Erythroid Progenitor Cells Research Articles

Erythroid progenitor cell modulates cancer immunity: Insights and implications.

The emergence of immunotherapies such as immune checkpoint blockade (ICB) has markedly enhanced cancer treatment outcomes for numerous patients. Nevertheless, the effectiveness of immunotherapy demonstrates substantial variation across different cancer types and individual patients. The immunosuppressive characteristics of the tumor microenvironment (TME) play a crucial role in contributing to this variation. Typically, people focus on cells with immunosuppressive functions in the TME, such as tumor-associated macrophages (TAMs), but research on TAMs alone cannot fully explain the complex structure and composition of the TME. Recent studies have reported that tumors can induce erythroid progenitor cells (EPCs) to exert immunosuppressive functions, not only acting within the TME but also secreting artemin in the spleen to promote tumor progression. In this review, we summarize the recent research on EPCs and tumors in recent years. We elucidate the mechanisms by which EPCs exert immunosuppressive functions in tumor-bearing conditions. In this review, we further propose potential therapeutic strategies targeting EPCs and emphasize the importance of in-depth exploration of the mechanisms by which EPCs regulate tumors and the immune system, as well as the significant clinical value of developing corresponding drugs.

Erythropoiesis and Gene Expression Analysis in Erythroid Progenitor Cells Derived from Patients with Hemoglobin H/Constant Spring Disease.

Hemoglobin H/Constant Spring (Hb H/CS) disease represents a form of non-deletional Hb H disease characterized by chronic hemolytic anemia that ranges from moderate to severe and may lead to transfusion-dependent thalassemia. To study the underlying mechanisms of this disease, we conducted an analysis of erythropoiesis and gene expression in erythroid progenitor cells derived from CD34+ hematopoietic stem/progenitor cells from patients with Hb H/CS disease and normal controls. Twelve patients with Hb H/CS disease and five normal controls were enrolled. Peripheral blood samples were collected to isolate CD34+ hematopoietic stem/progenitor cells for the analysis of cell proliferation and differentiation. Six samples from patients with Hb H/CS disease and three controls were subsequently studied for gene expression by next generation sequencing analysis. Erythroid progenitor cells derived from patients with Hb H/CS disease exhibited a trend towards increased rates of erythroid proliferation and decreased cell viability compared to those from controls. Moreover, erythroid progenitor cells derived from patients with Hb H/CS disease demonstrated delayed terminal differentiation. Gene expression profiling revealed elevated levels of genes encoding molecular chaperones, including the heat shock protein genes (HSPs) and the chaperonin containing TCP-1 subunit genes (CCTs) in the Hb H/CS disease group. In summary, erythroid progenitor cells derived from patients with Hb H/CS disease exhibit a trend towards heightened erythroid proliferation, diminished cell viability, and delayed terminal differentiation. Additionally, the increased expression of genes encoding molecular chaperones was observed, providing information on potential underlying pathophysiological mechanisms.

GATA1 transcription factor targets the gene expression of B19 virus in HEK293 cell line

Backgrond/Aim: B19 virus (B19V) is a single strand DNA virus that has specific tropism to erythroid progenitor cells (EPCs). The virus enters to the cells via P antigen and coreseptors and induces infection and cell apoptosis. GATA1 has a high expression in EPC and is a critical transcription factor for the cells development and differentiation. As human EPCs are the main target of the virus infection that have high expression of GATA-1 as the critical transcription factor, the aim of this study was to investigate the effect of GATA1 cotransfection with B19V genome on expression of the viral mRNAs in HEK293 as non-permissive cell line to the virus that had no mRNA expression of GATA-1. Methods: HEK293 cells were transfected with pHI0 plasmid containing B19V genome and the plasmid of GATA1 genome. The quantity of B19V mRNAs (NS1, 7.5 kDa, 11 kDa) expression was evaluated after 24h of transfection. Results: The results showed a statistically significant increase in fold change expression of (NS1 ∽ 12.3, VP1 ∽ 27.6, 11kb protein ∽ 38) in cotransfected cells with GATA1 and B19 plasmids compare to control group (P<0.05). Conclusion: This research showed transfected cells with GATA1had elevation in expression of the B19V genes mRNAs in a non-permissive cell. This result may show a role of GATA1 as a critical transcription factor in support of the virus infection in EPCs. This suggests that GATA1 may potentially sport B19V replication or gene expression.

Parvovirus B19 in Rheumatic Diseases.

Parvovirus B19 (B19V) is a human pathogen belonging to the Parvoviridae family. It is widely diffused in the population and responsible for a wide range of diseases, diverse in pathogenetic mechanisms, clinical course, and severity. B19V infects and replicates in erythroid progenitor cells (EPCs) in the bone marrow leading to their apoptosis. Moreover, it can also infect, in an abortive manner, a wide set of different cell types, normally non-permissive, and modify their normal physiology. Differences in the characteristics of virus-cell interaction may translate into different pathogenetic mechanisms and clinical outcomes. Joint involvement is a typical manifestation of B19V infection in adults. Moreover, several reports suggest, that B19V could be involved in the pathogenesis of some autoimmune rheumatologic diseases such as rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), or vasculitis. This review provides basic information on the B19 virus, highlights characteristics of viral infection in permissive and non-permissive systems, and focuses on recent findings concerning the pathogenic role of B19V in rheumatologic diseases.

Single-cell proteo-transcriptomic profiling reveals altered characteristics of stem and progenitor cells in patients receiving cytoreductive hydroxyurea in early-phase chronic myeloid leukemia.

Hydroxyurea (HU) is frequently used in the early phase of chronic myeloid leukemia (CML) to achieve cytoreduction prior to tyrosine kinase inhibitor (TKI) therapy. However, its impact on CML stem and progenitor cells (SPC) remains largely unknown. This study utilized targeted proteo-transcriptomic expression data on 596 genes and 51 surface proteins in 60,000 CD14-CD34+ cells from chronic phase CML patients to determine effects of shortterm HU treatment (4-19 days) on CML SPC. Peripheral blood and bone marrow samples were obtained from 17 CML patients eligible for short-term HU treatment (three patients before and after HU; seven patients before HU; and seven patients after HU) and subjected to single-cell CITE-seq and/or flow cytometry analysis. The analysis revealed enhanced frequencies of hemoglobin-expressing (HBA1, HBA2, HBB) erythroid progenitor cells in blood and bone marrow following HU treatment. In addition, there was an accumulation of cell subsets with S/G2/M phase-related gene and protein expression, likely representing cells arrested in, or progressing slowly through, the cell cycle. The increased frequency of cells in S/G2/M phase after HU was observed already among the most immature leukemic stem cells (LSC), and patients with a high fraction of LSC in the S/G2/M phase showed poor responsiveness to TKI treatment. We conclude that short-term HU treatment entails differentiation of erythroid progenitor cells and alters the characteristics of LSC in CML. The results imply that studies of LSC and progenitor populations in CML should take effects of initial HU therapy into account.

MiR-1915-3p regulates megakaryocytic and erythroid differentiation by targeting SOCS4

BackgroundProper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation.MethodsWe utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance.ResultsIn this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p.ConclusionsmiR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs.

Gestational diabetes mellitus affects the differentiation of hematopoietic stem cells in neonatal umbilical cord blood.

There are abundant hematopoietic stem cells (HSCs) in cord blood. It is known that HSCs continue to differentiate to CLP, CMP and erythroid progenitor cells (EPC), EPC ultimately differentiated to platelets and erythrocytes. It has been reported that the proportion of HSCs in cord blood was higher than that in healthy pregnant women, so as the incidence of neonatal polycythemia in gestational diabetes mellitus (GDM) patients. We aimed to investigate whether the hyperglycemic and/or hyperinsulin environment in GDM patients has effects on the differentiation of HSCs into erythrocytes in offspring cord blood. In this study, we collected cord blood from 23 GDM patients and 52 healthy pregnant women at delivery. HSCs, CLP, CMP and EPCs in cord blood of the two groups were identified and quantified by flow cytometry. HSCs were sorted out and treated with glucose and insulin, respectively, and then, the changes of HSCs proliferation and differentiation were detected. Compared to healthy controls, HSCs, CMP and EPC numbers in cord blood from GDM group were significantly increased, while CLP cell number was decreased. The differentiation of HSCs into EPC was promoted after treatment with glucose or insulin. There were more HSCs in the cord blood of GDM group, and the differentiation of HSCs to EPCs was increased. These findings were probably caused by the high-glucose microenvironment and insulin medication in GDM patients, and the HSCs differentiation changes might be influencing factors of the high incidence of neonatal erythrocytosis in GDM patients.

A cellular reporter system to evaluate endogenous fetal hemoglobin induction and screen for therapeutic compounds.

Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.

Targeting erythroid progenitor cells for cancer immunotherapy.

Immunotherapy, especially immune checkpoint blockade therapy, represents a major milestone in the history of cancer therapy. However, the current response rate to immunotherapy among cancer patients must be improved; thus, new strategies for sensitizing patients to immunotherapy are urgently needed. Erythroid progenitor cells (EPCs), a population of immature erythroid cells, exert potent immunosuppressive functions. As a newly recognized immunosuppressive population, EPCs have not yet been effectively targeted. In this review, we summarize the immunoregulatory mechanisms of EPCs, especially for CD45+ EPCs. Moreover, in view of the regulatory effects of EPCs on the tumor microenvironment, we propose the concept of EPC-immunity, present existing strategies for targeting EPCs, and discuss the challenges encountered in both basic research and clinical applications. In particular, the impact of existing cancer treatments on EPCs is discussed, laying the foundation for combination therapies. The aim of this review is to provide new avenues for improving the efficacy of cancer immunotherapy by targeting EPCs.

Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages.

Conventional treatments exhibit various side effects on chronic stress anemia. Extramedullary stress erythropoiesis is a compensatory mechanism, which may effectively counteract anemia. Angelica sinensis polysaccharides (ASP) are the main active ingredient found in Angelica sinensis and exhibit antioxidant and hematopoietic effects. However, the effects of ASP on extramedullary stress erythropoiesis remain to be unclear. Here, we demonstrated the protective effects of ASP on chemotherapeutic drug 5-fluorouracil (5-FU)-induced decline in peripheral blood parameters such as RBC counts, HGB, HCT, and MCH, and the decline of BFU-E colony enumeration in the bone marrow. Meanwhile, ASP promoted extramedullary erythropoiesis, increasing cellular proliferation in the splenic red pulp and cyclin D1 protein expression, abrogating phase G0/G1 arrest of c-kit+ cells in mouse spleen. RT-qPCR and immunohistochemistry further revealed that ASP increased macrophage chemokine Ccl2 genetic expression and the number of F4/80+ macrophages in the spleen. The colony-forming assay showed that ASP significantly increased splenic BFU-E. Furthermore, we found that ASP facilitated glycolytic genes including Hk2, Pgk1, Pkm, Pdk1, and Ldha via PI3K/Akt/HIF2α signaling in the spleen. Subsequently, ASP declined pro-proinflammatory factor IL-1β, whereas upregulating erythroid proliferation-associated genes Gdf15, Bmp4, Wnt2b, and Wnt8a. Moreover, ASP facilitated EPO/STAT5 signaling in splenic macrophages, thus enhancing erythroid lineage Gata2 genetic expression. Our study indicated that ASP may improve glycolysis, promoting the activity of splenic macrophages, subsequently promoting erythroid progenitor cell expansion. Additionally, ASP facilitates erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling; suggesting it might be a promising strategy for stress anemia treatment.

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)—The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells

Background aimsEx vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. MethodsWe generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. ResultsMorphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5–7 days. ConclusionsOur results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGFβ Expression and Regulatory T-cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells.

CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy.

THE ROLE OF ERYTHROPOIETIN IN A RAT MODEL OF RENAL ISCHEMIA/ REPERFUSION INJURY

Objective: A powerful activator of erythroid progenitor cells, erythropoietin (EPO) is markedly elevated during hypoxia. A major cause of renal cell death is renal ischemia caused by artery blockage or organ transplantation, and reperfusion exacerbates the damage. The study aimed to investigate the effect of EPO treatment on renal injury following ischemia and reperfusion (I/R). Method: Thirty rats assigned to five groups of six rats each as control, EPO, ischemia, ischemia/reperfusion (I/R) and I/R+EPO.The renal tissue samples were evaluated in terms of hematoxylin-eosin (H&E) staining for histopathological changes, immunoexpression of Beclin-1 for autophagy, and the TUNEL assay for apoptosis. Results: The H&E staining showed the impairment in the tubular epithelium, glomerular and peritubular hemorrhage in the renal tissues of I/R group. Less histopathological changes were observed in I/R + EPO group. Renal tissue Beclin-1 immunoexpression and TUNEL positive cells were significantly increased in the I/R group compared with the others (p

A Review of Key Regulators of Steady-State and Ineffective Erythropoiesis.

Erythropoiesis is initiated with the transformation of multipotent hematopoietic stem cells into committed erythroid progenitor cells in the erythroblastic islands of the bone marrow in adults. These cells undergo several stages of differentiation, including erythroblast formation, normoblast formation, and finally, the expulsion of the nucleus to form mature red blood cells. The erythropoietin (EPO) pathway, which is activated by hypoxia, induces stimulation of the erythroid progenitor cells and the promotion of their proliferation and survival as well as maturation and hemoglobin synthesis. The regulation of erythropoiesis is a complex and dynamic interaction of a myriad of factors, such as transcription factors (GATA-1, STAT5), cytokines (IL-3, IL-6, IL-11), iron metabolism and cell cycle regulators. Multiple microRNAs are involved in erythropoiesis, mediating cell growth and development, regulating oxidative stress, erythrocyte maturation and differentiation, hemoglobin synthesis, transferrin function and iron homeostasis. This review aims to explore the physiology of steady-state erythropoiesis and to outline key mechanisms involved in ineffective erythropoiesis linked to anemia, chronic inflammation, stress, and hematological malignancies. Studying aberrations in erythropoiesis in various diseases allows a more in-depth understanding of the heterogeneity within erythroid populations and the development of gene therapies to treat hematological disorders.

Neuronal nitric oxide synthase required for erythropoietin modulation of heart function in mice.

Introduction: Erythropoietin (EPO) acts primarily in regulating red blood cell production mediated by high EPO receptor (EPOR) expression in erythroid progenitor cells. EPO activity in non-erythroid tissue is evident in mice with EPOR restricted to erythroid tissues (ΔEPORE) that become obese, glucose-intolerant, and insulin-resistant. In animal models, nitric oxide synthase (NOS) contributes to EPO activities including erythropoiesis, neuroprotection, and cardioprotection against ischemia-reperfusion injury. However, we found that extended EPO treatment to increase hematocrit compromised heart function, while the loss of neuronal NOS (nNOS) was protective against the deleterious activity of EPO to promote heart failure. Methods: Wild-type (WT) mice, ΔEPORE mice, and nNOS-knockout mice (nNOS-/-) were placed on a high-fat diet to match the ΔEPORE obese phenotype and were treated with EPO for 3weeks. Hematocrit and metabolic response to EPO treatment were monitored. Cardiac function was assessed by echocardiography and ultrasonography. Results: ΔEPORE mice showed a decrease in the left ventricular outflow tract (LVOT) peak velocity, ejection fraction, and fractional shortening, showing that endogenous non-erythroid EPO response is protective for heart function. EPO treatment increased hematocrit in all mice and decreased fat mass in male WT, demonstrating that EPO regulation of fat mass requires non-erythroid EPOR. EPO treatment also compromised heart function in WT mice, and decreased the pulmonary artery peak velocity (PA peak velocity), LVOT peak velocity, ejection fraction, and fractional shortening, but it had minimal effect in further reducing the heart function in ΔEPORE mice, indicating that the adverse effect of EPO on heart function is not related to EPO-stimulated erythropoiesis. ΔEPORE mice had increased expression of heart failure-associated genes, hypertrophic cardiomyopathy-related genes, and sarcomeric genes that were also elevated with EPO treatment in WT mice. Male and female nNOS-/- mice were protected against diet-induced obesity. EPO treatment in nNOS-/- mice increased the hematocrit that tended to be lower than WT mice and decreased the PA peak velocity but did not affect the LVOT peak velocity, ejection fraction, and fractional shortening, suggesting that nNOS is required for the adverse effect of EPO treatment on heart function in WT mice. EPO treatment did not change expression of heart failure-associated gene expression in nNOS-/- mice. Discussion: Endogenous EPO has a protective effect on heart function. With EPO administration, in contrast to the protective effect to the cardiac injury of acute EPO treatment, extended EPO treatment to increase hematocrit in WT mice adversely affected the heart function with a corresponding increase in expression of heart failure-associated genes. This EPO activity was independent of EPO-stimulated erythropoiesis and required EPOR in non-erythroid tissue and nNOS activity, while nNOS-/- mice were protected from the EPO-associated adverse effect on heart function. These data provide evidence that nNOS contributes to the negative impact on the heart function of high-dose EPO treatment for anemia.

Long noncoding RNA GATA2AS influences human erythropoiesis by transcription factor and chromatin landscape modulation

AbstractLong noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.

Abcb10 regulates murine hematopoietic stem cell potential and erythroid differentiation

Erythropoiesis in the adult bone marrow relies on mitochondrial membrane transporters to facilitate heme and hemoglobin production. Erythrocytes in the bone marrow are produced although the differentiation of erythroid progenitor cells that originate from hematopoietic stem cells (HSCs). Whether and how mitochondria transporters potentiate HSCs and affect their differentiation toward erythroid lineage remains unclear. Here, we show that the ATP-binding cassette (ABC) transporter 10 (Abcb10), located on the inner mitochondrial membrane, is essential for HSC maintenance and erythroid-lineage differentiation. Induced deletion of Abcb10 in adult mice significantly increased erythroid progenitor cell and decreased HSC number within the bone marrow (BM). Functionally, Abcb10-deficient HSCs exhibited significant decreases in stem cell potential but with a skew toward erythroid-lineage differentiation. Mechanistically, deletion of Abcb10 rendered HSCs with excess mitochondrial iron accumulation and oxidative stress yet without alteration in mitochondrial bioenergetic function. However, impaired hematopoiesis could not be rescued through the in vivo administration of a mitochondrial iron chelator or antioxidant to Abcb10-deficient mice. Abcb10-mediated mitochondrial iron transfer is thus pivotal for the regulation of physiologic HSC potential and erythroid-lineage differentiation.

Generation of induced pluripotent stem cell line (VRISGi004-A) from a healthy female donor by reprogramming erythroid progenitor cells

We generated a human induced pluripotent stem cell (hiPSC) line from erythroid progenitor cells (EPCs) of a 20-year-old female healthy donor using Sendai virus vector encoding Yamanaka factors OCT3/4, SOX2, c-MYC, and KLF4. The established hiPSCs showed a standard morphology and expression of typical undifferentiated stem cell markers, a normal karyotype (46, XX), and demonstrated potential for differentiation in vitro. Furthermore, they were successfully differentiated into cardiomyocytes that expressed cardiomyocyte-specific markers. The iPSC line and iPSC-derived cardiomyocytes will provide new avenues for future drug testing/development and personalized cell therapy for cardiovascular diseases (CVDs).

Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis.

Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the inhibitor SHIN1, and by AICAR supplementation. Mechanistically, the metabolically driven differentiation is independent of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine 5'-monophosphate-activated protein kinase (AMPK) and is instead mediated by protein kinase C. Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate deficiency-induced anemia.