HMGB1 alters the growth and differentiation of erythroid precursors. (A) CB-derived CD34+ HSPCs were differentiated to enucleated reticulocytes using a 3-phase liquid culture system (ii) in the presence or absence of 10 μg/mL HMGB1. Erythroid cell growth measured at indicated days of differentiation (i). (B) Cell pellets demonstrating the extent of hemoglobinization (i) and cytospins showing cell morphology and level of differentiation (ii) at D14 of culture. (C) A 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay on UT7-Epo cells after 72 hours in the presence of absence of HMGB1. (D) Representative flow cytogram of erythroid precursor maturation using GPA, α4-integrin, and band 3 surface expression at indicated days of culture. (E) Quantification of GPApos cells. (F) Quantification of erythroid precursor populations as a percentage of GPApos cells. Data represented as mean ± SEM. Unpaired, 2-tailed Student t test: *P < .05; **P < .01; ***P < .001; ns, not significant.