scholarly journals Transcriptional Regulation of Coenzyme Q Biosynthesis By TIF1γ Drives Erythropoiesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 152-152
Author(s):  
Marlies P. Rossmann ◽  
Karen Hoi ◽  
Victoria Chan ◽  
Julie R. Perlin ◽  
Elliott J. Hagedorn ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
De Novo ◽  
Coenzyme Q ◽  
Transcription Elongation ◽  
Erythroid Differentiation ◽  
Nucleotide Metabolism ◽  
Mitochondrial Metabolism ◽  
Hematopoietic Stem ◽  
Equity Ownership

Understanding the cell-autonomous as well as niche contributions governing erythropoiesis is critical for directed differentiation approaches of hematopoietic stem cells into differentiated red blood cells (RBCs) to treat blood disorders such as anemias and leukemias. Transcriptional intermediary factor 1 gamma (TIF1γ) is essential for erythropoiesis from zebrafish to mammals. Zebrafish moonshine mutant embryos defective for tif1γ do not make red blood cells (RBCs) due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. Loss of factors involved in transcription elongation control, PAF1 and spt5, rescues the moonshine RBC defect. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a high-content chemical suppressor screen in the bloodless moonshine mutant using 3,500 compounds. Among the suppressors, we identified leflunomide, an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme for de novo pyrimidine synthesis. Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar both rescue the formation of primitive erythroid cells in 61% (38/62) and 68% (50/74) of moonshine embryos, respectively. Blastula transplant experiments revealed that tif1γ, in addition to its cell-autonomous role, plays a role in the hematopoietic niche for RBC development. Through in-vivo metabolomics analyses we have identified nucleotide metabolism as the most significantly altered process in moonshine mutants, including elevated levels of uridine monophosphate and low levels of nicotinamide adenine dinucleotide (NAD+). Low NAD+ levels are accompanied by a reduced oxygen consumption rate in tif1γ-depleted embryos by Seahorse analysis. In support, genome-wide transcriptome analysis coupled with chromatin immunoprecipitation studies revealed genes encoding coenzyme Q (CoQ) metabolic enzymes as direct TIF1γ targets. DHODH is the only enzyme of the pyrimidine de novo synthesis pathway located on the inner mitochondrial membrane and its activity is coupled to that of the electron transport chain (ETC). Rotenone, a potent ETC complex I inhibitor reverses the rescue of the blood defect by DHODH inhibition in moonshine embryos. Since DHODH function is linked to mitochondrial oxidative phosphorylation via CoQ activity, we asked whether alterations in mitochondrial metabolism might be causal for the RBC defect in moonshine mutants. Indeed, treatment with the CoQ analog decylubiquinone results in rescue of βe3 globin expression in 26% (33/126) of moonshine embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases and consequences for erythroid differentiation protocols. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in Myelodysplastic Syndromes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 610-610 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Aaron Mulivor ◽  
R. Scott Pearsall ◽  
Ravindra Kumar
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Supportive Care ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Significant Proportion ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem

Abstract Abstract 610 Myelodysplatic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Patients develop peripheral blood cytopenias; however, the bone marrow shows increased proliferation and apoptosis. In addition to bone marrow apoptosis, a failure of differentiation contributes to reduced terminally differentiated blood cells. A significant proportion of patients with MDS will develop anemia that are refractory to treatment with recombinant human erythropoietin (EPO) and must rely on transfusions as supportive care. The use of blood transfusions as supportive care is associated with iron overload and significant morbidity. Therefore, alternative therapies to treat anemia in MDS patients are needed. Members of the TGFβ super family of signaling molecules have been implicated in erythropoiesis and represent alternative, EPO-independent targets for the treatment of anemia. ACE-536 is a soluble receptor fusion protein consisting of a modified Activin Receptor Type IIB extracellular domain linked to a human Fc domain. ACE-536 acts as a ligand trap to modulate the activity of TGFβ ligands and promote erythroid differentiation in an EPO independent manner. Subcutaneous administration of ACE-536 to C57BL/6 mice resulted in significant increases in hematocrit, hemoglobin and red blood cells compared to vehicle treated controls within four days. These effects were observed with concurrent treatment of an EPO neutralizing antibody, indicating that EPO is not directly responsible for the initial RBC response of ACE-536. BFU-E or CFU-E colony formation assays from bone marrow or spleen of mice 48 hours after ACE-536 were normal, indicating no effect on the erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by FACS analysis following 72 hours after RAP-536 (murine version of ACE-536) treatment revealed a decrease in basophilic erythroblasts and an increase in late stage poly-, ortho-chromatophilic and reticulocytes in bone marrow and spleen compared to vehicle treated mice. The data demonstrate that while EPO treatment increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. The efficacy of ACE-536 has been demonstrated in various animal models of acute and chronic anemia. In this study we investigated the effect of ACE-536 on anemia in mouse model of MDS. The NUP98-HOXD13 (NHD13) transgenic mouse carries a common translocation found in MDS patients. NHD13 mice develop anemia, neutropenia and lymphopenia at 4–7 months of age, with normal or hypercellular bone marrow. Starting at 4 months of age, mice were treated with RAP-536 (murine homolog of ACE-536) at 10 mg/kg or vehicle control twice per week for 8 months. Wild-type littermate controls were also dosed on the same schedule. As expected, at study baseline (mice 4 months of age), NHD13 mice had reduced RBC, Hb and HCT compared to wild-type littermates. The progression of anemia over the study period was reduced by treatment with RAP-536 compared to vehicle (HCT: −8.3% v. −22%, RBC: −13% v. −30%). Based on blood smear analyses, there was no indication of increased leukemic cells with ACE-536 treatment. Our data demonstrate that RAP-536 can increase hematology parameters through enhancing maturation of terminally differentiated red blood cells and can serve as a therapeutic molecule for the treatment of anemia. As anemia contributes significantly to the morbidity of patients with MDS, a mouse model was used to test the therapeutic efficacy of ACE-536 in this disease. We have shown that systemic administration of RAP-536 to MDS mice promotes increases in red blood cell mass without enhanced progression to AML. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. Disclosures: Suragani: Acceleron Pharma Inc: Employment. Mulivor:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment. Kumar:Acceleron Pharma Inc: Employment.

scholarly journals Mesenchymal stromal cells can be applied to red blood cells storage as a kind of cellular additive

2017 ◽  
Vol 37 (5) ◽  
Author(s):  
Yaozhen Chen ◽  
Jing Zhang ◽  
Shunli Gu ◽  
Dandan Yin ◽  
Qunxing An ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Storage Duration ◽  
Hematopoietic Stem ◽  
Diminished Capacity ◽  
Blood Banks ◽  
Citrate Phosphate

During storage in blood banks, red blood cells (RBCs) undergo the mechanical and metabolic damage, which may lead to the diminished capacity to deliver oxygen. At high altitude regions, the above-mentioned damage may get worse. Thus, more attention should be paid to preserve RBCs when these components need transfer from plain to plateau regions. Recently, we found that mesenchymal stromal cells (MSCs) could rescue from anemia, and MSCs have been demonstrated in hematopoietic stem cells (HSCs) transplantation to reconstitute hematopoiesis in vivo by us. Considering the functions and advantages of MSCs mentioned above, we are trying to find out whether they are helpful to RBCs in storage duration at high altitudes. In the present study, we first found that mice MSCs could be preserved in citrate phosphate dextrose adenine-1 (CPDA-1) at 4 ± 2°C for 14 days, and still maintained great viability, even at plateau region. Thus, we attempted to use MSCs as an available supplement to decrease RBCs lesion during storage. We found that MSCs were helpful to support RBCs to maintain biochemical parameters and kept RBCs function well on relieving anemia in an acute hemolytic murine model. Therefore, our investigation developed a method to get a better storage of RBCs through adding MSCs, which may be applied in RBCs storage as a kind of cellular additive into preservation solution.

scholarly journals Patient-Specific Adhesion of Sickle Red Blood Cells in Shear-Gradient Microscale Flow

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 964-964
Author(s):  
Erdem Kucukal ◽  
Jane A. Little ◽  
Umut A. Gurkan
Keyword(s):  
Shear Rate ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Flow Direction ◽  
Patient Specific ◽  
Shear Rates ◽  
Microscale Flow ◽  
Shear Gradient ◽  
Equity Ownership

Abstract The pathophysiology of sickle cell disease (SCD) involves altered biophysical properties of red blood cells (RBCs) and increased cellular adhesion, which can synergistically trigger recurrent and painful vaso-occlusive events in the microcirculatory network. RBC adhesion to the endothelial wall is heterogeneous and may initiate such occlusions by disrupting the local flow thus activating platelets and promoting subsequent cell-cell interactions. Moreover, these episodic events take place within a wide range of dynamically changing shear rates at the microscale. In order to better understand the role of shear rate on this process, we quantified shear-dependent RBC adhesion to endothelial proteins fibronectin (FN) and laminin (LN) utilizing a microfluidic system that can simulate physiologically relevant shear gradients of microcirculatory blood flow at a single flow rate. Whole blood samples were collected from 20 patients (10 males and 10 females) with homozygous SCD (HbSS). Samples were perfused through FN and LN immobilized shear-gradient microchannels (Fig. 1A) in which the shear rate continuously changes along flow direction. Computational simulations characterized the flow dynamics near the adherent RBCs (Fig. 1B). Based on the numerical results, a rectangular "field of interest (FOI)", along which the shear rate dropped approximately three-fold, was chosen for quantification of shear-dependent RBC adhesion. We observed changes in RBC adhesion to LN and FN in the shear gradient flow. Figure 1C and 1D show typical adhesion curves of surface adherent RBCs for an individual SCD sample within the FOI. To assess patient specific shear-dependent adhesion, we defined a parameter, "shear dependent adhesion rate (SDAR)", which is the slope of the adhesion curves based on normalized RBC adhesion numbers. A higher SDAR value was indicative of marked numbers of adherent RBCs that detach at higher shear rates whereas the effect of shear rate on RBC detachment was less for a lower SDAR. We observed an inverse relationship between SDAR and number of persistently adherent RBCs at high shear rates. Shear-dependent RBC adhesion to LN was heterogeneous among SCD patients. Patients with higher WBC counts constituted the low SDAR population with a threshold SDAR value of 60 (Fig. 1E, p=0.005, ANOVA). WBCs from patients with higher SDARs (and fewer persistently adhered cells) were all within the normal range. Patients in the low SDAR group also had significantly elevated absolute neutrophil counts (Fig. 1F, p=0.006, ANOVA), and ferritin levels (Fig. 1G, p=0.007, ANOVA). The mean ferritin level of those with low SDAR was nearly ten times greater than normal (mean= [3272.3 ± 791.9] μg/L vs. [784.5±219.6] μg/L). No white blood cell (WBC) adhesion was observed in the experiments. Here, we report a novel shear dependent adhesion ratio of sickle RBCs utilizing LN and FN functionalized microchannels. The approach presented here enabled us to create a shear gradient throughout the channel which may simulate the physiological flow conditions in the post-capillary venules. We further analyzed shear-dependent RBC adhesion in a patient specific manner and identified patient groups with low and high SDAR. The findings also suggested a link between lower shear dependent sickle RBC adhesion to LN and patient clinical phenotypes including inflammation and iron overload. Acknowledgments: This work was supported by grant #2013126 from the Doris Duke Charitable Foundation, National Heart Lung and Blood Institute R01HL133574, and National Science Foundation CAREER Award 1552782. Figure 1: Shear-dependent sickle RBC adhesion in microscale flow. (A) Macroscopic image of the shear-gradient microchannel with the arrow indicating flow direction. (B) Velocity and shear rate contours on a 2D plane above the bottom surface. The dashed rectangular area indicates the field of interest (FOI) where the experimental data were obtained. (C, D) Typical distribution of adherent deformable and non-deformable RBCs in LN and FN functionalized microchannels with the shear gradient. Dashed lines represent the adhesion curves and the corresponding equations were used to quantify shear dependent adhesion data. Shear-dependent RBC adhesion was lower (nSDAR<60) in patients with elevated white blood cell counts (E), absolute neutrophil counts (F), and serum ferritin levels (G). The dashed rectangles indicate the normal clinical values. Figure 1 Figure 1. Disclosures Little: Hemex Health: Equity Ownership. Gurkan: Hemex Health: Employment, Equity Ownership.

scholarly journals Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 21-24 ◽  
Author(s):  
OM Smith ◽  
SA Dolan ◽  
JA Dvorak ◽  
TE Wellems ◽  
F Sieber
Keyword(s):  
Stem Cells ◽  
Plasmodium Falciparum ◽  
Hematopoietic Stem Cells ◽  
Red Blood Cells ◽  
Human Blood ◽  
Blood Cells ◽  
Potential Usefulness ◽  
Hematopoietic Stem ◽  
Infected Blood ◽  
Merocyanine 540

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

scholarly journals MicroRNAs as Haematopoiesis Regulators

2013 ◽  
Vol 2013 ◽  
pp. 1-20 ◽  
Author(s):  
Ram Babu Undi ◽  
Ravinder Kandi ◽  
Ravi Kumar Gutti
Keyword(s):  
Blood Cells ◽  
Erythroid Differentiation ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Complex Process ◽  
Development And Differentiation ◽  
Multiple Myelomas

The production of different types of blood cells including their formation, development, and differentiation is collectively known as haematopoiesis. Blood cells are divided into three lineages erythriod (erythrocytes), lymphoid (B and T cells), and myeloid (granulocytes, megakaryocytes, and macrophages). Haematopoiesis is a complex process regulated by several mechanisms including microRNAs (miRNAs). miRNAs are small RNAs which regulate the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells. Evidence shows that miRNAs play an important role in haematopoiesis; for example, myeloid and erythroid differentiation is blocked by the overexpression of miR-15a. miR-221, miR-222, and miR-24 inhibit the erythropoiesis, whereas miR-150 plays a role in B and T cell differentiation. miR-146 and miR-10a are downregulated in megakaryopoiesis. Aberrant expression of miRNAs was observed in hematological malignancies including chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelomas, and B cell lymphomas. In this review we have focused on discussing the role of miRNA in haematopoiesis.

scholarly journals Peripheral Red Blood Cell Split Chimerism as a Consequence of Intramedullary Selective Apoptosis of Recipient Red Blood Cells in a Case of Sickle Cell Disease

2014 ◽  
Vol 6 (1) ◽  
pp. e2014066 ◽  
Author(s):  
Marco Marziali ◽  
Antonella Isgrò ◽  
Pietro Sodani ◽  
Javid Gaziev ◽  
Daniela Fraboni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Blood Cells ◽  
Marrow Transplant ◽  
Mixed Chimerism ◽  
Radical Cure ◽  
Hematopoietic Stem ◽  
Hematopoietic Chimerism

Allogeneic cellular gene therapy through hematopoietic stem cell transplantation is the only radical cure for congenital hemoglobinopathies like thalassemia and sickle cell anemia. Persistent mixed hematopoietic chimerism (PMC) has been described in thalassemia and sickle cell anemia. Here, we describe the clinical course of a 6-year-old girl who had received bone marrow transplant for sickle cell anemia. After the transplant, the patient showed 36% donor hematopoietic stem cells in the bone marrow, whereas in the peripheral blood there was evidence of 80%  circulating donor red blood cells (RBC). The analysis of apoptosis at the Bone Marrow  level suggests that Fas might contribute to the cell death of host erythroid precursors. The increase in NK cells and the regulatory T cell population observed in this patient suggests that these cells might contribute to the condition of mixed chimerism.

scholarly journals A Silicon-based Coral-like Nanostructured Microfluidics to Isolate Rare Cells in Human Circulation: Validation by SK-BR-3 Cancer Cell Line and Its Utility in Circulating Fetal Nucleated Red Blood Cells

Micromachines ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 132 ◽  
Author(s):  
Gwo-Chin Ma ◽  
Wen-Hsiang Lin ◽  
Chung-Er Huang ◽  
Ting-Yu Chang ◽  
Jia-Yun Liu ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Pregnant Women ◽  
Blood Cells ◽  
De Novo ◽  
Capture Rate ◽  
Genetic Diagnosis ◽  
Maternal Blood ◽  
Capture Efficiency ◽  
Nucleated Red Blood Cells ◽  
Silicon Based

Circulating fetal cells (CFCs) in maternal blood are rare but have a strong potential to be the target for noninvasive prenatal diagnosis (NIPD). “Cell RevealTM system” is a silicon-based microfluidic platform capable to capture rare cell populations in human circulation. The platform is recently optimized to enhance the capture efficiency and system automation. In this study, spiking tests of SK-BR-3 breast cancer cells were used for the evaluation of capture efficiency. Then, peripheral bloods from 14 pregnant women whose fetuses have evidenced non-maternal genomic markers (e.g., de novo pathogenic copy number changes) were tested for the capture of circulating fetal nucleated red blood cells (fnRBCs). Captured cells were subjected to fluorescent in situ hybridization (FISH) on chip or recovered by an automated cell picker for molecular genetic analyses. The capture rate for the spiking tests is estimated as 88.1%. For the prenatal study, 2–71 fnRBCs were successfully captured from 2 mL of maternal blood in all pregnant women. The captured fnRBCs were verified to be from fetal origin. Our results demonstrated that the Cell RevealTM system has a high capture efficiency and can be used for fnRBC capture that is feasible for the genetic diagnosis of fetuses without invasive procedures.

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