CHIR99021 hampers expansion and differentiation of cultured human EBs. (A) Examination of β-catenin protein levels in human EBs and HEK293T cells by western blot in response to 24-hour stimulation with either 10 or 100 ng/mL Wnt3a or 1 or 10 μM CHIR99021. (B) Relative β-catenin protein levels as defined by western blot quantified for EBs and (C) for HEK 293T cells. (D) Representative flow cytometry plots of erythroid differentiation, using CD71 and CD235a cell surface staining at t = 0 and 5 days after stimulation with CHIR99021 (10 μM) compared with control (DMSO)-treated cells. (E) Quantification of EB cell viability, as determined by cell counts, 5 days after stimulating cells with DMSO, or 1 μM or 10 μM CHIR99021 (n = 3; Student t test for DMSO vs 10 μM ChiR99021, ∗P = 0.049). (F) Calculated fold change in CD71⁻CD235a⁻, CD71⁺CD235a⁻, CD71⁺CD235a⁺, and CD71⁻CD235a⁺ defined cell stages of EB development, using CD71 and CD235a cell surface staining, 5 days after stimulating cells with 1 or 10 μM CHIR99021 as compared with control, treated cells with DMSO (n = 3; two-way ANOVA; ctrl vs 1 μM CHIR99021, P < .0001; and ctrl vs 10 μM CHIR99021, P < .0001).