Deciphering signalling pathway dynamics during cell-fate commitment in stem cells

Introduction

Understanding the identity and intensity of the specific extracellular signals that a cell experiences at different times during its differentiation is essential to develop advanced cellular therapies. However, uncovering the sequence of these signaling events, their intensities, timing, and relevance in development and disease is proving to be very challenging.

Project Proposal

Here, I propose to build i-SignalTrace: a CRISPR/Cas9-based molecular recorder with the capacity to store both lineage information and signaling pathway activity for multiple signals over time in single cells.

Methodology

By performing kinetic experiments and mathematical modeling, I will use i-SignalTrace to:

1. Extract the probability of signaling pathways to be activated in stem cells when subject to different extracellular signals.
2. Reconstruct the lineage tree of pathway activities during differentiation with single-cell resolution.

In combination with single-cell RNA sequencing, i-SignalTrace will make it possible to characterize transition and intermediate states along differentiation trajectories, and quantify the integration between extracellular signals and autonomous programs of gene expression.

Expected Outcomes

These results will allow:

• Predicting the differentiation trajectories that stem cells follow when subject to external perturbations.
• Deciphering the role of heterogeneity in signaling pathway activity during cell-fate commitment.

Applications

Using i-SignalTrace, I will identify missing or redundant signaling pathways induced during in vitro differentiation protocols. Therefore, I expect that exploitation of i-SignalTrace will allow establishing new criteria to design protocols to differentiate stem cells on demand.

Proof-of-Concept

As a proof-of-concept, I propose a framework to improve the functionality of monolayer-derived cardiomyocytes.

Conclusion

Taken together, i-SignalTrace will find applications in both fundamental developmental biology and translational regenerative medicine, which will benefit a much wider scientific community.