mTOR inhibition facilitates the conversion of CD133- to CD133+ cells.

<p><b>A</b>–<b>C</b> De novo generation of CD133+ cells after long-term single-cell culture of CD133- LPC-H12 cells. Single CD133- LPC-H12 cells were deposited by FACS into each well of 96-well plates with or without rapamycin (10 nM). After expansion, the single-cell derived LPC-H12 cells were analyzed by FACS. Schematic diagram shows FACS isolation and robotic plating of single CD133- cells into 96-well plates (A). Phase image of a single cell in one well after sorting (bottom). Bar = 10 µm. The percentages of the colonies containing CD133+ cells are shown in B (n = 10 per group). The proportions of CD133+ cells in each colony are shown in C. <b>D-G</b> Knockdown of mTOR expression enhanced the rate of conversion of CD133- to CD133+ cells. The percentages of CD133+ colonies derived from single CD133- LPC-H12 cells infected with retrovirus containing mTOR-sh2 and control, respectively, are shown in D (n = 12 per group). The proportions of CD133+ cells in each colony are shown in E. The expression of CD133 mRNA in each clone examined by Real-time PCR experiments in triplicate, and all samples were normalized to GAPDH expression (F). The result of RT-PCR is shown in G.</p>