Hyperactive variants improve CGS-mediated expansion from cord blood CD34⁺ cells. (A) Expansion of total cells. Cord blood-derived CD34⁺ cells were transduced with lentivectors expressing the wt, Y*, K*K*, and K*K*Y* variants of the CGS and expanded in serum-containing medium supplemented with 100 nM CID AP20187 and no additional cytokines. Cell numbers were determined by hemocytometer count with media changes on the indicated days, and are reported as a fold-expansion based on the starting cell number. The maximum fold-expansion is shown for the wt and Y* samples. P values are based on the t test across all time points. P = .16 for K*K* vs K*K*Y*. (B) Expansion of transduced cells. The percent of cells expressing vector GFP was also determined on the indicated days by flow cytometry as a means of determining the degree of expansion for the vector-transduced cells. Data represent the mean ± standard error from 4 independent experiments. P values are based on the t test across all time points. (C) Morphology of expanded cells. Representative Wright-Giemsa–stained cytospins are shown for the original cord blood mononuclear cells (CB MNC) used to prepare the CD34⁺ cells, and from day 14 and day 21 cultures expanded with the wt and Y* CGS vectors.