title: Multicolor staining of globin subtypes reveals impaired globin switching during erythropoiesis in human pluripotent stem cells.
abstract: Adult hemoglobin composed of α- and β-globin reflects a change from expression of embryonic ε- and fetal γ-globin to adult β-globin in human erythroid cells, so-called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ-globin with little β-globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of β-globin and the corresponding silencing of γ-globin at the single-cell level during cord blood CD34(+) cell-derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous β-globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ-globin. These hPSC-derived erythroid cells also displayed reduced expression of BCL11A-L. However, doxycycline-induced overexpression of BCL11A-L in selected hPSCs promoted γ-globin silencing. These results strongly suggest that impaired γ-globin silencing is associated with downregulated BCL11A-L in hPSC-derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan.
authors.forename: Kiyosumi
authors.initials: K
authors.lastname: Ochi
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Takayama
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Hirose
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan.
authors.forename: Tatsutoshi
authors.initials: T
authors.lastname: Nakahata
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Laboratory of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Terumo Company Ltd., Tokyo, Japan kojieto@cira.kyoto-u.ac.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Eto
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D018920:Coculture Techniques; D004920:Erythropoiesis; D061252:Feeder Cells; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D055554:Hemoglobin Subunits / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015335:Molecular Probes; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D012097:Repressor Proteins; D013997:Time Factors; D014162:Transfection; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism*; D055545:gamma-Globins / Q000235:genetics / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; C486563:BCL11A protein, human; D015415:Biomarkers; D002352:Carrier Proteins; D055554:Hemoglobin Subunits; D015335:Molecular Probes; D009687:Nuclear Proteins; D012097:Repressor Proteins; D055544:beta-Globins; D055545:gamma-Globins
keywords: BCL11A; Embryonic stem cells; Erythrocyte; Globin switching; iPS cells