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.

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.

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.

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.

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.

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).

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.

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.

Aferição da taxa de eritroblastos e dos parâmetros do equilíbrio ácido-básico no sangue da veia umbilical no nascimento: capacitação da enfermagem na aplicação da metodologia / Measurement of the erythroblast level and acid-base paramethers in the umbilical vein blood at delivery: nursing qualification in the methodology aplication

Objetivo: Identificar os fatores que dificultam a aferição da taxa dos eritroblastos e dos parâmetros do estado ácido-básico no sangue da veia umbilical e avaliar as repercussões de um treinamento sobre a efetividadeda metodologia. Pacientes e Métodos: Ensaio clínico observacional, com 1204 parturientes divididas em três amostras...