Fig. 1.

General outline of the study. First: A high-content screening (HCS) assay based on H1 hESCs and NTera-2 cells was developed, to explore pluripotency and differentiation. Several morphometrical features, as well as intensity-related measurements (from OCT4 and Cyclin B1 staining), were obtained from nuclei and cytoplasm compartments. Second: The HCS assay was used in a miR functional screen in 96-well plates to investigate the effects of 31 miRs. Following transfection and culture, images were acquired with an ImageXpress micro XLS HCS system (Molecular Devices). Third: Following image and data analysis (with CellProfiler and KNIME softwares, respectively), the multiparametric phenotypic profile describing the functional effect of each miR was submitted to hierarchical clustering (using Cluster 3.0 and Java Treeview), allowing the identification of miRs with similar functional effects (pro-pluripotency or pro-differentiation). Fourth: TargetScan was used to identify predicted targets shared by miRs in the same cluster (i.e., inducing similar phenotypes). Signaling pathways and biological processes enriched for these shared targets were identified using the DAVID tool