Detection of residual undifferentiated cells contained in differentiating cell cultures.

(A) Differentiation scheme of 253G1 cells into MSCs is shown. (B) Expression levels of undifferentiated cell markers (OCT3/4, NANOG and LIN28) in each cell culture were determined using qRT-PCR. Relative mRNA expression levels are presented as ratios to the level of that in 253G1 cells at Day 1. Results are the mean ± SD (n = 3). (C) Numbers of the forming colonies derived from residual undifferentiated cells in differentiating cell culture at Day 6 or Day 14 are shown. Experiments were carried out in duplicate. Data are present as raw data in each well (shown by plots) or the mean of well 1 and well 2 (shown by bar graphs). (D) Phase contrast images of forming colonies derived from residual undifferentiated cells are shown. Cells at Day 6 of differentiation (EBs) were dissociated into single cells by Accutase and cultured on laminin-521-coated wells in Essential 8 medium (5×104/well). After 4 days of culture, small clusters emerged and then started to grow rapidly. Finally, they formed colonies that were positive for TRA-1-60 (shown by immunofluorescence staining, green). Arrowheads indicate a colony derived from same origin. Scale bars, 500 µm.