Small molecules identified in the OCT4-GFP screening assay were not confirmed in secondary assays.

<p>Three treatments are compared to assess changes in survival of dissociated hESCs: 0.1% DMSO (negative control; <b>A,D</b>), 10 µM HA-1077 (positive control; <b>B,E</b>), and 10 µM AST 5588603 (representative of small molecules identified in OCT4-GFP screening assay; <b>C,F</b>). <b>A-C:</b> Readout from the Acumen microplate cytometer. The wells treated with HA-1077 and AST 5588603 had similar numbers of GFP positive objects (quantified in <b>G</b>). <b>D-F:</b> Alkaline phosphatase staining of cells treated as described. Pluripotent cells are stained with a red color. Wells treated with HA-1077 (<b>E</b>) contain significantly more pluripotent cells than cells treated with DMSO or any other candidate compounds. <b>G:</b> Quantification of GFP signal detected in (<b>A-</b>C). <b>H:</b> Quantification of alkaline phosphatase staining in (<b>D-F</b>) by calculating what percent of the well area stained positive for alkaline phosphatase. Data shown as mean ±SD, ***: p<0.001.