(A) Experimental timeline used in the screen. (B) Representative flow cytometric analyses of cell population used in HDMA screening, with percentages marked. Dot plots of CD90+ stromal cell content from 15 d digestion of differentiated hPSCs; dot plots of Ki67 and cTnT expression in both 15 d digestion of differentiated hPSCs and cells replated for 2 d prior to assay startpoint. (C) Representative phase contrast micrographs of CMs before and after seeding, and cultured for up to 3 d with drug treatments, as indicated. Scale bars for indicated objective magnification: 4×, 200 μm; 10×, 200 μm; 20×, 100 μm. (D) Matrix of design culture conditions in each of the 81 column pairs in the array, for the screened factors: CHIR99021 (CHIR), μM; Purmorphamine (Pm), μM; IGF-1, ng/mL; FGF-2, ng/mL. DMSO was included so as to be at a uniform total concentration of 0.05% v/v in the CHIR channels, acting as a vehicle control. (E) Tile-scan confocal image of the entire HDMA (~91 × 28 mm) treated for 24 h and used in further analysis, after fixation and in situ immunostaining for Ki67, cTnT and DNA. (F) Higher-magnification confocal image of subsection of replicate HDMA treated for 3 d, showing cell population arrangement in arrayed culture chambers. (G) Confocal image showing detail of individual HDMA chambers from replicate HDMA treated for 3 d, demonstrating presence of various proliferating (Ki67+) and non-proliferating (Ki67−) myocyte (cTnT+) and nonmyocyte (cTnT−) populations. Scale bar: 200 μm.