Human Erythroid Progenitor Research Articles

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development

AbstractAbstract 3188To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as KitposCD71highTer119neg; KitnegCD71highTer119neg; and KitnegCD71highTer119pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages. Disclosures:No relevant conflicts of interest to declare.

Distinct functions of erythropoietin and stem cell factor are linked to activation of mTOR kinase signaling pathway in human erythroid progenitors

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis. The concerted effects of these two growth factors are responsible for guiding initial commitment, expansion and differentiation of progenitors. The aim of the study was to identify signaling elements pertinent to translational control and elucidate whether both cytokines can contribute to protein translation providing some functional redundancy as seen with respect to apoptosis. The current study focused on non-apoptotic functions of SCF mediated through mTOR/p70S6 leading to protein translation and cell proliferation. We utilized a human primary erythroid progenitors and erythroblasts that are responsive to EPO and SCF to investigate the activation of mTOR/p70S6 kinases and their downstream effectors, the pathway primarily responsible for protein translation. We showed that mTOR, p70S6 kinases and their downstream signaling elements 4EBP1 and S6 ribosomal protein are all activated by SCF but not by EPO in primary erythroid progenitors. We also found that SCF is the sole contributor to activation of the protein translational machinery and activation of mTOR/p70S6 pathway is confined to the proliferative phase of erythroid differentiation program. Altogether these results demonstrate that unlike the survival function which is supported by both EPO and SCF protein translation essential for proliferation is governed by only SCF.

The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators ofmicrotubule behavior has been studied mainly incultured cells. We show here that absence of murine CLASP2 invivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization ofmicrotubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

MiRNA and tropism of human parvovirus B19

Parvovirus B19 has an extreme tropism for human erythroid progenitors. Here we propose the hypothesis explaining the tropism of human parvovirus B19. Our speculations are based on experimental results related to the capsid proteins VP1 and VP2. These proteins were not detectable in nonpermissive cells in course of these experiments, although the corresponding mRNAs were synthesized. Our interpretation of these results is an inhibition of translation in nonpermissive cells by human miRNAs. We bring support to our hypothesis and propose detailed experimental procedure to test it.

Human Parvovirus B19 DNA Replication Induces a DNA Damage Response That Is Dispensable for Cell Cycle Arrest at Phase G 2 /M

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells

Background In many countries, the available supply of transfusable red blood cells (RBCs) is often inadequate. This problem has stimulated considerable research around the world into the development of a robust method for in vitro production of transfusable RBCs. Some possible methods are under active investigation such as the production of RBCs from hematopoietic stem/progenitor cells and from embryonic stem (ES) or induced Pluripotent Stem (iPS) cells. Although it may be feasible to produce RBCs from their immediate precursor cells, it is not easy to obtain sufficient numbers of these precursor cells. One solution to the latter problem may be to establish immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro; these immortalized cells would form a very valuable resource. We previously developed a robust method for generating immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse ES cells, and successfully established several immortalized erythroid progenitor cell lines that we designated Mouse ES cell Derived‐Erythroid Progenitor (MEDEP) cell lines. Although the precise characteristics of these cell lines varied, each of them could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Transplantation of MEDEP cells into mice suffering from acute anemia resulted in transient proliferation of the cells, which subsequently differentiated into functional RBCs and significantly ameliorated the acute anemia.Aims Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated RBCs.Methods We sought to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of human ES cells and iPS cells using a similar method as for establishment of MEDEP cell lines (Hiroyama et al., PLoS ONE 3: e1544, 2008).Results Using the original method developed in mice, we failed to establish immortalized human erythroid progenitor cell lines. This may have been due to the low efficiency of inducing erythroid cells from ES and iPS cells. It was previously reported that enforced expression of the transcription factor TAL1, which plays essential roles in early hematopoiesis and differentiation of erythroid cells and megakaryocytes, improved the efficiency of induction of hematopoietic cells from ES cells of the common marmoset. We therefore enforced expression of TAL1 in human iPS cells and found that this resulted in a significant improvement in the rate of production of hematopoietic cells, in particular of erythroid cells. However, our attempts to establish immortalized human erythroid progenitor cell lines from iPS cells expressing Tal1 were unsuccessful. Recently, it was reported that enforced expression of HPV16‐E6/E7 in CD36‐positive erythroid cells aided the establishment of immortalized human erythroid progenitor cell lines. We decided to adopt this strategy with some modifications and succeeded in establishing immortalized human erythroid progenitor cell lines able to produce enucleated RBCs.Summary/Conclusions We have successfully established immortalized Human Umbilical Cord Blood Derived‐Erythroid Progenitor (HUDEP) cell lines and Human iPS cell Derived‐Erythroid Progenitor (HiDEP) cell lines that are able to produce enucleated RBCs.

The role of WDR5 in silencing human fetal globin gene expression

Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established. To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP. We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program. Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny

BackgroundIt has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny.MethodsHematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells.ResultsIn microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH).ConclusionsThis broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes.

Erythropoietin-driven signaling ameliorates the survival defect of DMT1-mutant erythroid progenitors and erythroblasts

Hypochromic microcytic anemia associated with ineffective erythropoiesis caused by recessive mutations in divalent metal transporter 1 (DMT1) can be improved with high-dose erythropoietin supplementation. The aim of this study was to characterize and compare erythropoiesis in samples from a DMT1-mutant patient before and after treatment with erythropoietin, as well as in a mouse model with a DMT1 mutation, the mk/mk mice. Colony assays were used to compare the in vitro growth of pre-treatment and post-treatment erythroid progenitors in a DMT1-mutant patient. To enable a comparison with human data, high doses of erythropoietin were administered to mk/mk mice. The apoptotic status of erythroblasts, the expression of anti-apoptotic proteins, and the key components of the bone marrow-hepcidin axis were evaluated. Erythropoietin therapy in vivo or the addition of a broad-spectrum caspase inhibitor in vitro significantly improved the growth of human DMT1-mutant erythroid progenitors. A decreased number of apoptotic erythroblasts was detected in the patient's bone marrow after erythropoietin treatment. In mk/mk mice, erythropoietin administration increased activation of signal transducer and activator of transcription 5 (STAT5) and reduced apoptosis in bone marrow and spleen erythroblasts. mk/mk mice propagated on the 129S6/SvEvTac background resembled DMT1-mutant patients in having increased plasma iron but differed by having functional iron deficiency after erythropoietin administration. Co-regulation of hepcidin and growth differentiation factor 15 (GDF15) levels was observed in mk/mk mice but not in the patient. Erythropoietin inhibits apoptosis of DMT1-mutant erythroid progenitors and differentiating erythroblasts. Ineffective erythropoiesis associated with defective erythroid iron utilization due to DMT1 mutations has specific biological and clinical features.

Ex-vivo generation of human red cells for transfusion

The present article reviews the recent data concerning the generation of red blood cells from haematopoietic stem cells using laboratory culture and discusses the potential for generating cultured red cells in sufficient quantity for use in transfusion practice. Functional human reticulocytes have been generated from adult peripheral blood haematopoietic stem cells in laboratory culture without the use of heterologous feeder cells and their viability was demonstrated in vivo. Human erythroid progenitor cells lines have been produced from cord and human induced pluripotent stem cell (hiPSC) haematopoietic progenitors. Availability of cultured human red cells from haematopoietic stem cells in the quantities required for transfusion therapy would have a major impact on healthcare provision worldwide. Recent studies provide cause for optimism that this ambitious goal is achievable. Functional adult reticulocytes have been made in culture and shown to survive in vivo. Erythroid progenitor cell lines have been derived from cord blood and from human induced pluripotent stem cells, suggesting that large-scale culture of erythroid cell lines and their differentiation to reticulocytes will be possible. Significant problems remain. More efficient enucleation and induction of maturation to an adult phenotype will be required in order to exploit high proliferative capacity of human embryonic stem cells and hiPSCs. Novel bioengineering solutions will be required to generate cultured red cells in the large quantities required, and in this context, use of synthetic three-dimensional scaffolds to mimic the bone marrow niche holds great promise for the future.

Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

BackgroundThe fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation.ResultsWe confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified.The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.ConclusionsThe transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.

Novel Pathways to Erythropoiesis Induced by Dimerization of Intracellular C-Mpl in Human Hematopoietic Progenitors

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl.

Unique Requirements for Lipoproteins Derived From Human Plasma for Maximal Expansion of Human Erythroid Cells Ex-Vivo Under Humanized Conditions

Abstract 1257Ex-vivo expanded erythroid cells (EBs) have been proposed as alternative transfusion products. We have recently formulated humanized media (HEMAdef) that sustain amplification of EBs from cord blood as efficiently as media containing fetal bovine serum (HEMAser) and 10-times greater from adult blood (Migliaccio et al, Cell Transplantation, 2010; 19:453–469). However, while a minority (14.1±5.3%) of EBs generated under HEMAser conditions contain alterations of their membrane which produce cell protrusions (see Figure), membrane abnormalities were observed in 22.7±7.1% of EBs generated under HEMAdef conditions. [Display omitted] The membrane of the red cells consists of a lipid-bilayer attached to the cytoskeleton by integral membrane proteins. The physical-chemical properties of these membranes rely on maintenance of the integrity of the lipid bilayer as disruption of this layer leads to shorten red cell survival or cell removal from the circulation. EBs internalize apolipoprotein-lipid complexes (lipoproteins) present in the environment. Lipoproteins are classified on the basis of floating density into high density lipoproteins [HDL, 1.063<ρ<1.210, ∼50:50 (in dry weight) complex of apoliprotein A-I (apoAI; 32%) and apoAII (10%) with cholesterol esters (15%)], low density lipoproteins [LDL, 1.019<ρ<1.063, ∼20:80 complex of apoB100 (18%) with cholesterol esters (40%)] and very low density lipoproteins [VLDL, 0.950<ρ<1.006, ∼8:82 complex of apoB100 (4%), apoE and apoCI, CII and CIII (4%) with triglycerides (55–65%)]. In addition, all lipoproteins contain 20% of phospholipids. In culture, lipids are provided by lipoproteins present in fetal bovine serum or by synthetic vesicles containing animal (egg cholesterol) and/or vegetal (soybean lecithin) lipids conjugated with albumin (bovine or human). In spite of emerging associations between genetic apolipoprotein gene deficiency and red cell membrane abnormalities, the role played by individual lipoproteins in erythroid cell differentiation is unknown.The aim of this study was to analyze the effect of lipoproteins purified from human plasma on the growth and maturation of EBs generated by mononuclear cells (MNC) from adult blood. HDL, LDL and VLDL were purified according to published methods (Havel RJ et al, J Clin Inv 1955; 34:1345) and their purity assessed on the basis of apoprotein/lipid content analyses. HDL, LDL, VLDL and an artificial mixture of the three fractions (total plasma lipoprotein fraction, TPLF) were used at a concentration of 20 mM/mL which had been previously determined to induce optimal EBs proliferation in preliminary concentration/MTT proliferation curves both under HEMAser and HEMAdef conditions.In semisolid assay, none of the fractions had an effect on the growth of CFU-GM-derived colonies. By contrast, LDL and VLDL both increased by 0.5 and 2-fold, respectively, the numbers of BFU-E derived colonies generated by adult blood MNC in serum-supplemented cultures (78±8 and 113±15 vs 57±9, p<0.01) while only LDL increased the numbers of BFU-E-derived colonies under serum-deprived conditions (64±8 vs 39±5, p<0.05). The various lipoprotein fractions did not improve the overall FI in HEMAser cultures (FI ∼20 by day 14 in all conditions) but increased the frequency of EBs with membrane abnormalities (from 14% to 20–27%). LDL and VLDL also increased by 2-fold the total numbers of progenitor cells observed at day 10 (total progenitor cells/culture 756±104 and 1064±292 vs 542±54, p<0.01–0.05). Under HEMAdef conditions, LDL and VLDL increased by 3-2-fold the FI (95±10 and 64±14 vs 27±6, p<0.01–0.05) by day 14 but did not affect the frequency of EBs with membrane abnormalities which remained high (∼30%). By contrast TPLF modestly increased the FI over those observed in HEMAser cultures (40±12 vs 20±14 in HEMAdef + TPLF vs HEMAser, p<0.05) but greatly reduced the frequency of EBs with membrane abnormalities (14±6% vs 28±7%, p<0.05). The frequency of mature CD36posCD235ahigh EBs observed by day 14 was ∼38–50% in HEMAser and 8–16% in HEMAdef.Human LDL and VLDL increase cloning efficiency and ex-vivo expansion of human adult erythroid progenitors in liquid culture but impair membrane assembly which is improved by TPLF. These results suggest that a balanced combination of lipoproteins, in concentrations still to be identified, may be required to observe maximal expansion of morphologically normal human EBs under HEMAdef conditions. 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.

Induction of Fetal Hemoglobin by Inactivation of HDAC1 or HDAC2 without Altering Cellular Proliferation

Abstract 354Histone deacetylase (HDAC) inhibitors are effective inducers of fetal hemoglobin, and prior studies have shown that selective inactivation of HDAC1 or HDAC2 is sufficient to induce fetal hemoglobin in vitro. In our current work, we demonstrate that HDAC1 and HDAC2 are attractive targets for clinical translation for two reasons: 1) Selective inhibition will decrease off-target effects that currently limit the use of hydroxyurea and pan-HDAC inhibitors, and 2) HDAC inhibitors induce fetal hemoglobin in various preclinical models, and they can be combined with hydroxyurea to achieve further fetal hemoglobin induction. To investigate off-target effects, we selectively inactivated HDAC1, HDAC2 or HDAC3 in human erythroid progenitor cells, and examined the effect of each knockdown on cellular cytotoxicity and cell cycle progression. Although knockdown of HDAC3 negatively influenced growth, selective knockdown of HDAC1 or HDAC2 had no effect on expansion of erythroid progenitors. In addition, knockdown of HDAC2 does not block cell cycle progression. These data support the possibility that an HDAC1- or HDAC2-specific inhibitor may offer a therapeutic advantage by reducing side effects, while maintaining robust HbF induction. Armed with this knockdown data, we are now investigating HDAC inhibitor compounds of various selectivity in in vitro and in vivo models. To perform optimal clinical trials, and ultimately to benefit the most sickle cell disease patients, it would be ideal to combine HDAC inhibitor treatment with hydroxyurea. A combination treatment approach may ameliorate some of the limitations of hydroxyurea use, such as the unpredictable effect on fetal hemoglobin levels, and the lack of benefit in beta thalassemia patients. First, we combined HDAC2 inactivation with hydroxyurea treatment in vitro. Human bone marrow-derived CD34+ cells were infected with lentiviruses containing an shRNA targeting either HDAC2 or a luciferase control gene. The cells were then treated on day 4 of erythroid differentiation with hydroxyurea (10–20 uM dose). Compared to the untreated luciferase control samples, we observed a 7–9-fold increase in gamma-globin expression in the untreated HDAC2-knockdown samples, a 2.5-fold increase in the hydroxyurea-treated luciferase control samples, and a trend toward an additive effect on gamma-globin induction in the cells where HDAC2 knockdown was combined with hydroxyurea treatment. To investigate the effects of HDAC inhibitors in vivo, we administered compounds to BCL11A conditional knockout transgenic mice (by erythroid-selective EpoR-GFP Cre) containing the human beta-globin locus. As reported previously, BCL11A inactivation powerfully de-repressed gamma-globin expression, and administration of an HDAC inhibitor, SAHA, led to a further elevation of gamma-globin mRNA. We now demonstrate that administration of another pan-HDAC inhibitor, panobinostat (LBH589), results in an additional 1.5- to 2.5-fold increase in gamma-globin mRNA relative to pre-treatment baseline. We are currently evaluating the combination of panobinostat and hydroxyurea in these mice to confirm that the compounds have an additive effect in vivo as well as in vitro. Taken together, these experiments indicate that inhibiting HDAC1 or HDAC2 is a promising therapeutic approach to increasing fetal hemoglobin levels in patients with beta-hemoglobinopathies, both alone and in combination with hydroxyurea. Disclosures:Bradner:Acetylon: .

“RHEX-26”, a Novel EPO/EPOR-Regulated Mediator of Human Erythroid Progenitor Cell Expansion

Abstract 682Erythropoietin (EPO) and its receptor (EPOR) serve well as an informative paradigm for understanding hematopoietic growth factor effects and receptor action mechanisms. Recently, interest in EPO/EPOR investigations has been heightened based on the clinical emergence of novel EPO mimetics, and by reported EPO/EPOR activities in modulating cellular immunity, diabetic susceptibility, retinopathy and the cytoprotection of select non-hematopoietic tissues. To better understand EPOR action modes, we presently have applied a global phosphoproteomics approach to specifically discover new targets of EPO/EPOR-regulated tyrosine phosphorylation. Using an EPO-dependent erythroid progenitor cell model, our LC-MS/MS approach was first validated for known EPO/EPOR-targets (e.g., JAK2, STATs, SHIPs, PLCgamma, GABs). Second, among certain known targets, new phosphorylation sites were identified. Third, several novel major downstream targets of EPO/EPOR were discovered. One (a previously uncharacterized open reading frame here designated as “RHEX26”) was induced ∼20-fold in tyrosine-phosphorylation by EPO treatment. Quantitative RT-PCR analyses of tissues and primary hematopoietic cells indicate RHEX26 expression to be erythroid- restricted. Antibodies prepared to RHEX26 and tyrosine phosphorylated-RHEX26 confirmed strong EPO-induced phosphorylation; cell fractionation and Western blot studies demonstrated membrane residence of this 26 kDa phosphoprotein; and immunofluorescence microscopy showed sharp RHEX26 sub-localization as an integral plasma membrane protein. In functional analyses, lentiviral shRNA knockdown of RHEX26 proved to markedly inhibit the EPO-dependent expansion of erythroid progenitor cells (clonal colony-forming analyses demonstrated >80% specific inhibition), while effects on cell viability were limited. Mechanistically, RHEX26 is coupled (in part) to GRB2, with co-immunoprecipitation observed specifically upon EPO exposure, but without dependency on RHEX26 tyrosine phosphorylation. Finally, genealogy analyses intriguingly indicate RHEX26 to be well-conserved only in H. sapiens and primates, while no ortholog is present in the rat, mouse, or zebrafish. RHEX26, as a novel EPO/EPOR 26 kDa target, therefore is proposed to comprise an important new Regulator of Human Erythroid eXpansion. Disclosures:Leu:Affymax, Inc.: Employment, own stock in Affymax, Inc. Young:Affymax, Inc.: Employment, own stock in Affymax, Inc. Schatz:Affymax, Inc.: Employment, own stock in Affymax, Inc. Green:Affymax, Inc.: Employment, own stock in Affymax, Inc.

Does Quantitative Heterogeneity of Human Fetal Hemoglobin (Hb F) Reveal Friends or Foes of KLF1 in Globin Gene Switching ?

Abstract 1092The chemical heterogeneity of fetal hemoglobin (Hb F) due to variable ratios of the Gγ and Aγ globin subunits reflects genetic complexity because of common dimorphisms such as Hb F Sardegna (or Aγ75(E19) Ile>Thr; also known as AγT) in Caucasians, and common variants such the Gγ globin variant, Hb F Malta I (or Gγ117(G19) His>Arg) that is in tight linkage disequilibrium with the β globin variant Hb Valletta (or β87(F3) Thr>Pro) and is found in 1.8% of neonates from Malta. Comprehensive and integrated maternal and neonatal testing has led to the finding of triple compound heterozygotes with Hb F Malta I in association with Hb F Sardegna and Hb Valletta in whom all globin genes that are functional in the transition from fetal to adult Hemoglobin profiles are genetically tagged, and quantifiable with High Performance Liquid Chromatography in the neonate or mRNA in the adult in the context of diverse XMNI −158 C>T Gγ globin and (AT)XTY −540 β globin haplotypes. The interaction between XMNI and (AT)XTY revealed “conditional” cis-trans interplay that appeared to be under developmental control. A family with members carrying Hb F Malta I in association with a rare form of the Hereditary Persistence of Fetal Hemoglobin has also been found. The genetic cause has been traced to haplo-insufficiency of the putative erythroid master regulator KLF1, and, as confirmed by functional assays in vitro. However, levels of Hb F expression varied considerably (3.3% – 19.5%) while a second family from Malta with the same KLF1 mutation (p.K288X) had normal Hb F indicating interplay of KLF1 with modifying genes. These data, together with comparative expression profiling of human erythroid progenitors, indicated a small set of additional gene products that may interact positively (friends) or negatively (foes) at the level of commitment or expression in globin gene switching with significant effects on the Mean Corpuscular - Hb F. Whole genome sequencing on critically informative family members currently in progress may further uncover the complex genetic interactions in developmental globin gene control. Disclosures:No relevant conflicts of interest to declare.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.