SHP2 knockdown in CD34⁺ cells results in significantly reduced progenitor proliferation in response to GF stimulation. (A) CD34⁺DsRED⁺ cells were cultured in 96-well plates with graded concentrations of a GF combination (SCF, GM-CSF, G-CSF, IL-3, and erythropoietin) including for 72 hours, and the number of cells was analyzed using an MTS assay. Representative results showing inhibition of SHP2 expression significantly reduced cell proliferative response to GF stimulation. (B) CD34⁺DsRED⁺ cells were cultured for 7 days in high concentrations of GFs as used in CFC culture and expansion in cell numbers evaluated. Data are mean ± SEM of 3 experiments. **P < .01, SHP2 shRNA-transduced cells compared with sh-Ctrla. (C) CD34⁺DsRED⁺ cells were labeled with CFSE and cultured in low, physiologic concentrations of GF for 3 days. Cell division was evaluated on the basis of reduction of CFSE fluorescence intensity. A proliferation index was calculated based on the using ModFit LT 3.0 software. Data are mean ± SEM of 3 experiments. *P < .05. Representative ModFit analysis results from 1 experiment are shown. (D) CD34⁺DsRED⁺ cells (1 × 10⁴) were placed in methylcellulose CFC culture, and colonies were counted after 14 days culture. Data are mean ± SEM of 3 experiments. Knockdown of SHP2 significantly reduced colony formation ability of CB CD34⁺ cells. *P < .05 for sh-Shp2-1, and **P < .01 for sh-Shp2-2 compared with sh-Ctrla.