Characterization of SALL4-transduced HSC cells. (A) Growth curves of CD34⁺ cells transduced with SALL4A, SALL4B, or GFP and cultured in media containing 75% less cytokines. After transduction, 50 000 cells of each group were cultured in stringent conditions in which normal cytokine concentrations were decreased by 75%. HSCs transduced with SALL4A or SALL4B continued to survive and expand over 7 days, whereas control cells growth halted at day 5. (B) Fold expansion of CD34⁺/CD38⁻ cells 14 days after infection of lenti-SALL4A or -SALL4B versus control. Cells transduced with SALL4A demonstrated a 368-fold increase of CD34⁺/CD38⁻ cells over control, whereas those transduced with SALL4B showed a 384-fold increase. Values are means + SD. (C) Phenotypic analysis of SALL4-induced hematopoietic stem cells 31 days after lentiviral infection. Human-specific antibodies CD34-PE and CD38-APC were used to compare SALL4-transduced HSCs versus 3-day control cells. Thirty-one days after lentiviral infection, the aged SALL4-induced cells continued to demonstrate similar phenotypic ratios compared with control cells for CD34⁺/C38⁻. FLOW analysis was carried out on 3 separate samples. Therefore, many of these aged cells still attained progenitor characteristics and had the ability to differentiate into various cells lines. (D) Thirty-one-day old SALL4-induced HSCs attain blastlike morphology. Aged 31-day-old SALL4-induced HSCs were Wright-Giemsa–stained. Many cells showed blastlike morphology including large nuclei and scant cytoplasm (100×). These cells represented a population of undifferentiated cells still visible 31 days after SALL4-lentiviral infection and expansion.