Spatial organization of cell type markers is consistent with early, maintained cell type determination in cardiomyocyte differentiation.

We performed single-molecule RNA fluorescence in situ hybridization (RNA FISH) targeting multiple cell type markers on cardiac differentiated human induced pluripotent stem (hiPS) cells in their differentiation wells—specifically, we probed for the expression of TNNT2 for cardiomyocytes, LUM for fibroblasts, EPCAM for epithelial cells, ISL1 which is expressed in cardiac progenitors, and WT1 for epicardium. We stained nuclei with DAPI and imaged multiple positions throughout each well, capturing expression of each marker gene in at least 17 images. Markers were often imaged simultaneously though not all combinations were tested. To determine marker patch size, we counted the number of nuclei associated with cell patches displaying marker gene expression within each image.