Establishment and Maintenance of Human CSCs

The establishment of the human CSCs used in this study (GS-Y03, PANC-1 CSLC, and A2780 CSC) has been described [17 (link), 34 (link), 35 ]. These cell lines were maintained under the monolayer stem cell culture condition reported previously [17 (link), 34 (link), 35 ]. In brief, cells were cultured on collagen-I-coated dishes (IWAKI, Tokyo, Japan) in the stem cell culture medium (DMEM/F12 medium supplemented with 1% B27 [Gibco-BRL, Carlsbad, CA, USA], 20 ng/mL EGF and FGF2 [Peprotech, Inc., Rocky Hill, NJ, USA], D-(+)-glucose [final concentration, 26.2 mM or 5 mM where indicated as such], L-glutamine [final concentration, 4.5 mM], 100 units/mL penicillin and 100 μg/mL streptomycin). In principle, the stem cell culture medium was changed every 3 days, and EGF and FGF2 were added to the culture medium every day. Throughout the study, the cell number was determined using a hemocytometer, and the cell viability was examined by the dye exclusion method (0.2% trypan blue). Cell viability (%) was defined as 100 x ‘the number of viable cells’/(‘the number of viable cells’ + ‘the number of dead cells’).