Colony-forming Unit-erythroid Progenitors Research Articles

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming "units" known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown.To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors.To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. DisclosuresNo relevant conflicts of interest to declare.

Mercury Chloride Impacts on the Development of Erythrocytes and Megakaryocytes in Mice.

Inorganic mercury (Hg2+) is a highly toxic heavy metal. The aim of this study was to investigate the impact of Hg2+ on the development of erythrocytes and megakaryocytes. B10.S mice (H-2s) and DBA/2 mice (H-2d) were administrated with 10 μM HgCl2 or 50 μM HgCl2 via drinking water for four weeks, and erythro-megakaryopoiesis was evaluated thereafter. The administration of 50 μM HgCl2 increased the number of erythrocytes and platelets in B10.S mice, which was not due to a reduced clearance for mature erythrocytes. The administration of 50 μM HgCl2, but not 10 μM HgCl2, increased the number of progenitors for erythrocytes and megakaryocytes in the bone marrow (BM) of B10.S mice, including erythroid-megakaryocyte progenitors (EMPs), burst-forming unit-erythroid progenitors (BFU-Es), colony-forming unit-erythroid progenitors (CFU-Es), and megakaryocyte progenitors (MkPs). Moreover, 50 μM HgCl2 caused EMPs to be more proliferative and possess an increased potential for differentiation into committed progenies in B10.S mice. Mechanistically, 50 μM HgCl2 increased the expression of the erythropoietin receptor (EPOR) in EMPs, thus enhancing the Jak2/STAT5 signaling pathway to promote erythro-megakaryopoiesis in B10.S mice. Conversely, 50 μM HgCl2 did not impact erythro-megakaryopoiesis in DBA/2 mice. This study may extend our current understanding for hematopoietic toxicology of Hg.

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

Single Cell Resolution of Glucocorticoid Effects on Erythroid Progenitor Cells

To generate the billions of new erythrocytes required on a daily basis, erythroid progenitor cells must exponentially increase in number before undergoing terminal differentiation. A limited number of cell divisions occur during erythropoietin (EPO)-regulated erythroid terminal differentiation, but the principal regulation of erythroid transit-amplification occurs earlier in erythropoiesis between the burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) stages of development. The importance of this EPO-independent early erythropoietic process is highlighted in Diamond-Blackfan Anemia (DBA). DBA is characterized by pure red cell aplasia, loss of BFU-E and CFU-E progenitors in the bone marrow, and severe anemia despite high circulating EPO levels. The only known effective medical therapy for DBA also provides insight into the regulation of erythroid transit-amplification. In patients responsive to glucocorticoid treatment, there are increased numbers of BFU-E and CFU-E progenitors in the bone marrow, and ex vivo culture studies indicate that the synthetic glucocorticoid dexamethasone (Dex) predominantly increases proliferative capacity of BFU-Es, with minimal effect on proliferative capacity of CFU-Es. These findings led to a prevailing model that glucocorticoids increase BFU-E proliferative capacity by stimulating several rounds of self-renewal cell divisions. However, a limitation of this model is that there is no mechanistic explanation for how BFU-Es regulate the fate choice of undergoing a self-renewal cell division versus a differentiation cell division in the presence or absence of glucocorticoids. In this study, we address this question by examining progression of early erythroid progenitor development at single cell resolution, and subsequently elucidate the true mechanistic nature of glucocorticoid-induced erythroid progenitor proliferative capacity amplification. By performing single cell transcriptome profiling (scRNAseq) of primary-isolated mouse fetal liver BFU-Es, CFU-Es, and their developmental intermediates, we identify a continuum of transcriptomic states during erythroid transit-amplification when performing principle component analysis (PCA) on transcriptomes of individual cells. We show that ex vivo culture of primary-isolated BFU-Es in serum free media supplemented with stem cell factor, insulin-like growth factor 1, and EPO results in developmental progression along the transcriptome continuum when performing scRNAseq and PCA on cultured BFU-Es. The addition of Dex into this culture system does not result in self-renewal of BFU-Es at the transcriptome level, but rather still results in developmental progression, albeit to less of a degree per cell division than BFU-Es cultured without Dex. We additionally show that the continuum of transcriptome states in erythroid transit-amplification is reflective of a continuum of functional states, with developmental progression characterized by decreasing proliferative capacity and decreasing glucocorticoid-responsiveness. Lastly, through manual separation of daughter cells resulting from a BFU-E cell division, we demonstrate that BFU-E cell division is a symmetric process at the transcriptome level, both with and without the addition of Dex. Our results clarify the nature of how glucocorticoids amplify BFU-E proliferative capacity. As opposed to stimulating a finite number of BFU-E self-renewal cell divisions, glucocorticoids decrease the extent of progression through the erythroid transit-amplifying developmental continuum per cell division. Thus, a decreased rate of progression through the developmental continuum is associated with an increased number of transit-amplifying cell divisions prior to terminal differentiation. These findings are important not only for the rational development of glucocorticoid-alternatives for treating DBA, but also for all bone marrow failure syndromes characterized by progenitor cell hypoplasia. DisclosuresNo relevant conflicts of interest to declare.

PPARα Agonists and TGFβ Inhibitors Stimulate Red Blood Cell Production by Enhancing Self-Renewal of BFU-E Erythroid Progenitors

The production of red blood cells (RBCs) in mammals is tightly regulated by erythropoietin (Epo), which stimulates erythropoiesis by promoting survival, proliferation, and terminal differentiation of the colony-forming unit erythroid progenitors (CFU-Es). Several acute and chronic anemias, including hemolysis, severe trauma-induced anemia and genetic bone marrow failure disorders such as Diamond-Blackfan anemia (DBA), are not treatable with Epo because the CFU-E erythroid progenitors that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain adequate RBC production. Treatment of Epo-resistant anemias requires a drug that acts earlier than Epo in erythropoiesis and that enhances the formation of CFU-Es. One attractive approach is to devise strategies to promote self-renewal of the upstream burst-forming unit erythroid progenitors (BFU-Es). We recently showed that, like transient amplifying cells in other stem-cell rooted developmental pathways, BFU-Es can undergo a limited number of self- renewal divisions before entering a differentiation pathway leading to formation of CFU-Es. Over time BFU-E self-renewal increases the number of CFU-Es and, after ~2-3 additional days, results in increased numbers of reticulocytes produced from each initiating BFU-E. We showed that glucocorticoids increase the probability that a BFU-E undergoes a self-renewal division and over time increase the numbers of red cells formed from a BFU-E. Recently we showed that activation of two other nuclear receptor/transcription factors - PPARα (by the lipid - lowering drugs fenofibrate and GW7647) and HIF1α (by two clinically- tested Prolyl Hydroxylase 2 (PH2) inhibitors) synergize with low corticosteroid concentrations to further enhance BFU-E self-renewal and red cell production. "Early" BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do those forming small BFU-E colonies. In order to understand the mechanism underlying this heterogeneity, we conducted single cell transcriptome analysis on BFU-E cells purified from mouse embryos. Our analyses showed that there are two principal subgroups of mouse BFU-E cells and that expression of the Type III TGFβ receptor (TGFβ RIII) is markedly elevated in "late" relative to "early" BFU-Es. Expression of TGFβ RIII is correlated with that of GATA1, a gene encoding an erythroid transcription factor induced during the BFU-E to CFU-E transition. Both mouse and human BFU-E sub populations (TGFBR310%lo) expressing the 10% lowest amount of surface TGFβ RIII are indeed enriched for early BFU-Es, and are significantly more responsive to glucocorticoid stimulation, which promotes BFU-E self-renewal, as compared to the total BFU-E population. The TGFBR310%lo BFU-E subpopulation presumably represents earlier BFU-Es with maximal capacity for self-renewal. Consistent with this notion, signaling by the TGFβ receptor kinases RI and RII increases during the transition from early (TGFBR310%low) to late (TGFBR310%hi) BFU-Es and then decreases in CFU-E cells. Blocking TGFβ signaling by receptor kinase inhibitors increases TGFBR310%lo BFU-E cell self-renewal and increases total erythroblast production, suggesting the use of this type of drug in treating EPO unresponsive anemias. Disclosures No relevant conflicts of interest to declare.

PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.

SummaryMany acute and chronic anemias, including hemolysis, sepsis, and genetic bone marrow failure diseases such as Diamond-Blackfan Anemia (DBA), are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production 1,2,3–5,6,7,8,9. Treatment of these anemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently we showed that glucocorticoids specifically stimulate self-renewal of the early erythroid progenitor, the burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells 10,11. Here we demonstrate that activation of the peroxisome proliferator-activated receptor alpha (PPARα) by PPARα agonists, GW7647 and fenofibrate, synergizes with glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures both of mouse fetal liver BFU-Es and of mobilized human adult CD34+ peripheral blood progenitors, the latter employing a new and effective culture system that generates normal enucleated reticulocytes. While PPARα−/− mice show no hematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPARα agonists facilitate recovery of wild-type mice, but not PPARα−/− mice, from PHZ-induced acute hemolytic anemia. We also showed that PPARα alleviates anemia in a mouse model of chronic anemia. Finally, both in control and corticosteroid-treated BFU-E cells PPARα co-occupies many chromatin sites with GR; when activated by PPARα agonists, additional PPARα is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPARα agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPARα agonists we used may improve the efficacy of corticosteroids in treating Epo resistant anemias.

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.

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.

Podocalyxin selectively marks erythroid-committed progenitors during anemic stress but is dispensable for efficient recovery

Podocalyxin expression on Ter119(+) erythroblasts is induced following administration of erythropoietin (Epo) or phenylhydrazine treatment, but is notably absent on committed erythroid progenitors during homeostatic red cell turnover. Following high-dose Epo administration in vivo, podocalyxin surface expression is upregulated, in part, via a signal transducers and activators of transcription 5-dependent pathway and this expression has been postulated to play a role in the release of reticulocytes from hematopoietic organs into the periphery under conditions of increased erythropoietic rate. Here we have thoroughly addressed this hypothesis and further examined the expression profile of podocalyxin during Epo-induced erythroblast expansion and stress erythropoiesis. Following Epo induction, progenitor cells were sorted to characterize podocalyxin expression during stress. In addition, as podocalyxin-deficient mice die perinatally, we used chimeric mice reconstituted with wild-type or podocalyxin-deficient hematopoietic cells to analyze differences in response to high dose Epo administration and chemically induced anemia. Podocalyxin surface expression is rapidly upregulated in response to stress and marks early erythroid progenitors and erythroblasts. Despite loss of podocalyxin, chimeras exhibit normal basal erythropoiesis and no differences in erythroid progenitor proportions in the spleen and marrow in response to Epo. Further, podocalyxin is dispensable for efficient recovery from models of anemia. We demonstrate that podocalyxin is a highly specific marker of stress-induced blast-forming unit erythroid and colony-forming unit erythroid progenitors in mouse bone marrow and spleen. In addition, our findings suggest that podocalyxin is not necessary for efficient erythroblast expansion, erythroid differentiation, or reticulocyte release in response to Epo stimulation in vivo.

The Prolactin Receptor Rescues EpoR−/− Erythroid Progenitors and Replaces EpoR in a Synergistic Interaction With c-kit

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR−/−progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR−/− progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR−/− and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.© 1998 by The American Society of Hematology.

Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)-/- fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation in vitro by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred in vivo before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in (Epo-R)-/- fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF/KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.