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  • The reporter lines allowed us to

    2018-10-20

    The reporter lines allowed us to observe in real time that colonies with greater than 300 ciprofloxacin displayed slightly higher expression levels of the reporter at the colonies’ outermost few cell layers (Fig. 1A, F, fluorescence profiles across colonies shown in Supplementary Fig. 2M). This is in agreement with previous observations of higher levels ciprofloxacin of TGFβ/activin signalling and higher expression of pluripotency markers on the edges of the human pluripotent stem cell colonies (Hough et al., 2009). We demonstrate that removal of the puromycin selection causes the emergence of a population of differentiated, OCT4-negative cells (Fig. 2H). It appears that these spontaneouslydifferentiated cells have a cell non-autonomous effect on the directed neuroepithelial conversion of the whole population, resulting in a 33% drop in the numbers of neuroectodermallyconverted cells as measured by the expression of PAX6 protein (Fig. 2H and Supplementary Fig. 2D). The difference in correlation between EGFP and TRA-160 as compared to that between EGFP and SSEA4 or TG30 is consistent with the observations that extinction dynamics of TRA-1-60 with differentiation are substantially faster than that of TG30 or SSEA4 (Fig. 3D and Supplementary Fig. 2). The dynamic response range and sensitivity of the reporter is in fact superior to TRA-1-60, as can be judged from the more rapid and pronounced downregulation of EGFP than TRA-1-60 following RA-induced differentiation (Fig. 3D and Supplementary Fig. 2C). Similarly, when titrating concentration combinations of FGF2 and TGFβ1, the expression of reporter EGFP corresponded much more dynamically to variations in these pluripotency-maintaining factors than TRA-1-60 (Fig. 3E). The following are the supplementary data related to this article.
    Acknowledgments We would like to thank StemCore (http://www.stemcore.com.au) at the University of Queensland for provision of hES cell lines and logistical support with teratoma and karyotype analyses. We also would like to acknowledge the funding provided by the Australian Stem Cell Centre and the ARC Centre of Excellence “Stem Cells Australia”.