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.

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).

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.

The impact of erythroblast enucleation efficiency on the severity of anemia in patients with myelodysplastic syndrome.

Anemia is the most common manifestation in myelodysplastic syndrome (MDS) patients, but the cause of ineffective hematopoiesis is not fully understood. Enucleation is an important event in the maturation process of erythroblasts. According to a series of morphological phenotypes of the pathological development of MDS erythroblasts, we speculate that there may be enucleation disorders. To verify this hypothesis, we cultured MDS bone marrow CD34+ cells in vitro and induced erythroblast development. The results showed that erythroblast enucleation in MDS was significantly lower than that in the normal group, and the rate of enucleation was positively correlated with hemoglobin concentration. Risk stratification of MDS was performed to further analyze the differences in enucleation among the normal group, low-middle risk group and high-risk group. The results showed that the enucleation rate of the high risk group was higher than that of the low-middle risk group but still lower than that of the normal group. Moreover, the expression of pERK and pAKT in MDS erythroblasts in the high risk group was higher than that in the normal group, while the expression of pERK and pAKT in the low-middle risk group was lower than that in the normal group. Furthermore, the enucleation of MDS was positively correlated with the phosphorylation degree of ERK and AKT. In conclusion, this study reveals that the enucleation of erythroblasts is one of the possible causes of anemia in MDS. Video Abstract.

Gene expression changes in sickle cell reticulocytes and their clinical associations.

Transcriptional changes in compensatory erythropoiesis in sickle cell anemia (SCA) and their disease modulation are unclear. We detected 1226 differentially expressed genes in hemoglobin SS reticulocytes compared to non-anemic hemoglobin AA controls. Assessing developmental expression changes in hemoglobin AA erythroblasts for these genes suggests heightened terminal differentiation in early erythroblasts in SCA that diminishes toward the polychromatic to orthochromatic stage transition. Comparison of reticulocyte gene expression changes in SCA with that in Chuvash erythrocytosis, a non-anemic disorder of increased erythropoiesis due to constitutive activation of hypoxia inducible factors, identified 453 SCA-specific changes attributable to compensatory erythropoiesis. Peripheral blood mononuclear cells (PBMCs) in SCA contain elevated proportions of erythroid progenitors due to heightened erythropoiesis. Deconvolution analysis in PBMCs from 131 SCA patients detected 54 genes whose erythroid expression correlated with erythropoiesis efficiency, which were enriched with SCA-specific changes (OR = 2.9, P = 0.00063) and annotation keyword "ubiquitin-dependent protein catabolic process", "protein ubiquitination", and "protein polyubiquitination" (OR = 4.2, P = 7.5 × 10-5). An erythroid expression quantitative trait locus of one of these genes, LNX2 encoding an E3 ubiquitin ligase, associated with severe pain episodes in 774 SCA patients (OR = 1.7, P = 3.9 × 10-5). Thus, erythroid gene transcription responds to unique conditions within SCA erythroblasts and these changes potentially correspond to vaso-occlusive manifestations.

Plasmodium falciparum infection of human erythroblasts induces transcriptional changes associated with dyserythropoiesis.

During development down the erythroid lineage, hematopoietic stem cells undergo dramatic changes to cellular morphology and function in response to a complex and tightly regulated program of gene expression. In malaria infection, Plasmodium spp parasites accumulate in the bone marrow parenchyma, and emerging evidence suggests erythroblastic islands are a protective site for parasite development into gametocytes. Although it has been observed that Plasmodium falciparum infection in late-stage erythroblasts can delay terminal erythroid differentiation and enucleation, the mechanism(s) underlying this phenomenon are unknown. Here, we apply RNA sequencing after fluorescence-activated cell sorting of infected erythroblasts to identify transcriptional responses to direct and indirect interaction with P falciparum. Four developmental stages of erythroid cells were analyzed: proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. We found extensive transcriptional changes in infected erythroblasts compared with that in uninfected cells in the same culture, including dysregulation of genes involved in erythroid proliferation and developmental processes. Although some indicators of cellular oxidative and proteotoxic stress were common across all stages of erythropoiesis, many responses were specific to cellular processes associated with developmental stage. Together, our results evidence multiple possible avenues by which parasite infection can induce dyserythropoiesis at specific points along the erythroid continuum, advancing our understanding of the molecular determinants of malaria anemia.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

Crosstalk between terminal erythropoiesis and granulopoiesis within their common niche: the erythromyeloblastic island.

PURPOSE OF REVIEW: The identity of the erythroblastic island (EBI) macrophage (Mϕ) has been under investigation for decades since it was recognized as the first hematopoietic niche 'nursing' terminal erythropoiesis. This review will focus on the current insights to the characteristics and the role of the EBI Mϕ balancing terminal erythropoiesis and granulopoiesis. RECENT FINDINGS: While the EBI has long been known as the niche for erythroid precursors, significant advancements in biology research technologies, including optimization of EBI enrichment protocols, single-cell ribonucleic acid sequencing, and imaging flow cytometry, have recently revealed that granulocytic precursors co-exist in this niche, termed erythromyeloblastic island (EMBI). More importantly, the balance noted at baseline between terminal granulopoiesis and erythropoiesis within EBIs/EMBIs is altered with diseases affecting hematopoiesis, such as stress erythropoiesis and inflammatory conditions causing anemia of inflammation. The role of the EMBI niche has yet to be fully investigated mechanistically, however, a notable degree of transcriptional and cell surface marker heterogeneity has been identified for the EMBI Mϕ, implicating its plasticity and diverse function. SUMMARY: Terminal erythropoiesis and granulopoiesis are regulated within the EMBI. Investigations of their balance within this niche in health and disease may reveal new targets for treatment of diseases of terminal hematopoiesis.

EHBP1L1, an apicobasal polarity regulator, is critical for nuclear polarization during enucleation of erythroblasts.

Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. epidermal growth factor receptor substrate 15 homology domain-binding protein 1-like 1 (EHBP1L1) is an adapter protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in nonepithelial cells, remains unknown. Here, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization before enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and nonepithelial cells.

CD169-CD43 interaction is involved in erythroblastic island formation and erythroid differentiation.

CD169, a specific marker for macrophages, is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family which acts as an adhesion molecule implicated in cell-cell interaction via sialylated glycoconjugates. Although CD169+ macrophages have been found to participate in erythroblastic island (EBI) formation and support erythropoiesis under homeostasis and stress, the exact role of CD169 and its counter receptor in EBI remains unknown. Herein, we generated CD169-CreERT knock-in mice and investigated the function of CD169 in EBI formation and erythropoiesis using CD169-null mice. EBI formation was impaired in vitro by both blockade of CD169 using anti-CD169 antibody and deletion of CD169 on macrophages. Furthermore, CD43 expressed by early erythroblasts (EB) was identified as the counter receptor for CD169 in mediating the EBI formation via surface plasmon resonance and imaging flow cytometry. Interestingly, CD43 was proven to be a novel indicator of erythroid differentiation due to the progressive decrease of CD43 expression as EB mature. Although CD169-null mice did not display defects in bone marrow (BM) EBI formation in vivo, CD169 deficiency impeded BM erythroid differentiation probably via CD43 under stress erythropoiesis, in concert with the role of CD169 recombinant protein in hemin-induced K562 erythroid differentiation. These findings have shed light on the role of CD169 in EBI under steady and stress erythropoiesis through binding with its counter receptor CD43, suggesting that CD169-CD43 interaction might be a promising therapeutic target for erythroid disorders.

The NFIA-ETO2 fusion blocks erythroid maturation and induces pure erythroid leukemia in cooperation with mutant TP53.

The NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver-derived EBs. However, NFIA-ETO2-expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions 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. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

The inner nuclear membrane protein NEMP1 supports nuclear envelope openings and enucleation of erythroblasts.

Nuclear envelope membrane proteins (NEMPs) are a conserved family of nuclear envelope (NE) proteins that reside within the inner nuclear membrane (INM). Even though Nemp1 knockout (KO) mice are overtly normal, they display a pronounced splenomegaly. This phenotype and recent reports describing a requirement for NE openings during erythroblasts terminal maturation led us to examine a potential role for Nemp1 in erythropoiesis. Here, we report that Nemp1 KO mice show peripheral blood defects, anemia in neonates, ineffective erythropoiesis, splenomegaly, and stress erythropoiesis. The erythroid lineage of Nemp1 KO mice is overrepresented until the pronounced apoptosis of polychromatophilic erythroblasts. We show that NEMP1 localizes to the NE of erythroblasts and their progenitors. Mechanistically, we discovered that NEMP1 accumulates into aggregates that localize near or at the edge of NE openings and Nemp1 deficiency leads to a marked decrease of both NE openings and ensuing enucleation. Together, our results for the first time demonstrate that NEMP1 is essential for NE openings and erythropoietic maturation in vivo and provide the first mouse model of defective erythropoiesis directly linked to the loss of an INM protein.

The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity.

During erythroid differentiation, the maintenance of genome integrity is key for the success of multiple rounds of cell division. However, molecular mechanisms coordinating the expression of DNA repair machinery in erythroid progenitors are poorly understood. Here, we discover that an RNA N6-methyladenosine (m6A) methyltransferase, METTL16, plays an essential role in proper erythropoiesis by safeguarding genome integrity via the control of DNA-repair-related genes. METTL16-deficient erythroblasts exhibit defective differentiation capacity, DNA damage and activation of the apoptotic program. Mechanistically, METTL16 controls m6A deposition at the structured motifs in DNA-repair-related transcripts including Brca2 and Fancm mRNAs, thereby upregulating their expression. Furthermore, a pairwise CRISPRi screen revealed that the MTR4-nuclear RNA exosome complex is involved in the regulation of METTL16 substrate mRNAs in erythroblasts. Collectively, our study uncovers that METTL16 and the MTR4-nuclear RNA exosome act as essential regulatory machinery to maintain genome integrity and erythropoiesis.

Mitochondria transfer mediates stress erythropoiesis by altering the bioenergetic profiles of early erythroblasts through CD47.

Intercellular mitochondria transfer is a biological phenomenon implicated in diverse biological processes. However, the physiological role of this phenomenon remains understudied between erythroblasts and their erythroblastic island (EBI) macrophage niche. To gain further insights into the mitochondria transfer functions, we infused EBI macrophages in vivo into mice subjected to different modes of anemic stresses. Interestingly, we observed the occurrence of mitochondria transfer events from the infused EBI macrophages to early stages of erythroblasts coupled with enhanced erythroid recovery. Single-cell RNA-sequencing analysis on erythroblasts receiving exogenous mitochondria revealed a subset of highly proliferative and metabolically active erythroid populations marked by high expression of CD47. Furthermore, CD47 or Sirpα blockade leads to a decline in both the occurrence of mitochondria transfer events and their mediated erythroid recovery. Hence, these data indicate a significant role of mitochondria transfer in the enhancement of erythroid recovery from stress through the alteration of the bioenergetic profiles via CD47-Sirpα interaction in the early stages of erythroblasts.

Discovery of N-arylcinnamamides as novel erythroblast enucleation inducers.

Derivation of mature red blood cells (RBCs) from stem cells in vitro is a promising solution to the current shortage of blood supply, in which terminal enucleation is the rate-limiting step. Here we discovered two cinnamamides B8 and B16 showed potential activities of enhancing the enucleation of erythroblasts through the screening of "in-house" compound library. Subsequently, twenty-four N-arylcinnamamides were rationally designed and synthesized on the basis of the structure of B8 and B16, in which N-(9H-carbazol-2-yl)cinnamamide (KS-2) significantly elevated the percentage of reticulocytes in the cultured mouse fetal liver cells in vitro (relative enucleation = 2.43). The underlying mechanism of KS-2 in promoting mouse erythroid enucleation is accelerating the process of cell cycle exit via p53 activation in late stage erythrocytes. These results strongly suggest that compound KS-2 is worthy of further study as a potential erythrocyte enucleation inducer.