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.
journal_name
Stem Cells Transl Medjournal_title
Stem cells translational medicineauthors
Ochi K,Takayama N,Hirose S,Nakahata T,Nakauchi H,Eto Kdoi
10.5966/sctm.2013-0216subject
Has Abstractpub_date
2014-07-01 00:00:00pages
792-800issue
7eissn
2157-6564issn
2157-6580pii
sctm.2013-0216journal_volume
3pub_type
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