Hyperactive CGS variant improves expansion of both mature and primitive cells. Expansion cultures were established with cord blood CD34⁺ cells transduced with lentivectors expressing the wt or Y* CGS as for Figure 2. Cell aliquots were analyzed by immunofluorescent staining and flow cytometry for (A) erythroid cells (CD235a⁺/CD41a⁻), (B) megakaryocytic cells (CD41a⁺/CD42b⁺), (C) primitive stem/progenitor cells (CD34⁺), and (E) bipotent erythroid/megakaryocytic precursor cells (CD235a⁺/CD41a⁺), later determined to be PEMs. Data represent the mean ± standard error (SE) from 4 independent experiments for the fold-expansion based on the starting number of total cells. P values are based on the t test across all time points. The maximum fold-expansion is shown for the wt and Y* samples based on each parameter. (D) Histograms showing the fold expansion of progenitor colony-forming cells present in the cord blood CD34⁺ expansion cultures transduced with the Y* lentivector and expanded for the indicated days. Colonies were detected by plating cell aliquots in methylcellulose supplemented with a broad cytokine cocktail, and consisted predominantly of primitive and mature erythroid colonies, granulocyte-macrophage colonies, and low levels of megakaryocyte colonies and colonies with mixed phenotypes. Data represents the mean ± SE from 3 independent experiments. (F) Histograms showing the relative frequency of the indicated cell populations present in the cord blood CD34⁺ cultures transduced with the wt or Y* lentivectors and expanded for 7, 14, and 21 days.