Pluripotency and self-renewal are distinctive properties of embryonic stem cells (ESCs). microRNAs (miRs) play important roles in the post-transcriptional control of gene expression, by targeting multiple mRNA, and have been implicated in the maintenance of pluripotent cells characteristics. Many miRs effects are attributed to cell cycle regulation, in line with the intimate link between cell-cycle regulation, self-renewal, pluripotency and differentiation. We will systematically investigate the roles of all currently annotated human miRs (2,588 mature miRs) in these characteristics, using automated High-Content Screening fluorescence microscopy. Images derived from pluripotent cells transfected with microRNA mimics will be analyzed, in order to quantify biological features reflecting, cell cycle status (based on Cyclin B1 staining and Hoechst/DNA content analysis), pluripotency (based on Oct4 staining) and differentiation (cell morphology). Single-cell data will be combined into multiparametric population profiles describing miR induced phenotypes. Profiles will be clustered to identify microRNA groups with common effects. Enrichment functional analysis of predicted miR targets will be carried out to identify biological pathways and processes mediating the phenotypes observed. The primary objective of the present FAPESP-BPE application is to allow the applicant to develop specific image and data analysis pipelines, under the guidance of Dr. Marc Bickle (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden-Germany). For this, images from a pilot focused screen (30 miRs) will be used in the development and optimization of the analysis pipelines. Once established, the pipelines will be adapted to the full library screen, which will be carried by the PhD student Ildercílio Lima (supervised by Dr. Panepucci, FAPESP 2013/27061-0), in collaboration with Dr. Miguel Mano (ICGEB, Trieste-Italy). In view of the complexity of the analytical approaches, this fellowship will bring important immediate and precisely defined benefits for the success of this project (a subproject of the Center for Cell Therapy - CTC, a CEPID FAPESP).
News published in Agência FAPESP Newsletter about the scholarship: