# Single cell transcriptional perturbome in pluripotent stem cell models

## TL;DR

The paper introduces iPS2-seq, a new platform for studying gene function in human stem cells at the single-cell level, revealing insights into cardiac development and gene effects.

## Contribution

iPS2-seq is a novel platform for single-cell loss-of-function screening in human pluripotent stem cells with multi-omic profiling capabilities.

## Key findings

- iPS2-seq distinguishes true perturbation effects from genetic and epigenetic variability in hiPSC derivatives.
- Multi-omic profiling reveals and corrects ZIC1⁺ clonal differentiation bias.
- SMAD2 knockdown redirects cardiac progenitors toward fibroblast and epicardial fates.

## Abstract

Functional genomics screens in human induced pluripotent stem cells (hiPSCs) remain challenging despite their transformative potential. We developed iPS2-seq: an inducible, clone-aware screening platform that enables phenotype-agnostic, single-cell resolved dissection of loss-of-function effects in hiPSC derivatives, including complex multicellular models such as organoids. iPS2-seq distinguishes true perturbation effects from genetic and epigenetic variability. It supports pooled and arrayed formats, integrates with microfluidic or split-pool single-cell RNA sequencing, and extends to multi-omic profiling of chromatin and proteins. A dedicated pipeline, catcheR, streamlines design and analysis. The platform enables stage-specific follow-up dissection of screen hits. We demonstrate this by targeting congenital heart disease-associated genes in monolayer cardiomyocytes and organoids. This reveals that epigenetic neuroectodermal priming interferes with germ layer differentiation in specific clones. Accounting for this bias, we show that SMAD2 controls cardiac progenitor specification, with knockdown redirecting cells toward fibroblast and epicardial fates. iPS2-seq unlocks rigorous functional genomics in hiPSC-based models.

iPS2-seq is an inducible, clone-aware platform for single-cell loss-of-function screening in human pluripotent stem cells, enabling integrated transcriptomic, epigenetic, and proteomic dissection of gene function across differentiation stages.

Multi-omic profiling reveals and corrects ZIC1⁺ clonal differentiation bias.Pooled screening maps gene functions during cardiac development in 2D and 3D models.Arrayed studies identify the role of SMAD2 in cardiac–epicardial development.Stage-specific validation pinpoints SMAD2 function in cardiac progenitor specification.

Multi-omic profiling reveals and corrects ZIC1⁺ clonal differentiation bias.

Pooled screening maps gene functions during cardiac development in 2D and 3D models.

Arrayed studies identify the role of SMAD2 in cardiac–epicardial development.

Stage-specific validation pinpoints SMAD2 function in cardiac progenitor specification.

iPS2-seq is an inducible, clone-aware platform for single-cell loss-of-function screening in human pluripotent stem cells, enabling integrated transcriptomic, epigenetic, and proteomic dissection of gene function across differentiation stages.