Mature Red Cells Research Articles

Papers of note in Science 357 (6350)

This week’s articles uncover mechanisms of drug action, a way bacteria can help defend against the flu, and a ubiquitin ligase that facilitates the maturation of red blood cells.

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.

Human Cord Blood and Bone Marrow CD34+ Cells Generate Macrophages That Support Erythroid Islands.

Recently, we developed a small molecule responsive hyperactive Mpl-based Cell Growth Switch (CGS) that drives erythropoiesis associated with macrophages in the absence of exogenous cytokines. Here, we compare the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded CD34+ cells harvested from cord blood, marrow or G-CSF-mobilized peripheral blood. Results indicated that macrophage based erythroid islands could be generated from cord blood and marrow CD34+ cells but not from G-CSF-mobilized CD34+ cells. Additional studies suggest that the deficiency resides with the G-CSF-mobilized CD34+ derived monocytes. Gene expression and proteomics studies of the in vitro generated erythroid islands detected the expression of erythroblast macrophage protein (EMP), intercellular adhesion molecule 4 (ICAM-4), CD163 and DNASE2. 78% of the erythroblasts in contact with macrophages reached the pre reticulocyte orthochromatic stage of differentiation within 14 days of culture. The addition of conditioned medium from cultures of CD146+ marrow fibroblasts resulted in a 700-fold increase in total cell number and a 90-fold increase in erythroid cell number. This novel CD34+ cell derived erythroid island may serve as a platform to explore the molecular basis of red cell maturation and production under normal, stress and pathological conditions.

Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells.

Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion‐transmitted infections and complications can arise from immunological incompatibility. Clinically‐compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel‐like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC‐derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen‐inducible KLF1‐ERT2 expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic‐like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886–897

Spatiotemporal Mapping of Erythroid, Stromal, and Osteogenic Niche Formation to Support Physiologic Red Cell Production in a Three-Dimensional Hollow Fibre Perfusion Bioreactor

Current in vitro human erythroid culture platforms require abnormally high cytokine supplementation and use lower cell density (<106/mL) compared with that present in bone marrow during physiologic erythropoiesis. These in vitro culture conditions limit extracellular interactions, are not dynamic, and exhaust stem and progenitor cell pools, thereby limiting culture longevity. We have developed a three-dimensional hollow fibre perfusion bioreactor (HFBR) comprised of a collagen-coated polyurethane scaffold which surrounded ceramic hollow fibres (HFs) and expanded dense inocula of human umbilical cord blood (CB) mononuclear cells (MNCs; >107/mL) when perfused with cytokine-free media in long-term culture. In order to study the role of this manufactured HFBR microenvironment on spatiotemporal physiologic erythropoiesis, we now extend our previous reports by implementing 5-fold less cytokine concentrations than those used in typical ex vivo erythropoietic cultures. Herein, we show a >107 red cell harvest from the HFBR culture over 28-days with spontaneous expansion of stromal cells, maintenance of erythroid progenitor pools, and formation of stromal and erythroid cell niches in defined areas within the HFBR structure with differential in situ production of 23 growth factors varying over time.The 5.25 mL HFBR scaffold was inoculated with 108 CBMNCs and HFs were rapidly perfused (20 mL/h) with serum-free StemSpan medium gradually supplemented with a cytokine gradient of decreasing SCF (50 - 0 ng/mL) and increasing EPO (0 - 0.3 U/mL) over 28 days in order to maintain progenitor cells whilst inducing erythropoiesis. Quantitative confocal microscopy analyses of HFBR sections demonstrated that DAPI+ CBMNCs maintained high cell density (>107/mL), and high viability (>80%), while more than 107 enucleated cells were filtered through HFs over the 28-day culture. Inside the HFBR, hematopoietic progenitor cells were maintained (total of 3.1·106 CD34+ and 5.5·106 CKIT+ MNCs) while erythroid cells were expanded across various stages of maturation (28-day total increase of 1.2·107 EPOR+, 1.8·107 CD71+, and 2.3·107 CD235a+ MNCs); CD235a+mature red cell phenotypes were enriched 10-fold in the HF filtrate over 28 days.Stromal cells expanded and differentiated during the 28-day HFBR culture with a total increase of mesenchymal stem cell marker Stro-1 (2.2·107 cells), pre-osteoblast marker osterix (OSx; 1.6·107 cells), and mature osteoblast marker osteopontin (OPN; 0.5·107 cells). Expression of human collagen-1, fibronectin, and laminin-2 was detected by microscopy, while enzyme-linked immunoassays on HFBR filtrate detected 23 multilineal, unsupplemented cytokine profiles including interleukins produced primarily from day 0-12 (IL-6, IL-10, IL-21) as well as colony stimulating factors and stromal growth factors which increased in production from day 20-28 (G-CSF, GM-CSF, EGF, VEGF, Ang-2, PDGF, FGF-β). Using a novel confocal microscopy computational analyses that we have developed, DAPI+MNCs were found to self-associate into expanding 50-500µm clusters throughout the 28-day culture which increased local cell density 10-20 fold, representing niche-like areas. At day 14 and 28, MNCs formed clustered niches far from HFs which expressed hypoxic (HIF1a, PIMO), stromal, and erythroid markers (Stro-1, OSx, collagen-1, laminin-2, VCAM-1, CD45, EPOR: >1400µm from HFs). At day 28, 3-fold more MNC clusters formed near HFs and were comprised of hematopoietic progenitor and erythroid phenotypes (CD45, CD34, CKIT, CD235a, CD71: <700µm from HFs).Our data suggested that the dense inoculation of CBMNCs in a serum-free HFBR platform using physiologic concentrations of SCF and EPO enabled the long-term simultaneous differentiation of human erythroid, stromal, and osteogenic lineages, and the generation of an ex vivo erythroid inductive environment. This environment maintained multilineal progenitors, enabled harvest of mature erythrocytes, generated cytokine support in situ, and formed interactive cell niches which could be quantitatively mapped in spatiotemporal zones. The HFBR we have developed may represent a more physiologically-relevant culture system to study ex vivo erythropoiesis and could potentially provide a platform for translational cell expansion protocols. DisclosuresNo relevant conflicts of interest to declare.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.

: We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs. As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

Anemic syndrome in patients with community-acquired pneumonia

Community-acquired pneumonia remains a most widespread acute infectious disease of socio-economic significance all over the world. Up to 30% of the patients present with anemia responsible for the unfavourable prognosis and elevated mortality. Not infrequently, anemia is not diagnosed during the hospital stay und therefore remains uncorrected. Severe anemia results in enhanced hypercapnia and slowed maturation of red blood cells in the bone marrow which facilitates the development of ischemic syndrome. Hepcidin, a mediator of inflammation and iron-regulatory hormone, plays an important role in the clinical course of community-acquired pneumonia. Hepsidin production increases during inflammation; it suppresses erythtropoiesis and depletes the iron depot leading to so-called anemia of inflammation. Hypoxia and anemia activate erythtropoiesis, and the released erythropoietin inhibits hepsidin production. During pneumonia resolution, hepsidin promotes recovery from anemia by activating iron absorption. The curreni literature contains few data on the use of hepcidin as a diagnostic marker of anemia. The necessity oftreating anemia in patients with pneumonia under hospital conditions is a matter of discussion. Direct involvement of hepcidin in iron metabolism creates a prerequisite for the treatment of anemia. Medicamental suppression of its activity by stimulating erythtropoiesis can facilitate normalization of iron metabolism and restoration of hemoglobin level.

Erythropoietin: a new perspective in cardiovascular therapy

Erythropoietin is a hormone produced by the kidney, which regulates proliferation, differentiation and maturation of red cells. Recombinant human EPO (rH-EPO) is well known to correct anaemia in patients with chronic renal failure in terminal stage. However, recent studies showed the existence of several not haematopoietic effects of erythropoietin. EPO receptors have been found to be expressed in several tissues, included the cardiovascular system. An increase in cardiac systolic function has been observed in patients with chronic heart failure treated with EPO. Other beneficial effects appear to be related to the pro-angiogenic properties on endothelial cells and could be useful for treatment of ischemic heart disease. These findings suggest that EPO could provide potential therapeutic benefits in the management of cardiovascular diseases beyond anaemia correction. This review focuses its attention on the pleiotropic effects of EPO and its future promising applications in cardiovascular pathology.

Relationship between macrophages and erythropoiesis

Macrophages have two major roles in regulating the dynamic equilibrium in erythropoiesis, promoting the differentiation and maturation of nucleated red blood cells into reticulocytes and removing old red blood cells. A recent mouse study has demonstrated that the phenotype of macrophages in erythroblastic islands is CD169+ VCAM-1+ ER-HR3+ CD11b+ F4/80+ Ly-6G+. Molecular connections between erythroid progenitor cells and central macrophages help to maintain the function and integrity of erythroblastic islands. New research advances in Kruppel-like factor 1 (KLF1) provide new evidence for the important role of macrophages in erythroblastic islands. Macrophages play an important role in erythropoiesis both in sickness and in health, and provide a potential targeted therapy for diseases such as polycythemia vera and beta-thalassemia in the future.

Mathematical Modelling of Erythropoiesis in Patients with End Stage Renal Disease: New Insights into Marrow Functioning

BACKGROUNDAnemia is a well-recognized complication of chronic kidney disease (CKD). Shortened red blood cell (RBC) survival, uremic inhibitors of erythropoiesis and a relative deficiency of erythropoietin have been proposed as mechanisms of anemia of CKD. In normal individuals a low level of circulating erythropoietin (EPO) produced by the nephrons (10-30 mUnits/mL) is sufficient to maintain erythropoiesis. CKD patients do not have elevations of serum EPO levels that are seen in healthy individuals with comparable degree of anemia. The US FDA approved recombinant EPO in 1989. Since then, treatment with supra-physiologic doses of EPO has become the mainstay of anemia management in CKD. It has been estimated that at any given time 90% of prevalent dialysis patients require erythropoiesis-stimulating agents.Undifferentiated pluripotent stem cells, in the bone marrow are capable of producing committed erythroid colony forming units (CFU-E) under the influence of EPO. These cells ultimately differentiate into RBCs that are released into the circulation. Because of finite non-zero cell maturation and cell replication times there is a delay between EPO administration and RBC production. In healthy individuals the time needed for cell maturation in the marrow ranges from 6-10 days. The maturation of red cells in marrow of CKD patients has not been studied. We hypothesize that this maturation time is prolonged in patients with CKD. By identifying the existence of a delay in cell maturation we can begin to investigate the factors determining the delay. We may also be able to identify new targets for treatments of anemia in kidney disease by targeting cell maturation pathways.METHODSMathematically, erythropoiesis can be formulated as a time model taking the form of a simple differential equation. Delay differential equations can be used to incorporate the delay in cell proliferation depicted by τ.The circulating erythrocytes are removed from the circulation at a rateγ. Therefore, 1/γ represents the life span of the erythrocyte. η and θ are shape parameters in the model and β (rate of RBC production) is a constant. This model was first proposed by Mackey and Glass and is popularly known as the Mackey-Glass Model. Monthly hemoglobin values were collected for 50 end stage renal disease patients on peritoneal dialysis over a period of 24-36 months. Individual models for each patient were developed in Mathematica v. 9.0. To obtain the best fit τ was progressively varied from 1-8 weeks till a good fit was achieved. γ was maintained in the range that represents expected RBC survival in these patients (range 7-14 weeks).RESULTSA representative model is shown in figure 1 below. We found τ to be significantly prolonged up to 6 weeks (mean 4 weeks, range: 1-6 weeks) in patients receiving peritoneal dialysis. This has important implications. A prolonged delay of up to 6 weeks in the effect of EPO can give rise to pseudo EPO-resistance. This phenomenon is clearly observed in clinical practice. Reactionary increase or reduction in EPO doses to apparent lack of response to EPO, leads to fluctuations in hemoglobin, a phenomenon that has been called hemoglobin cycling by clinical epidemiologists and has been related to mortality and cardiovascular outcomes.Recognition of the delay in cell maturation within the marrow which may show wide variation amongst patients (range 1-6 weeks); calls for individualizing drug treatment protocols rather than using population based algorithms.We also found evidence that patients with chronic renal failure are still capable of increasing their rate of red cell production in response to an increase in the rate of cell loss [Fig 2(a)]. Interestingly we found that the delay in cell maturation (τ) is prolonged when the basal erythropoietic rate (β) are increased [Fig 2(b)]. This effect is paradoxical since normal physiologic response to anemia should shorten cell transit times in face of increased demand. This leads us to hypothesize that cells may get arrested during their maturation. Increased levels of several inflammatory mediators may explain the maturation arrest. A lack of clear understanding of the factors affecting the maturation delay makes management of ESA-resistance challenging. New therapeutic approaches that target cellular pathways resulting in arrest of cellular maturation in the marrow may provide an avenue for new drug development. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MicroRNAs in erythropoiesis and red blood cell disorders

MicroRNAs (miRNAs) are 19-24 nucleotide non-coding ribonucleic acids binding DNA or RNA and controlling gene expression via mRNA degradation or its transcription inhibition. Erythropoies is a multi step differentiation process of erythroid progenitors to nucleate red blood cells. Maturation, proliferation and differentiation of red blood cells is affected by erythroid factors, signaling pathways in niche of hematopoietic cells, transcription factors as well as miRNAs. Expression of different types of miRNAs during erythroid development provides a background for the study of these molecules to control erythroid differentiation and maturation as well as their use as diagnostic and prognostic markers to treat erythroid disorders like thalassemia, sickle cell disease and erythrocyte enzyme deficiencies. In this paper, with reference to biosynthesis of miRNAs, their function in normal and anemic erythropoiesis has been investigated. The target molecule of each of these miRNAs has been cited in an attempt to elucidate their role in erythropoiesis.

Evaluation of erythrocyte and reticulocyte parameters as indicative of iron deficiency in patients with anemia of chronic disease

ObjectiveThe aim of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters to identify three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency.MethodsPeripheral blood cells from 117 adult patients with anemia were classified according to iron status, inflammation, and hemoglobinopathies as: iron deficiency anemia (n = 42), anemia of chronic disease (n = 28), anemia of chronic disease associated with iron deficiency anemia (n = 22), and heterozygous β-thalassemia (n = 25). The percentage of microcytic erythrocytes, hypochromic erythrocytes, and the levels of hemoglobin in both reticulocytes and mature red cells were determined. Receiver operating characteristic analysis was used to evaluate the accuracy of the parameters in differentiating anemia.ResultsThere was no difference between the groups of iron deficiency and anemia of chronic disease associated with absolute iron deficiency for any of the parameters. The percentage of hypochromic erythrocytes was the best parameter to identify absolute iron deficiency in patients with anemia of chronic disease (area under curve = 0.785; 95% confidence interval: 0.661–0.909 with sensitivity of 72.7%, and specificity of 70.4%; cut-off value 1.8%). The formula microcytic erythrocyte count minus hypochromic erythrocyte count was very accurate to differentiate iron deficiency anemia from heterozygous β-thalassemia (area under curve = 0.977; 95% confidence interval: 0.950–1.005 with a sensitivity of 96.2%, and specificity of 92.7%; cut-off value 13.8).ConclusionThe erythrocyte and reticulocyte indices are moderately good to identify absolute iron deficiency in patients with anemia of chronic disease.

Comment on: Evaluation of erythrocyte and reticulocyte parameters as indicative of iron deficiency in patients with anemia of chronic disease.

The article on the evaluation of the effectiveness of mature red cell and reticulocyte parameters under three conditions: iron deficiency anemia, anemia of chronic disease (ACD), and anemia of chronic disease associated with absolute iron deficiency by Torino et al.1 is very valuable. Automated reticulocyte counts are widely used in the clinical laboratory due to their greater precision, accuracy and reproducibility compared to those obtained using the microscope. The most important benefit of automated methods is the greater precision of counts. By analyzing a much greater number of reticulocytes (more than ten thousand), the statistical error is minimized.2 Visual microscopy is still recommended as the comparability method for reticulocytes, despite studies showing that the variation coefficient (VC) ranges from 20% to 40%. Fully automated methods have eliminated inter-observer variability and subjectivity and substantially reduced turnaround time. Automated methods employ a wide variety of reagents for reticulocyte RNA and these show different sensitivity on binding to RNA.3 Therefore, although automated flow cytometric analysis has led to a significant advance in reticulocyte counting, some limitations still persist in comparability across different laboratories

English

Chronic kidney disease (CKD) is characterized by gradual and permanent loss of kidney function. One of the most common complications of CKD is Anaemia. 1,3 Anaemia often appears earlier in course of CKD and worsens with disease progression. 1,2 Erythropoietin is a hormone synthesized in the kidney is deficient in the majority of patients of CKD thereby predisposing them for developing anaemia. Erythropoiesis stimulating agents ESAs) first became clinically available in 1989 and has contributed to the advances in the management of CKD related anemia. The present study highlights the correction of anaemia with erythropoietin (Eprex) in population having CKD on MHD and anaemia. Based on GFR CKD is divided into five stages from 1-5. Patients often become anaemic at stage 3 or 4 CKD but anaemia may develop as early as stage 2. And it is present in the majority of patients with stage 5 disease.2,4 The objective of this study is to evaluate efficacy of administration of 10000.U. Epotin alfa sc for once a week as initiation therapy in patients with anaemia of CKD/ESRD on MHD for 28 weeks of duration of treatment. It is a prospective study done at Central Hospital, South Central Railway, Secunderabad on patients with ESRD or stage 4 on maintenance hemodialysis and receiving subcutaneously EPO (Eprex). Serum feritin and %transferrin saturation are estimated before the initiation of EPO(Eprex) & if the levels are below normal, IV IRON 100 mg is given once weekly and oral elemental Iron 200 to 400 mg to every to normalize the serum ferritin and % transferrin saturation levels. Hemoglobin level estimation has done before and after the initiation of the EPO (Eprex) and once in weekly and four weekly to see the response more than 1gm in four weeks to achieve the target hemoglobin level (11 to 12 grams) in 28 weeks. In the present study target hemoglobin level (11-12g/dl) was achieved in 42 out of 50 patients under consideration (84%) in 28 weeks of duration of treatment and not achieved for 8 amongst 50(16%) patients. Increase in the hemoglobin level of 1/dl is seen in 13(26%),>1 g/dl in 29(58%) and <1 g/dl in 8(16%) patients. INTRODUCTION: Chronic kidney disease (CKD) is characterized by gradual and permanent loss of kidney function that worsens as it progresses from stages 1-5. One of the most common complications of CKD is Anaemia. 1,3 which is defined as hemoglobin (Hb) concentration below 13.0 g/dl for adult males and post-menopausal women, and a hemoglobin below 12.0 g/dl for premenopausal women, is a major component of CKD and is common in all stages but becomes more pronounced at the later stages of kidney failure. Anaemia is associated with increased morbidity in these patients. Erythropoietin is a hormone synthesized in the kidney responsible for red blood cell maturation in the bone marrow. It is deficient in the majority of patients with advanced kidney disease thereby predisposing them to anaemia. Until the late 1980s, blood transfusions and androgen

Erythroid Adhesion Molecules in Sickle Cell Anaemia Infants: Insights Into Early Pathophysiology

Sickle cell anaemia (SCA) results from a single mutation in the β globin gene. It is seldom symptomatic in the first semester of life. We analysed the expression pattern of 9 adhesion molecules on red blood cells, in a cohort of 54 SCA and 17 non-SCA very young infants of comparable age (median 144days, 81–196). Haemoglobin F (HbF) level was unsurprisingly elevated in SCA infants (41.2%±11.2) and 2–4 fold higher than in non-SCA infants, yet SCA infants presented significantly decreased Hb level and increased reticulocytosis. Cytometry analysis evidenced a specific expression profile on reticulocytes of SCA infants, with notably an increased expression of the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3, both in percentage of positive cells and in surface density. No significant difference was found on mature red cells. Our findings demonstrate the very early onset of reticulocyte membrane modifications in SCA asymptomatic infants and allow an insight into the first pathological changes with the release of stress reticulocytes expressing a distinctive profile of adhesion molecules.

CD47 Is Dependent on the Actin Cytoskeleton for Its Membrane Stability Prior to Protein 4.2 Expression during Early Erythroblast Differentiation

CD47 is a ubiquitously expressed ‘Marker of Self’ that protects cells from phagocytosis, through recognition by SIRPα on macrophages (Oldenborg et al Science 2000). CD47 was originally isolated on ovarian tumour cells (Poels et al J Natl Cancer Inst 1986) and has subsequently been detected on leukemic stem cells, where increased CD47 levels ensure immune evasion (Jaiswal et al Cell 2009). CD47 is also a ‘Marker of Self’ on red cells, but is reduced at the cell surface in certain patients with Hereditary Spherocytosis.In red cells, ~60% of CD47 is connected to the cytoskeleton (Dahl et al Blood 2004). Cytoskeletal connectivity of CD47 in the red cell membrane is dependent on the band 3 complex associated protein 4.2, demonstrated by an ~80% reduction in CD47 levels in protein 4.2 null red cells (Mouro-Chanteloup et al Blood 2003). Previous work (van den Akker et al Haematologica 2009) established that CD47 becomes dependent on protein 4.2 at the basophilic erythroblast stage (48 hours post-differentiation), but it is unknown what interactions support CD47 membrane stability prior to protein 4.2 expression during expansion and early erythroid differentiation.CD47 mRNA is alternatively spliced giving rise to four potential isoforms. The most abundant isoforms are form 2, expressed in all bone-marrow derived cells, and form 4 (and form 3), found predominantly in neural tissues (Reinhold et al J Cell Sci 1995). CD47 isoform 2 is the only form expressed on mature red cells, but we hypothesized that expression of other CD47 isoforms with different trafficking or binding characteristics could explain the independence of CD47 prior to band 3 complex assembly. Using specific polyclonal antibodies to multiple CD47 isoforms, we demonstrate that isoform 2 is expressed prior to and throughout in vitroerythroid differentiation. CD47 isoforms 3 and 4 were detected by western blotting until the late polychromatic erythroblast stage (96 hours post-differentiation), but only CD47 isoform 2 was detected at the cell surface.Therefore, we next hypothesised that CD47 must interact with another protein or exhibit different trafficking characteristics to maintain its membrane stability early during terminal differentiation. To identify a candidate protein or associated protein complex, CD47 was immunoprecipitated from expanding erythroblasts (Exp), proerythroblasts (T0), and basophilic erythroblasts (T48), and analysed via Nano-LC mass spectroscopy. In Exp and T0 erythroblasts, CD47 pulled down actin and multiple actin-associated proteins. These interactions were not observed in T48 erythroblasts, corresponding to the time during terminal differentiation when CD47 is dependent on protein 4.2. To confirm a dependence on actin for CD47 membrane stability, well-characterised drugs that disrupt actin dynamics were employed. CD47 expression at the cell membrane, as judged by flow cytometry, was markedly reduced within 30 minutes using actin stabilising drugs (Cytochalasin D (5µM): Exp 13.7±5.4% versus T48 0.5±5.7%; Latrunculin A (1µM): Exp 18.9±3.5% versus T48 9.9±5.9%, of the DMSO control), and destabilising drug (Jasplakinolide (1µM): Exp 24.2±1.9% versus T48 -6±1.8%, of the DMSO control), until the basophilic erythroblast stage. In K562 cells, which predominantly express CD47 isoforms 3 and 4, a larger actin dependency is observed (37±14.9% reduction in CD47 with Cytochalasin D versus a DMSO control) suggesting that dependence on actin by CD47 is not isoform specific.In summary, we propose a role for actin in the maintenance of CD47 at the cell surface before and during early erythroid differentiation. We have shown that CD47 isoform 2 is the major isoform present at the cell surface and that this version is initially dependent on the actin cytoskeleton for its membrane stability by an as yet undetermined mechanism. Once band 3 complex assembly initiates at the surface of the basophilic erythroblast (48 hours post-differentiation), CD47 is selectively incorporated via an interaction with protein 4.2, and is preferentially retained whilst the actin cytoskeleton remodels. In addition to explaining how CD47 expression is maintained during the formation of the red cell membrane, this work raises the possibility that the dependence on actin by CD47 for its membrane stability in hematopoietic stem cells may be exploited for the development of therapeutics that render the leukemic cells susceptible to phagocytosis. DisclosuresNo relevant conflicts of interest to declare.

Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation

Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin ET74A T393A). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored.We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation. DisclosuresNo relevant conflicts of interest to declare.

A Systems Approach Identifies Essential FOXO3 Functions in Erythroblast Enucleation Process

Elucidating mechanisms that regulate terminal erythroblast maturation may serve to improve the treatment of anemias as well as the generation of red blood cells in vitro. Key steps in red blood cell maturation are chromatin condensation and expulsion of the nucleus (or enucleation) from late-stage erythroblasts. The transcriptional program that coordinates these steps is poorly defined. The expression and transcriptional activity of FOXO3 increase with terminal erythroblast maturation suggesting a potential function for FOXO3 in this process. Using comparative transcriptomic analysis of primary freshly isolated wild type and Foxo3-/- erythroblasts at distinct stages of maturation, we found that many genes involved in DNA packaging-related pathways were highly downregulated in Foxo3-/- erythroblasts, raising the possibility that FOXO3 might be involved in regulating chromatin condensation and/or the enucleation process. Foxo3-/- enucleating cells were also significantly fewer than wild type bone marrow erythroblasts according to in vivo DNA staining by DRAQ5. qRT-PCR analysis confirmed the reduced expression of genes implicated in the control of chromatin condensation and/or enucleation including Mxi1, Riok3, Trim58, Rac GTPase I and II at most if not all stages of maturation in Foxo3-/- erythroblasts. Immunoprecipitation of endogenous FOXO3 in wild type but not in Foxo3-/- bone marrow erythroblasts recovered the regulatory regions of both Mxi1 and Riok3 in vivo suggesting that FOXO3 controls directly the expression of these genes in erythroblasts. To investigate more directly the potential impact of loss of FOXO3 on the enucleation process, we examined bone marrow erythroblasts at high magnification by confocal fluorescence microscopy. Wild-type enucleating erythroblasts were identified by a gap in the bright TER119 membrane staining and displayed a dumbbell-shaped nucleus, with a neck located at the TER119 sorting boundary of the nascent reticulocyte, so that the cells used a single direction for nuclear extrusion. However, Foxo3 mutant erythroblasts exhibited multiple nuclear necks accompanied by multiple sorting boundaries with each lobe extruding in a different direction away from the nascent reticulocyte, suggesting defective polarization of Foxo3 mutant erythroblasts during enucleation. As a result 48% of the Foxo3-/-enucleating erythroblasts displayed abnormal enucleation morphologies. Next using imaging flow cytometry we further characterized the potential enucleation defect in Foxo3-/- erythroblasts. Segregation of TER119+ erythroblasts according to their cell body and nuclear size into distinct stages of maturation (pro-, basophilic, polychromatic and orthochromatic) showed a relative increase in late erythroid precursors in Foxo3-/- bone marrow. It also revealed that the Foxo3 mutant erythroblasts were specifically accumulated at the orthochromatic stage of maturation. The percentage of orthochromatic erythroblasts containing an asymmetric nucleus was quantified using the delta centroid, a measure of the distance between the center of the cell body and center of the DRAQ5-stained nucleus. This analysis revealed a lower percentage of asymmetric enucleating erythroblasts in the Foxo3-/- as compared to wild-type bone marrow. Using this approach lobular nuclei were distinguished from circular nuclei and demonstrated that odd nuclei were present with remarkably greater frequency in Foxo3-/- than in wild type orthochromatic erythroblasts. These combined analyses support the notion that FOXO3 is critical for the erythroblast enucleation process and may be implicated in polarization of erythroblasts leading to enucleation. The direct ability of FOXO3 to rescue the defective Foxo3 mutant erythroblast enucleation was shown by ectopic expression MSCV-IRES-GFP (MIG)-FOXO3 as compared to empty control vector in wild type and Foxo3-/- bone marrow-derived erythroid progenitors. Restoration of FOXO3 in GFP-positive Foxo3-/-erythroblasts rescued the enucleation rate to similar levels as controls after 72 hours of retroviral transduction and significantly enhanced the levels of expression of Mxi1, Riok3 and Trim58 in Foxo3-/-erythroblasts. Collectively, these findings show that FOXO3 is an essential component of the transcriptional program that regulates terminal erythroblast maturation and is implicated in the enucleation process. DisclosuresNo relevant conflicts of interest to declare.

Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored.Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets.Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members.Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.