progenitor cultures became more acidic in the presence of HMGB1 suggesting that HMGB1 may also influence progenitor metabolic processes.Our data demonstrate that HMGB1 negatively affects erythropoiesis at EPO-dependent and -independent stages of erythroid differentiation through decreased CFU-E numbers and apoptosis of erythroblasts, and also favor increased myeloid differentiation of HSPCs. Contrary to classical inflammatory mediators, HMGB1 relies on a novel mechanism, involving downstream EPO effectors, to mediate erythroid dysfunction. Ongoing pharmacologic and genetic screens of EPO signaling axes, putative HMGB1 receptor(s), and progenitor metabolic profiling are currently underway to better understand how HMGB1 causes erythropoietin refractory anemia in the setting of inflammation. DisclosuresNo relevant conflicts of interest to declare.

SETD2 is essential for terminal differentiation of erythroblasts during fetal erythropoiesis

SET domain-containing 2 (SETD2), the primary methyltransferase for histone 3 lysine-36 trimethylation (H3K36me3) in mammals, is associated with many hematopoietic diseases when mutated. Previous works have emphasized its role in maintaining adult hematopoietic stem cells or tumorigenesis, however, whether and how SETD2 regulates erythropoiesis during embryonic development is relatively unexplored. In this study, using a conditional SETD2 knockout (KO) mouse model, we reveal that SETD2 plays an essential role in fetal erythropoiesis. Loss of Setd2 in hematopoietic cells ablates H3K36me3, and leads to anemia with a significant decrease in erythroid cells in the peripheral blood at E18.5. This is due to impaired erythroblast differentiation in both spleen and liver. We also find increased proportions of nucleated erythrocytes in the blood of Setd2 KO embryos. Lastly, we ascribe embryonic erythropoiesis-related genes Vegfc, Vegfr3, and Prox1, as likely downstream targets of SETD2 regulation. Our study reveals a critical role of SETD2 in fetal erythropoiesis that precedes adult hematopoiesis, and provide unique insights into the defects in erythroid lineages, such as anemia.

GATA factor-regulated solute carrier ensemble reveals a nucleoside transporter-dependent differentiation mechanism.

Developmental-regulatory networks often include large gene families encoding mechanistically-related proteins like G-protein-coupled receptors, zinc finger transcription factors and solute carrier (SLC) transporters. In principle, a common mechanism may confer expression of multiple members integral to a developmental process, or diverse mechanisms may be deployed. Using genetic complementation and enhancer-mutant systems, we analyzed the 456 member SLC family that establishes the small molecule constitution of cells. This analysis identified SLC gene cohorts regulated by GATA1 and/or GATA2 during erythroid differentiation. As >50 SLC genes shared GATA factor regulation, a common mechanism established multiple members of this family. These genes included Slc29a1 encoding an equilibrative nucleoside transporter (Slc29a1/ENT1) that utilizes adenosine as a preferred substrate. Slc29a1 promoted erythroblast survival and differentiation ex vivo. Targeted ablation of murine Slc29a1 in erythroblasts attenuated erythropoiesis and erythrocyte regeneration in response to acute anemia. Our results reveal a GATA factor-regulated SLC ensemble, with a nucleoside transporter component that promotes erythropoiesis and prevents anemia, and establish a mechanistic link between GATA factor and adenosine mechanisms. We propose that integration of the GATA factor-adenosine circuit with other components of the GATA factor-regulated SLC ensemble establishes the small molecule repertoire required for progenitor cells to efficiently generate erythrocytes.

Efficient Enucleation and In Vivo Circulation of Differentiated Human Erythroblasts Derived from Peripheral Blood Mononuclear Cells after Extensive Expansion

Transfusion of red blood cells (RBCs) is a standard and indispensable cellular therapy commonly used globally in elective and emergency surgery and for treating anemic patients who need frequent transfusion. However, the current donor-based supply is often insufficient to meet the increasing need especially in pandemic and natural disasters. Thus that, ex vivo production of functional cultured RBCs in a laboratory setting is highly desirable and has been pursued in the past decades. Significant progress has been made in the differentiation of mature human erythrocytes from CD34+ HSPCs and human pluripotent stem cells (PSCs), however, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex-vivo has been challenging. We and others have also tried an alternative approach to directly and extensively expand or immortalize erythroid progenitors (erythroblasts) from a postnatal source of individual patients or desirable donors. Here we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts derived from peripheral blood mononuclear cells (PBMCs) while reserving the high efficiency of enucleation after erythroblast differentiation. Being encouraged by previous studies including those with mouse marrow cells, we examined if the BMI1 gene expression is present and critical to human erythroblast proliferation. We observed that the human BMI1 gene is expressed in proliferating erythroblasts as in CD34+ HSPCs, but decreases in late-stage erythroblasts undergoing differentiation. Using a short hairpin RNA-mediated knockdown approach, we found that BMI1 expression is essential to maintain a proliferative state of human erythroblasts established in culture. We next tested whether the ectopic expression of human BMI1 as a transgene can prolong the expansion of human erythroblasts in culture, which only lasts for 3-4 weeks before spontaneous differentiation and ceasing proliferation. The BMI1-transduced erythroblasts continued to proliferate for at least 60 days, resulting in a 1012-fold expansion of erythroblasts established from PBMCs of several adult donors; we termed them as "extensively expanded erythroblasts" or E3 cells. To investigate the capacity of BMI1-E3 cells to terminally differentiate into reticulocytes, a standard maturation medium was applied to replace the serum-free expansion medium at various time points after expansion. We found that these E3 cells are capable of efficient terminal maturation, yielding ~50% enucleated erythrocytes after ex vivo differentiation induction. Moreover, similar results were seen on erythroblasts derived from PBMCs of two adult patients with sickle cell disease. To broaden and enhance the clinical applications of these culture-expanded erythroblasts, we confirmed the feasibility of genetic manipulation on E3 cells by inserting a transgene or ablating an endogenous gene such as CD55. Finally, we examined whether human erythrocytes differentiated from BMI1-E3 cells have the capacity to circulate (and possibly further mature) in vivo using an improved mouse model. E3-derived cultured RBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Interestingly, percentages of enucleated erythrocytes derived from the BMI1-E3 maturation products increased in the circulation in mice, to reach from ~57% at transfusion to approximately 72-84% after transfusion. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex-vivo. Disclosures No relevant conflicts of interest to declare.

SF3B1-Mutant Myelodysplastic Syndrome with Ringed Sideroblasts (MDS-RS) at the Single-Cell Level

The biological mechanisms of abnormal terminal erythroid differentiation (TED) in MDS-RS and SF3B1 mutations (SF3B1MT) are largely unknown. This gap in understanding, which has dramatically delayed the design of second-line approaches for SF3B1MT patients whose disease has failed hypomethylating agent (HMA) therapy, is primarily due to a lack of studies molecularly characterizing how SF3B1MTaffect distinct stages of erythropoiesis. Here, we dissected at the single-cell level the cellular and transcriptomic changes induced by SF3B1MT in cells undergoing erythroid differentiation and elucidated how HMA therapy can overcome SF3B1MT-defective erythropoiesis. We first analyzed the expression profile of the lineage-negative CD34+ stem and progenitor (HSPC) compartment. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs isolated from 2 healthy donors (HDs) and 5 untreated MDS-RS patients with SF3B1MT revealed cell clusters driven by the cells' differentiation potential that we defined based on the differential expression of validated lineage-specific transcriptional factors and cellular markers. Whereas the HD HSPCs had equally distributed erythroid/megakaryocyte and myeloid/lymphoid differentiation trajectories, the SF3B1MT HSPCs had a predominant erythroid differentiation route (Fig. 1a). Differential expression analysis revealed that the SF3B1MT HSPCs undergoing erythroid differentiation were characterized by the expression of genes involved in translation, oxidative phosphorylation, and cell cycle progression, which underlines these cells' metabolic and proliferative activation. Consistent with these findings, scRNA-seq of bone marrow (BM) mononuclear cells (MNCs) from the same samples showed that SF3B1MT MNCs had a predominant population of erythroid cells at the expense of B lymphocytes and myeloid cells (Fig. 1b). An analysis of the erythroid cluster distribution inside the erythroid compartment showed that the frequency of early-stage maturation (BFU-E [cluster #18], CFU-E [#9]) was lower in the SF3B1MT erythroblasts than in the HD erythroblasts, owing to a significantly increased frequency of SF3B1MT cells in the latest stages of TED (proerythroblasts [#4]), basophilic normoblasts [#12], polychromatophilic normoblasts [#7], orthochromatic normoblasts [ONs; #1/11], pre-reticulocytes [#13]). These data suggest that SF3B1MT enhance HSPC differentiation towards the erythroid lineage but arrest erythroblasts at the last step of their maturation by inhibiting the transition of ONs to pre-reticulocytes, resulting in the accumulation of TED cells in the BM. Transcriptomic analysis of SF3B1MT ONs revealed a significant upregulation of genes regulating heme metabolism, including those involved in EIF2AK1's response to impaired heme production, and of major effectors of cell cycle checkpoint activation (e.g., CHK1, CDKN1A, GADD45). These data are consistent with previous studies showing that the SF3B1MT-induced defective accumulation of iron in erythroblasts' mitochondria leads to activated cell stress response, cell cycle arrest, and apoptosis. Of note, the growth differentiation factor 11 receptors ACVR1B, TGFBR1, and ACVR1C were not expressed at any step of erythroid differentiation, which challenges this factor's role as a target of luspatercept-induced differentiation of late-stage erythroblasts. To evaluate how HMA therapy can overcome inefficient erythropoiesis, we performed scRNA-seq of HSPCs and BM MNCs isolated from SF3B1MT MDS-RS patients before any therapy and at the time of hematological response to HMA therapy. HMA therapy did not reduce the aberrant differentiation of SF3B1MT HSPCs towards the erythroid lineage but temporarily induced the TED of SF3B1MT ONs into reticulocytes and these cells' release into peripheral blood. Accordingly, blast progression following the initial HMA therapy-induced hematological response coincided with the further expansion of the erythroid-primed cells arising from the earliest stem cell population (Fig. 1c), which suggests that failure to eradicate or genetically correct SF3B1MT stem cells leads to disease relapse. In conclusion, our results elucidate how SF3B1MT molecularly affect distinct stages of erythropoiesis and have implications for developing approaches that achieve lasting hematological remission in patients with MDS-RS. Disclosures Garcia-Manero: Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy; Merck: Research Funding; AbbVie: Honoraria, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding. Colla:Amgen: Other: Unspecified.

Lysophosphatidic acid receptors 2 and 3 regulate erythropoiesis at different hematopoietic stages

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

Substitution of Thr572 to Ala in mouse c-Myb attenuates progression of early erythroid differentiation

The expression level of transcription factor c-Myb oscillates during hematopoiesis. Fbw7 promotes ubiquitin-mediated degradation of c-Myb, which is dependent on phosphorylation of Thr572. To investigate the physiological relevance of Fbw7-mediated c-Myb degradation, we generated mutant mice carrying c-Myb-T572A (TA). Homozygous mutant (TA/TA) mice exhibited a reduction in the number of peripheral red blood cells and diminished erythroblasts in bone marrow, presumably as a result of failure during erythroblast differentiation. We found that c-Myb high-expressing cells converged in the Lin−CD71+ fraction, and the expression of c-Myb was higher in TA/TA mice than in wild-type mice. Moreover, TA/TA mice had an increased proportion of the CD71+ subset in Lin− cells. The c-Myb level in the Lin−CD71+ subset showed three peaks, and the individual c-Myb level was positively correlated with that of c-Kit, a marker of undifferentiated cells. Ultimately, the proportion of c-Mybhi subgroup was significantly increased in TA/TA mice compared with wild-type mice. These results indicate that a delay in reduction of c-Myb protein during an early stage of erythroid differentiation creates its obstacle in TA/TA mice. In this study, we showed the T572-dependent downregulation of c-Myb protein is required for proper differentiation in early-stage erythroblasts, suggesting the in vivo significance of Fbw7-mediated c-Myb degradation.

Overexpression of miR-669m inhibits erythroblast differentiation

MicroRNAs (miRNAs), one of small non-coding RNAs, regulate many cell functions through their post-transcriptionally downregulation of target genes. Accumulated studies have revealed that miRNAs are involved in hematopoiesis. In the present study, we investigated effects of miR-669m overexpression on hematopoiesis in mouse in vivo, and found that erythroid differentiation was inhibited by the overexpression. Our bioinformatic analyses showed that candidate targets of miR-669m which are involved in the erythropoiesis inhibition are A-kinase anchoring protein 7 (Akap7) and X-linked Kx blood group (Xk) genes. These two genes were predicted as targets of miR-669m by two different in silico methods and were upregulated in late erythroblasts in a public RNA-seq data, which was confirmed with qPCR. Further, miR-669m suppressed luciferase reporters for 3′ untranslated regions of Akap7 and Xk genes, which supports these genes are direct targets of miR-669m. Physiologically, miR-669m was not expressed in the erythroblast. In conclusion, using miR-669m, we found Akap7 and Xk, which may be involved in erythroid differentiation, implying that manipulating these genes could be a therapeutic way for diseases associated with erythropoiesis dysfunction.

Cdc42 regulates cell polarization and contractile actomyosin rings during terminal differentiation of human erythroblasts

The molecular mechanisms involved in the terminal differentiation of erythroblasts have been elucidated by comparing enucleation and cell division. Although various similarities and differences between erythroblast enucleation and cytokinesis have been reported, the mechanisms that control enucleation remain unclear. We previously reported that dynein and microtubule-organizing centers mediated the polarization of nuclei in human erythroblasts. Moreover, the accumulation of F-actin was noted during the enucleation of erythroblasts. Therefore, during enucleation, upstream effectors in the signal transduction pathway regulating dynein or actin, such as cell division control protein 42 homolog (Cdc42), may be crucial. We herein investigated the effects of the Cdc42 inhibitor, CASIN, on cytokinesis and enucleation in colony-forming units-erythroid (CFU-Es) and mature erythroblasts (day 10). CASIN blocked the proliferation of CFU-Es and their enucleation in a dose-dependent manner. Dynein adopted an island-like distribution in the cytoplasm of non-treated CFU-Es, but was concentrated near the nucleus as a dot and co-localized with γ-tubulin in CASIN-treated cells. CASIN blocked the accumulation of F-actin in CFU-Es and day 10 cells. These results demonstrated that Cdc42 plays an important role in cytokinesis, nuclear polarization and nuclear extrusion through a relationship with dynein and actin filament organization during the terminal differentiation of erythroblasts.

Parallel bimodal single-cell sequencing of transcriptome and chromatin accessibility.

Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)–centered upstream regulatory networks for various cell states.

Transformation Mechanisms of the Nfia-ETO2 Fusion Gene Associated with Pediatric Pure Acute Erythroleukemia

Pure erythroleukemia (PEL) is a very aggressive, but poorly understood form of acute myeloid leukemia characterized by malignant accumulation of erythroid progenitor cells. A novel t(1;16)(p31;q24) chromosomal translocation leading to expression of a fusion between the nuclear factor I A (NFIA) and the ETO2 transcriptional co-regulator (a.k.a. CBFA2T3 or MTG16) has been identified in pediatric patients with PEL. Based on the function of the fusion partners, we hypothesized that NFIA-ETO2 (N-E) might initiate PEL by interfering with erythroid differentiation. To investigate its function, we cloned a full-length ORF and retrovirally expressed N-E in primary mouse bone marrow (BM)- and fetal liver (FL)-derived erythroblasts (EB). N-E expression significantly increased proliferation and blocked differentiation of EB. N-E expressing BM-derived hematopoietic stem and progenitor cells (HSPC) could be plated in erythropoietin (EPO)-containing methylcellulose (MC) for up to 3 rounds. Expression of N-E deletion mutants lacking the NFIA DNA-binding, the ETO2 NHR2 or NHR4 (ΔNHR4) transcriptional repression domains were unable to block erythroid differentiation. Notably, interfering with the ETO2-NHR2 oligomerization domain by overexpressing a competing peptide overcame the N-E-mediated differentiation block. Transplantation of N-E-expressing BM-derived HSPC into irradiated syngenic mice did not induce any disease suggesting the need of genetic cooperation. As TP53 gain-of-function (GOF) mutations are molecular hallmarks of PEL, we explored functional cooperation by using a conditional TP53R248Q allele. Interestingly, the TP53 status did not affect EB in vitro proliferation or differentiation. However, N-E expression increased proliferation of TP53R248Q+ EB and resulted in the formation of abnormal round and dense colonies in MC that could be serially propagated. In addition, transplantation of N-E-expressing TP53R248Q+ EB into irradiated recipients induced a transplantable PEL-like disease after a median latency of 4 months. Symptomatic mice presented with anemia, thrombocytopenia, multi-organ tumor cell infiltration and increased white blood cell counts. To better understand the molecular mechanism, we compared the gene expression signatures before and 24 hours after induced differentiation of FL-derived EB expressing WT or the inactive ΔNHR4 N-E mutant, in presence or absence of TP53R248Q. Principal component analysis (PCA) revealed a clear separation between the transcriptomes of WT EB expressing either the active or the inactive ΔNHR4 N-E (PC1:54.7%) and by their erythroid differentiation stage (PC2:9.07%). Overall, we observed 3753 (FDR&amp;lt;0.05, logFC&amp;gt;1.5) differentially expressed genes. Many of the significantly higher expressed genes (2313/3753) were related to hematopoietic stemness (GSEAs, p&amp;lt;0.001). Almost 10% of the significantly lower expressed genes (92/1440) were linked to the erythroid lineage development and to erythropoietic targets of NFIA or the erythroid master regulator GATA1. Interestingly, we also found reduced expression of genes encoding for ETO2-interacting transcription factors including TAL1 and KLF1. Despite a critical role on disease progression, PCA showed only minimal changes in the N-E expression signature in presence or absence of TP53R248Q with only 12 genes differently expressed (FDR&amp;lt;0.05, logFC&amp;gt;1). These genes were previously shown to be oncogenic mediators of TP53-GOF mutations, related to metabolism and transcriptional regulation. Interestingly, the signature of differentially expressed genes in N-E transformed FL-derived EB were significantly differentially expressed in tumor cells from pediatric but not adult PEL patients (p=0.00045), indicating the pediatric origin of the fusion. Collectively, we found that the PEL-associated N-E fusion blocks erythroid differentiation, and cooperates with a TP53-GOF mutation to induce a PEL-like disease in mice that phenocopies the human disease. Mechanistically, its activity seems to correlate with repression of erythroid regulatory genes controlled by the fusion partners NFIA, ETO2, and the erythroid master regulator GATA1. Disclosures No relevant conflicts of interest to declare.