VPA induces a rapid acquisition of the HSC phenotypic markers. (A) The number of viable TNCs generated throughout 7 days of culture of UCB-CD34⁺ cells with either cytokines alone or a combination of VPA with cytokines, as measured by acridine orange/propidium iodide staining (n = 16). X-axis represents days of the culture, and PC denotes the uncultured UCB cells. (B) Cell division history of CD34⁺ cells incubated with cytokines alone (gray) or VPA (red), as determined by loss of CFSE fluorescence intensity. Green histogram represents the CFSE fluorescence intensity of the CD34⁺ cells before treatment with VPA. (C) Percentage of quiescent CD34⁺ cells (G₀), as determined by pyronin and Hoescht staining (n = 8). (D-E) Percentage and absolute numbers of CD34⁺CD90⁺ cells generated throughout 7 days of culture with cytokines alone or VPA, as determined by flow cytometry (n = 21). (F) Representative of flow cytometry plots of CD34⁺CD90⁺ cells present in cultures initiated with highly purified CD34⁺CD90⁻ cells after 24 hours of treatment with either cytokines alone (gray) or VPA (red) (n = 4). (G) Percentage of CD34⁺CD90⁺ cells generated from CD34⁺CD90⁻ cells and determined by flow cytometry (n = 4). “n” is the number of biological replicates. Error bars with standard error of the mean. ****P ≤ .0001; ***P ≤ .001 were determined by negative-binomial models for panels A and E, β models for panel D, and 2-way ANOVA for panels C and G. ns, not significant.