Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

Area of Science:

• Cell biology
• Stem cell research
• Molecular mechanisms of reprogramming

Background:

• Induced pluripotent stem cells (iPSCs) are generated by expressing OCT4, SOX2, KLF4, and cMYC (OSKM).
• Cellular senescence is a barrier to efficient iPSC reprogramming.
• Identifying senescence modulators is crucial for improving reprogramming efficiency.

Purpose of the Study:

• To identify novel regulators of senescence induced by OSKM.
• To elucidate the mechanism by which mechanistic target of rapamycin (mTOR) influences reprogramming-induced senescence.
• To explore the interplay between senescence and reprogramming using integrated screening and single-cell analysis.

Main Methods:

• Genome-wide shRNA screen in human fibroblasts expressing OSKM to identify senescence modulators.
• Integration of single-cell RNA sequencing (scRNA-seq) with shRNA screening.
• Analysis of cyclin-dependent kinase inhibitors (CDKIs) and senescence-associated secretory phenotype (SASP) following mTOR modulation.

Main Results:

• Identified novel mediators of OSKM-induced senescence, including validation of CDKN1A.
• Uncovered a dual role for mTOR in regulating reprogramming: inhibition prevents senescence by reducing CDKIs but also impairs beneficial SASP.
• Demonstrated that mTOR's complex effects on reprogramming are mediated through its influence on senescence pathways.

Conclusions:

• Combining functional screens with scRNA-seq accelerates the discovery of pathways controlling complex biological processes like reprogramming.
• Regulation of senescence is a key mechanism through which mTOR impacts iPSC generation.
• Understanding these pathways offers potential strategies to enhance reprogramming efficiency.