Cell-to-cell heterogeneity measurement using Shannon entropy.

<p>(A) A Shannon entropy was calculated for each time-point for each gene. Boxplots represent the distribution of the entropy values; (B) Gene entropy variation: for each gene (i.e., lines), we represented the difference between entropy values at two consecutive time-points (Δ-entropy) using a color gradient code. Negative and null delta entropies (i.e., for a given time-point, the entropy value for these genes decreased or does not change, compared to the earlier time-point) are colored in blue and green. Positive delta entropies are colored in orange or red; (C) We assessed the significance of the differences between any pair of time-point through a Wilcoxon test. The robustness of the result was assessed by performing subsampling. The barplot shows the results as the percentage of 1,000 iterations for which a significant difference (<i>p</i>-value < 0.05) was detected. Data for this figure can be found at osf.io/k2q5b.</p

Analysis of single-cell data averaged over pseudo-populations.

<p>We separated single-cells into three pseudo-populations with around one-third of single cells for each time-point. We then calculated the average gene expression over each pseudo-population, and analyzed the resulting averaged data using multivariate statistical methods. (A) Principal component analysis (PCA); (B) Hierarchical cluster analysis (HCA).</p

Evolution of physiological differentiation parameters.

<p>(A) T2EC were induced to differentiate for 24 and 48 h and subsequently seeded back in self-renewal conditions. Cells were then counted every day for 5 d. The green curve represents the growth of cells induced to differentiate for 24 h and the orange curve indicates the growth of cells induced to differentiate for 48 h. The data shown are the mean +/− standard deviation calculated on the basis of three independent experiments for the time-points 72 h and 96 h and four experiments for all other time-points. The growth ratio was computed as the cell number divided by the total cells at day 0. The significance of the difference between growth ratios at 24 h and 48 h was calculated using a Wilcoxon test. (B) The boxplots of the mean size observed were based on four independent experiments, each using 50,000 cells, using FSC_A as a proxy for cell size. All of the variances were compared by pairs using the F test and the * indicates when the variances were significantly different. Data for this figure can be found at osf.io/k2q5b.</p

Analysis of bulk-cell gene expression during the differentiation process.

<p>Gene expression data were produced by RT-qPCR in triplicate from three independent T2EC populations collected at five differentiation time-points (0 h, 8 h, 24 h, 48 h, 72 h). The expression level of 110 genes (18 invariants, 50 down-regulated and 42 up-regulated) was analyzed by two different multivariate statistical methods: (A) Principal component analysis (PCA), and (B) Dendogram resulting from hierarchical cluster analysis (HCA). The dots in (A) and leaves in (B) indicate the different cell populations and the colors indicate the differentiation time-points at which they were collected.</p

Initial expression waves analysis.

<p>Genes are sorted according to the time of the first significant expression variation. The first wave corresponds to genes with a significant variation detected during 0 h and 2 h. The second wave corresponds to genes with a significant variation detected during 2 h and 4 h but without significant variation detected earlier. Genes labeled in red belong to the group of genes associated with sterol synthesis. Significant variations (-*-) are detected by non-parametric Mann-Whitney test (<i>p</i>-value < 0.05) if the test is positive in more than 90% of 1,000 bootstrap samples. Genes prefixed by * have a significant variation between 0 h and 8 h detected in both experiments (0 to 72 h, as well as 0 to 8 h). The probability of having 6 genes over 7 (in the first and second waves) belonging to the 10 sterol cluster genes among all 90 genes is estimated to <i>p</i> = 1.8 × 10⁻⁶ with the hypergeometric probability density function. Data for this figure can be found at osf.io/k2q5b.</p

Exploration of potential cofounding factors.

<p>(A) Correlation of the CTCF gene with the rest of the 91 genes, at all six time-points. (B) FACS analysis of the cell cycle repartition at 0 and 8 h of differentiation. The difference between the two distributions was found not to be statistically significant (<i>p</i> = 0.18 using a Wilcoxon test). (C and D): calculation of the entropy content per cluster of cells re-organized using either WANDERLUST (C) or TSCAN algorithm (D). (E and F) In silico comparison of the effect of a synchronous versus an asynchronous differentiation process on the evolution of entropy. Data for this figure (C to F) can be found at osf.io/k2q5b.</p

Gene expression-based discrimination between self-renewing and differentiating individual cells.

<p>Single-cell gene expression data were analyzed considering only self-renewing cells and cells induced to differentiate since 72 h. (A) Principal component analysis (PCA); (B) Hierarchical cluster analysis (HCA) was used to sort single-cells picked up at 0 h and 72 h of the differentiation process according to similarity measurement; (C) Two-dimensional representation of the contribution of each variable (gene) to the inertia. The direction of the arrows displays the contribution of that variable to the underlying component. The colored genes highlight genes of interest and genes that contributed the most to the PCA outcome, associated with self-renewal (blue) and the erythroid differentiation process (red).</p