Quantifying Synchrony and Tempo of Mouse Embryonic Stem Cells Differentiation

The aim of this thesis is the study of the differentiation process of mouse embryonic stem cells (mESCs), in particular to develop an early protocol for quantifying the synchrony and tempo of early embryonic development. mESCs were used as a model for this process in mammals: the cells were differentiated by removing pluripotency-promoting factors (2iL) from the culture medium (N2B27) and the expression of the naive, formative and neuroectoderm markers (Nanog, Oct6 and Sox1 respectively) was monitored over time at the single-cell level using immunofluorescence and fluorescence microscopy. Subsequently, image analysis was conducted to quantify markers expression at the single-cell level. This information was then used to define quantitative parameters that are able to describe differentiation synchrony, based on the distribution of cells across differentiation stages using information entropy, and tempo, based on the speed at which cells moved on to their next differentiation stage. These parameters provide a way to screen for mutants or treatments that modify these characteristics and identify the molecular mechanisms governing this process, critical to normal embryogenesis.