Multicolor Staining of Globin Subtypes Reveals Impaired Globin Switching During Erythropoiesis in Human Pluripotent Stem Cells

• Published in: Stem cells translational medicine Vol. 3; no. 7; pp. 792 - 800
• Main Authors: Ochi, Kiyosumi, Takayama, Naoya, Hirose, Shoichi, Nakahata, Tatsutoshi, Nakauchi, Hiromitsu, Eto, Koji
• Format: Journal Article
• Language: English
• Published: Durham, NC, USA AlphaMed Press 01.07.2014; Oxford University Press
• Subjects: Antigens, CD34 - metabolism; BCL11A; beta-Globins - genetics; beta-Globins - metabolism; Biomarkers - metabolism; Blood; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Differentiation; Cloning; Coculture Techniques; Embryonic stem cells; Embryonic Stem Cells/Induced Pluripotent Stem (iPS) Cells; Embryos; Erythroblasts; Erythrocyte; Erythroid cells; Erythropoiesis; Feeder Cells; Fetuses; Flow cytometry; Flow Cytometry - methods; Fluorescent Antibody Technique; gamma-Globins - genetics; gamma-Globins - metabolism; Gene Expression Regulation, Developmental; Gene Silencing; Globin switching; Hematopoietic Stem Cells - metabolism; Hemoglobin; Hemoglobin Subunits - genetics; Hemoglobin Subunits - metabolism; Humans; iPS cells; Molecular Probes; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Pluripotency; Pluripotent Stem Cells - metabolism; R&D; Research & development; Science; Stem cells; Time Factors; Transfection; Japan; Erythrocyte; Embryonic stem cells; BCL11A; Globin switching; iPS cells
• ISSN: 2157-6564; 2157-6580
• DOI: 10.5966/sctm.2013-0216

The authors examined impaired globin switching of embryonic ε‐ and fetal γ‐globin to adult β‐globin in human erythroid cells by optimizing multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. Results showed that impaired γ‐globin silencing is associated with downregulated BCL11A‐L in human pluripotent stem cell‐derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro. 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.