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. The differentiation process has classically been seen as a stereotyped program leading from one progenitor toward a functional cell. This vision was based upon cell population-based analyses averaged over millions of cells. However, new methods have recently emerged that allow interrogation of the molecular content at the single-cell level, challenging this view with a new model suggesting that cell-to-cell gene expression stochasticity could play a key role in differentiation. We took advantage of a physiologically relevant avian cellular model to analyze the expression level of 92 genes in individual cells collected at several time-points during differentiation. We first observed that the process analyzed at the single-cell level is very different and much less well ordered than the population-based average view. Furthermore, we showed that cell-to-cell variability in gene expression peaks transiently before strongly decreasing. This rise in variability precedes two key events: an irreversible commitment to differentiation, followed by a significant increase in cell size variability. Altogether, our results support the idea that differentiation is not a “simple” series of well-ordered molecular events executed identically by all cells in a population but likely results from dynamical behavior of the underlying molecular network.

Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations. In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from time-stamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-by-one through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. Together, these results demonstrate WASABI versatility and ability to tackle some general gene regulatory networks inference issues. It is our hope that WASABI will prove useful in helping biologists to fully exploit the power of time-stamped single-cell data.

Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations. In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from time-stamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-by-one through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a fascinating new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. In conclusion, WASABI is a versatile algorithm which should help biologists to fully exploit the power of time-stamped single-cell data.

Background: Inference of Gene Regulatory Networks (GRNs) is a difficult and long-standing question in Systems Biology. Numerous approaches have been proposed with the latest methods exploring the richness of single-cell data. One of the current difficulties lies in the fact that many methods of GRN inference do not result in one proposed GRN but in a collection of plausible networks that need to be further refined. In this work, we present a Design of Experiment strategy to use as a second stage after the inference process. It is specifically fitted for identifying the next most informative experiment to perform for deciding between multiple network topologies, in the case where proposed GRNs are executable models. This strategy first performs a topological analysis to reduce the number of perturbations that need to be tested, then predicts the outcome of the retained perturbations by simulation of the GRNs and finally compares predictions with novel experimental data. Results: We apply this method to the results of our divide-and-conquer algorithm called WASABI, adapt its gene expression model to produce perturbations and compare our predictions with experimental results. We show that our networks were able to produce in silico predictions on the outcome of a gene knock-out, which were qualitatively validated for 48 out of 49 genes. Finally, we eliminate as many as two thirds of the candidate networks for which we could identify an incorrect topology, thus greatly improving the accuracy of our predictions. Conclusion: These results both confirm the inference accuracy of WASABI and show how executable gene expression models can be leveraged to further refine the topology of inferred GRNs. We hope this strategy will help systems biologists further explore their data and encourage the development of more executable GRN models.

Rationale. Bioinformatics workflows increasingly need to integrate heterogeneous biomedical data, including experimental assay outputs, patient-derived model metadata, mechanistic models and molecular-pathology information. In glioblastoma (GBM), such integration can help connect treatment responses measured in patient-derived 3D cultures with molecular context and mechanistic representations of tumor metabolism, while maintaining a clear distinction between experimental observations and model-derived hypotheses. Results. We describe a standards-based workflow that integrates patient-derived GBM drug-response screens with a machine-readable QSP model, MGMT promoter methylation metadata and an explicit evidence-tier framework. MGMT-promoter methylation was confirmed in all patient-derived 3D cultures (Ge258, Ge518 and Ge904). Phenformin (PTF) showed the most pronounced single-agent activity, with a concentration-dependent reduction in viability, while 2-deoxy-D-glucose (2-DG) produced a more moderate effect. Among the higher-order combinations, MTF + BPTES + 2-DG + TMZ produced the most pronounced combination response and a statistically significant reduction in viability under the tested conditions. These findings are interpreted as treatment-response and prioritization signals rather than definitive evidence of pharmacological synergy or clinical efficacy. Availability and implementation. The workflow is designed around open and independently checkable data and model files, including raw plate exports, processed block-level tables, molecular metadata, an SBML/SED-ML/COMBINE model package, an evidence-tier table and a next-experiment protocol. Together, these components provide a reproducible framework for integrating computational predictions with experimental evidence and prioritizing testable drug-combination hypotheses.