In vitro β-thal erythroid precursors show reduced cell proliferation, persistence of PKM expression, and abnormal metabolomic and proteomic profiles compared with healthy cells. (A) May-Grunwald-Giemsa staining for erythroblast morphology (upper) of CD34⁺-derived erythroid precursors on day 14 of culture from HCs and patients with β-thal (cod β039) showing irregular nuclear shape and chromatin condensation. One representative image from 5 with similar results is shown. Original magnification, 100×. Quantification of abnormal erythroblasts is shown in supplemental Figure 1A. Cell growth (lower) of the erythroid precursors, as in upper panel. Data are mean ± standard error of the mean (SEM; n = 5). ∗P < .05 (compared with HC cells). (B) PKLR and PKM gene expression normalized on HBA1 expression (upper) in erythroid precursors on days 11 and 14 of culture from HCs and patients with β-thal. Data are mean ± SEM (n = 3-5). ∗P < .05 (compared with 11th day); °P < .05 (compared with HC cells). (C) Western blot analysis with specific antibodies against PKLR and PKM2 of erythroid precursors, as in panel A, on day 14 of culture. Catalase was used as loading control. One representative immunoblot of 3 others with similar results. Densitometric analysis is shown in supplemental Figure 1B. (D) Metabolomics profile of erythroid precursors, after 14 days in culture, from HCs and patients with β-thal (cod β039). Heat maps show the top 50 significant metabolites (see also supplemental Figure 8). (E) Heat map focusing on glutathione homeostasis, purine metabolites, glycolysis, Krebs cycle (tricarboxylic acid cycle [TCA]), and PPP. (F) A summary overview of the metabolic pathways in panel E; blue-white-red indicate lower to higher values of a given metabolite in β-thal erythroblasts compared with healthy cells.