SHP2 knockdown inhibits CD34⁺ cell differentiation. (A) CD34⁺DsRED⁺ cells were cultured with high concentrations of GFs (IL-3 + SCF + G-CSF + GM-CSF + Epo), and cell differentiation toward both myeloid (CD11b⁺ and CD14⁺) and erythroid lineages (GPA⁺) was analyzed. SHP2 shRNA-transduced CB CD34⁺ cells generated significantly lower amounts of both myeloid and erythroid lineage cells during the culture compared with sh-Ctrla. Representative flow cytometric analyses from day 4 culture. (B) Total myeloid and erythroid cells generated from CD34⁺DsRED⁺ cells (2 × 10⁵) after 7 days of culture. Data are mean ± SEM for 3 experiments. **P < .01 compared with sh-Ctrla. (C) Inhibition of CD34⁺ cell differentiation to myeloid and erythroid lineage after SHP2 knockdown. Inhibition is calculated relative to control shRNA-expressing cells after 4 days of culture with high concentrations of GFs. Data are mean ± SEM for 3 experiments. (D) Effect of SHP2 knockdown on retention of CD34⁺ cells during GF culture. The number of CD34⁺ cells is shown. Data are mean ± SEM of 3 experiments. (E) CD34⁺DsRED⁺ cells were cultured with high concentrations of GFs (IL-3 + SCF + G-CSF + GM-CSF + Epo) for 7 days, and CD34⁺DsRED⁺ cells were purified from cultured cells by flow cytometry. The selected CD34⁺DsRED⁺ cells were cultured with high concentrations of GFs for an additional 7 days, and cell expansion was evaluated based on cell number. Data are mean ± SEM of 3 experiments.