Human Erythroid Progenitor Research Articles

RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs

AbstractDiamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin−CD34+CD38+CD45RA−CD123−CD71+CD41a−CD105−CD36− bone marrow cells as EEP giving rise to BFU-E, and Lin−CD34+/−CD38+CD45RA−CD123−CD71+CD41a−CD105+CD36+ cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.

Increase of microRNA-210, decrease of raptor gene expression and alteration of mammalian target of rapamycin regulated proteins following mithramycin treatment of human erythroid cells.

Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells.

In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors

AbstractTissue hypoxia induces a systemic response designed to increase oxygen delivery to tissues. One component of this response is increased erythropoiesis. Steady-state erythropoiesis is primarily homeostatic, producing new erythrocytes to replace old erythrocytes removed from circulation by the spleen. In response to anemia, the situation is different. New erythrocytes must be rapidly made to increase hemoglobin levels. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best characterized in the mouse, where it is extramedullary and utilizes progenitors and signals that are distinct from steady-state erythropoiesis. In this report, we use an in vitro culture system that recapitulates the in vivo development of stress erythroid progenitors. We identify cell-surface markers that delineate a series of stress erythroid progenitors with increasing maturity. In addition, we use this in vitro culture system to expand human stress erythroid progenitor cells that express analogous cell-surface markers. Consistent with previous suggestions that human stress erythropoiesis is similar to fetal erythropoiesis, we demonstrate that human stress erythroid progenitors express fetal hemoglobin upon differentiation. These data demonstrate that similar to murine bone marrow, human bone marrow contains cells that can generate BMP4-dependent stress erythroid burst-forming units when cultured under stress erythropoiesis conditions.

Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis.

Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.

Human parvovirus B19: a mechanistic overview of infection and DNA replication.

Human parvovirus B19 (B19V) is a human pathogen that belongs to genus Erythroparvovirus of the Parvoviridae family, which is composed of a group of small DNA viruses with a linear single-stranded DNA genome. B19V mainly infects human erythroid progenitor cells and causes mild to severe hematological disorders in patients. However, recent clinical studies indicate that B19V also infects nonerythroid lineage cells, such as myocardial endothelial cells, and may be associated with other disease outcomes. Several cell culture systems, including permissive and semipermissive erythroid lineage cells, nonpermissive human embryonic kidney 293 cells and recently reported myocardial endothelial cells, have been used to study the mechanisms underlying B19V infection and B19V DNA replication. This review aims to summarize recent advances in B19V studies with a focus on the mechanisms of B19V tropism specific to different cell types and the cellular pathways involved in B19V DNA replication including cellular signaling transduction and cell cycle arrest.

TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1.TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10.The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis.

Discovery of Novel EPO/EPOR/JAK2 Targets and Signal Transduction Factors Via Proteomic-Based Interrogations of Post-Translational Motif Modifications

Although EPO/EPOR/JAK2 actions have been intensely studied, the majority of investigations to date have relied heavily upon conventional approaches, extant reagents and murine cell models. We therefore hypothesized that important EPOR/JAK2 targets consequently remain undiscovered, and by applying (phospho)proteomic based interrogations, have uncovered intriguing sets of new candidate mediators of EPO’s effects on human erythroid progenitor cells. Specifically, following [-] vs [+] EPO challenge, total cellular tryptic or Glu-C peptides were generated, and those post-translationally modified (PTM) due to EPO at phosphotyrosine residues (p-Y), p-TPP motifs, and MAPK plus ATK signaling nodes were isolated, and identified by duplicate tandem LC-MS/MS. EPO targets furthermore were confirmed via parallel analyses using an EPOR agonist. Known EPO/EPOR/JAK2 targets first were validated (eg., JAK2, STAT5, GAB1-3, SHIP1-3, PLCgamma, p85alpha-PI3K, LNK, SHC1, SPRY1) (and further defined with regards to specific isoforms, plus known vs novel sites of EPO-induced PTM). Beyond this, newly discovered EPO/EPOR/JAK2 regulated targets included those within the following five categories: 1] Cytoskeletal targets unexpectedly as erythroid alpha and beta spectrins (pY2332, pY15) together with pY modulation of Beta-Adducin at a calmodulin binding site (plus EPO-regulated PTM of Calmodulin per se); 2] Select EPO- regulated transcription factors, including 100-fold p-Y modulation of KLF5 at a novel transcriptional activation motif; 3] EPO/EPOR/JAK2 modulation of two new protein tyrosine phosphatase targets as PTPN18 and PTPN7; 4] Rapid EPO-induction of a notable number of novel upstream molecular adaptors including C1ORF186/”RHEX”, C1ORF150, DOK1, IRS2, SH2D2A, STAM2, SPRY2,4, DLG1,3, FRS2, AMOTL1, RAI14, CTNND1, EPS15, HGS, ITSN2, and WASL – with two novel ORF factors as C1ORF186/RHEX and C1ORF150 as prime targets in human erythroid progenitors, but absent from rat, mouse, and lower vertebrate genomes. [For C1ORF186/RHEX, select cell and molecular biological properties will be summarized]. And 5] highlighted additional novel targets (eg, transporters, metabolic regulators) that, together with the above, further emphasize a previously underestimated sophistication of EPOR/JAK2 signal transduction circuits, certain of which are proposed to be central to EPO and ESA effects on anemia (and potentially, hypertensive side-effects). DisclosuresNo relevant conflicts of interest to declare.

Micro RNA Profiling to Identify Regulator of Fetal Hemoglobin Expression in Sickle Cell Disease

Elevated fetal hemoglobin (HbF) expression ameliorates the clinical severity of sickle cell disease (SCD) by inhibiting hemoglobin S polymerization. Differences in HbF levels are attributed to inherited DNA genetic variations that regulate γ-globin transcription; however the role of microRNA (miRNA) genes in HbF regulation has not been investigated using clinical samples. miRNAs are small non-protein-coding RNA molecules that negatively regulate gene expression through inhibition of mRNA translation. Our goal is to identify miRNA genes with altered expression in sickle cell patients with elevated HbF levels, to elucidate mechanisms of γ-globin gene regulation. After IRB approval, peripheral blood was collected from SCD patients (not on hydroxyurea therapy), followed in the pediatric and adult Sickle Cell Clinics at Georgia Regents University. HbF levels measured by high performance liquid chromatography and complete blood and reticulocyte counts were obtained. Twelve blood samples, six each from SCD subjects with high HbF (19.9±2.1%) or low HbF (4.4±0.9%) levels were analyzed. We observed more severe anemia and higher reticulocyte counts in the low HbF group. After Histopaque separation, red blood cells were processed on a MACS column with anti-CD71 antibody to isolate reticulocytes, followed by total RNA extraction using Trizol. RNA (750ng) was hybridized to a genome-wide miRCURY LNA microRNA Array (Exiqon) containing 1,921 human probes. The microarray raw data were collected on an Agilent G2565BA Microarray Scanner System and normalized by Model-Based Background Correction and Principle Component Analysis. Characterization of miRNA profiles for low HbF compared to high HbF groups identified 327 differentially expressed genes including multiple miR-144 isoforms. We subsequently explored the function of miR-144 because it targets Nrf2 which mediates drug-induced HbF expression and Nrf2 has an antioxidant protective effect in SCD. Therefore, we conducted supervised learning of the normalized microarray data based on miR-144 expression. Interestingly, in the low HbF group we observed two subphenotypes: 1) associated with 8-fold increased miR-144 expression (3 subjects) and 2) associated with no change in miR-144 level (3 subjects) when compared to the high HbF group suggesting a role of miR-144 in HbF regulation. In the supervised learning analysis, there were 62 up-regulated and 33 down-regulated miRNAs (>2-fold; p<0.05) in the first subphenotype. We hypothesized that miRNAs up-regulated in the low HbF group might silence known γ-globin trans-activators. By TargetScan and Miranda analysis 7 miRNAs were predicted to target γ-globin including miR-96 a known negative regulator. There were 4 miRNAs predicted to target Nrf2 and 12 miRNAs that target other transcription factors such as KLF1, KLF4, BCL11A, and GATA2.To define a functional role of miR-144 we conducted studies in adult CD34+ stem cells grown in a two-phase culture system containing Stem Cell Factor (50ng/mL), Interleukin-3 (10ng/mL) and Erythropoietin (4IU/mL). At day 8 in culture, miR-144 mimic, miR-144 antagomir (inhibitor) or scrambled control (100nM, 200nM, and 300nM) were transfected using a Nucleofector System. After 72 hr incubation, RT-qPCR was conducted to measure γ-globin and Nrf2 mRNA levels. miR-144 mimic or antagomir at 100-300nM concentrations had no significant effect on γ-globin mRNA levels. By contrast, flow cytometry analysis using a FITC-anti-γ-globin antibody in erythroid cells treated with miR-144 mimic, produced a 30-70% decrease in HbF positive cells (p<0.05). On the contrary, we observed a 1.8-fold increase in HbF positive cells mediated by 300nM miR-144 antagomir. Evidence that miR-144 targets Nrf2 was established when antagomir treatment increased Nrf2 expression 1.4-fold (p<0.05). Final studies using day 8 erythroid progenitors treated with Nrf2 siRNA demonstrated a 40% decrease in γ-globin mRNA supporting a role of Nrf2 on γ-gene expression. In summary, the miRNA profiles associated with HbF expression in SCD patients combined with functional studies in human primary erythroid progenitors, support a role for miR-144 in γ-globin regulation. These findings will be expanded to a pre-clinical SCD mouse model to develop miR-144 as a potential therapeutic option. DisclosuresNo relevant conflicts of interest to declare.

Exploring the Potential Usefulness of 5-Aminolevulinic Acid for X-Linked Sideroblastic Anemia

(Background) Congenital sideroblastic anemia (CSA) is an inherited disease; it is a microcytic type of anemia characterized by bone marrow sideroblasts with excess iron deposition in the mitochondria. The most common form of CSA is XLSA (X-linked sideroblastic anemia), which is attributed to mutations in the X-linked gene ALAS2 (erythroid-specific 5-aminolevulinate synthase). ALAS2 encodes the first and rate-limiting enzyme involved in heme biosynthesis in erythroid cells, which utilizes glycine and acetyl-coenzyme A to form 5-aminolevulinic acid (ALA) and also requires pyridoxal 5′-phosphate (PLP, vitamin B6) as a cofactor. Based on the evidence that half of the XLSA cases were unresponsive to PLP (Ohba et al. Ann Hematol 2013), ALA supplementation could emerge as an alternative therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. As a preclinical study, we focused our study on the effect of ALA on human erythroid cells. Furthermore, we investigated the molecular mechanism by which ALA is transported into erythroid cells.(Method) Human K562 erythroid cells as well as human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2013) were used for the analysis. We investigated the effects of ALA (0.01, 0.1, and 0.5 mM for 72 h) on heme content, hemoglobinization, and erythroid-related gene expression. Heme content was determined fluorometrically at 400 nm (excitation) and 662 nm (emission). Small interfering RNA (siRNA)-mediated knockdown of ALAS2 was conducted using Amaxa Nucleofector™ (Amaxa Biosystems, Koln, Germany). For transcription profiling, Human Oligo chip 25K (Toray, Tokyo, Japan) was used for control and ALAS2 siRNA-treated HiDEP cells. Gamma-aminobutyric acid (GABA) (Sigma, St. Louis, MO, USA) was used at concentrations of 10 and 20 mM.(Results) First, we demonstrated that ALA treatment resulted in significant dose-dependent accumulation of heme in K562 cells. Concomitantly, the treatment substantially induces erythroid differentiation as assessed using hemoglobin (benzidine) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes such as the globin genes (HBA and HBG) and the heme oxygenase 1 (HMOX1) gene in K562 cells. To investigate the mechanism by which ALA was transported into erythroid cells, we conducted quantitative RT-PCR analysis for previously reported ALA transporters, including SLC15A1, SLC15A2, SLC36A1, and SLC6A13 (Frølund et al. Br J Pharmacol 2010; Ahlin et al. Drug Metab Dispos 2009; Moretti et al. Br J Cancer 2002). The analysis revealed that SLC36A1 was abundantly expressed in K562 and HiDEP cells. Thus, GABA was added to K562 cells to competitively inhibit SLC36A1-mediated transport (Frølund et al. Br J Pharmacol 2010). GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells.Next, siRNA-mediated knockdown of ALAS2 in HiDEP cells resulted in a significant decrease in the expression of globin genes as well as HMOX1; however, ringed sideroblasts were not observed. Microarray analysis revealed >2-fold up- and down-regulation of 38 and 68 genes caused by ALAS2 knockdown, respectively. The downregulated gene ensemble included globins (HBZ, HBG, HBE, HBD, and HBM) as well as genes involved in iron metabolism (ferritin heavy chain 1: FTH1, transferrin receptor: TFRC and glutaredoxin-1: GLRX5). Gene ontology analysis revealed significant enrichment of cellular iron ion homeostasis (p = 0.000076), cell division (p = 0.00062), DNA repair (p = 0.0006) and translation (p = 0.018), implying that heme was involved in various biological processes in erythroid cells. Interestingly, ALA treatment significantly improved the consequences of ALAS2 knockdown-mediated downregulation of HBA, HBG, and HMOX1.(Conclusion) ALA appears to enter into erythroid cells mainly by SLC36A1 and utilized to generate heme precursor. Thus,ALA may represent a novel therapeutic option for CSA, particularly for cases harboring ALAS2 mutations. DisclosuresFujiwara:Chugai Pharmaceutical, CO., LTD.: Research Funding.

Role of Iron-Responsive Mitochondrial Metabolism and ROS in Anemia

Iron and erythropoietin (Epo) are intimately linked regulators of erythropoiesis. Moderate iron restriction suppresses erythropoiesis at the Epo-dependent, CFU-E stage, without induction of apoptosis and without suppression of other hematopoietic cell lineages. Iron modulates Epo bioactivity in patients with iron deficiency anemia (IDA) and patients with anemia of chronic disease and inflammation (ACDI). To conserve iron when supplies are low, this erythroid iron restriction response reduces iron consumption by suppressing erythropoiesis. The erythroid iron sensor is unknown. Aconitases are multifunctional iron-sulfur cluster proteins localized in the cytosol (Aco1) and mitochondria (Aco2) that convert citrate into isocitrate. We have shown that iron restriction inhibits Aco2 enzymatic activity leading to suppression of erythropoiesis in vitro, and these effects are reversed by isocitrate. Isocitrate corrects IDA in mice and ACDI in rats (Bullock GC, et al. Blood. 2010;116:97-108; Richardson CL, et al. J Clin Invest. 2013 Aug 1;123(8):3614-3623). Iron restriction also alters the cross-talk between transferrin receptor and Epo receptor signaling pathways. These results suggest that Aco2 is an iron-responsive regulator of erythropoiesis. We are investigating the downstream molecular signaling mechanisms by which iron restriction induced-inactivation of Aco2 suppresses erythropoiesis. Our novel preliminary data show that mitochondrial oxidative metabolism rates change over time during erythropoiesis and that iron restriction reduces erythroid mitochondrial metabolism 4 to 7-fold compared to iron replete controls. This iron restriction induced change in respiration is associated with a significant, 1.5 to 3-fold, increase in mitochondrial superoxide production without a corresponding increase in hydrogen peroxide. Importantly, these mitochondrial alterations are reproduced by direct inhibition of aconitase with fluoroacetate (FA) and are not due to changes in mitochondrial number. Further, isocitrate reverses the effects of iron restriction or aconitase inhibition on mitochondrial metabolism and attenuates superoxide production. Based on these data and the known role of reactive oxygen species (superoxide/hydrogen peroxide) in Epo signaling, we propose the overarching hypothesis that iron restriction inhibits mitochondrial aconitase which, in turn, alters erythroid mitochondrial metabolism and ROS signaling resulting in suppression of erythropoiesis (Figure 1). We show for the first time bioenergetics profiles from iron restricted and iron replete primary human erythroid progenitor cells undergoing erythropoiesis. We also show that moderate levels of iron restriction cause mitochondrial dysfunction and alterations in mitochondrial ROS in differentiating erythroid progenitors. The clinical relevance of this project lies in its potential for the development of new iron-free agonists and antagonists of red blood cell production. Agonists may benefit patients with anemia due to iron deficiency or chronic inflammation and antagonists may benefit patients with myeloproliferative neoplasms.Figure 1:Proposed mechanism of iron-dependent regulation of erythropoiesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Non-Coding RNAs Transcribed from ERV-9 LTR Retrotransposons Regulate Erythropoiesis

Long noncoding RNAs (lncRNAs) regulate diverse cellular processes in development, differentiation and malignancy. In human cells, over 80% of the lncRNAs contain retrotransposon sequences transcribed from Alu, L1 and LTR retrotransposons, which comprise ~40% of the human genome. The functional significance of the retrotransposon lncRNAs is largely unknown. The human genome contains ~4000 copies of the ERV-9 LTR retrotransposon, which exhibits strong enhancer activity and initiates synthesis of ERV-9 lncRNAs in erythroid progenitor cells. Recently, we discovered that depletion of the ERV-9 lncRNAs in human erythroid progenitor cells cultured ex vivo from peripheral blood CD34+ cells inhibited ex vivo erythropoiesis. Whole genome RNA sequencing (RNA-seq) found that depletion of ERV-9 lncRNAs significantly suppressed transcription of 608 genes including ~50 key erythroid genes. We hypothesize that the ERV-9 lncRNAs together with the ERV-9 LTR act in cis to regulate transcription of these key erythroid genes and other genes to set up a transcriptional network that promotes erythropoiesis.In the human b-globin gene locus, we showed previously that the ERV-9 LTR retrotransposon performs a beneficial biological function: The ERV-9 LTR enhancer binds NF-Y and GATA-1 and -2 to assemble an LTR-pol II transcription complex, which transcribes long, noncoding RNAs (lncRNAs) from the LTR R-U5 regions through the downstream locus control region (LCR) and intergenic DNAs to reach and activate transcription of b-globin gene 70 kb away. In this tracking and transcription (T&T) mechanism of long-range LTR enhancer function, the ERV-9 lncRNAs could be merely by-products of the tracking and transcribing process of the LTR complex without any functional significance. However, we found recently that depletion of the ERV-9 lncRNAs suppressed transcription of the entire human globin gene locus, diminished occupancies of NF-Y, GATA-1 and -2 and pol II at the ERV-9 LTR and reduced the looping frequency of the ERV-9 LTR with the globin gene locus in erythroid progenitor cells. Thus, the ERV-9 lncRNAs acted in cis to facilitate assembly of the LTR-pol II complex and modulate long-range LTR enhancer function in transcriptional activation of b-globin gene. Genome-wide, whether the ERV-9 lncRNAs transcribed from other gene loci perform similar biological function is under investigation. DisclosuresNo relevant conflicts of interest to declare.

Recombinant scAAV2 Vector-Mediated Ex Vivo Transduction of Primary Human Hematopoietic Stem Cells from a β-Thalassemia Patient and Human β-Globin Gene Expression in a Murine Xenograft Model in Vivo

Background: The most severe form of β-thalassemia, β°-thalassemia major, is characterized by the complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors have been used successfully to achieve clinical efficacy in one patient, although clonal expansion of a myeloid cell population also occurred in this patient which was associated with the activation of a cellular proto-oncogene, HMGA2. We reasoned that recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), might offer a safer alternative. We have previously documented that although the conventional single-stranded (ss) AAV2 vectors mediated β-globin gene transfer and expression in primary human fetal liver cells and in human HSCs from patients with β-thalassemia patients in vitro, the level of transgene expression was sub-optimal. In the present study, we investigated whether double-stranded self-complementary (sc) AAV2 vectors could overcome this limitation.Methods: Human HSCs, obtained from a β-thalassemia homozygous patient, were mock-transduced or transduced with recombinant scAAV2-β-globin vectors at 5×104 vgs/cell, followed by i.v. injection into sub-lethally irradiated NOD/SCID mice (2.65 cGy total body irradiation), which were also pre-treated with 200 µg purified anti-IL2RB/CD122 monoclonal antibody. Recipient mice were sacrificed 12 weeks post-transplantation. Bone marrow cells from recipient mice were analyzed by BFU-E assays. Human β-globin gene expression in human erythroid progenitor cells from transplanted mice was evaluated by RT-PCR.Results: Pre-treatment of NOD/SCID mice with anti-CD122 antibody improves engraftment of human HSCs in bone marrow of receipt mice. Human β-actin (538-bp) and β-globin (272-bp) transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that recombinant scAAV2-β-globin–transduced HSCs from a patient with β-thalassemia were successfully transduced and transplanted in these mice and that human β-globin gene was transcriptionally active 12 weeks post-transplantation.Conclusion: Our results indicate that human HSCs from β-thalassemia patients can be efficiently transduced by recombinant scAAV2-β-globin vectors followed by expression of normal human β-globin gene. These studies provide the proof-of-concept that scAAV2 vector-mediated gene transfer into human HSCs might be a potentially safer alternative approach for gene therapy of β-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

Single Cell Transcriptome Profiling of Highly Purified Human Megakaryocyte-Erythroid Progenitors (MEP) Reveals New Insights into the MEP Fate Decision

Megakaryocyte-Erythroid Progenitors (MEPs) are bipotent cells capable of generating megakaryocytic (Mk) or erythroid (E) progeny. However, neither the cell fate-determining componentry nor the initial molecular consequences of lineage specification have been defined. To elucidate this, it is critical to rigorously purify MEP from primary cell sources. Unfortunately, existing purification strategies to do this fail to yield pure, bipotent cells. To improve upon existing approaches for the enrichment of primary human MEPs from G-CSF mobilized peripheral blood (MPB) and BM, we used the cell surface markers CD36 and CD110 in order to further enrich MEP from CD34+CD38+Lin-Flt3-CD45RA- cells. We then quantitated the Mk and E potential of those cells using single cell colony assays. Using this approach demonstrated that CD36/CD110 selection led to an increase of biphenotypic MEP (assessed as CFU-Mk/E) from ~15% to ~40% of colonies that grew. However, it was unclear from colony assay data alone whether or not the heterogeneity of the underlying population was accurately reflected. To address this, we subjected the FACS-sorted MEP-enriched population to single cell mRNA deep sequencing using the Fluidigm C1 platform. For comparison to MEP, we also performed single cell deep sequencing of CD34+CD38+CD41+Flt3- and CD34+CD38+Flt3-CD36+ cells, which are highly enriched for megakaryocyte progenitors (MkP) and erythroid progenitors (ErP), respectively. A total of 150 single cells were captured and sequenced with an average of 3 million reads per cell (1x100bp sequencing). The mRNA deep sequencing data was analyzed by a combination of gene and cell bi-clustering approach to identify both transcripts and cells that exhibited shared or differential patterning. Initial expression patterns and cell groups were identified using stringent expression filtering for transcripts that exhibited >10 FPKM in at least one cell, and iteratively defined and refined based on known E, Mk, and other hematopoietic genes, and then extended for all strongly expressed transcripts. For the MkP and ErP groups, the resulting clusters of cells expressed genes indicative of commitment to E or Mk differentiation. In contrast, within the MEP-enriched population, while a few cells clustered with MkP and ErP, the vast majority of cells fell into distinct subsets of uncommitted cells, supporting the idea that the MEP-enriched population was unique and distinct from MkP or ErP. Analysis of the gene expression patterns from the MEP, ErP and MkP revealed two remarkable trends.First, the transcription factors GATA1 and GATA2 showed distinct expression patterns in the different clusters of cells; there was a subset of MEP that had high GATA2 expression with little to no GATA1 expression (GATA2 cluster), and an opposite cluster containing high GATA1 expression and low or absent GATA2 expression (GATA1 cluster). The genes most positively correlated with GATA2 expression were also low or absent in the GATA 1 cluster. Closer analysis revealed that the GATA 1 cluster cells were predominantly erythroid and megakaryocyte committed, while the GATA2 cluster appeared uncommitted. A third cluster was present, containing intermediate expression of both GATA1 and GATA2. This cluster is as yet undefined, but appears to contain both MkP and MEP, suggesting a possible link between these two cell types. The second pattern we noted was that the genes in the GATA1 cluster correlated very strongly with cell cycle activity and cell cycle progression while the GATA2 cluster geneset had very low cell cycle activity. This suggested that the commitment of the MEP to E or Mk fates could not be unlinked from their cell cycling status. Such a finding could only be ascertained using single cell sequencing. Using single cell sequencing also provided us with a gene expression signature for primary human MkP, something which was not possible before because there is no reliable way to sort pure human MkP. Regarding GATA1 and GATA2 clusters, real time RT-PCR analysis of primary human ErP, MkP, and MEP point to a scenario where the ratio of GATA2/GATA1 is critical to determining the E vs. Mk fate decision. These findings will be further addressed in future studies aiming to understand the link between cell cycle and the MEP fate decision. Our new findings will help clarify genetic events critical for the E/Mk fate decision. DisclosuresNo relevant conflicts of interest to declare.

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.

Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations.

A cell-based high-throughput screen for novel chemical inducers of fetal hemoglobin for treatment of hemoglobinopathies.

Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.

Characterization of transcription factor networks involved in umbilical cord blood CD34+ stem cells-derived erythropoiesis.

Fetal stem cells isolated from umbilical cord blood (UCB) possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs) associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs) with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1) and 30 TFs were activated by day 56 (Profile-2). Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1) and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34+ stem cells including prolonged γ-globin expression combined with unique TFNs support novel mechanisms controlling the γ/β-globin switch during UCB-derived erythropoiesis.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor

AbstractIn adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Human fetal globin gene expression is regulated by LYAR.

Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.