Isoquinoline-based intrinsic fluorescence assessment of erythropoiesis-stimulating agent, Roxadustat (FG-4592), in tablets: applications to content uniformity and human plasma evaluation.

In the present study, a first validated and green spectrofluorimetric approach for its assessment and evaluation in different matrices was investigated. After using an excitation wavelength of 345 nm, Roxadustat (ROX) demonstrates a highly native fluorescence at an emission of 410 nm. The influences of experimental factors such as pH, diluting solvents, and different organized media were tested, and the most appropriate solvent choice was ethanol. It was confirmed that there was a linear relationship between the concentration of ROX and the relative fluorescence intensity in the range 60.0-1000.0 ng ml-1, with the limit of detection and limit of quantitation, respectively, being 17.0 and 53.0 ng ml-1. The mean recoveries % [±standard deviation (SD), n = 5] for pharmaceutical preparations were 100.11% ± 2.24%, whereas for plasma samples, they were 100.08 ± 1.08% (±SD, n = 5). The results obtained after the application of four greenness criteria, Analytical Eco-Scale metric, NEMI, GAPI, and AGREE metric, confirmed its eco-friendliness. In addition, the whiteness meter (RGB12) confirmed its level of sustainability. The International Council for Harmonisation (ICH) criteria were used to verify the developed method through the study in both spiked plasma samples and content uniformity evaluation. An appropriate standard for various applications in industry and quality control laboratories was developed.

[Mitochondrial metabolism and erythroid differentiation].

The transcription factor GATA-1 is essential for erythroid differentiation. Recently, FAM210B, which encodes a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1. To clarify the role of FAM210B, we depleted endogenous FAM210B in human iPS-derived erythroid progenitor (HiDEP-1) cells, and found that erythroid differentiation was more pronounced in the FAM210B depleted cells. Comprehensive metabolite analysis revealed a decline in mitochondrial function accompanied by increased lactate production, indicative of anaerobic glycolysis. Mass spectrometry revealed that FAM210B could interact with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. This review will discuss the potential association between mitochondrial metabolism and erythropoiesis.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation.

BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation. METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1. RESULTS: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment. CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

The possible role of the vasopressin system in hematopoiesis.

Vasopressin is a pleiotropic hormone that controls body fluid homeostasis. Vasopressin has also been proposed to be involved in erythropoiesis, thrombocyte activity and inflammation. However, whether increasing vasopressin is associated with changes in hematopoietic markers is not known. To evaluate this gap of knowledge we measured the vasopressin marker copeptin and markers of erythropoiesis (erythrocyte count, hemoglobin (Hb), red blood cell distribution width (RDW), mean corpuscular volume (MCV), erythrocyte volume fraction (EVF)), leukocyte count (total count, lymphocytes, neutrophils) and thrombocyte count in 5312 participants from the Swedish CArdioPulmonary bioImage Study (SCAPIS). The associations between increasing copeptin tertile and the hematopoietic markers were analyzed in multivariate linear regression analyses. We found that increasing copeptin tertile was significantly (p < 0.001) associated with increasing erythrocytes, RDW, EVF, Hb, leukocytes and neutrophils after adjustment for age, sex, current smoking, prevalent diabetes, hypertension, creatinine, body mass index and physical activity. Increasing copeptin tertile was, however, not associated with change in MCV, lymphocyte or thrombocyte count. In conclusion, we found that increasing copeptin levels are positively associated with markers of erythropoiesis and leukocyte count in the general population. These results warrant further research on possible mechanistic effects of vasopressin on hematopoiesis.

The crucial role of NRF2 in erythropoiesis and anemia: Mechanisms and therapeutic opportunities.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor crucial in cellular defense against oxidative and electrophilic stresses. Recent research has highlighted the significance of NRF2 in normal erythropoiesis and anemia. NRF2 regulates genes involved in vital aspects of erythroid development, including hemoglobin catabolism, inflammation, and iron homeostasis in erythrocytes. Disrupted NRF2 activity has been implicated in various pathologies involving abnormal erythropoiesis. In this review, we summarize the progress made in understanding the mechanisms of NRF2 activation in erythropoiesis and explore the roles of NRF2 in various types of anemia. This review also discusses the potential of targeting NRF2 as a new therapeutic approach to treat anemia.

Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis.

During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.

Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19.

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

Impact of anemia requiring transfusion or erythropoiesis-stimulating agents on new-onset cardiovascular events and mortality after continuous renal replacement therapy.

Anemia is common in critically ill patients undergoing continuous renal replacement therapy (CRRT). We investigated the impact of anemia requiring red blood cell (RBC) transfusion or erythropoiesis-stimulating agents (ESAs) on patient outcomes after hospital discharge in critically ill patients with acute kidney injury (AKI) requiring CRRT. In this retrospective cohort study using the Health Insurance Review and Assessment database of South Korea, 10,923 adult patients who received CRRT for 3 days or more between 2010 and 2019 and discharged alive were included. Anemia was defined as the need for RBC transfusion or ESAs. Outcomes included cardiovascular events (CVEs) and all-cause mortality after discharge. The anemia group showed a tendency to be older with more females and had more comorbidities compared to the control group. Anemia was not associated with an increased risk of CVEs (adjusted hazard ratio [aHR]: 1.05; 95% confidence interval [CI]: 0.85-1.29), but was associated with an increased risk of all-cause mortality (aHR: 1.41; 95% CI 1.30-1.53). For critically ill patients with AKI requiring CRRT, anemia, defined as requirement for RBC transfusion or ESAs, may increase the long-term risk of all-cause mortality.

Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Developmental regulation of primitive erythropoiesis.

PURPOSE OF REVIEW: In this review, we present an overview of recent studies of primitive erythropoiesis, focusing on advances in deciphering its embryonic origin, defining species-specific differences in its developmental regulation, and better understanding the molecular and metabolic pathways involved in terminal differentiation. RECENT FINDINGS: Single-cell transcriptomics combined with state-of-the-art lineage tracing approaches in unperturbed murine embryos have yielded new insights concerning the origin of the first (primitive) erythroid cells that arise from mesoderm-derived progenitors. Moreover, studies examining primitive erythropoiesis in rare early human embryo samples reveal an overall conservation of primitive erythroid ontogeny in mammals, albeit with some interesting differences such as localization of erythropoietin (EPO) production in the early embryo. Mechanistically, the repertoire of transcription factors that critically regulate primitive erythropoiesis has been expanded to include regulators of transcription elongation, as well as epigenetic modifiers such as the histone methyltransferase DOT1L. For the latter, noncanonical roles aside from enzymatic activity are being uncovered. Lastly, detailed surveys of the metabolic and proteomic landscape of primitive erythroid precursors reveal the activation of key metabolic pathways such as pentose phosphate pathway that are paralleled by a striking loss of mRNA translation machinery. SUMMARY: The ability to interrogate single cells in vivo continues to yield new insights into the birth of the first essential organ system of the developing embryo. A comparison of the regulation of primitive and definitive erythropoiesis, as well as the interplay of the different layers of regulation - transcriptional, epigenetic, and metabolic - will be critical in achieving the goal of faithfully generating erythroid cells in vitro for therapeutic purposes.

Saracatinib prompts hemin-induced K562 erythroid differentiation but suppresses erythropoiesis of hematopoietic stem cells.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

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.

Macrocytic anemias.

PURPOSE OF REVIEW: Over the last century, the diseases associated with macrocytic anemia have been changing with more patients currently having hematological diseases including malignancies and myelodysplastic syndrome. The intracellular mechanisms underlying the development of anemia with macrocytosis can help in understanding normal erythropoiesis. Adaptations to these diseases involving erythroid progenitor and precursor cells lead to production of fewer but larger red blood cells, and understanding these mechanisms can provide information for possible treatments. RECENT FINDINGS: Both inherited and acquired bone marrow diseases involving primarily impaired or delayed erythroid cell division or secondary adaptions to basic erythroid cellular deficits that results in prolonged cell division frequently present with macrocytic anemia. SUMMARY OF FINDINGS: In marrow failure diseases, large accumulations of iron and heme in early stages of erythroid differentiation make cells in those stages especially susceptible to death, but the erythroid cells that can survive the early stages of terminal differentiation yield fewer but larger erythrocytes that are recognized clinically as macrocytic anemia. Other disorders that limit deoxynucleosides required for DNA synthesis affect a broader range of erythropoietic cells, but they also lead to macrocytic anemia. The source of macrocytosis in other diseases remains uncertain.

Signaling networks guiding erythropoiesis.

PURPOSE OF REVIEW: Cytokine-mediated signaling pathways, including JAK/STAT, PI3K/AKT, and Ras/MAPK pathways, play an important role in the process of erythropoiesis. These pathways are involved in the survival, proliferation, and differentiation function of erythropoiesis. RECENT FINDINGS: The JAK/STAT pathway controls erythroid progenitor differentiation, proliferation, and survival. The PI3K/AKT signaling cascade facilitates erythroid progenitor survival, proliferation, and final differentiation. During erythroid maturation, MAPK, triggered by EPO, suppresses myeloid genes, while PI3K is essential for differentiation. Pro-inflammatory cytokines activate signaling pathways that can alter erythropoiesis like EPOR-triggered signaling, including survival, differentiation, and proliferation. SUMMARY: A comprehensive understanding of signaling networks is crucial for the formulation of treatment approaches for hematologic disorders. Further investigation is required to fully understand the mechanisms and interactions of these signaling pathways in erythropoiesis.

Generation of red blood cells from induced pluripotent stem cells.

PURPOSE OF REVIEW: Human induced pluripotent stem cells (iPSCs) are an attractive source to generate in-vitro-derived blood for use as transfusable and reagent red cells. We review recent advancements in the field and the remaining limitations for clinical use. RECENT FINDINGS: For iPSC-derived red blood cell (RBC) generation, recent work has optimized culture conditions to omit feeder cells, enhance red cell maturation, and produce cells that mimic fetal or adult-type RBCs. Genome editing provides novel strategies to improve cell yield and create designer RBCs with customized antigen phenotypes. SUMMARY: Current protocols support red cell production that mimics embryonic and fetal hematopoiesis and cell yield sufficient for diagnostic RBC reagents. Ongoing challenges to generate RBCs for transfusion include recapitulating definitive erythropoiesis to produce functional adult-type cells, increasing scalability of culture conditions, and optimizing high-density manufacturing capacity.

Zinc Supplementation Enhances the Hematopoietic Activity of Erythropoiesis-Stimulating Agents but Not Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitors.

Since zinc is involved in many aspects of the hematopoietic process, zinc supplementation can reduce erythropoiesis-stimulating agents (ESAs) in patients undergoing hemodialysis. However, it remains unclear whether hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have similar reduction effects. HIF-PHI stabilizes HIF, which promotes hematopoiesis, although HIF-1α levels are downregulated by zinc. This study aimed to investigate the effect of zinc supplementation on the hematopoietic effect of HIF-PHI in patients undergoing hemodialysis. Thirty patients undergoing maintenance hemodialysis who underwent periods of treatment with roxadustat or darbepoetin alfa during the past 3 years were retrospectively observed. Participants who underwent periods with and without zinc supplementation were selected, with nine treated with darbepoetin alfa and nine treated with roxadustat. Similarly to the ESA responsiveness index (ERI), the hematopoietic effect of zinc supplementation was determined by the HIF-PHI responsiveness index (HRI), which was calculated by dividing the HIF-PHI dose (mg/week) by the patient's dry weight (kg) and hemoglobin level (g/L). Zinc supplementation significantly increased ERI (p < 0.05), but no significant change was observed (p = 0.931) in HRI. Although zinc supplementation did not significantly affect HRI, adequate zinc supplementation is required to alleviate concerns such as vascular calcification and increased serum copper during the use of HIF-PHI.

Pas de deux: the coordinated coupling of erythroid differentiation with the cell cycle.

PURPOSE OF REVIEW: Recent work reveals that cell cycle duration and structure are remodeled in lock-step with distinct stages of erythroid differentiation. These cell cycle features have regulatory roles in differentiation, beyond the generic function of increasing cell number. RECENT FINDINGS: Developmental progression through the early erythroid progenitor stage (known as colony-forming-erythroid, or 'CFU-e') is characterized by gradual shortening of G1 phase of the cycle. This process culminates in a key transcriptional switch to erythroid terminal differentiation (ETD) that is synchronized with, and dependent on, S phase progression. Further, the CFU-e/ETD switch takes place during an unusually short S phase, part of an exceptionally short cell cycle that is characterized by globally fast replication fork speeds. Cell cycle and S phase speed can alter developmental events during erythroid differentiation, through pathways that are targeted by glucocorticoid and erythropoietin signaling during the erythroid stress response. SUMMARY: There is close inter-dependence between cell cycle structure and duration, S phase and replication fork speeds, and erythroid differentiation stage. Further, modulation of cell cycle structure and speed cycle impacts developmental progression and cell fate decisions during erythroid differentiation. These pathways may offer novel mechanistic insights and potential therapeutic targets.

Erythropoiesis and estimated fluid volume regulation following initiation of ipragliflozin treatment in patients with type 2 diabetes mellitus and chronic kidney disease: A post-hoc analysis of the PROCEED trial.

AIMS: To analyse the changes in erythropoietic and estimated fluid volume parameters after the initiation of ipragliflozin, a sodium-glucose co-transporter 2 inhibitor, in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD). METHODS: This was a post-hoc analysis of the PROCEED trial, which evaluated the effect of 24-week ipragliflozin treatment on endothelial dysfunction in patients with T2DM and CKD. We evaluated the changes in erythropoietic and estimated fluid volume parameters from baseline to 24 weeks post-treatment in 53 patients who received ipragliflozin (ipragliflozin group) and 55 patients with T2DM and CKD without sodium-glucose co-transporter 2 inhibitors (control group), a full analysis set of the PROCEED trial. RESULTS: The increases in haemoglobin [estimated group difference, 0.5 g/dl; 95% confidence interval (CI), 0.3-0.8; p < .001], haematocrit (estimated group difference, 2.2%; 95% CI, 1.3-3.1; p < .001) and erythropoietin (estimated log-transformed group difference, 0.1; 95% CI, 0.01-0.3; p = .036) were significantly greater in the ipragliflozin group than those in the control group. Ipragliflozin treatment was significantly associated with an increase in erythropoietin, independent of the corresponding change in haemoglobin (ß = 0.253, p < .001) or haematocrit (ß = 0.278, p < .001). Reductions in estimated plasma volume (estimated group difference, -7.94%; 95% CI, -11.6 to -4.26%; p < .001) and estimated extracellular volume (estimated group difference, -181.6 ml; 95% CI, -275.7 to -87.48 ml; p < .001) were significantly greater in the ipragliflozin group than those in the control group. CONCLUSIONS: Erythropoiesis was enhanced and estimated fluid volumes were reduced by ipragliflozin in patients with T2DM and CKD. CLINICAL TRIAL: PROCEED trial (registration number: jRCTs071190054).