Decoding the network logic governing resetting of pluripotency

Theme: World-Class Underpinning Bioscience

The pluripotent ground state of embryonic stem cells (ESCs) is governed by a self-reinforcing interaction network of transcription factors1. The dynamics of this molecular circuitry underlie the remarkable cellular plasticity of ESCs, while dissolution of the circuitry is required for differentiation. Combinations of factors within this network can induce somatic cells to acquire pluripotency2,3, a process called molecular reprogramming. Experimental and computational efforts have led to circuitry mapping of the key players in maintenance of the naïve state. However, how this molecular circuitry is launched and fully connected during reprogramming remains unclear.

This project is a cross-disciplinary collaboration to address systematically how cells transit from an established identity to the pluripotent ESC state at a molecular network level. The multi-step, heterogeneous and asynchronous nature of the reprogramming process presents technical challenges to molecular delineation over time. This project is designed to overcome these challenges by using a minimal reprogramming system and integrating quantitative single-cell gene expression profiling at defined reprogramming stages with computational network synthesis and modelling. This approach will transform single-cell snapshots of network status into reconfiguring network trajectories along the reprograming progression timeline. It is anticipated that this original approach will reveal new molecular principles in how cells transit between identities.

Objective 1 - Characterise and utilise a dual-reporter experimental platform to dissect stages of reprogramming. Our experimental model is resetting of post-implantation epiblast derived epiblast stem cells (EpiSCs) to naïve ESCs4. This is reprogramming, but within the pluripotent compartment, potentially resembling the final stages of reprogramming from somatic cells. A new EpiSC line was recently established within our laboratory that carries two knock-in fluorescent reporters, GFP and mKO, driven by Oct4 and Rex1 promoters respectively. The dual reporter system will allow the isolation of cells at different resetting stages because Rex1 is specific to the naïve ESC state while Oct4 is expressed in all pluripotent cells. This experimental system will be fully characterised in terms of reporter expression dynamics and the functional clonogenic capacity of individual subpopulations marked by reporter expression combinations.

Objective 2 - Analyse single-cell expression of transcription factor network players at different stages of reprograming. Based on the combinations of dual-reporter status, up to 2,000 individual cells from different reprogramming stages will be isolated and profiled for 56 genes of interest using a multiplex OpenArray real-time PCR platform.

Objective 3 - Automated model synthesis from experimental data to delineate network reconfigurations over time. An approach based on logical modelling and automated computational reasoning, which was established to investigate steady-state ESC self-renewal behaviour1,5, will be extended and applied to identify trajectories of network reconfiguration6. Single-cell expression data will be used to constrain and synthesise network snapshots corresponding to individual cells at different stages of reprogramming. Analysis of network reconfiguration will explain the conversion from EpiSC to the naïve state over time.

Objective 4 - Predict and experimentally validate network perturbations. Based on synthesised models, predictions will be formulated on reprogramming behaviours, such as the effect of loss or gain of function of one or more components. These predictions will be tested experimentally and the results used for iterative refinement of the model set.