A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC...

Area of Science:

• Stem cell biology
• Developmental biology
• Genomics

Background:

• Optimizing stem cell differentiation protocols is challenging due to time and labor intensity.
• Current methods often fail to comprehensively analyze all relevant signaling pathways.

Purpose of the Study:

• To introduce barcodelet single-cell RNA sequencing (barRNA-seq) for systematic exploration of cellular perturbations.
• To investigate combinatorial effects of signaling pathways on embryonic stem cell (ESC) differentiation.
• To develop a data-driven framework for identifying signaling perturbations that drive specific cell fates.

Main Methods:

• Development and application of barRNA-seq for simultaneous manipulation of up to seven developmental pathways.
• Analysis of gene expression changes in ESCs during germ layer and mesodermal specification.
• Creation of a data-driven framework to predict cell fate based on signaling perturbations.

Main Results:

• Uncovered combinatorial effects of signaling pathway activation on gene expression during ESC differentiation.
• Identified specific signaling perturbations that drive cells towards annotated gastrulation fates.
• Successfully guided ESC differentiation into a notochord-like population using the developed framework.

Conclusions:

• barRNA-seq provides a powerful tool for high-throughput screening of signaling pathway combinations.
• The data-driven framework enables precise control over stem cell differentiation.
• This approach has broad utility for understanding cooperative signaling in cell fate acquisition.