In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a “simple” program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned).