Figure 5.

Long-term depletion of differentiation factor(s) secreted from differentiated cancer cells lead to the loss of vascular differentiation properties of miPS-LLCcm cells. (a) miPS-LLCcm cells were cultured in the medium containing puromycin for 1 week to eliminate differentiated cells. Following a week, cells were cultured in puromycin-free medium to allow differentiated cells to grow. This cycle was repeated three times. The ability to differentiate into vascular endothelial cells at the end of each cycle was evaluated by in vitro tube formation assay (scale bar 200 µm). (b) The expression levels of VEGFR2 in indicated cells were quantified by real-time RT-PCR. The ability of differentiation into endothelial cells was deprived. (c) After third round selection and differentiation, the resultant culture consisted of GFP-positive cells with stem-like morphology and GFP-negative differentiated cells, indicating the stem cells still possessed the differentiation capacity (scale bar 200 µm). (d) Sphere-forming ability of conditioned medium from miPS-LLCcm cells After third round selection (CM-3^rdad) were analyzed by sphere formation assay with/without 20 µM DAPT (*p < 0.05; **p < 0.01, Student's t-test). Data are the results of three independent experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]