Technologies for Stem Cell Research

Poster

F-2129 - MAPPING CELLULAR REPROGRAMMING VIA POOLED OVEREXPRESSION SCREENS WITH PAIRED FITNESS AND SINGLE CELL RNA-SEQUENCING READOUT

6/22/2018
18:00 - 19:00

Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) is critical to discovering novel reprogramming factors for differentiation to diverse lineages, and to study the subtle and heterogenous effects of reprogramming. This has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, we use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. We further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and whole gene families. From the transcriptomic responses, we built genetic co-perturbation networks to identify key altered gene modules. Strikingly, from the network analysis we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, while from the fitness responses we identified ETV2 as a driver of reprogramming towards an endothelial-like state. These findings highlight the effectiveness of SEUSS in understanding the effects of genetic perturbations, furthering our understanding of reprogramming, and in discovering novel methods of differentiating hPSCs.


 

Udit Parekh

University of California, San Diego, California, United States

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