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1
Piperno, Alberto, Stefania Galimberti, Raffaella Mariani, Sara Pelucchi, Giulia Ravasi, Carolina Lombardi, Grzegorz Bilo, et al. "Modulation of hepcidin production during hypoxia-induced erythropoiesis in humans in vivo: data from the HIGHCARE project." Blood 117, no. 10 (March 10, 2011): 2953–59. http://dx.doi.org/10.1182/blood-2010-08-299859.
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AbstractIron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Forty-seven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting.
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Cerdan, Chantal, Anne Rouleau, and Mickie Bhatia. "VEGF-A165 augments erythropoietic development from human embryonic stem cells." Blood 103, no. 7 (April 1, 2004): 2504–12. http://dx.doi.org/10.1182/blood-2003-07-2563.
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Abstract Combinations of hematopoietic cytokines and the ventral mesoderm inducer BMP-4 have recently been shown to augment hematopoietic cell fate of human embryonic stem cells (hESCs) during embryoid body (EB) development. However, factors capable of regulating lineage commitment of hESC-derived hematopoiesis have yet to be reported. Here we show that vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development from hESCs. Effects of VEGF-A165 were dependent on the presence of hematopoietic cytokines and BMP-4, and could be augmented by addition of erythropoietin (EPO). Treatment of human EBs with VEGF-A165 increased the frequency of cells coexpressing CD34 and the VEGF-A165 receptor KDR, as well as cells expressing erythroid markers. Although fetal/adult globins were unaffected, VEGF-A165 induced the expression of embryonic zeta (ζ) and epsilon (ϵ) globins, and was accompanied by expression of the hematopoietic transcription factor SCL/Tal-1. In addition to promoting erythropoietic differentiation from hESCs, the presence of VEGF-A165 enhanced the in vitro self-renewal potential of primitive hematopoietic cells capable of erythroid progenitor capacity. Our study demonstrates a role for VEGF-A165 during erythropoiesis of differentiating hESCs, thereby providing the first evidence for a factor capable of regulating hematopoietic lineage development of hESCs.
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Socolovsky, Merav, Hyung-song Nam, Mark D. Fleming, Volker H. Haase, Carlo Brugnara, and Harvey F. Lodish. "Ineffective erythropoiesis in Stat5a−/−5b−/− mice due to decreased survival of early erythroblasts." Blood 98, no. 12 (December 1, 2001): 3261–73. http://dx.doi.org/10.1182/blood.v98.12.3261.
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Abstract Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a−/−5b−/− mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a−/−5b−/− mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a−/−5b−/− mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a−/−5b−/− hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-xLand increased apoptosis in Stat5a−/−5b−/−early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.
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Hopfer, Olaf Joachim, Martina Komor, Claudia Freitag, Maximilian Mossner, Dieter Hoelzer, Eckhard Thiel, and Wolf-Karsten Hofmann. "Epigenetic Dysregulation of GATA1 but Not Downstream Notch Effectors is Involved in MDS Dyserythropoiesis." Blood 112, no. 11 (November 16, 2008): 1652. http://dx.doi.org/10.1182/blood.v112.11.1652.1652.
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Abstract Myelodysplastic syndromes (MDS) are mainly characterized by dyserythropoiesis resulting in anemia. So far, this pathological hallmark is incompletely understood. Notch signalling has recently been linked to impaired erythropoiesis and megakaryopoiesis of CD34+ progenitor cells, however its role in MDS is unclear. On the other hand, in MDS demethylating therapy results in decreased transfusion dependency, indicating aberrant methylation of key erythroid genes. Therefore, we have analyzed Notch pathway elements and its association with the key erythroid factors GATA1 and BCLxl and examined the methylation of CpGs flanking cis-regulatory elements (including N-box suppressor binding site for HES1 and GATA box) of the GATA1 erythroid promoter in differentiating CD34+ cells selected from MDS patients. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO. Cell harvest was at days 0, 4, 7 and 11. RNA-expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). GPI was used as a housekeeping gene. DNA methylation at 7 CpGs of the GATA1 gene promoter was quantitatively analyzed by Pyrosequencing (Pyro Mark ID, Biotage, Uppsala, Sweden) of bisulfite treated genomic DNA at any specific time point. In normal erythropoietic cells, RNA expression of GATA1 and of BCLxl was steadily up regulated, particularly during late erythropoietic differentiation. In contrast, during MDS erythropoiesis a loss of typical up regulation of GATA1 (day 11: 2.08 vs. 0.11; p=0.001) and BCLxl (day 11: 7.46 vs. 0.16; p=0.0005) was observed. Notch ligand DLK1 showed increased expression during erythropoiesis particularly in high risk MDS as compared to normal controls (days 4–11: 0.38 vs. 0.03; p=0.02). Furthermore, expression of HES1 was increasing during the course of normal erythropoietic differentiation but not in lineage specific cells from MDS patients (day 11: 0.01 vs. 0.006; p=0.1). No hypomethylation of CpGs flanking repressor HES1 binding site within the 5′-GATA region was detected in MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up regulation in contrast to MDS erythropoiesis (day 11: 26% vs. 58%; p=0.00004). Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fail to be up regulated during MDS erythropoiesis. The higher residual 5′-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggests a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS. However, a transcriptional activation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 and a hypomethylation of its binding site could not be detected in MDS.
5
Colancecco, Alessandra, Luisa Ronzoni, Lorena Duca, Laura Sonzogni, Isabella Nava, Giovanna Graziadei, and Maria Domenica Cappellini. "In Vitro GDF15 Expression During Thalassemic Erythroid Differentiation and Maturation." Blood 118, no. 21 (November 18, 2011): 5285. http://dx.doi.org/10.1182/blood.v118.21.5285.5285.
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Abstract Abstract 5285 INTRODUCTION: The growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to be related to ineffective or apoptotic erythropoiesis. GDF-15 levels were found to be significantly elevated in sera of patients with β thalassemia major (TM), however little is known about the GDF15 expression during thalassemic erythropoiesis. Non-transfusion dependent thalassemia intermedia (NTDT) represents the model of thalassemic erythropoiesis not affected by transfusions. AIM: To determine the GDF15 gene expression profile during normal and thalassemic erythroid differentiation in standard cultures and under different iron conditions, from CD34+ of normal and thalassemia intermedia subjects. METHODS: After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with NTDT by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. Each culture was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4 mM) as iron chelating agent and ferric ammonium citrate (FAC 100 mM) at day 0 of culture. The expression profiling of GDF15 gene was evaluated at baseline, day 7 and day 14 by real-time PCR (2̂-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit (R&D Systems, Minneapolis, MN). RESULTS: GDF15 expression and secretion increased significantly during erythroid differentiation either in normal and in NTDT cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were higher (althoug not statistically significant) compared to control (Table 1). At day 14 in control cultures GDF15 expression is up-regulated by DFO and down-regulated by FAC addition. In NTDT GDF15 expression was influenced by iron addition in cultures, but was not increased by iron depletion (Table 2). CONCLUSIONS: GDF15 levels in erythroid cultures are related to the erythropoietic stage of differentiation. In NTDT cultures the GDF15 gene profile and protein levels in supernatants are higher than in normal cultures. GDF15 levels seem to be modulated by iron in normal cultures whereas in NTDT cultures they seem to be independent from iron availability. This in vitro study supports that signals different than iron, such as erythropoietic stress, could be the major factor regulating GDF15 expression. Disclosures: No relevant conflicts of interest to declare.
6
Dumitriu, Bogdan, Michael R. Patrick, Jane P. Petschek, Srujana Cherukuri, Ursula Klingmuller, Paul L. Fox, and Véronique Lefebvre. "Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development." Blood 108, no. 4 (August 15, 2006): 1198–207. http://dx.doi.org/10.1182/blood-2006-02-004184.
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Abstract Erythropoiesis, the essential process of hematopoietic stem cell development into erythrocytes, is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver, neonatal spleen, and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen, short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype, demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation, including hemoglobinization, cell condensation, and enucleation, and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells.
7
Labbaye, C., M. Valtieri, U. Testa, A. Giampaolo, E. Meccia, P. Sterpetti, I. Parolini, E. Pelosi, D. Bulgarini, and YE Cayre. "Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture." Blood 83, no. 3 (February 1, 1994): 651–56. http://dx.doi.org/10.1182/blood.v83.3.651.651.
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Abstract All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte- macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid- suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.
8
Labbaye, C., M. Valtieri, U. Testa, A. Giampaolo, E. Meccia, P. Sterpetti, I. Parolini, E. Pelosi, D. Bulgarini, and YE Cayre. "Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture." Blood 83, no. 3 (February 1, 1994): 651–56. http://dx.doi.org/10.1182/blood.v83.3.651.bloodjournal833651.
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All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte- macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid- suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.
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Wiles, M. V., and G. Keller. "Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture." Development 111, no. 2 (February 1, 1991): 259–67. http://dx.doi.org/10.1242/dev.111.2.259.
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When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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Hopfer, Olaf J., Martina Komor, Ina S. N. Koehler, Claudia Freitag, Dieter Hoelzer, Eckhard Thiel, and Wolf-Karsten Hofmann. "GATA and BCLxl Downregulation in Erythropoiesis during In Vitro Lineage Specific Differentiation of MDS Hematopoietic Progenitor Cells Is Not Induced by Activated Notch Pathway." Blood 110, no. 11 (November 16, 2007): 4118. http://dx.doi.org/10.1182/blood.v110.11.4118.4118.
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Abstract Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.
11
Paulson, Robert F., Jie Xiang, and Sneha Hariharan. "Epo Dependent Signaling In Macrophages Regulates Stress Erythropoiesis." Blood 122, no. 21 (November 15, 2013): 938. http://dx.doi.org/10.1182/blood.v122.21.938.938.
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Abstract Steady State erythropoiesis occurs in the bone marrow and is primarily homeostatic. In response to anemic stress the need for new erythrocytes quickly outpaces the erythropoietic capacity of steady state erythropoiesis. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best understood in mice where this process is primarily extra-medullary occurring in the adult spleen and liver and in the fetal liver during development. Stress erythropoiesis utilizes progenitors and signals that are distinct from steady state erythropoiesis. Using a variety of experimental systems, we have developed a model for stress erythropoiesis during the recovery from anemic stress. This recovery can be divided into three stages. Amplification of progenitors that exhibit stem cell properties, the induction of a signal that promotes the switch from amplifying stress progenitors to differentiating stress progenitors and the final stage where stress progenitors rapidly differentiate into new erythrocytes. We have identified specific stress progenitor populations at each stage on this process as well as the signals that regulate the amplification, the switch to differentiation and differentiation of stress erythroid progenitors. Here we show that macrophage dependent signals play key roles at each stage of stress erythropoiesis. The transition from amplifying stress erythroid progenitors to differentiating stress erythroid progenitors is mediated by Epo dependent signaling in macrophages which changes the signals made by the macrophage microenvironment from those that promote amplification (Wnt family factors) to those that promote differentiation (PGE2). This paradigm is true for murine and human stress erythroid progenitors. This analysis reveals a dynamic interplay between progenitor cells, the macrophage microenvironment and hypoxic tissues in vivo during the recovery from anemic stress. Disclosures: No relevant conflicts of interest to declare.
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Egan, Daniel N., Zhantao Yang, John Phillips, and Janis L. Abkowitz. "Inducing iron deficiency improves erythropoiesis and photosensitivity in congenital erythropoietic porphyria." Blood 126, no. 2 (July 9, 2015): 257–61. http://dx.doi.org/10.1182/blood-2014-07-584664.
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Key Points Iron deficiency results in symptom improvement in CEP and could be considered a novel therapeutic approach for this disease. CEP marrow cells demonstrated improved growth and erythroid differentiation in vitro under conditions of relative iron restriction.
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Ziegler, Benedikt L., Robert Müller, Mauro Valtieri, Christa P. Lamping, Christian A. Thomas, Marco Gabbianelli, Christina Giesert, Hans-Jörg Bühring, Lothar Kanz, and Cesare Peschle. "Unicellular-Unilineage Erythropoietic Cultures: Molecular Analysis of Regulatory Gene Expression at Sibling Cell Level." Blood 93, no. 10 (May 15, 1999): 3355–68. http://dx.doi.org/10.1182/blood.v93.10.3355.410k30_3355_3368.
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In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34+ cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34+CD38− cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.
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Carrier, Ewa, Shermila Kausal, and Anand S. Srivastava. "Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells." Blood 106, no. 11 (November 16, 2005): 4255. http://dx.doi.org/10.1182/blood.v106.11.4255.4255.
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Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.
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Agheli, Aref, Boris Avezbakiyev, William Steier, Madhumati Kalavar, Chi Chen, and Zili He. "In-Vivo Study of Erythropoietic Pathway Genes Regulation and Differentiation by Prolonged Simultaneous Administration of Glucocorticoids with Epoietin in Animal Modules." Blood 118, no. 21 (November 18, 2011): 4798. http://dx.doi.org/10.1182/blood.v118.21.4798.4798.
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Abstract Abstract 4798 Objectives: The role of steroids in mammalian erythropoiesis has not well defined. We have previously reported our observation on three human cases in which there was a synergism and accelerated response to the Erythropoietic Stimulating Agents (ESA) with simultaneous low and physiologic dose administration of glucocorticoids. In the current study, we investigated the additive effects of different dose schedules of steroids on hematopoietic effects of ESA in animal modules. Methods: A total of 74, four-weeks old male Sprague-Dawley rats were randomized to 6 groups; (A) control, (B) therapeutic doses of either erythropoietin, [Procrit Epoetin Alfa, 100 UI/kg], or (C) dexamethasome (300 mcg/kg), as well as combination of erythropoietin (Epoetin Alfa, 100 UI/kg) with (D) low, [25 mcg/kg], (E) physiologic, [300 mcg/kg], and (F) high, [2.5 mg/kg] doses of dexamethasone through abdominal hypodermal injection three times a week for a total of four weeks. At the conclusion of the study, peripheral blood sample, and Bone marrow mononuclear cells were collected through femur flushing. The samples were lysed and stored in RNA denaturation buffer at –80°C until use. Expressions of multiple hematopoietic major genes were assessed by real-time RT-PCR. Amplification data were processed using ΔΔCt method. Hemoglobin concentration and other CBC parameters were measured at the reference lab. Results: Mean hemoglobin concentrations were significantly higher in groups D (20.76 g/dl, 95% CI 20.08–21.45), E (20.45 g/dl, 95% CI, 19.97–20.94), and F (20.99 g/dl, 95% CI 20.55–21.42), compared to the controlled groups A, B, C (14.57, 15.68, 19.23 g/dl respectively) with two-tailed p-value of <.0001. (Figure-1) Real time RT-PCR based gene expression profiling of major hematopoietic regulators revealed robot increases of JAK2 gene expression in groups of animals treated with EPO only, or even higher increase with EPO plus either low or physiologic doses of dexamethasome. Similarly, GATA-1 levels are increased in groups treated with EPO only, or EPO with low or physiologic doses of dexamethasome. c-kit and NFkB1 expression levels are markedly higher in EPO plus dexamethasome groups. In contrast, the levels of EPOR are generally reduced in all groups receiving ESA. (Figure -2) Conclusion: The findings in this study is suggestive that simultaneous administration of ESAs with glucocorticoids is associated with significant additive elevation of the hemoglobin concentration; however, higher dose of dexamethasone is associated with more frequent adverse side effects such as significant weight loss. It is also suggested that the erythropoietic effect of steroid is concerted by up-regulation of the multiple erythropoietic gene expressions, such as JAK2, GATA-1, c-Kit, and NFkB1, while down regulations of EPOR is uniformly seen in the Epo-treated groups. This novel finding could be clinically utilized to accelerate the erythropoietic response of the ESA in selected cases. Disclosures: No relevant conflicts of interest to declare.
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Modepalli, Susree, Anna Eastman, Chloe Shaw, Shangqin Guo, Shilpa M. Hattangadi, and Gary M. Kupfer. "Novel Fluorescent Timer Tool Enables Characterization of Erythropoietic Differentiation Based on Differential Cell Cycling Speeds." Blood 136, Supplement 1 (November 5, 2020): 27–28. http://dx.doi.org/10.1182/blood-2020-141666.
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Erythropoietic proliferation and differentiation are coordinated and regulated by a complex compendium of molecular components and networks. Understanding the underlying mechanisms and the dependence of erythroid maturation on cell-cycle behavior can provide a detailed insight into normal and ineffective erythropoiesis. The dynamic cell cycle speed of erythroid progenitors reflects the erythron's response to external stimuli, such as severe anemia or bleeding. Aberrant cell cycle speed also defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. Current methods to resolve cell cycle length heterogeneity at a single-cell level in real-time present with limitations, including cellular toxicity, insufficient intensity, and dilution over subsequent cell divisions. We utilized a unique live-cell reporter of cell cycle speed using a histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein. The FT protein emits blue fluorescence when newly synthesized and matures into a stable red fluorescent protein over 1.2 hours. The fusion protein thus distinguishes faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence. Knock-in mice expressing H2B-FT from a universally active locus under the control of a dox inducible promoter were previously generated and characterized. We successfully characterized the stress erythropoietic response of the spleen and bone marrow (BM) after inducing hemolytic anemia by phenylhydrazine (PHZ) administration in these transgenic mice. Flow cytometric investigation of successive stages of erythroblasts revealed that all stages of erythroblasts maintain rapid cell division after the hemolytic insult (****p<0.0001, Mann-Whitney test) and not only early progenitors, as previously thought. We also observed that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. Most importantly, we observed that the last nucleated cell stage, orthochromatic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate the anemia. Blue-red (BR) profiles of the different erythroblasts from the PHZ-treated animals showed a marked distribution into fast-cycling (high blue fluorescence) and slow-cycling (high red fluorescence) subpopulations. Histograms of normalized BR ratios revealed significantly differentially cycling subpopulations in the polychromatic erythroblasts from spleen and orthochromatic erythroblasts from BM under stress. Mass spectrometric analysis of the differentially cycling subpopulations sorted from the respective erythroblasts shows upregulation of genes encoding cell cycle related and phospho-proteins. We are currently performing comparative analyses with openly available proteomic data. With the Erythropoietin (Epo) model for inducing stress erythropoiesis, we do find a modest increase in blue-red ratios for each of the erythroblast populations in Epo-treated timer mice as compared to the PHZ model. A recent study on steroid resistance in DBA reported that dexamethasone (dex) treatment of peripheral blood progenitors caused the specific upregulation of p57Kip2 leading to higher expansion and accelerated erythroid differentiation. We will utilize in vitro human CD34+ primary cell culture to assess the erythropoietic response to known treatments of anemia of chronic kidney disease and Diamond-Blackfan Anemia, like Epo and dex, respectively. These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. We highlight the use of a sophisticated fluorescent system that can help elucidate the role of cell cycle speed in stress hematopoiesis, and determine the mechanistic pathways acting at single-cell or population level. Further phosphoproteomic investigation can lead to identification of discrete molecular targets regulating erythroid cell proliferation and differentiation with potential therapeutic implications. The tool can aid in answering important questions delineating cell cycle dynamics as the cause or consequence of erythroid differentiation in normal and pathophysiological conditions. Disclosures No relevant conflicts of interest to declare.
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De Maria, R., U. Testa, L. Luchetti, A. Zeuner, G. Stassi, E. Pelosi, R. Riccioni, N. Felli, P. Samoggia, and C. Peschle. "Apoptotic Role of Fas/Fas Ligand System in the Regulation of Erythropoiesis." Blood 93, no. 3 (February 1, 1999): 796–803. http://dx.doi.org/10.1182/blood.v93.3.796.
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Abstract The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.
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De Maria, R., U. Testa, L. Luchetti, A. Zeuner, G. Stassi, E. Pelosi, R. Riccioni, N. Felli, P. Samoggia, and C. Peschle. "Apoptotic Role of Fas/Fas Ligand System in the Regulation of Erythropoiesis." Blood 93, no. 3 (February 1, 1999): 796–803. http://dx.doi.org/10.1182/blood.v93.3.796.403k23_796_803.
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The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.
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Hu, Ping, Angel Nebreda, Helmut Hanenberg, Garrett Kinnebrew, Mircea Ivan, Mervin C. Yoder, Marie-Dominique Filippi, Hal E. Broxmeyer, and Reuben Kapur. "P38α/JNK Signaling Restrains Erythropoiesis By Suppressing Ezh2-Mediated Epigenetic Silencing of Bim." Blood 132, Supplement 1 (November 29, 2018): 3837. http://dx.doi.org/10.1182/blood-2018-99-117098.
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Abstract A remarkable feature of erythropoiesis is the coordination of proliferation, differentiation and apoptosis of erythroid cells to precisely achieve erythropoietic homeostasis to avoid anemia and polycythemia. Anemia is a common disease arising from various causes, including Myelodysplastic syndromes, thalassemia, cancer chemotherapy, chronic kidney disease and hemorrhage. The pro-erythropoietic factor erythropoietin (EPO) is often employed for anemia therapy. However, questions have been raised about the safety of EPO given its potential for tumor promotion in cancer-related anemia. Moreover, many acute and chronic anemias, including hemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anemia are untreatable with EPO. To overcome these hurdles, new molecular mechanisms need to be identified that physiologically restrain erythropoiesis by acting as molecular brakes to prevent over-active erythropoiesis caused by pro-erythropoietic signals. Inhibiting these restraining mechanisms could provide alternative approaches to treat anemia in an EPO-independent fashion. P38 MAPK (Mitogen-activated protein kinase) is an important pathway involved in diverse biological processes. P38 modulates cell proliferation, controls cell survival and decides cell fate during differentiation. P38 pathway functions mainly by phosphorylating and activating important transcription factors in response to different stimuli, including ATF2, CREB, and MEF2. There are four members within the P38 MAPK family, including P38α, P38β, P38γ, and P38δ. These members are encoded by different genes and have different tissue expression patterns. Among them, P38α is ubiquitously expressed. P38α modulates the function of different cell types. There are two distinct developmental defects reported in global P38α knockout mice by two separate groups using different mouse strains. One displayed embryonic death with highly anemic appearance due to reduced EPO production and another showed even earlier embryonic lethality due to placental developmental defects. In a P38α conditional mice model in which Cre recombinase was expressed in the whole mouse embryo but not in the placenta by crossing to MORE-Cre mice, no anemia or EPO defects were observed. However, the intrinsic and cell autonomous role of P38α in adult steady-state or stressful erythropoiesis has not been established. Loss of P38α causes activation of JNK in the liver. P38 inhibitors are in clinical trials and have the potential for the treatment of human disease. Therefore, it is important to understand the down-stream targets and functional outcomes induced by P38α deficiency. Using primary human erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs) and P38α conditional knockout mice, we find that P38α acts as a molecular brake during anemia recovery through integrating apoptotic signals and by shortening the lifespan of erythroblasts to prevent potential over-active erythropoiesis caused by pro-erythropoietic signaling. Loss of P38α in erythroblasts activates JNK through augmented Map3k4 via a negative feedback circuit revealed by gene expression profiling. Functionally, JNK serves as a pro-survival signal independent of EPO by compromising Bim expression via stabilizing the epigenetic silencer Ezh2 in erythroblasts. JNK-controlled Cdk1 activity modulates full interaction of Ezh2 to the E3 ligase Smurf2 through multiple threonine phosphorylation sites within Ezh2. Our findings identify a key signaling cascade involving P38α/JNK/Cdk1/smurf2/Ezh2/Bim in fine tuning stress erythropoiesis. We propose that inhibition of P38α may provide an alternative strategy for the treatment of anemia. Disclosures No relevant conflicts of interest to declare.
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Sen, Saswati, Hanming Wang, Chi Lan Nghiem, Kim Zhou, Janice Yau, Chetankumar S. Tailor, Meredith S. Irwin, and Yigal Dror. "The ribosome-related protein, SBDS, is critical for normal erythropoiesis." Blood 118, no. 24 (December 8, 2011): 6407–17. http://dx.doi.org/10.1182/blood-2011-02-335190.
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AbstractAlthough anemia is common in Shwachman- Diamond syndrome (SDS), the underlying mechanism remains unclear. We asked whether SBDS, which is mutated in most SDS patients, is critical for erythroid development. We found that SBDS expression is high early during erythroid differentiation. Inhibition of SBDS in CD34+ hematopoietic stem cells and early progenitors (HSC/Ps) and K562 cells led to slow cell expansion during erythroid differentiation. Induction of erythroid differentiation resulted in markedly accelerated apoptosis in the knockdown cells; however, proliferation was only mildly reduced. The percentage of cells entering differentiation was not reduced. Differentiation also increased the oxidative stress in SBDS-knockdown K562 cells, and antioxidants enhanced the expansion capability of differentiating SBDS-knockdown K562 cells and colony production of SDS patient HSC/Ps. Erythroid differentiation also resulted in reduction of all ribosomal subunits and global translation. Furthermore, stimulation of global translation with leucine improved the erythroid cell expansion of SBDS-knockdown cells and colony production of SDS patient HSC/Ps. Leucine did not reduce the oxidative stress in SBDS-deficient K562 cells. These results demonstrate that SBDS is critical for normal erythropoiesis. Erythropoietic failure caused by SBDS deficiency is at least in part related to elevated ROS levels and translation insufficiency because antioxidants and leucine improved cell expansion.
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Tamary, Hannah, Hanna Shalev, Galit Perez-Avraham, Meira Zoldan, Itai Levi, Dorine W. Swinkels, Toshihiko Tanno, and Jeffery L. Miller. "Elevated growth differentiation factor 15 expression in patients with congenital dyserythropoietic anemia type I." Blood 112, no. 13 (December 15, 2008): 5241–44. http://dx.doi.org/10.1182/blood-2008-06-165738.
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Abstract Congenital dyserythropoietic anemia (CDA) is a rare group of red blood cell disorders characterized by ineffective erythropoiesis and increased iron absorption. To determine whether growth differentation factor 15 (GDF15) hyper-expression is associated with the ineffective erythropoiesis and iron-loading complications of CDA type I (CDA I), GDF15 levels and other markers of erythropoiesis and iron overload were studied in blood from 17 CDA I patients. Significantly higher levels of GDF15 were detected among the CDA I patients (10 239 ± 3049 pg/mL) compared with healthy volunteers (269 ± 238 pg/mL). In addition, GDF15 correlated significantly with several erythropoietic and iron parameters including Hepcidin-25, Ferritin, and Hepcidin-25/Ferritin ratios. These novel results suggest that CDA I patients express very high levels of serum GDF15, and that GDF15 contributes to the inappropriate suppression of hepcidin with subsequent secondary hemochromatosis.
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Lenox, Laurie E., John M. Perry, and Robert F. Paulson. "BMP4 and Madh5 regulate the erythroid response to acute anemia." Blood 105, no. 7 (April 1, 2005): 2741–48. http://dx.doi.org/10.1182/blood-2004-02-0703.
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Abstract Acute anemia initiates a systemic response that results in the rapid mobilization and differentiation of erythroid progenitors in the adult spleen. The flexed-tail (f) mutant mice exhibit normal steady-state erythropoiesis but are unable to rapidly respond to acute erythropoietic stress. Here, we show that f/f mutant mice have a mutation in Madh5. Our analysis shows that BMP4/Madh5-dependent signaling, regulated by hypoxia, initiates the differentiation and expansion of erythroid progenitors in the spleen. These findings suggest a new model where stress erythroid progenitors, resident in the spleen, are poised to respond to changes in the microenvironment induced by acute anemia.
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Rademacher, Marlena, Hartmut Kuhn, and Astrid Borchert. "Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7795. http://dx.doi.org/10.3390/ijms22157795.
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Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.
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Millot, Sarah, Valerie Andrieu, Philippe Letteron, Sigismond Lasocki, and Carole Beaumont. "Erythropoietin Injections Stimulate Spleen BMP4-Dependant Stress Erythropoiesis in a Mouse Model of Generalized Inflammation." Blood 114, no. 22 (November 20, 2009): 1983. http://dx.doi.org/10.1182/blood.v114.22.1983.1983.
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Abstract Abstract 1983 Poster Board I-1005 Introduction: In mouse, acute anemia leads to the rapid expansion and differentiation of stress erythroid progenitors in the spleen. It has been shown that these progenitors respond to BMP4, Stem Cell Factor and hypoxia and differentiate into stress BFU-E. These are sensitive to high levels of erythropoietin (EPO) and rapidly expand in the spleen, allowing rapid recovery from the anemia (JM Perry et al., Blood 2009, 113:911-918). Inflammation is known to inhibit growth and differentiation of erythroid progenitors and to suppress EPO synthesis in the kidney. However, the effect of pro-inflammatory cytokines on this stress erythropoiesis response is not known. We have recently developed a mouse model of zymosan-induced generalized inflammation and shown that stimulation of erythropoiesis by repeated blood withdrawal or injections of erythropoietin favours iron mobilization from tissue iron stores (S. Lasocki et al., CCM 2008, 36:2388-2394), suggesting that EPO treatment may be beneficial provided effective erythropoiesis can be elicited. Objectives: The aim of our study was to assess the impact of EPO injections on the stress erythropoietic response in this mouse model of chronic inflammation. Methods: Mice (C57BL/6) received a single intraperitoneal injection of zymosan at day 1 (Z1) followed by four consecutive daily injections of EPO at day 5, 6, 7 and 8. Mice were analyzed one day (Z9EPO1), four days (Z12EPO4) or nine days (Z17EPO9) after the final injection and compared to controls, Z alone or EPO alone. Double Ter119/CD71 labelling was used to analyze the different stages of erythroblast differentiation by FACS, in bone marrow and spleen in the different conditions. Spleen BMP4 expression was followed by RT-qPCR and immunohistochemistry. Serum EPO levels were measured by ELISA and haematological parameters were recorded. Results: In the inflammatory condition, bone marrow erythropoiesis is suppressed and does not respond to EPO injections. There is a concomitant increased in the percentage of apoptotic Ter119+ cells. In the spleen, inflammation increases spleen size but only moderately stimulates the percentage of erythroblasts. However, EPO injections lead to a 10-fold increase in the percentage of immature erythroblasts at Z9EPO1, followed three days later (Z12EPO4) by a similar increase in the proportion of mature erythroblasts. This finally results in increased reticulocytes and haemoglobin concentration. In the spleen, BMP4 mRNAs are not stimulated by inflammation but significantly increased by EPO injections, both in normal mice and mice with Zymosan-induced inflammation. The protein BMP4 is expressed by erythroid precursors and stromal cells. Double labelling with F4/80 and BMP4 clearly shows that spleen macrophages are the BMP4-expressing cells following EPO injections in mice with a generalized inflammation. Conclusion: In mouse, bone marrow erythropoiesis is repressed by inflammation as it has been shown for human erythropoiesis and it does not respond to EPO injections. By contrast, spleen stress erythropoiesis is strongly stimulated by injections of EPO despite the presence of inflammation. This results from a strong increase in BMP4 synthesis by spleen macrophages. BMP4 is known to be stimulated by acute anemia but our study is the first report of a direct effect of EPO injections on BMP4 expression in the spleen and of the identification of macrophages as the stromal cells producing BMP4. It will be of interest to find out if bone marrow macrophages in humans can synthesize BMP4 and also contribute to a medullar stress erythropoietic response. Disclosures: No relevant conflicts of interest to declare.
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Armeanu, S., H. J. Bühring, M. Reuss-Borst, C. A. Müller, and G. Klein. "E-cadherin is functionally involved in the maturation of the erythroid lineage." Journal of Cell Biology 131, no. 1 (October 1, 1995): 243–49. http://dx.doi.org/10.1083/jcb.131.1.243.
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Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.
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Srivastava, A. S., S. Kaushal, and E. Carrier. "Dexamethasone enhances erythropoietic differentiation of embryonic stem cells." Biology of Blood and Marrow Transplantation 12, no. 2 (February 2006): 107–8. http://dx.doi.org/10.1016/j.bbmt.2005.11.331.
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Durkin, JP, JM Biquard, JF Whitfield, N. Morardet, J. Royer, P. Macdonald, R. Tremblay, JD Legal, R. Doyonnas, and JP Blanchet. "The identification and characterization of a novel human differentiation-inhibiting protein that selectively blocks erythroid differentiation." Blood 79, no. 5 (March 1, 1992): 1161–71. http://dx.doi.org/10.1182/blood.v79.5.1161.1161.
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Abstract We have isolated a novel inhibitor of erythropoietic differentiation from the plasma of a patient suffering from idiopathic pure red cell aplasia. This differentiation-inhibiting protein (DIP) specifically blocked the differentiation of human burst-forming unit-erythroid (BFU- E), but not colony-forming unit-erythroid (CFU-E) cells. DIP also blocked the maturation of murine BFU-E cells, but not CFU-E or CFU- granulocyte-macrophage cells, and it inhibited the dimethyl sulfoxide (DMSO)-induced differentiation of Friend murine erythroleukemia cells (FLC) at levels between 10(-10) and 10(-12) mol/L. DIP activity was not detectable in the plasma of normal, healthy subjects. Unlike other known inhibitors of hematopoiesis, DIP appears to directly inhibit erythropoietic differentiation, because it did not affect the proliferation of untreated FLC and it effectively blocked FLC hemoglobinization without affecting the ability of the blocked cells to proliferate. DIP blocked FLC differentiation only when added to the culture medium within 1 hour of inducing the cells with DMSO, suggesting that the protein inhibited an early, but critical, DMSO- induced cellular process. DIP appears to be at least partially responsible for the patient's anemia, and its unique activity suggests a role in the early development of some erythroleukemias.
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Durkin, JP, JM Biquard, JF Whitfield, N. Morardet, J. Royer, P. Macdonald, R. Tremblay, JD Legal, R. Doyonnas, and JP Blanchet. "The identification and characterization of a novel human differentiation-inhibiting protein that selectively blocks erythroid differentiation." Blood 79, no. 5 (March 1, 1992): 1161–71. http://dx.doi.org/10.1182/blood.v79.5.1161.bloodjournal7951161.
Full textAbstract:
We have isolated a novel inhibitor of erythropoietic differentiation from the plasma of a patient suffering from idiopathic pure red cell aplasia. This differentiation-inhibiting protein (DIP) specifically blocked the differentiation of human burst-forming unit-erythroid (BFU- E), but not colony-forming unit-erythroid (CFU-E) cells. DIP also blocked the maturation of murine BFU-E cells, but not CFU-E or CFU- granulocyte-macrophage cells, and it inhibited the dimethyl sulfoxide (DMSO)-induced differentiation of Friend murine erythroleukemia cells (FLC) at levels between 10(-10) and 10(-12) mol/L. DIP activity was not detectable in the plasma of normal, healthy subjects. Unlike other known inhibitors of hematopoiesis, DIP appears to directly inhibit erythropoietic differentiation, because it did not affect the proliferation of untreated FLC and it effectively blocked FLC hemoglobinization without affecting the ability of the blocked cells to proliferate. DIP blocked FLC differentiation only when added to the culture medium within 1 hour of inducing the cells with DMSO, suggesting that the protein inhibited an early, but critical, DMSO- induced cellular process. DIP appears to be at least partially responsible for the patient's anemia, and its unique activity suggests a role in the early development of some erythroleukemias.
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Chong, Zhao Zhong, Jing-Qiong Kang, and Kenneth Maiese. "Hematopoietic Factor Erythropoietin Fosters Neuroprotection through Novel Signal Transduction Cascades." Journal of Cerebral Blood Flow & Metabolism 22, no. 5 (May 2002): 503–14. http://dx.doi.org/10.1097/00004647-200205000-00001.
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In addition to promoting the survival, proliferation, and differentiation of immature erythroid cells, erythropoietin and the erythropoietin receptor have recently been shown to modulate cellular signal transduction pathways that extend beyond the erythropoietic function of erythropoietin. In particular, erythropoietin has been linked to the prevention of programmed cell death in neuronal systems. Although this work is intriguing, the underlying molecular mechanisms that serve to mediate neuroprotection by erythropoietin are not well understood. Further analysis illustrates that erythropoietin modulates two distinct components of programmed cell death that involve the degradation of DNA and the externalization of cellular membrane phosphatidylserine residues. Initiation of the cascades that modulate protection by erythropoietin and its receptor may begin with the activation of the Janus tyrosine kinase 2 protein. Subsequent downstream mechanisms appear to lead to the activation of multiple signal transduction pathways that include transcription factor STAT5 (signal transducers and activators of transcription), Bcl-2, protein kinase B, cysteine proteases, mitogen-activated protein kinases, proteintyrosine phosphatases, and nuclear factor-κB. New knowledge of the cellular pathways regulated by erythropoietin in neuronal environments will potentially solidify the development and initiation of therapeutic strategies against nervous system disorders.
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Dmytriyeva, Oksana, Stanislava Pankratova, Irina Korshunova, and Peter S. Walmod. "Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects." Mediators of Inflammation 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/1346390.
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The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobisin vitropromotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.
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Feigenson, Marina, Remya Nathan, Christopher Materna, Alana Gudelsky, Evan Lema, Claire C. Tseng, Ffolliott Fisher, Jasbir Seehra, and Jenn Lachey. "Ker-050, a Novel Inhibitor of Tgfβ Superfamily Signaling, Induces Red Blood Cell Production By Promoting Multiple Stages of Erythroid Differentiation." Blood 136, Supplement 1 (November 5, 2020): 34. http://dx.doi.org/10.1182/blood-2020-140364.
Full textAbstract:
Diseases such as myelodysplastic syndrome (MDS) and myelofibrosis (MF) are characterized by ineffective hematopoiesis resulting in one or multiple cytopenias. Disease-causing defects occur across multiple cell lineages and stages of hematopoiesis, making development of an effective treatment for all patients challenging. Current treatment options to address anemia in these diseases target discreet stages in erythropoiesis, whereas defects leading to ineffective hematopoiesis can occur throughout the pathway. Therefore, a treatment that more globally modulates hematopoiesis has the potential to treat broad patient groups. The TGFβ superfamily plays a key role in regulating hematopoiesis; as signaling via SMAD2/3 activation results in cell quiescence, inhibiting precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA ligand trap, promotes hematopoiesis through inhibition of ligands that signal though SMAD2/3, including activins and GDFs. In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to robust, rapid and sustained increases in red blood cells (RBCs), hemoglobin (HGB) and platelets, supporting an effect on the multiple stages of hematopoiesis. Here, we characterize the time course of KER-050-mediated effects on RBC production and changes in erythroid precursor cell populations in mice to characterize the mechanism of action of KER-050 on erythropoiesis. Mice treated with a single dose of a research form of KER-050 (RKER-050, 10mg/kg) had increased RBCs, HGB and hematocrit (HCT) (+8%, +9%, +7%, respectively) 12 hours after administration compared to vehicle-treated mice (VEH), and this effect was further increased on Day 7. There was also a reduction in the number of enucleated erythroid cells in the bone marrow and a parallel increase in the percent of immature reticulocytes (RET) in peripheral blood, suggesting an increased outflux of RET into circulation. This observation is consistent with RKER-050 promoting the maturation of late stage erythroid precursors leading to increases in RBCs, HGB and HCT as early as 12 hours post treatment and remaining 14 days post a single dose. In studies evaluating the effect of RKER-050 on bone marrow erythroid progenitors, a 2-fold increase in late orthochromatic erythroblasts/RETs (EryC) at Days 2 and 7 post-dose was observed. These data are consistent with RKER-050 promoting maturation and release of late-stage erythroid precursors into circulation. RKER-050 also elicited effects on early progenitors. Day 2 post-dose, there was a 2-fold increase in CFU-Es, with a 46% decrease in poly-erythrochromatic/early orthochromatic erythroid precursors (EryB) at Day 4, as compared to VEH. Day 7 post treatment, both CFU-Es and the EryB population returned to VEH levels. The increase in early progenitors appears to replenish the polychromatic erythroblasts (as shown by the return to VEH level of EryB precursors at Day 7), allowing for continued supply of maturing RETs. Consistent with this hypothesis, RKER-050-mediated changes in erythroid precursors continued to Day 14 with significantly increased early progenitor population (BFU-E +24%) and increased late stage erythroid precursors (EryB +40%, EryC 7-fold) while maintaining increased circulating RETs and RBCs. These data demonstrate that the RKER-050 treatment increases early progenitor cells which continue to mature and contribute to the overall upregulation of erythropoietic tone. Surprisingly, RKER-050 treatment resulted in a greater than 2-fold increase in serum levels of erythropoietin (Epo) at Days 4, 7 and 14. These counterintuitive results demonstrate that RKER-050 promotes erythropoiesis while at the same time increasing Epo. This effect may result in a feed-forward effect on the system and result in a sustained upregulation of erythropoietic tone. Overall, these data demonstrate that KER-050 stimulates terminal maturation of late-stage erythroid precursors, expands the early stage precursor population and progresses precursors through erythropoiesis. Additionally, KER-050 increases Epo within the milieu of elevated RBCs. The ability of KER-050 to target multiple stages along the erythropoiesis cascade makes it an appealing therapeutic candidate for diseases that cause anemia due to ineffective erythropoiesis, including MDS and MF, where defects can arise throughout the erythropoietic pathway. Disclosures Feigenson: Keros Therapeutics: Current Employment. Nathan:Keros Therapeutics: Current Employment. Materna:Keros Therapeutics: Current Employment. Gudelsky:Keros Therapeutics: Current Employment. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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Liu, Xue-Song, Xi-Hua Li, Yi Wang, Run-Zhe Shu, Long Wang, Shun-Yuan Lu, Hui Kong, et al. "Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver." Blood 110, no. 3 (August 1, 2007): 870–76. http://dx.doi.org/10.1182/blood-2007-01-068528.
Full textAbstract:
Abstract Palladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld−/− embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld−/− fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld−/− fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld−/− fetal liver was found disorganized. Palld−/− fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.
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Maetens, Marion, Gilles Doumont, Sarah De Clercq, Sarah Francoz, Pascal Froment, Eric Bellefroid, Ursula Klingmuller, Guillermina Lozano, and Jean-Christophe Marine. "Distinct roles of Mdm2 and Mdm4 in red cell production." Blood 109, no. 6 (November 14, 2006): 2630–33. http://dx.doi.org/10.1182/blood-2006-03-013656.
Full textAbstract:
Abstract Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. Here, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by intercrossing conditional Mdm4 and Mdm2 alleles to an erythroid-specific Cre (Er-GFP-Cre) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, Mdm4 only contributes to p53 regulation at a specific phase of the differentiation program.
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Schuetze, S., R. Paul, B. C. Gliniak, and D. Kabat. "Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells." Molecular and Cellular Biology 12, no. 7 (July 1992): 2967–75. http://dx.doi.org/10.1128/mcb.12.7.2967-2975.1992.
Full textAbstract:
Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.
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Schuetze, S., R. Paul, B. C. Gliniak, and D. Kabat. "Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells." Molecular and Cellular Biology 12, no. 7 (July 1992): 2967–75. http://dx.doi.org/10.1128/mcb.12.7.2967.
Full textAbstract:
Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.
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Carrancio, Soraya, Jennifer A. Markovics, Piu Wong, Carla Heise, Tom O. Daniel, Rajesh Chopra, and Victoria Sung. "Sotatercept Promotes Differentiation and Survival Of Erythroid Progenitors By Blocking Inhibitory Effects Of TGFβ Superfamily Members." Blood 122, no. 21 (November 15, 2013): 944. http://dx.doi.org/10.1182/blood.v122.21.944.944.
Full textAbstract:
Abstract Erythropoiesis, the process of cell proliferation and differentiation that produces erythrocytes, is a tightly regulated process, but apart from early progenitor development and the EPO-dependent response, very little is known about other molecular signals which control cellular fate during RBC production. Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept, an ActRIIA ligand trap, binds to and inhibits activin and other members of the TGFβ superfamily to induce a rapid increase in red cell number and hemoglobin in healthy volunteers. Pharmacological findings demonstrate that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through a mechanism distinct from EPO. We conducted the current study to evaluate if RAP-011 may stimulate expansion of a late-stage erythroblast population that is not normally expanded and/or may induce faster differentiation of erythroid precursors. In order to determine if RAP-011 promotes proliferation or differentiation during erythropoiesis, the number of cell divisions was quantified by CFSE staining. During in vitro erythroid differentiation, RAP-011 did not appear to alter the number of cell divisions; however, the percentage of cells that underwent the last division was higher in cultures treated with RAP-011, suggesting that the drug induced faster cellular maturation/differentiation. We also analyzed cell viability of GPA+ cells at the end of the differentiation process and observed that the percentage of apoptotic death was higher in control vs. RAP-011-treated cells. This suggests that RAP-011 may promote survival of late-stage precursors. To assess potential candidates which may mediate the erythropoietic effects of RAP-011, we selected three high affinity RAP-011 ligands, Activin A, Activin B and GDF-11, and proceeded to evaluate their effects on Smad signaling and on erythroid differentiation of human bone marrow progenitors. First, we observed that RAP-011 blocked ligand-induced Smad2/3 phosphorylation in the bone marrow-derived cells. Secondly, RAP-011 rescued activin A-induced inhibition of BFU-E colony formation. Finally, when mature CD36+ cells were differentiated in liquid media containing each of the three ligands, RAP-011 was able to reverse GDF-11- and Activin A-induced inhibition of of erythroid cell proliferation. GDF-11 and Activin A also significantly decreased the percentage of GPA-positive cells in culture, while significantly increasing the percentage of CD45-positive cells. Consistent with proliferation results, RAP-011 blocked these ligand effects. Treatment of CD36+ cells with Activin B did not alter growth or differentiation. These data suggest that GDF-11 and Activin A may contribute, in part, to the erythropoietic stimulatory effects of RAP-011. Several members of the TGFβ superfamily of ligands have been implicated as negative growth regulators, or “chalones”, functioning in homeostasis to maintain specific, mature tissue size. The results from our studies using the ActRIIA-Fc ligand trap, RAP-011, suggest that GDF-11 and Activin A, as well as other sotatercept ligands, may also be “chalones” for the blood, specifically regulating homeostasis of mature RBCs. We suggest that sotatercept increases red blood cell maturation and survival by blocking the negative growth regulation by TGFβ members. In pathologic states such as ineffective erythropoiesis, sotatercept may have an even greater impact than in the healthy, homeostatically-balanced environment. Disclosures: Carrancio: Celgene Corp.: Employment. Markovics:Celgene Corp.: Employment. Wong:Celgene Corp.: Employment. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Sung:Celgene Corp.: Employment.
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Steensma, David P., Julie C. Porcher, Antony Chadderton, Kevin Laubscher, Deb Jaworski, Joanne J. Lager, William Bell, Bryan E. Hoffman, and Connie L. Erickson-Miller. "DYRK3 Encodes an Inhibitor of Erythroid Differentiation and Is Expressed at High Levels in Patients with Anemia." Blood 110, no. 11 (November 16, 2007): 3661. http://dx.doi.org/10.1182/blood.v110.11.3661.3661.
Full textAbstract:
Abstract Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis. Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor. Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied. Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.
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Kim, MinJung, Yee Sun Tan, Wen-Chih Cheng, and Curt I. Civin. "MiR-144 and MiR-451 Regulate Human Erythropoiesis By Targeting RAB14." Blood 122, no. 21 (November 15, 2013): 942. http://dx.doi.org/10.1182/blood.v122.21.942.942.
Full textAbstract:
Abstract MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by inhibiting translation and/or by degrading of target mRNAs. MiR-144 and miR-451 are expressed from a single precursor transcript, encoded by the miR-144∼451 cluster on human chromosome 17. Expression levels of both miR-144 and miR-451 are upregulated during zebrafish, mouse, and human erythroid differentiation. Several mouse and zebrafish studies have knocked down miR-451 expression and observed decreased late erythroid differentiation. In contrast, miR-144 knockdown had no or smaller effects in zebrafish or mouse erythropoiesis, and there is no study of the role of both members of this cluster in primary human erythropoiesis. Therefore, we investigated the effects of lentiviral-mediated loss-of-function of miR-144 and/or miR-451 in human erythropoiesis. We first utilized the TF1 human erythroleukemia cell line as a model, and then tested primary human CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human donors. When either TF1 or CD34+ cells are cultured with erythropoietin (EPO), the expression of the CD71 (transferrin receptor) and CD235a (glycophorin A) erythroid markers increases, and the expression of the CD34 HSPC marker decreases. In TF1 or primary CD34+ cells transduced with either a miR-144 trap or a miR-451 trap (using a lentivirus containing GFP and a series of 8 tandem complementary miR binding sequences to knock down function of either miR [Ebert, Nature Method, 2007]), as compared to control vector, erythropoiesis was decreased, as evidenced by lower numbers of erythroid (CD34-CD71hiCD235ahi) cells generated during EPO-induced erythroid differentiation. RAB14 was predicted to be a target of both miR-144 and miR-451 by the TargetScan 6.2 target prediction algorithm, with 2 predicted miR-144 binding sites and 1 predicted miR-451 binding site in its 3’UTR. RAB14 was validated to be a target of miR-451 in human non-small cell lung cancer cells [Wang, Oncogene, 2011]. RAB proteins have been reported to have important roles in vesicle trafficking, signal transduction, and receptor recycling. Recently, RAB12 was implicated in constitutive degradation of the transferrin receptor (CD71) in mouse embryonic fibroblast (MEF) cells [Matsui, Traffic, 2011], but the role of RAB14 in human erythropoiesis is unknown. We found that RAB14 protein expression decreases early during EPO-induced erythroid differentiation of TF1 cells. Then we showed that RAB14 is a direct target of miR-144, using luciferase assays and Western blots. We confirmed that shRNA-mediated RAB14 knockdown increased human erythropoiesis, as compared to control vector-transduced cells. Finally, shRNA-mediated RAB14 knockdown protected cells from miR-144/miR-451 trap-mediated erythropoietic inhibition. In summary, miR-144 and miR-451 regulate human erythropoiesis by decreasing RAB14 expression, which in turn might have a role in regulating function of receptors involved in erythropoiesis, such as the transferrin receptor. Disclosures: No relevant conflicts of interest to declare.
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Ohneda, O., N. Yanai, and M. Obinata. "Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver." Development 110, no. 2 (October 1, 1990): 379–84. http://dx.doi.org/10.1242/dev.110.2.379.
Full textAbstract:
Mouse stromal cell lines (FLS lines), established from the livers of 13-day gestation mouse fetus, supported the proliferation and differentiation of the erythroid progenitor cells from mouse fetal livers and bone marrow in a semisolid medium in the presence of erythropoietin. A large erythroid colony of over 1000 benzidine-positive erythroid cells was developed from a single erythroid progenitor cell on the FLS cell layer after 4 days of culture. When in close contact with the layer, the erythroid progenitor cells divided rapidly with an average generation time of 9.6 h and mature erythroid cells, including enucleated erythrocytes, were produced. The present studies demonstrate that the microenvironment created by the stromal cells can support the rapid expansion of erythropoietic cell population in the fetal liver of mice.
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Blouin, Marie-José, Monique E. De Paepe, and Marie Trudel. "Altered Hematopoiesis in Murine Sickle Cell Disease." Blood 94, no. 4 (August 15, 1999): 1451–59. http://dx.doi.org/10.1182/blood.v94.4.1451.
Full textAbstract:
Abstract We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of ∼42%) and erythroid colony-forming units (CFU-E; of ∼23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (∼4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units–granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca+Lin−) were highly mobilized to the peripheral blood (∼4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.
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Blouin, Marie-José, Monique E. De Paepe, and Marie Trudel. "Altered Hematopoiesis in Murine Sickle Cell Disease." Blood 94, no. 4 (August 15, 1999): 1451–59. http://dx.doi.org/10.1182/blood.v94.4.1451.416k02_1451_1459.
Full textAbstract:
We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of ∼42%) and erythroid colony-forming units (CFU-E; of ∼23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (∼4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units–granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca+Lin−) were highly mobilized to the peripheral blood (∼4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.
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Fu, Rong, Hui Liu, Zonghong Shao, and Lijuan Li. "Preliminary Study of the Relationship Between EPO Receptor and Autoantibody on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia." Blood 116, no. 21 (November 19, 2010): 4436. http://dx.doi.org/10.1182/blood.v116.21.4436.4436.
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Abstract Abstract 4436 Objective To observe the relationship between EPO receptor (EPOR) and autoantibody(IgG) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's(BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP),and then explore the probable autoantigens of autoantibodies in IRP. Methods 42 newly diagnosed IRP patients (14 with IgG autoantibody on erythropoietic cells and 28 without this autoantibody) and 11 healthy donors as controls were enrolled in this study. EPOR on their nuclear erythrocyte were tested with flow cytometry. Their clinical data were also analyzed. Results (1) The level of EPOR of IgG(+) arm(1.59±0.87)% was significantly lower than that of IgG(-) arm (4.58±4.09)%(P<0.01), and the level of EPOR of IgG(-) arm was significantly higher than that of normal controls(2.27±1.76)%(P<0.05) G The level of EPOR of IRP patients was negatively correlated with their IgG autoantibody(r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(IgG); (2) Reticulocyte percentage of IgG(+) arm (1.55±0.64)% was significantly lower than that of IgG(-) arm (2.41±1.42)%(P<0.05). The level of EPOR of IRP patients was positively correlated with reticulocyte percentage(r=0.346,P=0.029); (3)The serum level of indirect bilirubin(IDBIL) of IgG(+) arm was normal. While there were six patients (21.4%) whose serum level of IDBIL were higher than those of normal controls in IgG(-) arm. Conclusion The autoantibody(IgG) of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. It could inhibit the proliferation and differentiation of erythropoiesis in early phase. Disclosures: No relevant conflicts of interest to declare.
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Capocasale, Renold J., Dorie A. Makropoulos, Jeffrey Arlen, Ram Achuthanandam, John Quinn, Amy L. Volk, and Peter J. Bugelski. "Role of Fas / FasL in Regulation of Basophillic Erythroblast Homeostasis." Blood 106, no. 11 (November 16, 2005): 4289. http://dx.doi.org/10.1182/blood.v106.11.4289.4289.
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Abstract Many studies have shown Fas- Fas Ligand (FasL) mediated apoptosis to be important in maturation and differentiation of erythroid precursors in vitro. To determine if there is a similar process regulating erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr) and FasL (gld) deficient mice. We postulated that deficiency of Fas or FasL should result in changes in red blood cell (RBC) parameters and/or decreased levels of apoptosis of erythroblasts. To test this hypothesis under steady state conditions, blood and bone marrow were collected from 10-week old C57Bl/6 control mice, B6.MRL-Tnfrsf6 lpr /J CD95 deficient mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology was studied using a Bayer Advia 120 and femoral bone marrow was analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria. Hematologic analysis revealed no differences in reticulocyte counts, RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to C57Bl/6 controls. Similarly, analysis of bone marrow revealed no differences in % of Ter-119bright CD71bright basophilic erythroblast (BEB), % apoptotic BEB (annexin V+, 7-AADdim) or % FasL+ BEB in either gld or lpr mice compared to control. As expected, lpr mice expressed 10 fold fewer Fas+ BEB while similar levels were observed in gld mice compared to controls. To test our hypothesis under stimulated conditions, control, lpr and gld mice received a single s.c. dose of 10,000 units of recombinat human erythropoietin (rhEPO). Bone marrow samples were collected 48 hours after dosing and blood samples 4, 8 and 16 days after dosing. Hematologic analysis revealed no differences in the erythropoietic response among the three strains of mice tested. Moreover, treatment with rhEPO had no effect on % Fas+ BEB in any strain, but induced a 2–5 fold increase in the % FasL+ BEB and a 2–3 fold increase in apoptotic BEB in all three strains. Based on our observations, we conclude Fas/FasL is unlikely to play a pivotal role in regulating erythroid homeostasis.
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Temprine, Kelsey, Amanda Sankar, Costas Lyssiotis, and Yatrik Shah. "Metabolomic Characterization of Red Blood Cell Differentiation." Blood 136, Supplement 1 (November 5, 2020): 35. http://dx.doi.org/10.1182/blood-2020-137175.
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Background: Erythropoiesis is the highly coordinated multi-step process by which multipotent hematopoietic stem cells differentiate into mature enucleated red blood cells (RBCs). As erythroid cells become more terminally differentiated, they undergo changes in morphology and gene expression, start synthesizing hemoglobin, commit to an irreversible loss of proliferation, and eventually expulse their nuclei and other cytoplasmic organelles. Thus, RBCs must rely on their proteome and metabolome for proper function. The RBC proteome is estimated to contain 2,800 proteins, including a variety of receptors and transporters that allow RBCs to uptake xenobiotics or endogenous metabolites as they circulate for ∼120 days. Furthermore, they are metabolically active with glycolysis, nucleotide catabolism, and glutathione metabolism as the major pathways supporting cell survival and function. However, it is unclear how the metabolome is altered during erythropoiesis, what role metabolites play in normal erythropoiesis, and if dysregulation of metabolites contributes to diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Methods: Four models of erythropoiesis were used in this study. 1) Mice were treated with phenylhydrazine (Phz) to induce acute hemolysis followed by erythropoietic recovery, leading to an increase in circulating reticulocytes. 2) Mice were lethally irradiated and transplanted with wild-type or sickle cell bone marrow, leading to anemic profiles in sickle cell chimeras. 3) The mouse erythroleukemic (MEL) cell line was treated with DMSO to induce differentiation. 4) The human erythroleukemic (K562) cell line was treated with sodium butyrate to induce differentiation. For the in vivo mouse models, blood was collected from control and treated animals, and complete blood count (CBC) analysis was performed. For the in vitro cell culture models, the mRNA levels of β-globin were measured by Q-RT-PCR in control and differentiated cells, and the degree of hemoglobinization was determined visually and via staining for heme. In addition, metabolites were extracted from the collected RBCs and erythroleukemic cell lines, and a Snapshot LC/MS metabolomic platform was used to identify commonly altered metabolites. Results: We first validated our four models of erythropoiesis. Treatment with Phz decreased the number of total RBCs while increasing the RBC distribution width, indicating an increased number of reticulocytes (more immature RBCs) in circulation. Similar results were seen in the sickle cell chimeras. Treatment of MEL and K562 cells with DMSO and sodium butyrate, respectively, resulted in increased expression of β-globin, increased levels of heme, and increased red color. Then, using our Snapshot metabolomic platform, we identified global changes in RBC metabolism during erythropoiesis. Analyses of the commonly altered metabolites in the in vitro and in vivo models revealed an increase in amino acid, mitochondrial, and urea cycle metabolism during erythropoiesis. L-aspartate levels were particularly upregulated, especially in DMSO-treated MEL cells. We are now investigating the role of aspartate in the regulation of erythropoiesis. Conclusions: We defined how the metabolome was altered in multiple in vitro and in vivo models of erythropoiesis and identified global changes in RBC metabolism between the different models. Specifically, we found that L-aspartate was upregulated during RBC differentiation in all four models. Aspartate is an amino acid that plays a role in many processes in cells, including nucleotide biosynthesis, redox homeostasis, and amino acid biosynthesis. We hypothesize that aspartate metabolism is critical for RBC differentiation and that its dysregulation exacerbates disease of ineffective erythropoiesis, such as sickle cell anemia and β-thalassemia. We are currently testing its role in inducing hemoglobinization and in regulating the commitment of erythroid progenitor cells to an irreversible loss of proliferation. Overall, we believe that understanding the precise mechanisms by which cellular metabolism plays a role in proper RBC differentiation may lead to better therapies for diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Disclosures No relevant conflicts of interest to declare.
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Sen, Saswati, Hanming Wang, Sally-lin Adams, Janice Yau, Kim Zhou, and Yigal Dror. "Mechanisms of Erythropoietic Failure in Shwachman Diamond Syndrome Caused by Loss of Ribosome-Related Protein SBDS." Blood 114, no. 22 (November 20, 2009): 3203. http://dx.doi.org/10.1182/blood.v114.22.3203.3203.
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Abstract Abstract 3203 Poster Board III-140 Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. We hypothesize that SBDS protects early erythroid progenitors from apoptosis by promoting ribosome biosynthesis and translation. During early erythroid differentiation of human K562 cells and primary CD133+ cells, a prominent upregulation of SBDS by RT-qPCR was found. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and a mild reduction in proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. Both cell expansion and translation defects were rescued upon re-introduction of SBDS in K562 cells. Interestingly, leucine partly corrected the cell expansion and translational defects of non-differentiating SBDS-deficient K562 cells, while differentiating SBDS-deficient K562 cells showed improved cell expansion in the presence of additional translation stimulators such as IGF-1. SBDS-knockdown CD133+ cells showed increased BFU-E colony formation under conditions with leucine and a combination of leucine and IGF-1 treatment. Although the erythroid cell expansion defect in K562 cells is independent of p53 as these cells do not express the gene, an upregulation of TAp73, was found in resting SBDS deficient K562 cells. However expression of TAp73 was lost during differentiation. DNp63 was also not upregulated in SBDS-deficient K562 erythroid cells. These results demonstrate that the role of SBDS in non-differentiated cells versus differentiated cells represents two dynamic scenarios and that SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation through a pathway independent of p53 family members. Disclosures No relevant conflicts of interest to declare.
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Makita, Takako, Stephan A. Duncan, and Henry M. Sucov. "Retinoic acid, hypoxia, and GATA factors cooperatively control the onset of fetal liver erythropoietin expression and erythropoietic differentiation." Developmental Biology 280, no. 1 (April 2005): 59–72. http://dx.doi.org/10.1016/j.ydbio.2005.01.001.
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Miller, Jonathan, Ryan Hiltenbrand, Tamara Lamprecht, Aman Seth, Sherif Abdelhamed, Ilaria Iacobucci, Charles G. Mullighan, and Jeffery M. Klco. "NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation." Blood 134, Supplement_1 (November 13, 2019): 3775. http://dx.doi.org/10.1182/blood-2019-130768.
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Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p<0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p<0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p<0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p<0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal. To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p<0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p<0.001), and 2.6-fold increase in erythroid markers (p<0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p<0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver. To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p<0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p<0.05). In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.
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Eymard, Nathalie, Nikolai Bessonov, Olivier Gandrillon, Mark J. Koury, and Vitaly Volpert. "The Role Of Spatial Organization Of Cells In Erythropoiesis." Blood 122, no. 21 (November 15, 2013): 2189. http://dx.doi.org/10.1182/blood.v122.21.2189.2189.
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Abstract The functional unit of definitive mammalian erythropoiesis, the erythroblastic island, consists of a central macrophage surrounded by adherent erythroid progenitor cells at the colony-forming unit/proerythroblast (CFU-E/Pro-EB) stages of differentiation and their differentiating progeny, the erythroblasts. Central macrophages display on their surface or secrete various growth or inhibitory factors that influence the fate of the surrounding erythroid cells. CFU-E/Pro-EBs have three possible fates: a) expansion of their numbers without differentiation, b) differentiation through the erythroblast stages into reticulocytes that are released into the blood, c) death by apoptosis. CFU-E/Pro-EB fate is under the control of a complex intracellular molecular network that is highly dependent upon environmental conditions in the erythroblastic island. Direct examination of erythroblastic island function in vivo has been limited in mice and unfeasible in humans. In order to assess the functional role of spatial organization coupled with the complex network behavior in erythroblastic islands, we developed hybrid discrete-continuous models of erythropoiesis. A mathematical model was developed in which the cells of the erythroblastic island are considered as individual physical objects, intracellular regulatory networks are modeled with ordinary differential equations, and extracellular concentrations of cytokines or hormones are modeled by partial differential equations. The concentrations of the cytokines Fas-ligand and bone morphogenetic protein-4, which are produced locally in the erythroblastic island, and the hormones erythropoietin and glucocorticosteroid hormone, which are produced at remote locations in the body, are included in the model. We used the model in simulations that investigated the impact of an important difference between humans and mice in which mature late-stage erythroblasts produce the most Fas-ligand in humans, and early-stage erythroblasts produce the most Fas-ligand in mice. Although the global behaviors of the erythroblastic islands in both species were similar, differences were found, including a relatively slower recovery time of hematocrits and erythrocyte numbers to their baselines following the development of acute anemia in humans as compared to mice. These simulation results with the model were consistent with the more rapid recovery to baseline in mice that were bled to about one-half of their normal hematocrit compared to two patients who had acute blood loss to about one-half of their respective baseline hematocrits and recovered without erythrocyte transfusions. Our modeling approach was also very consistent with the previously reported results of in vitro cultures, where the central macrophages in reconstituted erythroblastic islands of mice had a strong impact on the dynamics of erythroid cell proliferation. The spatial organization of cells in erythroblastic islands is important for the normal, stable functioning of mammalian erythropoiesis, both in vitro and in vivo. Our model of a simplified molecular network controlling erythroid progenitor cell decision and fate provides a realistic functional unit of mammalian erythropoiesis that integrates factors within the microenvironment of the erythroblastic island with those of circulating regulators of erythropoiesis. Our model highlights the need for proper inclusion of the spatial relationships of erythropoietic cells and allowing decisions to be made at the level of individual erythroid cells in the modeling process. Disclosures: Koury: Keryx Biopharmaceuticals, Inc.: Consultancy; TG Therapeutics, Inc.: Consultancy.
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Takahashi, T., K. Ozawa, K. Takahashi, S. Asano, and F. Takaku. "Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation." Blood 75, no. 3 (February 1, 1990): 603–10. http://dx.doi.org/10.1182/blood.v75.3.603.603.
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Abstract B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus- induced transient aplastic crisis.
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Takahashi, T., K. Ozawa, K. Takahashi, S. Asano, and F. Takaku. "Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation." Blood 75, no. 3 (February 1, 1990): 603–10. http://dx.doi.org/10.1182/blood.v75.3.603.bloodjournal753603.
Full textAbstract:
B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus- induced transient aplastic crisis.