Primary Erythroid Progenitor Cells Research Articles

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Preclinical studies of Flonoltinib Maleate, a novel JAK2/FLT3 inhibitor, in treatment of JAK2V617F-induced myeloproliferative neoplasms

Janus kinase 2 (JAK2) hyperactivation by JAK2V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 μM) without prominent cytotoxicity (CC50 = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Transcriptional silencing of fetal hemoglobin expression by NonO.

Human fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells. We showed that NonO bound to the octamer binding motif, ATGCAAAT, of the γ-globin proximal promoter, resulting in inhibition of γ-globin transcription. Depletion of NonO resulted in significant activation of γ-globin expression in K562, HUDEP-2, and primary human erythroid progenitor cells. To confirm the role of NonO in vivo, we further generated a conditional knockout of NonO by using IFN-inducible Mx1-Cre transgenic mice. We found that induced NonO deletion reactivated murine embryonic globin and human γ-globin gene expression in adult β-YAC mice, suggesting a conserved role for NonO during mammalian evolution. Thus, our data indicate that NonO acts as a novel transcriptional repressor of γ-globin gene expression through direct promoter binding, and is essential for γ-globin gene silencing.

SIRT1 Increases the Expression of Fetal Hemoglobin Genes in Human Adult and Cord Blood Erythroblasts

Introduction: High fetal hemoglobin (HbF, α2γ2) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes (HBG) in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. Many efforts have been made to develop new HbF inducers. HDAC1 and HDAC2, the class I histone deacetylases, suppress HbF gene transcription, and histone deacetylase inhibitors induce HbF expression. We therefore wanted to study whether SIRT1, a NAD-dependent class III histone deacetylase, also played a role in suppressing HBG expression. Unexpectedly, we found that SIRT1 was an inducer of HBG expression.Methods: We evaluated the role of SIRT1 in fetal globin gene regulation by ectopic expression or knockdown of SIRT1 and treatment with small molecule SIRT1 activators SRT2104 and SRT1720, in primary erythroid progenitor cells isolated from both cord blood and adult peripheral blood, CD34+ HUDEP-1 and HUDEP-2 cell lines that express fetal and adult hemoglobins, and K562 cells, using qRT-PCR, flow cytometry, immunoblotting, chromatin immunoprecipitation, and chromatin conformation capture assays.Results: We identified SIRT1 as a new inducer of γ-globin. Ectopic expression of SIRT1 in cord blood CD34+ cells increased HBG mRNA 4-fold and the g-globin protein level 2.5-fold compared with vector control (p<0.05). SIRT1 knockdown in cord blood CD34+ cells decreased HBG mRNA 8-fold and γ-globin protein level 5-fold compared with a scrambled shRNA control (p<0.05). Stable SIRT1 knockdown in K562 cells decreased HBG mRNA levels by 11-fold (p<0.01).To evaluate the effect of small molecule SIRT1 agonists on HbF gene expression, we treated erythroid progenitor cells from cord blood or normal adult blood and HUDEP-1 and HUDEP-2 cells and analyzed HBG mRNA, HbF-cells and g-globin protein levels. In cord erythroid progenitor cells, SRT12104 at 100nM, 500nM, and 2uM concentrations induced a mean increase of 2.4-fold, 3.4-fold and 8.1-fold in HBG mRNA, respectively, compared with vehicle treated controls (p<0.01). SRT1720 at 500nM and 5uM concentrations induced a mean increase of 2.1-fold and 4.4-fold in HBG mRNA levels above the controls, respectively (p<0.05). In adult progenitor cells, SRT2104 at 500nM and 2uM induced a mean increase of 2.6-fold and 4.1-fold HBG mRNA respectively (p<0.05), and induced an increase of 2.7% and 6.5% HbF-cells respectively (p<0.01). g-Globin was increased 5-fold and 3-fold by exposure to SRT2104 at 2uM and SRT1720 at 2.5uM respectively. Incubation of HUDEP-1 cells with 500nM and 2uM SRT2104 induced HBG expression 3.3-fold (p<0.01) and 6.2-fold (p<0.05) respectively; SRT1720 at 2.5uM induced HBG expression 8.4-fold (p<0.01), compared with controls. F-cells were elevated by 15.0 % and 17.4 % with SRT2104 treatment at 500nM and 2uM respectively (p<0.05), compared with controls. HUDEP-2 cells treated with SRT2104 at 500 nM and 2 uM had 1.8-fold and 2.5-fold induction of HBG expression (p<0.05). Treatment with SRT1720 at 2.5uM induced HBG expression 4.2-fold (p<0.05) compared with controls. These findings show that SIRT1 is an HBG inducer and that SIRT1 activators can reactivate silenced HBG expression in adult progenitor cells.SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, and promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2 . SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a.Conclusions: SIRT1 is an activator of HBG transcription. Our data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at G\u03b3-globin gene promoter

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Drugs modulating stochastic gene expression affect the erythroid differentiation process.

To better understand the mechanisms behind cells decision-making to differentiate, we assessed the influence of stochastic gene expression (SGE) modulation on the erythroid differentiation process. It has been suggested that stochastic gene expression has a role in cell fate decision-making which is revealed by single-cell analyses but studies dedicated to demonstrate the consistency of this link are still lacking. Recent observations showed that SGE significantly increased during differentiation and a few showed that an increase of the level of SGE is accompanied by an increase in the differentiation process. However, a consistent relation in both increasing and decreasing directions has never been shown in the same cellular system. Such demonstration would require to be able to experimentally manipulate simultaneously the level of SGE and cell differentiation in order to observe if cell behavior matches with the current theory. We identified three drugs that modulate SGE in primary erythroid progenitor cells. Both Artemisinin and Indomethacin decreased SGE and reduced the amount of differentiated cells. On the contrary, a third component called MB-3 simultaneously increased the level of SGE and the amount of differentiated cells. We then used a dynamical modelling approach which confirmed that differentiation rates were indeed affected by the drug treatment. Using single-cell analysis and modeling tools, we provide experimental evidence that, in a physiologically relevant cellular system, SGE is linked to differentiation.

Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

BackgroundA robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells.ResultsPrimary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation.ConclusionsGenetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products.

Synthesis of 3-(Imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one Derivatives and Study of Their Antiviral Activity against Parvovirus B19.

Parvovirus B19 (B19V) is a human pathogenic virus associated with a wide range of clinical conditions. Currently, there are no recognized antiviral drugs for B19V treatment; therefore, efforts in the search for compounds inhibiting B19V replication are now being pursued. Coumarins (chromen-2-ones) are considered a privileged structure for designing novel orally bioavailable and non-peptidic antiviral agents. To further contribute to the development of new drugs against B19V, our research was focused on the synthesis, characterization and evaluation of antiviral activity of some new 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one derivatives. The effects of the synthesized compounds on cell viability and viral replication were investigated by employing two relevant cellular systems, the myeloblastoid cell line UT7/EpoS1 and primary erythroid progenitor cells (EPCs). Some of the tested compounds showed inhibitory activity both on cell viability and on viral replication, depending on the cellular system. These results suggest that the mechanism involved in biological activity is sensitive to small structural changes and that it is possible to direct the activity of the 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one core.

LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells

Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells.We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD.Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients. DisclosuresNo relevant conflicts of interest to declare.

Soluble Guanylate Cyclase Modulators Effect Fetal Hemoglobin Levels through a Non-cGMP-Protein Kinase G Signaling Pathway

Sickle cell anemia results from a point mutation in both alleles of the β-globin gene. This homozygous mutation ultimately leads to a structural alteration of the hemoglobin protein that promotes polymerization of the mutant sickle hemoglobin tetramer (HbS) upon deoxygenation. HbS polymerization results in rigid, sickle-shaped RBCs with increased cell-to-cell adhesion properties. These sticky and rigid RBCs are prone to become trapped in small capillary networks leading to ischemia-reperfusion injury, endothelial damage and the hallmark pain crisis of sickle cell anemia. Neonates are protected from deoxy-HbS polymerization by high levels of fetal hemoglobin (HbF). HbF is composed of two α-globin and two γ-globin subunits(α2γ2), The γ-globin molecule cannot interact with deoxy-βS-globin polymers, which makes HbF an effective inhibitor of deoxy-HbS polymerization. The level of HbF required to reduce the symptoms of sickle cell anemia is 20-25%, but levels as low as 9% can prolong red cell survival. Treatment with hydroxyurea (HU) induces HbF and reduces the hematologic and clinical consequences of sickle cell anemia. Basal and inducible HbF levels are important in predicting the severity of sickle cell anemia and are highly phenotypically variable among patients, leading to varied responses to treatment. Patients are also variably susceptible to HU-induced cytopenias, which limits the use of HU in certain patients. HU is currently the only FDA-approved HbF-inducer for the treatment of sickle cell anemia and there is clearly a need for alternative HbF-inducers. Understanding the signaling pathways that regulate HbF induction will lead to novel therapeutic targets for sickle cell anemia. The soluble guanylate cyclase/cyclic guanosine monophosphate-dependent protein kinase (sGC/PKG) signaling pathway potentially links HU to the induction of HbF expression. In this study we investigated the direct role of sGC in HbF induction using novel pharmacologic modulators of sGC. Nitric oxide (NO) activates sGC by binding to the ferrous iron (Fe2+) in the active site heme moiety. Once activated, sGC converts GTP to cGMP, which in-turn activates PKG. Reactive oxygen species (ROS) oxidize the active site heme of sGC leading to NO-insensitivity. We tested the ability of a novel sGC activator, BAY 58-2667, to induce γ-globinin primary and immortalized (HUDEP-2) human erythroid progenitor cells. BAY 54-6544 binds to heme-free inactivated sGC to restore its guanylyl cyclase activity independent of NO. We also tested the ability of the sGC stimulator, BAY 41-2272, to induce γ-globinin primary and HUDEP-2 human erythroid progenitor cells. BAY 41-2272, binds to the ferrous iron at the active site of non-oxidized sGC to stimulate guanylyl cyclase activity in a synergistic manner with NO. We compared g-globin mRNA and protein expressionin the primary and immortalized human erythroid progenitors after treatment with different concentrations and combinations of BAY 54-6544, BAY 41-2667 and HU. We also evaluated g-globin induction in cellstreated with the pan-phosphodiesterase inhibitor IBMX and a synthetic cGMP analog. Although we see robust induction of cGMP and activation of PKG with all treatments, we only see significant induction of g-globin expression in the HU treated cells. This data suggests that the induction of HbF occurs through a non-sGC/PKG-dependent signaling pathway. These data demonstrate a very limited induction of γ-globin by BAY 54-6544 and BAY 41-2667 that appears to be disproportionate to, and independent of, cGMP/PKG signaling. These data also demonstrate, for the first time, that HU treatment of the immortalized HUDEP-2 cell line induces γ-globin expression more consistently than in primary erythroid progenitors. DisclosuresNo relevant conflicts of interest to declare.

Cell sources for large‐scale manufacture of red blood cells

Owing to the high demand for blood production to replace donated blood, there have been many developments in the production methods of red blood cells (RBCs) in the past decades. Although cord blood or adult haematopoietic stem cells were shown to mature into functional reticulocytes in vitro, their limited proliferative capacity led to the development of new strategies that use pluripotent stem cells or cell lines that have been immortalized using gene modification techniques. This research highlights embryonic stem cells, induced pluripotent stem cells and immortalized cell lines as possible sources of RBCs. Despite the low enucleation efficiency of erythroblasts derived from these pluripotent stem cells, recent studies have shown the possibility of an increased yield of final enucleated red cell products. In addition, primary erythroid early progenitor cells could be used as a cell source by culture conditioning or the exogenous overexpression of adequate genes for self‐renewal and proliferation. The strategy of directly establishing immortalized erythroid progenitor cell lines is suitable for the manufacture of RBCs, since these cell lines are easy to maintain and mature rapidly. In this review, different approaches for the manufacture of erythrocytes, using more expandable precursor sources, are discussed.

In Vivo Effects of LSD1 Inhibition By Small Chemical Inhibitors in Sickle Cell Mice

For the past decades the principal strategy for treating the β-globinopathies [sickle cell disease (SCD) and β-thalassemia] has been to devise methods for increasing the levels of fetal hemoglobin (HbF) in adult erythroid cells, as a strong clinical literature has demonstrated that high HbF levels ameliorate disease pathophysiology.We originally reported the lysine-specific histone demethylase 1 (LSD1) as a new protein involved in the regulation of the fetal γ-globin genes. Inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine in primary human erythroid progenitor cells induces HbF to therapeutic levels. Thus, inhibition of LSD1 activity presents a novel exploratory avenue as a therapeutic strategy to treat SCD. Subsequent studies using another inhibitor RN-1 showed that RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with sickle cell disease.Here we report studies designed to examine the in vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 on HbF synthesis and erythroid physiology in SCD mice. In control SCD mice treated with DMSO only, the number of HbF positive cells was about 2.5% of total in RBC, while in SCD mice exposed to GSK-LSD1, or OG-L002, there was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment (up to 8%, or 6% respectively). Furthermore, sickled RBCs and reticulocytes are significantly reduced while the number of mature erythrocytes increased markedly in the peripheral blood of GSK-LSD1, or OG-L002 treated SCD mice. These findings suggest that GSK-LSD1 and/or OG-L002 could be potentially new HbF inducers based on LSD1 inhibition. We believe that these experiments will be of wide general interest and will be the foundation for development of small molecule therapeutics with which to treat hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context:Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis.Objective:To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients.Design, Setting, and Patients:Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points.Results:After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient–derived HEPs.Conclusions:Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation ofproliferation.

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

EPO Modulation of Thioredoxin Interacting Protein (TXNIP), and Key Roles for TXNIP during EPO-Dependent Human Erythroid Progenitor Cell Growth and Development

Via contemporary investigatory approaches, important new EPO action mechanisms continue to be discovered. As recent illustrating examples, lineage tracking studies have demonstrated guiding effects of EPO on the developmental fate of myeloerythroid progenitors [J Exp Med. 211:181-8]. Profiling of EPO response genes in primary erythroid progenitor cells (EPCs) has uncovered a pathway of EPO cytoprotection against leached lysosomal cathepsins in stressed erythroblasts [J Exp Med. 210:225-32]. And in a context of iron metabolism, Erythroferrone has been identified as an EPO-induced inhibitor of hepcidin [Nat Genet. 46:678-84].In a unique approach to defining EPO's cell and molecular actions, our laboratory has launched post-translational modification based LC-MS/MS proteomic analyses in human erythroid precursor cell systems. In interrogations of novel EPO/EPOR targets modified within a category of "p-TPP" motifs, TXNIP proved to be rapidly phosphorylated >10 fold due to EPO at novel C-terminal p-T349 and p-S358 sites. In parallel, multi-fold increases in TXNIP levels also were observed within 20 minutes of EPO exposure. To gain functional insight into TXNIP's roles during EPO-dependent erythropoiesis, LOF studies were performed via lentiviral shRNA mediated inhibition of TXNIP. As analyzed first in UT7epo cells, TXNIP knockdown (>90% efficiency) attenuated cell proliferation ~2.5-fold with EPO-dose dependency. This was reflected further in selective attenuation of cell cycle progression at S-phase (with only limited effects of TXNIP knockdown on cell survival observed). LOF experiments were also performed in human bone marrow derived CD34+ primary hematopoietic progenitor cells. Following initial plating, hematopoietic progenitors transduced with TXNIP shRNA exhibited a limited transient lag in growth (as compared directly with shRNA-NT transduced controls), but by day 4 of culture, and thereafter, expanded at essentially normal levels. Subsequently, erythroid progenitors with inhibited TXNIP expression prematurely committed to a program of late erythroblast differentiation. Specifically, TXNIP knockdown resulted in elevated frequencies of GPA-high erythroblasts (33.4+/-1.3% vs controls at 6.9+/-1.1%, p = 0.0001) and decreased KIT-high expression (6.4+/-3.0% vs controls at 62.5+/-7.1%, p = 0.0002). In addition, visibly obvious increases in erythroblast hemoglobinization due to TXNIP knockdown also were observed.The present investigations thus employ a unique PTM LC-MS/MS approach to identify TXNIP as a new EPO/EPOR target modified at novel C-terminal sites upon EPO expression, and in association with an indicated stabilization of TXNIP. LOF studies further reveal pro-erythropoietic roles for TXNIP as a novel mediator of EPO-dependent erythropoiesis with important effects exerted on erythroid precursor cell proliferation, together with a key requirement for TXNIP in governing a transition to a program of late erythroblast differentiation. Significance is further underlined by the nature of TXNIP as an emerging target within beta cells for small molecule inhibition in type-2 diabetics, a group highly representative among anemic chronic renal disease patients [J Diabetes Res Apr 7 2015:801348]. In particular, based on our present findings, such inhibition of TXNIP would be predicted to further compromise erythropoiesis in this CKD patient population. DisclosuresNo relevant conflicts of interest to declare.

ASH1L: A Novel Beta-Globin Gene Regulator in Humans?

BACKGROUNDWe have previously described a unique English family with beta-thalassemia trait which was not linked to the β-globin gene locus (Thein, Wood, Wickramasinghe, & Galvin, 1993). This suggested involvement of a trans-acting factor required for full activation of the β-globin gene locus. Such a factor is likely to be a modulator of disease severity in sickle cell disease and beta-thalassemia which could provide insights for novel therapeutic targets in the beta-globinopathies. RESULTSWe applied whole genome scan (WGS) to 2 affected and 2 unaffected subjects of the English family. The familial segregation suggested a dominant transmission mode; WGS identified 15 genes as potentially causative to the phenotype, with four genes located on chromosome 1, four on chromosome 3, three on chromosome 20, and one on chromosome 6, chromosome 8, chromosome 10 and chromosome 19.Sanger sequence analysis on 23 family members spanning three generations, including the 4 individuals that were subjected to WGS, revealed that the 15 variants were not artefacts of the WGS and that all variants were present in the 2 affected but not in the 2 unaffected individuals. Furthermore we found that 4 of the 15 variants were consistently and uniquely present in all 9 affected but absent in the unaffected family members. We performed association linkage analysis using the 15 markers in the whole family, and confirmed that the phenotype was closely linked to the 4 genes that were inherited as a block spanning the centromere on chromosome 1. We concluded that the region containing these 4 genes most likely harbours the mutation causing the phenotype. Among the 4 candidate genes, 2 were not expressed in erythroid cells, but the other 2 - one encoding an integral membrane protein (LRIG2) and the other one encoding a methyl transferase (ASH1L)- were expressed in erythroid cells.Functional studies for these two genes were performed on primary human erythroid progenitor cells (hEPCs) in culture. In following the kinetics of the 2 candidates during differentiation of hEPCs, we observed that the expression of ASH1L increased at later stages of differentiation, where LRIG2 displayed a less dramatic change of expression. Moreover, ASH1L has previously been found to occupy transcribed chromatin domains and methylate histone tails in vitro (Gregory et al., 2007; Miyazaki et al., 2013; Tanaka et al., 2011). In undifferentiated mouse embryonic stem cells there is no ASH1L recruitment to the β-globin gene locus but upon erythroid differentiation the protein is recruited to the transcribed portion of the gene (Gregory et al., 2007). This suggests an involvement of ASH1L in beta-globin activation in erythroid lineages. We used shRNA lentiviruses to generate knock-down (KD) of ASH1L and obtained over 65-75% KD of the gene. In hEPCs treated with the shRNA lentivirus, we observed a slight decrease in beta-globin expression compared to the control hEPCs. The α/β-globin and α/(β+γ) globin ratios were also affected by the gene knock-down. ChIP-qPCR was performed to assess the enrichment of the ASH1L protein at β-globin promoter region. The results show that enrichment of ASH1L at the β-globin promoter correlates with the β-globin expression in cells. CONCLUSIONSThese results suggest that ASH1L is responsible for the phenotype observed in the English family and act in differentiating hEPCs as a trans-acting factor for full beta-globin gene activation. Further ChIP analysis to assess the binding of the protein to the beta-globin locus during hEPCs differentiation and under KD condition will provide us with a better understanding of the influence of the methyl transferase on β-globin activation. The replication of the patient mutation in vitro using CRISPR technology will provide the model to study fully the impact of the mutation on the phenotype described in the original paper. These findings could provide new insights for therapeutic targets for beta-globinopathies. DisclosuresNo relevant conflicts of interest to declare.

Activity of RN-1, an LSD-1 Inhibitor, on Fetal Globin Expression in Sickle Mice and Sickle Erythroid Progenitors

Sickle cell disease (SCD) is the most common monogenic disorder, afflicting millions worldwide, and causing hemolytic anemia and chronic organ damage from vaso-occlusion. Fetal hemoglobin (HbF) is an endogenous type of hemoglobin present in all humans during development, which is normally suppressed in infancy. Biochemical and clinical studies have shown that increased synthesis of HbF inhibits sickle hemoglobin (HbS) polymerization and reduces clinical severity. Concerted efforts have been made to induce the synthesis of HbF in adult erythroid cells with chemical inducers of HbF and through disruption of transcription factors in repressor complexes. As wide variability in individual responses to drug candidates have been observed in clinical trials, consistently effective HbF inducers are highly desired. We previously identified that Lysine-specific histone demethylase 1 (LSD1) is involved in the regulation of the fetal γ-globin genes, and inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine (TC) in primary human erythroid progenitor cells induces HbF to therapeutic levels. However, TC treatment has potentially problematic side effects, and at high concentrations decreases adult b-globin mRNAs and impairs erythroid maturation. We have now investigated another LSD1 inhibitor, RN-1, which is a cell-permeable TC analog that acts as a potent, irreversible inhibitor of LSD1 with a lower IC50 than TC. We investigated in vivo effects of RN-1 on γ-globin gene expression and erythroid physiology in a transgenic mouse model of SCD which expresses human α- and sickle β-globin, and has many genetic, hematologic, and pathophysiological features found in SCD patients, including irreversibly sickled RBCs, hemolytic anemia, high reticulocyte counts, hepatosplenomegaly and organ pathology. We found a robust increase in human fetal γ-globin (15-fold) and murine embryonic εY- and βH1-globin mRNAs (36 and 54-fold) and 4-fold increases in human HbF in SCD mice following repeated RN-1 treatment (at 10 μg/g body weight) within 4 weeks. Further, irreversibly sickled RBCs were significantly reduced, and RBC lifespan increased markedly in RN-1-treated SCD mice, leading to significantly decrease pathophysiologic indicators (hemolysis, splenomegaly, and organ necrosis) compared to untreated SCD mice. To begin to evaluate potential effects of RN-1 on erythroid progenitor cells from patients with SCD, peripheral blood from 5 adult SCD patients was cultured with RN-1 (0.07 to 0.25 μM) in a 2-phase progenitor assay, with mRNA analyzed on day 12 and F-reticulocytes on day 13-14 of the erythroid differentiation phase. RN-1 treated progenitors demonstrated a mean 3.4-fold higher g-globin mRNA (p=0.04) and 5% higher absolute F-reticulocytes than were observed in untreated progenitors from the same subject, with responses occurring in 5/5 subjects' assays. These preclinical studies provide additional evidence that modulating LSD-1 activity is a promising approach to inducing HbF expression as a mechanism to reduce clinical severity of SCD.Disclaimer: "Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health"R01 DK052962 10A1R42-HL-110727 DisclosuresNo relevant conflicts of interest to declare.

The investigation of resveratrol and analogs as potential inducers of fetal hemoglobin

Βeta-thalassemia, is a hemoglobinopathy characterized by reduced beta-globin chain synthesis, leading to imbalanced globin chain production, ineffective erythropoiesis and anemia. Increasing gamma-globin gene expression is a promising therapeutic approach as it reduces this imbalance by combining with the excess alpha globin chains and producing fetal hemoglobin (HbF). Furthermore, increased iron absorption and repeated blood transfusions lead to iron overload and tissue damage secondary to reactive oxygen species. Compounds exhibiting both antioxidant and HbF inducing activities are, therefore, highly desirable therapeutic agents. Resveratrol, a natural phytoalexin, combines these two activities but is also cytotoxic. Nine hydroxystilbenic resveratrol derivatives were synthesized in an attempt to identify compounds that retain the HbF-inducing and antioxidant activities of resveratrol but exhibit reduced cytotoxicity. Three derivatives (P1, P4 and P11) exhibited similar hemoglobin-inducing properties to resveratrol in K562 cells, however, only P11 showed reduced cytotoxicity. All three derivatives demonstrated variable HbF-inducing activity in primary erythroid progenitor cells from healthy donors. Resveratrol and P11 increased HbF induction significantly, with P11 having the highest activity. Additionally, P4 significantly increased progenitor numbers. A combinatorial treatment in K562 cells using resveratrol and decitabine resulted in a statistically significant increase in hemoglobin-inducing activity only above the level shown by resveratrol alone.