Colony-forming Unit-erythroid Cells Research Articles

Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells.

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

Disruption of the Malate-Aspartate Shuttle Leads to Anemia

Erythropoiesis, the process of differentiating multipotent hematopoietic stem cells (HSCs) into mature enucleated red blood cells (RBCs), is a metabolically intensive process, resulting in the generation of over 2 million new RBCs per second. Although anemia due to impaired erythropoiesis is a common condition, therapeutic options are often limited to RBC transfusions. However, the emergence of effective metabolite therapies, such as glutamine supplementation for sickle cell disease, underscores the potential of understanding metabolism for developing additional treatments. Two key enzymes in the malate-aspartate shuttle (MAS), glutamic-oxaloacetic transaminase 1 (GOT1) and 2 (GOT2), play crucial roles in transferring energy in the form of NADH from the cytosol into the mitochondria for oxidative phosphorylation. NADH cannot directly cross the mitochondrial membrane, so its electrons are transported on malate, which enters the mitochondria through a specific transporter. Within this shuttle, mitochondrial GOT2 utilizes glutamine to generate aspartate, which is then transported back into the cytoplasm to be metabolized by GOT1. Previous research has shown that aspartate-dependent nucleotide biosynthesis is essential for HSC regeneration. To investigate the roles of GOT1 and GOT2 in erythropoiesis, we crossed mice expressing tamoxifen-inducible erythroid-specific Cre recombinase ( Gata1-Cre ERT2 BAC transgenic mice) to Got1 or Got2 floxed lines, generating Got1 flox/flox;Gata1-Cre ERT2( Got1 CKO) or Got2 flox/flox;Gata1-Cre ERT2( Got2 CKO) mice, respectively. Upon tamoxifen administration to these mice, conditional Got1 or Got2 deletion occurs in megakaryocytic-erythrocytic progenitor (MEP) cells, which give rise to both RBCs and platelets. Complete blood count analysis revealed decreased hemoglobin levels and RBC counts, along with a reciprocal increase in platelet count in both Got1 and Got2 CKO mice. These results were unexpected, as previous studies showed that hematopoietic Got1 loss led to increased aspartate-driven nucleotide biosynthesis and HSC regeneration, while the opposite effects were observed in mice with hematopoietic Got2 loss. Although these enzymes are expected to have opposite effects on aspartate levels, the deletion of either enzyme disrupts the transfer of reducing equivalents between the cytosol and mitochondria. This results in NADH accumulation and reductive stress, which in turn leads to metabolic dysfunction, affecting glycolysis and oxidative phosphorylation. Therefore, the occurrence of anemia in mice with either Got1 or Got2 deletion in the erythroid compartment suggests that impaired redox balance, rather than aspartate-driven nucleotide biosynthesis, is likely the cause of the anemia. To further understand the basis of anemia in Got1 and Got2 CKO mice, we harvested bone marrow cells and used conventional cell surface markers to quantify MEPs, burst-forming unit erythroid cells (BFU-Es), colony-forming unit erythroid cells (CFU-Es), and terminally differentiated erythroblast populations. Compared to littermate controls with no deletion of Got1 and Got2, the absolute numbers of pre-CFU-Es and CFU-Es were higher in both Got1 and Got2CKO mice, while the absolute numbers of differentiated erythroblasts were lower in both mutant mice. These findings indicate a block in erythroid differentiation, which is currently under further investigation. Collectively, these preliminary data suggest a novel role for MAS in regulating erythropoiesis. Ongoing studies are actively exploring the mechanisms by which dysfunctional MAS leads to anemia.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

LKB1 Coordinates Metabolic Pathways to Maintain Homeostasis of Haematopoietic Stem Cells and Regulate Erythropoiesis

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells (HSCs) proliferate, differentiate and eventually form mature erythrocytes. Stem-cell maintenance and cell differentiation require homeostasis and coordination of multiple metabolisms. However, underlying mechanism of this coordination is poorly identified. Liver kinase B1 (LKB1) acts as evolutionarily conserved regulator to control cellular metabolism, cell polarity and proliferation during development and stress response. Considering that all the fundamental cellular processes regulated by LKB1 are present in erythropoiesis, we hypothesize that LKB1 may involve in orchestrating this coordination.To explore the role of LKB1 in entire erythropoiesis, we firstly crossed mice carrying loxP-flanked LKB1 alleles with EpoR-tdTomato-Cre mice, which can lead to cleavage in from HSCs to erythroblasts. LKB1fl/fl EPORcre mice developed exhaustion of HSCs, progressive severe anemia and ultimately lethality. Intriguingly, HSCs of LKB1fl/fl EPORcre mice exhibited a swiftly skewness toward the erythoid lineage when LKB1 is deleted. Nevertheless, erythroblasts of LKB1fl/fl EPORcre mice were significantly reduced. Further analysis showed that the colony forming ability of the colony-forming unit-erythroid (CFU-E) cells and subsequent terminal erythoid differentiation in LKB1fl/fl EPORcre mice were seriously impaired.In order to study erythropoiesis more specifically without the influence of stem cells, we next assessed the impact of deletion of LKB1 in the mouse erythropoietic system using GYPA-eGFP-Cre mice. LKB1fl/flGYPAcre mice showed mild anemia but had the normal lifespan.The Ter119 + cells were decreased while CFU-E cells were significantly increased. However, the colony forming ability of sorted CFU-E cells from LKB1fl/flGYPAcre mice were drastically reduced. In addition, LKB1fl/flGYPAcre mice exhibited disordered terminal erythropoiesis. Taken together, these results indicate that LKB1 may play multinomial roles in regulating stem-cell homeostasis and effective erythoid differentiation.Previous studies have shown that LKB1 controls energy metabolism via phosphorylating AMPK and regulating PGC-1 transcription to maintain homeostasis of HSCs. We found that both of p-AMPK and PGC-1 of LKB1fl/fl EPORcre mice were down-regulated in Lin - cells while Ter119 + cells have comparable p-AMPK and PGC-1. In this regard, LKB1fl/flGYPAcre mice, whether in Lin - cells or in Ter119 + cells, exhibited similar p-AMPK and PGC-1 level. Moreover, administration of ZLN005, an activator of PGC-1, partly rescued the anemia in LKB1fl/fl EPORcre mice but not in LKB1fl/flGYPAcre mice. These data imply that there is other underlying mechanism of LKB1 in erythropoiesis.To gain further mechanistic insight, we carried out proteomics analysis. Gene ontology analysis of differentially expressed proteins revealed that cholesterol metabolism related genes were significantly altered under LKB1 deficiency. We then found that LKB1 ablation led to a reduction of cholesterol level and diminishment of expression levels of primary cholesterol biosynthesis related genes. Moreover, the mature active form of SREBP2, the master transcriptional regulator of cholesterol biosynthesis, was prominently reduced. Golgi apparatus, in which SREBP2 is cleaved for activation, is intumescent or dispersed in erythroid cells of LKB1fl/fl EPORcre mice and LKB1fl/flGYPAcre mice. These results supported that loss of LKB1 impaired the cholesterol metabolism in erythropoiesis.To demonstrate Golgi apparatus-dependent cholesterol metabolism is essential for erythropoiesis, we treated LKB1fl/flGYPAcre mice with 2,3-oxidosqualene, the important intermediate in cholesterol synthesis and found that the phenotypes of LKB1fl/flGYPAcre mice were effectively restored. In parallel, 2,3-oxidosqualene treatment just slightly alleviated the anemia of LKB1fl/fl EPORcre mice. However, combination treatment with 2,3-oxidosqualene and ZLN005 was more effective in restoring phenotypes in LKB1fl/fl EPORcre mice, in contrast to ZLN005 alone.Thus, LKB1 serves as an essential metabolic regulator to coordinate energy metabolism in hematopoietic stem cells and lipid metabolism in erythoid cells, thereby maintaining homeostasis of haematopoietic stem cells and functional fitness of erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Cell-to-cell variability in JAK2/STAT5 pathway components and cytoplasmic volumes defines survival threshold in erythroid progenitor cells.

Survival or apoptosis is a binary decision in individual cells. However, at the cell-population level, a graded increase in survival of colony-forming unit-erythroid (CFU-E) cells is observed upon stimulation with erythropoietin (Epo). To identify components of Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signal transduction that contribute to the graded population response, we extended a cell-population-level model calibrated with experimental data to study the behavior in single cells. The single-cell model shows that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of Epo receptor (EpoR):JAK2 complexes and of SHP1, as well as the extent of nuclear import because of the large variance in the cytoplasmic volume of CFU-E cells. 24-118 pSTAT5 molecules in the nucleus for 120min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes is sufficient to convert a switch-like behavior at the single-cell level to a graded population-level response.

TET2 Deficiency Led to Expansion of Dysfunctional Erythroid Progenitors

Erythrocyte development is a complex process involving multiple developmental states that include erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells and erythroid precursors, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts in the bone marrow. Orthochromatic erythroblasts expel nucleus to become reticulocytes which mature into erythrocytes in blood stream. Defects at any of these developmental stages will lead to anemia. Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenia and ineffective hematopoiesis. Anemia is the defining cytopenia in the majority of patients with MDS, yet the molecular mechanisms for dyserythropoiesis in MDS remain largely unclear. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is the most common mutation in MDS.In the present study, we explored shRNA-mediated knockdown approach in human CD34+ cell to study the role of TET2 in human erythropoiesis. We show that TET2 is downregulated starting at the CFU-E stage. Growth curves of purified CFU-E cells show no difference between control and TET2 knockdown cells until day 6. An increased proliferation of TET2 knockdown-CFU-E cells is noted starting at day 7. Interestingly, while the growth of control CFU-E cells plateaued on day 11 with completion of terminal erythroid differentiation and enucleation, TET2 knockdown CFU-E cells continued their growth for additional 13 days. Flow cytometry and cytospin morphologic analyses revealed that by day 11 all control cells differentiated into GPA+ late stage erythroblasts or reticulocytes. In marked contrast, about 30 % of TET2 knockdown cells are still GPA- but CD71+, IL-3R-, CD34-, CD36+, phenotypically resembling erythroid progenitor CFU-E cells. Surprisingly, in contrast to freshly purified control and TET2 knockdown CFU-E cells that generate colonies in methylcellulose colony forming assays, these cells fail to give rise to CFU-E colonies. We thus termed these late proliferating cells as “TET2 knockdown marker CFU-E”. Further characterization of the “TET2 knockdown marker CFU-E” cells revealed that in contrast to control CFU-E, the “TET2 knockdown marker CFU-E” exhibit following distinct features: 1) they had lower proliferative potential; 2) their proliferation required both EPO and SCF (while control CFU-E cells only require EPO); 3) they underwent extensive apoptosis in the absence of SCF; 4) they failed to undergo terminal erythroid differentiation. These findings demonstrate that TET2 knockdown led to expansion of dysfunctional erythroid progenitors.To explore the underlying molecular mechanisms, we performed RNA-seq analysis on control CFU-E, TET2 knockdown CFU-E and “TET2 knockdown marker CFU-E” cells. Bioinformatics analysis revealed that while only about 50 genes were differentially expressed between control CFU-E and TET2 knockdown CFU-E, about 400 genes were differentially expressed between control CFU-E and “TET2 knockdown marker CFU-E”. Gene set enrichment analysis revealed upregulation of pathways associated with apoptosis/cell death. Notably, FAS (CD95), the cell surface death receptor previously shown to be upregulated in MDS bone marrow cells, was among the top five upregulated apoptosis associated genes. The increased surface expression of FAS was also noted in TET2-knockdown cells as assessed by flow cytometry. Furthermore, the increased surface expression of FAS was detected in primary bone marrow erythroblasts of MDS patients with TET2 mutation. Our findings uncovered previously unknown roles of TET2 and its potential underlying molecular mechanisms. As the phenotypic changes upon TET2 knockdown resemble the characteristic pathological changes of MDS, our findings suggest the involvement of TET2 deficiency in the pathophysiology of MDS. DisclosuresAli:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Raza:Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau; Genoptix: Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Janssen R&D: Research Funding.

Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells.

U2AF1 (U2AF35) is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP (small nuclear ribonucleoproteins) of the spliceosome. Here, we examined the function of U2AF1 in human erythropoiesis. First, we examined the expression of U2AF1 during in vitro human erythropoiesis and showed that U2AF1 was highly expressed in the erythroid progenitor burst‐forming‐unit erythroid (BFU‐E) cell stage. A colony assay revealed that U2AF1 knockdown cells failed to form BFU‐E and colony‐forming‐unit erythroid (CFU‐E) colonies. Our results further showed that knockdown of U2AF1 significantly inhibited cell growth and induced apoptosis in erythropoiesis. Additionally, knockdown of U2AF1 also delayed terminal erythroid differentiation. To explore the molecular basis of the impaired function of erythroid development, RNA‐seq was performed and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage, were involved, with the p53 signalling pathway showing the greatest involvement. Western blot analysis revealed an increase in the protein levels of downstream targets of p53 following U2AF1 knockdown. The data further showed that depletion of U2AF1 altered alternatively spliced apoptosis‐associated gene transcripts in CFU‐E cells. Our findings elucidate the role of U2AF1 in human erythropoiesis and reveal the underlying mechanisms.

TET2 deficiency leads to stem cell factor–dependent clonal expansion of dysfunctional erythroid progenitors

AbstractMyelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)–dependent hyperproliferation and impaired differentiation of human colony-forming unit–erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors “marker CFU-E” cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E

AbstractBurst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA−IL-3R−CD34+CD36−CD71low and CD45+GPA−IL-3R−CD34−CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45+GPA−IL-3R−CD34+CD36−CD71low cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45+GPA−IL-3R−CD34−CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.

Early propranolol administration to severely injured patients can improve bone marrow dysfunction.

Bone marrow (BM) dysfunction is common in severely injured trauma patients, resulting from elevated catecholamines and plasma granulocyte colony-stimulating factor (G-CSF) as well as prolonged mobilization of hematopoietic progenitor cells (HPCs). We have previously shown that propranolol (β-blocker [BB]) reduces HPC mobilization in a rodent model of injury and hemorrhagic shock. We hypothesize that BB would prevent BM dysfunction in humans following severe injury. Forty-five severely injured trauma patients were studied in a prospective, randomized pilot trial. Twenty-five patients received BB, and 20 served as untreated controls. The dose of propranolol was adjusted to decrease the heart rate by 10% to 20% from baseline. Blood was analyzed for the presence of HPC (blast-forming unit erythroid cells [BFU-E] and colony-forming unit erythroid cells) and G-CSF. Demographic data, Injury Severity Score (ISS), hemoglobin, reticulocyte number, and outcome data were obtained. The mean age of the study population was 33 years; 87% were male, with a mean ISS of 29. There is a significant increase in BFU-E in peripheral blood immediately following traumatic injury, and this mobilization persists for 30 days. The use of BB significantly decreases BFU-E and colony-forming unit erythroid cells at all time points. G-CSF is significantly elevated in both groups on admission; the use of BB decreases G-CSF levels by 51% as compared with 37% for controls. The average hemoglobin is nearly 1 g higher on the day of discharge with propranolol treatment (BB, 9.9 ± 0.4 g/dL vs. no BB, 9.1 ± 0.6 g/dL). Following severe trauma, early treatment with propranolol following resuscitation is safe. The use of propranolol blunts early tachycardia, reduces HPC mobilization, and results in a faster return to baseline of the G-CSF peak seen after injury. There is also a trend toward faster recovery and resolution of anemia. Propranolol may be the first therapeutic agent to show improved BM function after severe injury. Therapeutic study, level III.

From Hematopoietic Stem Cell to Erythroblast: Regulation of Red Cell Production At Multiple Levels

Abstract SCI-1 Hematopoietic stem cells (HSC) undergo self-renewal and also generate all types of blood cells, including red cells, myeloid cells, and all immune system cells. Formation of red cells from HSC involves multiple cellular stages, including early erythroid-specific progenitors (burst-forming unit-erythroid [BFU-E]) that respond to several growth factors and an erythropoietin (Epo)-responsive progenitor, and the colony-forming unit-erythroid (CFU-E) that in 3 days generates ∼30 reticulocytes. In adult animals most CFU-E cells undergo apoptosis. A short-term need for red cells is met by increased Epo production by the kidney, which rescues increasing numbers of CFU-Es from apoptosis and increases red cell production over a few days. Chronic stress, such as in certain types of anemias and inflammatory diseases, leads to a marked increase in numbers of HSC and many types of progenitor cells. I will focus most of my talk on stress-triggered BFU-E proliferation and formation of CFU-Es, since our very recent work showed how glucocorticoids – an important class of stress hormones – as well as HIF-1α stimulate red cell production. Acting synergistically these allow ∼300 times more CFU-Es and erythroblasts to be formed from each BFU-E. More specifically, these molecules stimulate limited self-renewal of BFU-Es during cell division, thus maintaining progenitor immaturity and allowing over time increased numbers of CFU-E cells and thus mature red cells to be formed. This explains why certain corticosteroids are useful in treating some non-Epo responsive anemias, and I will present a molecular dissection of glucocorticoid and HIF-1α action on BFU-E progenitors that has led to the identification of genes critical for self-renewal and that suggest other possible avenues for treatment of chronic anemias. Disclosures: No relevant conflicts of interest to declare.

Isolation and Functional Characterization of Human Erythroid Progenitors: BFU-E and CFU-E

Abstract 1028The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes. Disclosures:No relevant conflicts of interest to declare.

SWAP-70 regulates erythropoiesis by controlling 4 integrin

Background The regulation of normal and stress-induced erythropoiesis is incompletely understood. Integrin-dependent adhesion plays important roles in erythropoiesis, but how integrins are regulated during erythropoiesis remains largely unknown. To obtain novel insights into the regulation of erythropoiesis, we used cellular and molecular approaches to analyze the role of SWAP-70 and the control of integrins through SWAP-70. In addition, mice deficient for this protein were investigated under normal and erythropoietic stress conditions. We show that SWAP-70, a protein involved in cytoskeletal F-actin rearrangements and integrin regulation in mast cells, is expressed in hematopoietic stem cells and myeloid-erythroid precursors. Although Swap-70(-/-) mice are not anemic, erythroblastic differentiation is perturbed, and SWAP-70 is required for an efficient erythropoietic stress response to acute anemia and for erythropoietic recovery after bone marrow transplantation in irradiated mice. SWAP-70 deficiency impairs colony-forming unit erythroid development, while burst-forming unit erythroid development is normal, and significantly affects development of late erythroblasts in the spleen and bone marrow. The α(4) integrin is constitutively hyper-activated in Swap-70(-/-) colony-forming unit erythroid cells, which hyper-adhere to fibronectin. Blocking α(4) and β(1) integrin chains in vivo restored erythroblastic differentiation and the erythropoietic stress response in Swap-70(-/-) mice. Conclusions Our study reveals that SWAP-70 is a novel regulator of integrin-mediated red blood cell development and stress-induced erythropoiesis.

Identification of erythroid progenitors induced by erythropoietic activity inXenopus laevis

Oxygen is essential for the survival of animals. Red blood cells in the circulation, i.e. peripheral erythrocytes, are responsible for transporting oxygen to tissues. The regulation of erythropoiesis in vertebrates other than mammals is yet to be elucidated. Recently we identified erythropoietin, a primary regulator of erythropoiesis, in Xenopus laevis, which should enable us to identify target cells, including erythroid progenitors, and to investigate the production and development of erythroid cells in amphibians. Here, we established a semi-solid colony-forming assay in Xenopus laevis to clarify the existence of colony-forming unit-erythroid cells, the functional erythroid progenitors identified in vitro. Using this assay, we showed that recombinant xlEPO induces erythroid colony formation in vitro and detected an increased level of erythropoietin activity in blood serum during acute anemic stress. In addition, our study demonstrated the possible presence of multiple, non-xlEPO, factors in anemic serum supportive of erythroid colony formation. These results indicate that erythropoiesis mediated by erythropoietin is present in amphibian species and, furthermore, that the regulatory mechanisms controlling peripheral erythrocyte number may vary among vertebrates.

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.

Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies

We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2.