Canonical and Non-Canonical Functions of Erythropoietin and Its Receptor in Mature Nucleated Erythrocytes of Western Clawed Frog, Xenopus tropicalis.

Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, Xenopus tropicalis, mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in Xenopus mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene (cish) and suppressor of cytokine signaling 3 gene (socs3), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (odc1), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased odc1 expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates' nucleated and enucleated erythrocytes.

Production and stability of cultured red blood cells depends on the concentration of cholesterol in culture medium.

The production of cultured red blood cells (cRBC) for transfusion purposes requires large scale cultures and downstream processes to purify enucleated cRBC. The membrane composition, and cholesterol content in particular, are important during proliferation of (pro)erythroblasts and for cRBC quality. Therefore, we tested the requirement for cholesterol in the culture medium during expansion and differentiation of erythroid cultures with respect to proliferation, enucleation and purification by filtration. The low cholesterol level (22 µg/dl) in serum free medium was sufficient to expand (pro)erythroblast cultures. Addition of 2.0 or 5.0 mg/dL of free cholesterol at the start of differentiation induction inhibited enucleation compared to the default condition containing 3.3 mg/dl total cholesterol derived from the addition of Omniplasma to serum free medium. Addition of 5.0 mg/dl cholesterol at day 5 of differentiation did not affect the enucleation process but significantly increased recovery of enucleated cRBC following filtration over leukodepletion filters. The addition of cholesterol at day 5 increased the osmotic resistance of cRBC. In conclusion, cholesterol supplementation after the onset of enucleation improved the robustness of cRBC and increased the yield of enucleated cRBC in the purification process.

Mitochondrial regulation of erythropoiesis in homeostasis and disease.

Erythroid cells undergo a highly complex maturation process, resulting in dynamic changes that generate red blood cells (RBCs) highly rich in haemoglobin. The end stages of the erythroid cell maturation process primarily include chromatin condensation and nuclear polarization, followed by nuclear expulsion called enucleation and clearance of mitochondria and other organelles to finally generate mature RBCs. While healthy RBCs are devoid of mitochondria, recent evidence suggests that mitochondria are actively implicated in the processes of erythroid cell maturation, erythroblast enucleation and RBC production. However, the extent of mitochondrial participation that occurs during these ultimate steps is not completely understood. This is specifically important since abnormal RBC retention of mitochondria or mitochondrial DNA contributes to the pathophysiology of sickle cell and other disorders. Here we review some of the key findings so far that elucidate the importance of this process in various aspects of erythroid maturation and RBC production under homeostasis and disease conditions.

Mechanism of the apoptosis of bone marrow erythroblasts in rats under hypobaric hypoxia.

This study aimed to investigate the mechanism of the apoptosis of erythroblasts in rat bone marrow after the exposure to hypobaric hypoxia. Male SD rats were randomly divided into three groups. The hypoxic group was kept in a hypobaric hypoxia chamber at a simulated altitude of 5000 m for 7 and 28 days, respectively. The control group was kept at an altitude of 2260 m. We found that myeloid: erythroid (M:E) ratio was significantly lower after hypoxia exposure and the proportions of polychromatic erythroblasts and orthochromatic erythroblasts significantly increased compared to control group, along with significant increase in the proportion of CD71+ cells and apoptosis rate. The expression levels of caspase-3, Bax, and Cyt-C in CD71+ cells were higher after hypoxia exposure than those in control group, while there was no significant difference in the expression levels of TNFR and Fas. In conclusion, after exposure to hypobaric hypoxia the proliferation of peripheral blood and bone marrow erythroblasts in rats increased, and apoptosis also increased, indicating that bone marrow erythroblasts in rats is regulated by both proliferation and apoptosis, and the mitochondrial pathway is one of the important pathways for apoptosis.

Membrane-Bound Ferric Hemoglobin in Nucleated Erythrocytes of the Black Scorpionfish Scorpaena porcus, Linnaeus 1758.

The content of membrane-bound methemoglobin (MtHb) in nucleated erythrocytes was studied in the black scorpionfish Scorpaena porcus (Linnaeus, 1758) in vitro. Spectral characteristics were determined for a whole hemolysate, a hemolysate obtained by stroma precipitation (a clarified hemolysate), and a resuspended stroma. The MtHb proportion in the erythrocyte stroma was found to exceed 80% (6.20 ± 0.59 µM). Clarified hemolysates were nearly free of MtHb (0.5 ± 0.2 µM). Membrane-bound ferric hemoglobin did not affect the erythrocyte resistance to osmotic shock. The osmotic fragility range was determined using a LaSca-TM laser microparticle analyzer (BioMedSystems, Russia) to be 102-136 mOsm/kg, much the same as in other bony fish species. A nitrite load (10 mg/L) significantly increased the MtHb content in the blood. However, the membrane-bound ferric hemoglobin content did not change significantly, amounting to 6.34 ± 1.09 µM (approximately 95%). The finding suggested a functional importance for MtHb present in the plasma membrane of nucleated erythrocytes. Membrane-bound MtHb was assumed to neutralize the external oxidative load and the toxic effect of hydrogen sulfide in bottom water layers, where the species lives.

TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia.

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Identification of three mechanistic pathways for iron-deficient heart failure.

Current understanding of iron-deficient heart failure is based on blood tests that are thought to reflect systemic iron stores, but the available evidence suggests greater complexity. The entry and egress of circulating iron is controlled by erythroblasts, which (in severe iron deficiency) will sacrifice erythropoiesis to supply iron to other organs, e.g. the heart. Marked hypoferraemia (typically with anaemia) can drive the depletion of cardiomyocyte iron, impairing contractile performance and explaining why a transferrin saturation < ≈15%-16% predicts the ability of intravenous iron to reduce the risk of major heart failure events in long-term trials (Type 1 iron-deficient heart failure). However, heart failure may be accompanied by intracellular iron depletion within skeletal muscle and cardiomyocytes, which is disproportionate to the findings of systemic iron biomarkers. Inflammation- and deconditioning-mediated skeletal muscle dysfunction-a primary cause of dyspnoea and exercise intolerance in patients with heart failure-is accompanied by intracellular skeletal myocyte iron depletion, which can be exacerbated by even mild hypoferraemia, explaining why symptoms and functional capacity improve following intravenous iron, regardless of baseline haemoglobin or changes in haemoglobin (Type 2 iron-deficient heart failure). Additionally, patients with advanced heart failure show myocardial iron depletion due to both diminished entry into and enhanced egress of iron from the myocardium; the changes in iron proteins in the cardiomyocytes of these patients are opposite to those expected from systemic iron deficiency. Nevertheless, iron supplementation can prevent ventricular remodelling and cardiomyopathy produced by experimental injury in the absence of systemic iron deficiency (Type 3 iron-deficient heart failure). These observations, taken collectively, support the possibility of three different mechanistic pathways for the development of iron-deficient heart failure: one that is driven through systemic iron depletion and impaired erythropoiesis and two that are characterized by disproportionate depletion of intracellular iron in skeletal and cardiac muscle. These mechanisms are not mutually exclusive, and all pathways may be operative at the same time or may occur sequentially in the same patients.

Comparison Evaluation of Automated Nucleated Red Blood Cell Enumeration by Sysmex XN 1000 in Comparison With Microscopic Reference in Children Under 1 Year.

BACKGROUND: In newborns, elevated nucleated red blood cell (NRBC) levels can be associated with enhanced erythropoietic stress and might be predictive for adverse outcome. Also, the presence of NRBC in peripheral blood might lead to erroneous enumeration results of white blood cells in automated hematology analyzers. We aimed to assess the comparability of the Sysmex XN 1000 to manual slide reviews and correlation of NRBC with inflammation markers. METHODS: Specimens of 3397 children under 1 year were compared by automated and microscopic NRBC enumeration. Additionally, potential correlations between NRBC and age and inflammation markers were examined. RESULTS: Overall, there was good correlation (r = 0.97) between automated (range: 0%-3883%) and microscopic enumeration (range: 0%-3694%) of NRBC with high comparability up to a NRBC value of 200% and an increase in the variation between the two methods with increasing NRBC numbers. When 94 samples with ≤ 200% NRBC and ≥ 30% divergence between methods were separately reanalyzed with respect to overlapping cell populations in their scattergrams, Sysmex would have generated unrecognized incorrect automated results in 47 samples, corresponding to 1.4% of total study samples. NRBC counts were negatively correlated to age, but not to inflammation markers. CONCLUSION: Sysmex XN 1000 is highly precise in the enumeration of NRBC in children under 1 year up to counts of 200% and might replace time-intense manual counting in routine diagnostics. In the setting of neonatal and intensive care diagnostics, microscopic control and supervision of scattergrams are highly recommended for any automated NRBC enumeration processes.

Comparative analysis of feature-based ML and CNN for binucleated erythroblast quantification in myelodysplastic syndrome patients using imaging flow cytometry data.

Myelodysplastic syndrome is primarily characterized by dysplasia in the bone marrow (BM), presenting a challenge in consistent morphology interpretation. Accurate diagnosis through traditional slide-based analysis is difficult, necessitating a standardized objective technique. Over the past two decades, imaging flow cytometry (IFC) has proven effective in combining image-based morphometric analyses with high-parameter phenotyping. We have previously demonstrated the effectiveness of combining IFC with a feature-based machine learning algorithm to accurately identify and quantify rare binucleated erythroblasts (BNEs) in dyserythropoietic BM cells. However, a feature-based workflow poses challenges requiring software-specific expertise. Here we employ a Convolutional Neural Network (CNN) algorithm for BNE identification and differentiation from doublets and cells with irregular nuclear morphology in IFC data. We demonstrate that this simplified AI workflow, coupled with a powerful CNN algorithm, achieves comparable BNE quantification accuracy to manual and feature-based analysis with substantial time savings, eliminating workflow complexity. This streamlined approach holds significant clinical value, enhancing IFC accessibility for routine diagnostic purposes.

Unveiling the genetic landscape of suspected congenital dyserythropoietic anemia type I: A retrospective cohort study of 36 patients.

Congenital Dyserythropoietic Anemia type I (CDA I) is a rare hereditary condition characterized by macrocytic/normocytic anemia, splenomegaly, iron overload, and distinct abnormalities during late erythropoiesis, particularly internuclear bridges between erythroblasts. Diagnosis of CDA I remains challenging due to its rarity, clinical heterogeneity, and overlapping phenotype with other rare hereditary anemias. In this case series, we present 36 patients with suspected CDA I. A molecular diagnosis was successfully established in 89% of cases, identifying 16 patients with CDA I through the presence of 18 causative variants in the CDAN1 or CDIN1 genes. Transcriptomic analysis of CDIN1 variants revealed impaired erythroid differentiation and disruptions in transcription, cell proliferation, and histone regulation. Conversely, 16 individuals received a different diagnosis, primarily pyruvate kinase deficiency. Comparisons between CDA I and non-CDA I patients revealed no significant differences in erythroblast morphological features. However, hemoglobin levels and red blood cell count differed between the two groups, with non-CDA I subjects being more severely affected. Notably, most patients with severe anemia belonged to the non-CDA I group (82% non-CDA I vs. 18% CDA I), with a subsequent absolute prevalence of transfusion dependency among non-CDA I patients (100% vs. 41.7%). All patients exhibited reduced bone marrow responsiveness to anemia, with a more pronounced effect observed in non-CDA I patients. Erythropoietin levels were significantly higher in non-CDA I patients compared to CDA I patients. However, evaluations of erythroferrone, soluble transferrin receptor, and hepcidin revealed no significant differences in plasma concentration between the two groups.

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo.

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL-carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically-defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.

Deciphering the regulatory landscape of murine splenic response to anemic stress at single-cell resolution.

ABSTRACT: Stress erythropoiesis can be influenced by multiple mediators through both intrinsic and extrinsic mechanisms in early erythroid precursors. Single-cell RNA sequencing was conducted on spleen tissue isolated from mice subjected to phenylhydrazine and serial bleeding to explore novel molecular mechanisms of stress erythropoiesis. Our results showed prominent emergence of early erythroblast populations under both modes of anemic stress. Analysis of gene expression revealed distinct phases during the development of emerging erythroid cells. Interestingly, we observed the presence of a "hiatus" subpopulation characterized by relatively low level of transcriptional activities that transitions between early stages of emerging erythroid cells, with moderate protein synthesis activities. Moreover, single-cell analysis conducted on macrophage populations revealed distinct transcriptional programs in Vcam1+ macrophages under stress. Notably, a novel marker, CD81, was identified for labeling central macrophages in erythroblastic islands (EBIs), which is functionally required for EBIs to combat anemic stress. These findings offer fresh insights into the intrinsic and extrinsic pathways of early erythroblasts' response to stress, potentially informing the development of innovative therapeutic approaches for addressing anemic-related conditions.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Automated hematological cell count using sysmex XN-1000V in Testudo hermanni: Agreement with manual count.

Mediterranean area represents the main habitat of Testudo hermanni. Clinical signs of disease of these tortoises are non-specific, making the hematology results crucial in revealing underlying pathological conditions. However, accurate automated identification of blood cell populations is hampered by the presence of nucleated erythrocytes (NRBC) and thrombocytes (Thr), necessitating manual methods such as counting chambers. The aim of the study was to assess the performance of the novel automated hematology analyzer Sysmex XN-1000 V, which includes a a specific channel (WNR) for counting NRBC, in accurately identify and quantify the different blood cell populations of Testudo hermanni. Additionally, its agreement with manual counts was evaluated. Fifty heparinized blood samples were initially counted using the Neubauer improved chamber and then analysed twice with Sysmex XN-1000 V. Thirteen out of 50 samples were instrumentally counted again after 48 h to assess the inter-assay precision. All WNR scattergrams were re-analysed using an ad hoc gate panel to differentiate two populations: NRBCs (weak fluorescence signal) and WBC + Thr (high fluorescence signal). Sysmex XN-1000 V demonstrated optimal intra- and inter-assay precision for NRBCs (CV 0.98% ± 1.96; 1.31% ± 2.98) and moderate precision for WBC + Thr (CV 9.24% ± 16.61; 12.69% ± 10.35). No proportional nor constant errors were observed between the methods for both the populations. The instrumental NRBC counts were consistently slightly lower, while WBC + Thr counts were slightly higher compared to manual counts. These findings suggest that Sysmex XN-1000 V can be used for analyzing cell populations in heparinized blood of Testudo hermanni. However, specific instrumental reference intervals are suggested.

Nucleated Red Blood Cell Counts Differentiate Cardiac from Respiratory Causes of Cyanosis at Birth.

Tissue hypoxia increases erythropoietin production and release of immature erythrocytes that can be measured using nucleated red blood cell counts (nRBC). We hypothesized that hypoxia due to congenital heart disease (CHD) is chronic and is better tolerated than hypoxia due to respiratory disease (RD), which is an acute stress in newborns leading to higher nRBC. This study assesses the utility of nRBC as a marker to differentiate hypoxia due to CHD vs RD in term neonates. This was a single-center, retrospective study of term neonates with cyanosis from 2015 to 2022. Neonates < 37 weeks of gestation, with hypoxic-ischemic encephalopathy, and those with other causes of cyanosis were excluded. The patients were divided into 2 groups: cyanotic CHD and cyanotic RD. Clinical and laboratory data done within 12 h and 24-36 h after birth were collected. Data are represented as median and Interquartile range. Of 189 patients with cyanosis, 80 had CHD and 109 had RD. The absolute nRBC count at ≤ 12 h of age was lower in the CHD (360 cells/mm3) compared to RD group (2340 cells/mm3) despite the CHD group having significantly lower baseline saturations. A value of 1070 cells/mm3 was highly sensitive and specific for differentiating CHD from RD. The positive predictive value for this cut-off value of 1070 cells/mm3 was 0.94 and the negative predictive value was 0.89. The absolute nRBC is a simple screening test and is available worldwide. A nRBC < 1070 cells/mm3 in cyanotic newborns should hasten the search for CHD etiology with the possible need for prostaglandin therapy.

NRBC concentrations over time in neonates with moderate to severe neonatal encephalopathy with and without sentinel events.

OBJECTIVE: To study the serum concentrations of nucleated red blood cells (NRBC) over time in neonates with moderate to severe neonatal encephalopathy (NE). STUDY DESIGN: A retrospective cohort study with subjects subdivided into three groups: definite sentinel events (n = 52), probable sentinel events (n = 20) and no history of sentinel events (n = 63). Peak absolute NRBC and NRBC/100 WBC were compared between groups and with MRI Injury score, cord and admission pH/base deficit. RESULTS: Absolute NRBC peaked at 24.05 h after birth (CI: 15.30-32.79), 17.56 h after birth (CI: 7.35-27.77), and 39.81 h after birth (CI: 28.73-50.89) in each respective group. The peak in absolute NRBC correlated with the severity of injury in the grey matter in group 2 and white matter in groups 1 and 2. Higher peak absolute NRBC value correlated to a lower admission ABG pH. CONCLUSION: NRBC peak at 24 h after birth in neonates with sentinel events.

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells.

ABSTRACT: During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

Congenital dyserythropoietic anemia type II in a newborn with a novel compound heterozygous mutation in the SEC23B: a case report and review of the literature.

Congenital dyserythropoietic anemia type II (CDA II) refers to a group of extremely rare heterozygous disorders characterized by ineffective erythropoiesis and morphological abnormalities of erythrocytes and bone marrow erythroblasts. Six types of CDA with differing heterogenous genetic mutations have been identified to date. Due to the genetic and clinical heterogeneity of CDA, accurate diagnosis can be very challenging, especially with the clinical overlap observed between CDA and other dyserythropoietic diseases. A 1-month-old infant girl, born to a non-consanguineous family, presented with severe normocytic anemia that required transfusions every 2 to 3 weeks since birth, as well as jaundice. Whole exome sequencing revealed a novel compound heterozygosity in the SEC23B gene, thus establishing the diagnosis of CDA II. Analysis by multiple bioinformatics tools predicted that the mutant proteins were deleterious. Here, we report a novel variation in SEC23B that extends the mutation spectrum of SEC23B in the diagnosis of CDA II.