Enrichment of hESC-CMs by mBax_499 mRNA and modelling to predict yield. (a) Representative confocal images of hESC-CMs that were either untreated or treated with mBax_499 mRNA for 24 h and stained for F-actin filaments by phalloidin (red), counterstained with NucBlue (nucleus, blue) and sarcomeric α-actinin (green). Scale bar: 100 µm. (b) Quantification of the percentage of actinin fiber alignment along the major strain axis in randomly cropped confocal images of treated hESC-CMs (n = 27) and untreated hESC-CMs (n = 29) using the Image J software and the OrientationJ Distribution plugin. (c) Co-staining of cells with SIRPa-APC and Annexin V-FITC before treatment (left) and after 24 h treatment with mBax_499 mRNA (right). (d) Experimental results vs. model for outcome prediction of % hESC-CMs after treatment. (e) FACS analysis of cells stained with cTNT after three consecutive treatments with mBax_499 mRNA. (f) Utilizing the model for repetition of treatment prediction (one through four treatments) and comparison to experimental results of one, two and three repeated treatments with mBax_499 mRNA. Data are represented as mean (SD). p value was generated using two-tailed unpaired student’s t-test. *p < 0.05.