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      Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

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          Abstract

          Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10–15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

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          Red cell membrane: past, present, and future.

          As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types.
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            Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.

            Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.
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              Identification and characterization of a fibroblast marker: FSP1

              We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast- like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                14 July 2014
                : 9
                : 7
                : e100874
                Affiliations
                [1 ]School of Biochemistry, University of Bristol, Bristol, United Kingdom
                [2 ]Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
                [3 ]Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Filton, Bristol, United Kingdom
                [4 ]Blood Research Laboratory, National Health Service Blood and Transplant, John Radcliffe Hospital, Oxford, United Kingdom
                Indian Institute of Toxicology Research, India
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JF DJA KT MCW REG. Performed the experiments: KT MCW KJH REG. Analyzed the data: KT MCW REG AMT SFP KJH DJA JF. Contributed reagents/materials/analysis tools: LC. Wrote the paper: JF. Conceived the study: JF DJA. Designed the experiments: JF DJA KT MCW SFP REG. Provided practical advice: SFP LC. Prepared the figures: KT. Reviewed the paper: KT MCW AMT LC SFP DJA.

                Article
                PONE-D-14-10194
                10.1371/journal.pone.0100874
                4096399
                25019302
                41584424-2701-4427-b7c2-09459a08f21b
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 5 March 2014
                : 31 May 2014
                Page count
                Pages: 11
                Funding
                This work was supported by the Department of Health (England), NHSBT R&D and funding from Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteins
                Hemoglobin
                Proteomes
                Proteomics
                Protein Abundance
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                Red Blood Cells
                Erythroblasts
                Stem Cells
                Adult Stem Cells
                Hematopoietic Progenitor Cells
                Hematopoietic Stem Cells
                Induced Pluripotent Stem Cells
                Stem Cell Lines
                Medicine and Health Sciences
                Diagnostic Medicine
                Clinical Laboratory Sciences
                Transfusion Medicine
                Blood Transfusion
                Hematology
                Hematopoiesis
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All data is provided within the manuscript or in the supplementary files.

                Uncategorized
                Uncategorized

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