THPO/MPL/Bcl-xL signaling is critical for primitive AE cells. (A) shRNA-expressing lentivirus transduced AE cells were sorted at day 3, followed by plating in methylcellulose media. Colony numbers were scored after 14 days. Data shown indicates 1 representative experiment with initial seeding of 3 × 104 cells. After colony counting, cells were collected and the percentage of transduced cells (Venus+ cells) was determined by flow cytometry. The experiment was repeated 3 additional times with consistent results. (B) CD34-selected cells were plated in methylcellulose media in the absence of THPO. Colony numbers were scored after 14 days under a light microscope. Cells were then collected and replated with indicated input cell numbers. One representative experiment is shown. Results were confirmed in 3 additional AE clones. Data represents mean ± SD (n = 3; *P < .05). (C) CD34-selected cells were cocultured with MS5 in 96-well plates at the densities of 3000, 10 000, and 30 000 cells per well in 10 replicates. After 5 weeks, methylcellulose media containing THPO was added to each well, which was then scored for the absence or presence of colonies after 14 days. L-Calc software was used to calculate LTC-IC frequency.
