Fig. 1

Characterization of erythroblasts differentiated from UCB-derived CD34⁺ HSPCs. A Inhibition of OGT by OSMI-1 promoted the differentiation of CD34⁺ HSPCs into the more committed erythroid progenitors BFU-E as evaluated by CFU assay. Colonies were visualized and scored under an inverted microscope at 14 days of culture. Data are mean (n = 3). ^*P < 0.05 versus % BFU-E colony of nontreated control (CTL); two-sided Student’s t test. TG: thiamet G. B Schematic diagram of erythroid differentiation from CD34⁺ HSPCs in the three-stage erythroid culture system. C Representative morphology of differentiated cells on Wright-stained cytospin slides on various days of culture (days 4–15) showing different stages of erythroid differentiation, including basophilic normoblasts (Baso), polychromatic normoblasts (Poly), orthochromatic erythroblasts (Ortho), and reticulocytes (Retic). Scale bar = 20 μm. D (left) Western blot analysis of O-GlcNAc level along the time course of erythroid differentiation using anti-O-GlcNAc-specific antibody (RL2). Blots were reprobed with anti-β-actin antibody to establish a loading control (see also Additional file 2: Fig. S2 for the OGA and OGT levels). (right) Quantitative analysis of O-GlcNAc level by densitometry after normalization to the loading control is shown. Data are mean ± SD (n = 4). ^*P < 0.05 versus cells at the start of culture (day 0); two-sided Student's t test. E Different subsets of differentiated erythroid cells, stages I–V, based on their maturity using CD71 versus FSC together with CD235a (see also Additional file 2: Fig. S4 for the gating strategy). Stage V cells were confirmed to express CD235a. F Representative morphology of sorted stage I–V cells on Wright-stained cytospin slides. Scale bar = 20 μm