Number Of Erythroblasts Research Articles

Erythroferrone exacerbates iron overload and ineffective extramedullary erythropoiesis in a mouse model of β-thalassemia.

β-thalassemia is characterized by chronic hepcidin suppression and iron overload, even in patients who have not undergone transfusion. The HbbTh3/+ (Th3/+) mouse model of nontransfusion-dependent β-thalassemia (NTDBT) partially recapitulates the human phenotype but lacks chronic hepcidin suppression, progressive iron accumulation into adulthood, or the interindividual variation of the rate of iron loading observed in patients. Erythroferrone (ERFE) is an erythroid regulator that suppresses hepcidin during increased erythropoiesis. ERFE concentrations in the sera of patients with NTDBT correlate negatively with hepcidin levels but vary over a broad range, possibly explaining the variability of iron overload in patients. To analyze the effect of high ERFE concentrations on hepcidin and iron overload in NTDBT, we crossed Th3/+ mice with erythroid ERFE-overexpressing transgenic mice. Th3/ERFE-transgenic mice suffered high perinatal mortality, but embryos at E18.5 showed similar viability, appearance, and anemia effects as Th3/+ mice. Compared with Th3/+ littermates, adult Th3/ERFE mice had similarly severe anemia but manifested greater suppression of serum hepcidin and increased iron accumulation in the liver, kidney, and spleen. The Th3/ERFE mice had much higher concentrations of serum ERFE than either parental strain, a finding attributable to both a higher number of erythroblasts and higher production of ERFE by each erythroblast.Th3/+ and Th3/ERFE mice had similar red blood cell count and shortened erythrocyte lifespan, but Th3/ERFE mice had an increased number of erythroid precursors in their larger spleens, indicative of aggravated ineffective extramedullary erythropoiesis. Thus, high ERFE concentrations increase the severity of nontransfusional iron overload and ineffective erythropoiesis in thalassemic mice but do not substantially affect anemia or hemolysis.

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4β1 and macrophage αVβ3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

Terminal Erythroid Differentiation Is Regulated By Mitochondrial Translation Via Maintenance of Iron Homeostasis

Erythropoiesis is a process that continuously produces red blood cells, the most abundant cell type in the body which actively synthesize hemoglobin protein. Modification of transfer RNA (tRNA) regulates its function such as protein translation and other cellular processes (Chujo et al, FEBS J. 2021). Lack of mitochondrial tRNA (mt-tRNA) taurine modification mediated by mitochondrial tRNA translation optimization 1 (Mto1) has been recently shown to induce unfolded protein response (UPR) in ES cells (Fakruddin et al, Cell Rep. 2018). However, how UPR is induced by mt-tRNA dysfunction and its relevance in tissue homeostasis remains unclear. Since aberrant protein translation induces cellular stress, we hypothesized that Mto1 dysfunction might result in defective hematopoiesis, especially erythropoiesis which produces a substantial amount of protein such as hemoglobin via mitochondria. Yet, the role of any modification of mt-tRNAs in hematopoiesis including taurine has not been elucidated at all. To elucidate the biological function of Mto1 in hematopoiesis, we generated hematopoietic-specific Mto1 conditional KO (cKO) mice by a cross with Vav-Cre mice. The Mto1cKO mouse was embryonic lethal and the fetus had a paler appearance than the wild-type (WT) control, indicating severe anemia. Total fetal liver cellularity was significantly reduced and erythrocyte-related parameters were marked reduced in a peripheral blood parameter analysis in Mto1 cKO fetuses compared to WT. Further erythroblast subpopulation analysis revealed that absolute number of erythroblasts decreased significantly in almost all differentiation stages in Mto1 KO fetal liver compared to WT, especially most decrease in number at polychromatic erythroblast stage. Analysis in adult hematopoiesis using Mx1-Cre system also showed similar anemic phenotype in Mto1 cKO mouse. Consistent with these results, Mto1 mRNA expression and taurine modification levels were markedly up-regulated at the polychromatic erythroblast stage in the WT fetal liver, suggesting essential role of Mto1 in terminal erythroid differentiation at the polychromatic erythroblast stage. Previous study showed Mto1 dysfunction suppressed mitochondrial translation which impaired the formation of respiratory complexes (Fakruddin et al, Cell Rep. 2018). Consistent with this finding, Mto1 cKO fetal liver polychromatic erythroblasts showed marked suppression of complex-I formation. Impaired formation of complex-I which contains abundant iron-sulfur clusters is supposed to affect intracellular iron distribution, from mitochondria to cytoplasm. In line with this hypothesis, Mto1 cKO fetal liver polychromatic erythroblasts showed cytoplasmic iron accumulation which upregulated ALAS2 protein translation and heme biosynthesis. Proteome analysis of Mto1 cKO fetal liver polychromatic erythroblasts revealed significant upregulation of embryonic hemoglobin proteins expression compared with WT. Upregulation of heme and unstable embryonic hemoglobin biosynthesis is supposed to induce cellular stress. Consistent with this hypothesis, Xbp1 splicing-mediated UPR was significantly upregulated in Mto1 cKO fetal liver polychromatic erythroblasts. Moreover, both specific inhibitor of Xbp1 splicing and iron chelator rescued terminal erythroid differentiation in the Mto1 KO fetal liver in vitro, suggesting that Xbp1 splicing-mediated UPR via cytoplasmic iron overload derived from Mto1 deficiency is a cause of defect in terminal erythroid differentiation in Mto1 cKO mouse. This study demonstrated for the first time, an indispensable role of mt-tRNA taurine modification in erythroid terminal differentiation and suggests the biological role of mitochondria in iron homeostasis and erythropoiesis. Our findings propose a novel, non-energy-related molecular mechanism which links primary mitochondrial protein deficiency with erythroid differentiation block via imbalanced iron homeostasis. This important but unappreciated molecular mechanism might make a breakthrough by bringing a new biological aspect to hematology.

Erythroferrone Overexpression Impairs Survival and Greatly Increases Tissue Iron Loading in a Mouse Model of β-Thalassemia

Beta-thalassemia, caused by mutations in the β-globin gene, is characterized by ineffective erythropoiesis, anemia and associated multi-organ dysfunction. Like in other anemias with ineffective erythropoiesis, the erythroid hormone erythroferrone (ERFE) is pathologically overproduced. ERFE functions by suppressing the master iron regulatory hormone hepcidin, and the chronic suppression of hepcidin in β-thalassemia results in excessive iron absorption and iron overload, even in patients who do not receive erythrocyte transfusions. A major obstacle in studying the role of ERFE in β-thalassemia has been the lack of a mouse model that represents the wide range of ERFE concentrations observed in human patients (Ganz et al, Blood 2017). Our lab generated ERFE-transgenic mice with a range of ERFE overexpression (Coffey et al, Blood 2022) and showed that they developed dose-dependent iron overload and relative hepcidin deficiency. In the current study, the mid-range ERFE-overexpressing E(M) mice were crossed with HbbTh3/+ (Th3) mice, a widely used model of β-thalassemia, to generate mice with a single copy of both transgenes (T-E(M)), as well as littermate controls (Th3, WT, E(M)), to analyze the effect of ERFE on the pathophysiology of ineffective erythropoiesis. The offspring had a noticeably skewed genotypic ratio, with T-E(M) pups making up only <9% of surviving pups compared to 25% expected for each genotype. At embryonic day 18.5, however, there was an equal distribution of all expected genotypes, suggesting that the mortality in T-E(M) pups is postnatal. Surviving T-E(M) mice were analyzed at 16 weeks of age. They had similar erythrocyte parameters to the Th3 littermates , with the expected decrease in red blood cell count, hemoglobin and mean corpuscular volume. Despite the similarities in blood counts, we discovered a synergistic effect of the ERFE and Th3 transgenes on ERFE production in the T-E(M) mice as indicated by increased serum ERFE levels (Figure 1a) and increased Erfe mRNA levels in the bone marrow and spleen compared with both E(M) and Th3 littermates. The dramatic increase in serum ERFE in T-E(M) mice may result from multiplicative effects of erythroblast expansion in Th3 mice and transgenic ERFE overexpression in individual erythroblasts of T-E(M) mice. The large range of ERFE concentrations in human patients may also reflect the combined effects of erythroblast number and ERFE overproduction by erythropoietin-stimulated thalassemic erythroblasts, both of which vary depending on patient genetics. T-E(M) mice also have significantly larger spleens compared to Th3 littermates, suggesting that ERFE overexpression contributes to increased extramedullary hematopoiesis in T-E(M) spleens. In addition, T-E(M) mice had significantly higher iron levels in the liver (Figure 1b) as well as serum and kidney compared to Th3 littermates. Coupled with the inappropriately low serum hepcidin levels (compared to liver iron levels) in T-E(M) mice, even when compared to E(M) and Th3 littermates, these data indicate that excessive ERFE further increases iron loading in a mouse model of β-thalassemia. Our data demonstrate that elevated ERFE levels contribute to the pathogenesis of β-thalassemia by decreasing postnatal survival, increasing iron overload, and increasing extramedullary hematopoiesis. These results suggest that neutralizing excessive ERFE levels may have therapeutic benefits for postnatal development and prevention of iron overload in β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Multiomics Reveal Hypomethylating Agent Partially Restores Hematopoiesis in a LR-MDS Patient at the Cost of Leukemic Transformation

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis. Hypomethylating agents (HMA) such as decitabine and azacytidine (AZA) induce clinical responses in both lower-risk (LR) and higher-risk groups; however, the treatment eventually fails and can be followed by leukemic transformation. The mechanism underlying transient clinical benefit from low-dose HMA (restoration of hematopoiesis) is not fully understood. Here we used cutting-edge single-cell multi-omic approaches to describe the abnormal clonal architecture and transcriptome in LR-MDS and how they are altered following HMA therapy. Results First, we defined genomic differences between aged and young healthy hematopoietic stem and progenitor cells (HSCPs) by building a single-cell atlas of human HSCP with molecularly-defined concentrations of relevant CITE seq antibodies. 350k high-quality bone marrow nucleated cells were obtained from four young donors; varying race and gender. A 6-fold molecular titration of 278 CITE-seq antibodies determined those which are functional and which contribute to clustering. Next, we repeated the captures on samples from aged healthy people (n=3), MDS patients (n=4) and conducted differential analysis on cell states and transcriptomes. Compared to young marrow, healthy marrow from elderly donors revealed global upregulation of inflammatory signals and innate immunity. In erythroid progenitors (ERP) we identified abnormally high expression of the transcription factors KLF1 and GATA1, but lower than normal expression of their hemoglobin target genes. At the same time altered erythropoiesis in the elderly appeared to be compensated by increased frequency of multilineage progenitors and more mature erythroblasts mitigating clinical anemia. Comparing MDS to aged marrow, while most cell populations from MDS showed significant gene expression changes, the amplitude was dwarfed by abnormal mRNA processing gene expression in ERP and erythroblasts. Moreover, in de novo LR-MDS samples and an anemia sample (without known mutations) further downregulation of hemoglobin genes in the erythroid lineage was accompanied by decreased erythroblast numbers leading to anemia. To study the impact of AZA on LR-MDS, we longitudinally profiled a single MDS patient (pre-HMA, post-HMA, sAML transformation, cycle 2 VEN+AZA) (Panel 1). We found that 6 cycles of AZA treatment depleted HSC; whereas lymphoid-primed multipotential progenitors (LMPP), megakaryocyte-erythroid progenitors (MEP), and less mature erythroblasts were enriched after AZA therapy (Panel 2 left). Using genotyping of the transcriptome (GoT) (Panel 2 right), we defined cells with mutations to find that not only SRSF2-relevant pathways (eg. mRNA processing, and mRNA splicing) are molecularly corrected (downregulated) but also there are fewer mutant cells among primitive stem cells and lineage-committed progenitors. However, mutant ERP and erythroblasts accumulated and pathways were more active suggesting the blockade of differentiation at terminal erythropoiesis is not rescued, consistent with the clinical observation that the patient was still RBC transfusion-dependent despite platelet recovery. Notably, these mutant erythroid cells, while defective, never transformed to leukemia. The expansion of mutant multilineage progenitors and LMPP preceded AML transformation and were eliminated (as evidenced by flow and CITE-seq) following two cycles of VEN+AZA. Conclusions: Age-matched references are critical in differential analysis of hematological diseases like AML or MDS. With matched elderly donor data sets, we were able to molecularly isolate functional disruption of erythropoiesis and megakaryopoiesis underlying anemia and thrombocytopenia, respectively, observed in an LR-MDS patient. AZA rescued the dysregulated signals in SRSF2-mutated LR-MDS patient HSPCs, but not in ERP and erythroblasts, and therefore only partially restored hematopoiesis. However, as AZA drove differentiation of both wildtype and mutant HSCP, accumulation of mutant multilineage progenitors and LMPPs contributed to progression to AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Local and Systemic Overexpression of COMP-Ang1 Induces Ang1/Tie2-Related Thrombocytopenia and SDF-1/CXCR4-Dependent Anemia.

While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.

Оценка возможной генотоксичности трех кормовых добавок, рекомендуемых для аквакультуры, методом микроядерного теста на эритроцитах Danio rerio

Based on the internal study results there has been examined the effect of three feed additives of different functional status (butyric acid, organomineral chelate compounds, lycopene) on occurrence of micronuclei (MN) and other nuclear anomalies (NA) in Danio rerio erythrocytes. Evaluation of the genotoxicity of butyric acid didn’t show any genotoxic effect throughout the experiment. The highest frequency of MN occurrence was recorded on the 5th day of the experiment at a concentration of 1 mg/l and amounted to 0.28%. When using organomineral chelate compounds in feed composition, it was also not found that the threshold values for the occurrence of MN (5/1 000 cells) were exceeded. Lycopene showed the pronounced antigenotoxic properties expressed in a decrease in the occurrence of MN and NA up to the control values, which is significantly lower than in testing other feed additives. The data obtained helped to find out that when using all the studied feed additives on the 5th day of the experiment the frequency of occurrence of NA significantly increased and then decreased to the control values. This effect may be explained by three factors: adaptation of fish to a new diet; increased erythropoiesis and greater number of erythroblasts in the peripheral blood; high affinity of the chelating agent with trace element ions. The biosafety tests of three feed additives on the Danio rerio model object showed the absence of a genotoxic effect &#x0D; in the entire range of concentrations studied. Consequently, these functional additives can be recommended for including into food products.

Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling.

SUMMARYCHD8 is an ATP-dependent chromatin-remodeling factor whose monoallelic mutation defines a subtype of autism spectrum disorders (ASDs). Previous work found that CHD8 is required for the maintenance of hematopoiesis by integrating ATM-P53-mediated survival of hematopoietic stem/progenitor cells (HSPCs). Here, by using Chd8F/FMx1-Cre combined with a Trp53F/F mouse model that suppresses apoptosis of Chd8−/− HSPCs, we identify CHD8 as an essential regulator of erythroid differentiation. Chd8−/− P53−/− mice exhibited severe anemia conforming to congenital dyserythropoietic anemia (CDA) phenotypes. Loss of CHD8 leads to drastically decreased numbers of orthochromatic erythroblasts and increased binucleated and multinucleated basophilic erythroblasts with a cytokinesis failure in erythroblasts. CHD8 binds directly to the gene bodies of multiple Rho GTPase signaling genes in erythroblasts, and loss of CHD8 results in their dysregulated expression, leading to decreased RhoA and increased Rac1 and Cdc42 activities. Our study shows that autism-associated CHD8 is essential for erythroblast cytokinesis.

PB2204: THE ROLE OF HES6 IN NORMAL HUMAN HEMATOPOIESIS

Background: The Notch signaling pathway is an important regulator of normal hematopoiesis and is often involved in leukemia. Some members of the HES gene family of transcription factors, like HES1 and HES4, are known Notch targets and have important roles in hematopoiesis. HES6, on the other hand, does not seem to be Notch dependent and has been poorly studied in the hematopoietic system. Interestingly, in the context of neurogenesis, it has been shown that HES6 can inhibit HES1 function, raising the hypothesis that HES6 might influence canonical Notch signaling activity. Within the hematopoietic system, HES6 has been described to be overexpressed in BPDCN, a very rare and aggressive hematological malignancy of plasmacytoid dendritic cells (pDCs). Also other studies have suggested the involvement of HES6 in cancer, including glioma, colorectal cancer, prostate cancer and uveal melanoma, and this mostly relates to a possible role for HES6 in cell proliferation. Aims: Given that HES6 is poorly studies within the hematopoeitic system, this study aims to unravel the role of HES6 in normal human hematopoiesis. Methods: We analyzed published datasets to examine the expression pattern of HES6 in different blood cell types. Through shRNA-mediated knockdown, we studied the impact of HES6 knockdown on the differentiation of CD34+ hematopoietic stem cells from cord blood to various hematopoietic lineages using in vitro stromal co-cultures and colony-forming unit assays. These experiments were analyzed using flow cytometry for the evaluation of differentiation, and cell counts for the evaluation of cell proliferation. Results: HES6 is highly expressed in megakaryocyte-erythroid progenitors (MEPs) and to a lesser extent in NK cells, pDCs and T cells. When exploring the expression in developing T cells, we observed that HES6 expression is inversely correlated with the expression of HES1. Considering the high HES6 expression in MEPs, we particularly studied the role of this gene during erythroid/megakaryocyte development. In our stromal cultures, HES6 expression remained high in developing erythroblasts and was low in megakaryocytes. Upon HES6 knockdown in CD34+ cells, a significant reduction in the number of megakaryocytes (50-85%) and erythroblasts (75-85%) was observed. The effect of HES6 knockdown in the was significant, with a 55-80%.reduction of colony formation. The number of cells per colony was also reduced by approximately 50%. HES6 knockdown during the development of other lineages also appears to impact cell proliferation but this effect is less obvious and needs further exploration. Summary/Conclusion: Consistent with the high expression of HES6 in MEPs, we observed significantly reduced cell proliferation upon HES6 knockdown during erythroid/mekaryocyte differentiation, with the most distinct effect on erythroblasts. HES6 knockdown also seems to impact the differentiation potential of precursor cells towards the myeloid lineages, as shown through colony-forming assays. Although the impact of HES6 on other hematopoietic lineages needs to be explored further, our results do provide the first clear insights into the functional role of HES6 in human hematopoiesis.

Development of a method for assessing the nutritional supplements biosafety using the micronucleus test on Danio rerio erythrocytes

In this work, based on the results of our own investigation, we studied the effect of the lycopene nutritional supplement on the occurrence of micronuclei (MN) and other nuclear anomalies (NA) in Danio rerio erythrocytes. Lycopene showed pronounced antigenotoxic properties, expressed in a decrease in the frequency of MN and NA occurrence to control values, which is significantly lower than in the study of other nutritional supplements. The obtained data made it possible to reveal that on the 5th day of the experiment, the lycopene food supplement slightly increased the NA incidence, which then decreased to the control values. This effect may be due to two factors: 1) fish adaptation to a new feeding diet; 2) an increase in the rate of erythropoiesis and the number of erythroblasts in the peripheral blood. The lycopene tests on the Danio rerio model object showed the absence of a genotoxic effect in the entire range of concentrations studied. Thus, this functional nutritional supplement can be recommended for inclusion in food products.

Erythroblasts in the Vessels of the Placenta – An Independent Factor of Chronic Hypoxic Damage to the Fetus

The aim is a comparative histological study of the relative number of fetal erythroblasts in the vessels of the placentas from a full term pregnancy with a low and high risk of fetal hypoxic damage. Material and methods. Based on data on the course of pregnancy, the state of health of the mother and the fetus/newborn, as well as histological examination of the placenta, 388 archived placenta tissue samples were selected in 2 groups: a high risk group for chronic hypoxic damage to the fetus and a group without clinical and laboratory signs of fetal/newborn hypoxia. The relationship between the number of erythroblasts in the vessels of the placenta and chronic hypoxic damage to the fetus was analyzed. Results: The high risk of chronic hypoxic fetal damage is higher for placentas with ≥8 fetal erythroblasts in chorionic villi vessels (OR=3.175; 95% CI =1.921-5.248, p&lt;0.001), with maternal vascular malperfusion (OR=2.798; 95% CI = 1.506-5.164, p=0.001) and combined (cross) placental lesions (OR=2.245; 95%CI=1.246-4.046, p =0.007) with damage of ≥30% of placental tissue. Conclusion: 8 or more fetal erythroblasts in the lumen of the vessels of the placenta is an additional independent factor in chronic hypoxic damage to the fetus. These results are of practical importance for identifying a group of newborns with a high risk of chronic hypoxic damage in the perinatal period and stratification of the risk group in the postnatal period in order to reduce infant morbidity and mortality.

Zkscan3 affects erythroblast development by regulating the transcriptional activity of GATA1 and KLF1 in mice

ZKSCAN3 encodes a zinc-finger transcription factor that regulates the expression of important genes and plays a significant role in tumor development, pathogenesis, and metastasis. However, its biological functions under normal physiological conditions remain largely unknown. In our previous studies, using flow cytometry, we found that the deletion of Zkscan3 may cause abnormal erythropoiesis. In this study, we found that, in a Zkscan3 knockout mice model, the number of splenic early-stage (basophilic-erythroblasts) and late-stage (chromatophilic-erythroblasts to polychromatophilic-erythroblasts through orthochromatophilic-erythroblasts) erythroblasts increased, whereas the number of late erythroblasts in the bone marrow decreased. Moreover, the phenotype was exacerbated after treating mice with phenylhydrazine (PHZ), which causes severe hemolytic anemia. In the knockout mice treated with PHZ, the percentage of reticulocyte in the peripheral blood conspicuously increased, whereas MCHC and red blood cells decreased. Then, we performed RNA-seq and quantitative-polymerase chain reaction assay and found that the expression of GATA1 and Tiam1 in erythroblasts were upregulated, whereas KLF1 was downregulated. Luciferase assays showed that Zkscan3 inhibited the transcription of GATA1 and Tiam1 and promoted the expression of KLF1. Additionally, ChIP and CO-IP results confirmed that Zkscan3 directly interacts with GATA1 and inhibits its transcriptional activity in MEL cells. Our results demonstrate, for the first time, the significant role of Zkscan3 in physiological erythropoiesis through the interaction with GATA1, both at the DNA and protein level, and with Tiam1 and KLF1 at the DNA level.

MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients’ erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.

The Condensin II Complex Subunit NCAPH2 Is Essential for Erythropoiesis and Regulates Erythroid Gene Expression

Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts.To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value <0.05). As expected, the downregulated genes were enriched for pathways related to cell cycle, such as Mitotic Spindle Organization (adj pvalue 5e-42). The upregulated genes were enriched for a variety of pathways including p53 transcriptional network (adj pvalue 4e-10), neutrophil mediated immunity (2e-9), DNA-binding transcription factor (adj pvalue 7e-5), and regulation of erythrocyte differentiation (adj pvalue 5e-4). Intriguingly, 91/340 genes differentially expressed in Setd8 mutant erythroblats were also differentially expressed in NCAPH2 mutant cells, including genes typically repressed early in erythroid commitment, such as GATA2 and SPI1. Cut&Tag in CD34+ derived erythroblasts demonstrated occupancy of H4K20me1 at these loci. Mass spectrometry of proteins isolated via mono-mehtylated H4K20 peptides in erythroid extracts identified Condensin II components, supporting a model where the Condensin II complex directly interacts with H4K20me1. Together, these results demonstrate that the Condensin II complex is essential for erythropoiesis, and may work in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts. DisclosuresNo relevant conflicts of interest to declare.

Comparative Characterization of the Cellular Composition of the Black Scorpionfish (Scorpaena porcus L.) Hematopoietic Organs during the Spawning Season and the Period of Reproductive Inactivity

Hematopoiesis in teleosts has a number of characteristics that are not fully understood. In the present work, the cellular composition of the hematopoietic organs (head kidney and spleen) of the black scorpionfish during the spawning season and the period of reproductive inactivity was studied using light microscopy. The morphology and the percentage of blood cells were described. The head kidney was shown to be the main hematopoietic organ of the black scorpionfish: immature blood cells of all hematopoietic lines at the different stages of differentiation were observed there. They were divided into 3 clusters depending on the average cell diameter. Lymphocytes, thrombocytes and colony-forming cells, the precursors for all types of blood cells, were observed within the cluster of small cells. The intermediate-size cluster comprised blast forms (erythroblasts and blasts of white blood cells). The large-size cluster consisted of maturing granulocytes, monocytes, macrophages, plasma cells, and mature erythrocytes. The spleen mainly contained mature erythrocytes, showing signs of senescence, and erythrocyte ghosts. Therefore, it was concluded that the spleen of the black scorpionfish performs the function of depositing and utilizing erythrocytes. The study also demonstrated the seasonal dynamics of hematopoiesis. The increase in the number of erythroblasts was recorded in the head kidney of spawning individuals. Erythroblasts were also found in the spleen, in spite of their total absence in the reproductively inactive fish. Consequently, the spleen of the black scorpionfish is an organ of secondary erythropoiesis, which functions when the hematopoietic capacity of the kidneys is insufficient

Apoptosis Imaging in Oncology by Means of Positron Emission Tomography: A Review.

To date, a wide variety of potential PET-apoptosis imaging radiopharmaceuticals targeting apoptosis-induced cell membrane asymmetry and acidification, as well as caspase 3 activation (substrates and inhibitors) have been developed with the purpose of rapidly assessing the response to treatment in cancer patients. Many of these probes were shown to specifically bind to their apoptotic target in vitro and their uptake to be enhanced in the in vivo-xenografted tumours in mice treated by means of chemotherapy, however, to a significantly variable degree. This may, in part, relate to the tumour model used given the fact that different tumour cell lines bear a different sensitivity to a similar chemotherapeutic agent, to differences in the chemotherapeutic concentration and exposure time, as well as to the different timing of imaging performed post-treatment. The best validated cell membrane acidification and caspase 3 targeting radioligands, respectively 18F-ML-10 from the Aposense family and the radiolabelled caspase 3 substrate 18F-CP18, have also been injected in healthy individuals and shown to bear favourable dosimetric and safety characteristics. However, in contrast to, for instance, the 99mTc-HYNIC-Annexin V, neither of both tracers was taken up to a significant degree by the bone marrow in the healthy individuals under study. Removal of white and red blood cells from the bone marrow through apoptosis plays a major role in the maintenance of hematopoietic cell homeostasis. The major apoptotic population in normal bone marrow are immature erythroblasts. While an accurate estimate of the number of immature erythroblasts undergoing apoptosis is not feasible due to their unknown clearance rate, their number is likely substantial given the ineffective quote of the erythropoietic process described in healthy subjects. Thus, the clinical value of both 18F-ML-10 and 18F-CP18 for apoptosis imaging in cancer patients, as suggested by a small number of subsequent clinical phase I/II trials in patients suffering from primary or secondary brain malignancies using 18F-ML-10 and in an ongoing trial in patients suffering from cancer of the ovaries using 18F-CP18, remains to be proven and warrants further investigation.

Single-cell analysis of erythropoiesis in Rpl11 haploinsufficient mice reveals insight into the pathogenesis of Diamond–Blackfan anemia

Rpl11 haploinsufficient mice develop a macrocytic anemia similar to patients with DBA. Here, we fully characterize this model from clinical and pathophysiological perspectives. Early erythroid precursors have increased heme content and high cytoplasmic reactive oxygen species, impairing erythroid differentiation at the colony-forming unit-erythroid (CFU-E)/proerythroblast stage and subsequently. Using single-cell analyses that link a cell's surface protein expression to its total transcriptome and unbiased analyses, we found GATA1, GATA1 target gene, and mitotic spindle pathway gene transcription were the pathways that decreased the most. Expression of ribosome protein and globin genes was amplified. These changes, as well as the other transcriptional changes that were identified, closely resemble findings in mice that lack the heme export protein FLVCR and, thus, suggest that heme excess and toxicity are the primary drivers of the macrocytic anemia. Consistent with this, treating Rpl11 haploinsufficient mice with corticosteroids increased the numbers of earliest erythroblasts but failed to overcome heme toxicity and improve the anemia. Rpl11 haploinsufficient mice uniquely upregulated mitochondrial genes, p53 and CDKN1A pathway genes, and DNA damage checkpoint genes, which should contribute further to erythroid marrow failure. Together our data establish Rpl11 haploinsufficient mice as an excellent model of DBA that can be used to study DBA pathogenesis and test novel therapies.

Red cell manufacturing using parallel stirred-tank bioreactors at the final stages of differentiation enhances reticulocyte maturation.

The aim of this study was to develop a robust, quality controlled, and reproducible erythroid culture system to obtain high numbers of mature erythroblasts and red blood cells (RBCs). This was achieved using a fully controlled stirred-tank bioreactor by the design of experiments (DOE) methods in the serum-free medium by defining the appropriate culture parameters. Human cord blood CD34+ cells were first cultured in static flasks and then inoculated to stirred-tank bioreactors. Cell diameter was gradually decreased and final RBC yields were significantly higher when cells were inoculated at sizes smaller than 12 μm. The larger immature cells in the basophilic stage did not survive, while smaller mature erythroid cells were successfully expanded at high agitation speeds, demonstrating that appropriate seeding timing is critical. A high inoculation cell density of 5 × 106 cells/ml was achieved reaching 1.5 × 107 cells/ml. By using DOE analysis fitted to precise stages of erythropoiesis, we were able to acquire the optimal culture parameters for pH (7.5), temperature (37°C), dissolved oxygen, agitation speed (500 rpm), inoculation timing (cell diameter 12-13 μm), media feeding regimen, and cell seeding density (5 × 106 cells/ml). The final pure RBCs showed appropriate functions compared with fresh donor RBCs, confirming that manufacturing mature RBCs with reproducibility is possible.

TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis

AbstractThe impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34+ hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin Ainterm) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin Adim erythroid cells nor mature (day 14) glycophorin Abright erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis.

The donor of carbon monoxide (CORM-2) affects the level of serum immunoglobulins and the state of the bone marrow during the immune response in mice

Toxic carbon monoxide in small concentrations has pro-apoptotic, anti-allergic, vasodilator effects, and stimulates angiogenesis. The problem with the therapeutic use of low doses of carbon monoxide is that it is difficult to dose. To control the amount and gradual release of carbon monoxide, non-toxic preparation is used - CO donor based on carbonyl compound of ruthenium (CORM-2).The aim – is to identify the effect of CORM-2 on the level of immunoglobulins in the blood serum and bone marrow of mice under conditions of inducing an immune response.Materials and methods. 3 groups of 15 white laboratory mice each were formed. Induction of the immune response was due to the intraperitoneal administration of xenogenic red blood cells. The first experimental group on the first day of immunization received CORM-2 (20 mg/kg), group No. 2 – on the 5th day after immunization (period of the productive phase). The control group consisted of immunized animals that did not receive CORM-2. The amount of IgA, IgM, and IgG in blood serum was determined by ELISA on the 2nd and 6th day after immunization. At the end of the experiment, bone marrow cell populations were counted.Results. After the injection of CORM-2 during the induction phase of the immune response, it inhibits the production of immunoglobulins. In comparison with the control, the level of IgA and IgG is reduced, but the amount of IgM remains unchanged. In the bone marrow, the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, increased. At the same time, the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes decreased. The use of CORM-2 during the productive phase caused a decrease in the level of IgM and IgG with a simultaneous increase in the level of IgA. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes decreased.Conclusions. The impact of the CORM-2 on the inductive phase of the immune response leads to inhibition of the production of immunoglobulins. The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, but at the same time, an increase in the level of IgA was observed. After the injection of CORM-2, in the bone marrow, the number of monocytes, lymphocytes, and plasma cells increased. The results indicate that CORM-2 is able to modulate the immune response.