IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data.

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs allow simulation of fundamental hypotheses governing developmental programs and help accelerate the design of strategies to control stem cell fate. We first describe the architecture of IQCELL. Next, we apply IQCELL to scRNA-seq datasets from early mouse T-cell and red blood cell development, and show that the platform can infer overall over 74% of causal gene interactions previously reported from decades of research. We will also show that dynamic simulations of the generated GRN qualitatively recapitulate the effects of known gene perturbations. Finally, we implement an IQCELL gene selection pipeline that allows us to identify candidate genes, without prior knowledge. We demonstrate that GRN simulations based on the inferred set yield results similar to the original curated lists. In summary, the IQCELL platform offers a versatile tool to infer, simulate, and study executable GRNs in dynamic biological systems.

High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.

Inferring Gene Regulatory Networks and Predicting the Effect of Gene Perturbations via IQCELL.

IQCELL is a platform that infers Boolean gene regulatory networks from single-cell RNA sequencing data. Boolean networks can be simulated under normal and perturbed conditions. In this chapter, we provide a detailed guideline for implementing IQCELL from a raw dataset. The steps include processing data, inferring informative genes, inferring gene regulatory network, and simulating the resulted network under normal and perturbed conditions.

Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification.

The hypoblast is an essential extraembryonic tissue set aside within the inner cell mass in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naive pluripotent stem cell (PSC) to hypoblast differentiation proceeds via reversion to a transitional ICM-like state from which the hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signaling is critical for hypoblast specification. Revisiting FGF signaling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro, the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mice and humans. Overall, this study demonstrates the utility of human naive PSC-based models in elucidating the mechanistic features of early human embryogenesis.

Development of a Virtual Cell Model to Predict Cell Response to Substrate Topography.

Cells can sense and respond to changes in the topographical, chemical, and mechanical information in their environment. Engineered substrates are increasingly being developed that exploit these physical attributes to direct cell responses (most notably mesenchymal stem cells) and therefore control cell behavior toward desired applications. However, there are very few methods available for robust and accurate modeling that can predict cell behavior prior to experimental evaluations, and this typically means that many cell test iterations are needed to identify best material features. Here, we developed a unifying computational framework to create a multicomponent cell model, called the "virtual cell model" that has the capability to predict changes in whole cell and cell nucleus characteristics (in terms of shape, direction, and even chromatin conformation) on a range of cell substrates. Modeling data were correlated with cell culture experimental outcomes in order to confirm the applicability of the virtual cell model and demonstrating the ability to reflect the qualitative behavior of mesenchymal stem cells. This may provide a reliable, efficient, and fast high-throughput approach for the development of optimized substrates for a broad range of cellular applications including stem cell differentiation.