Target discovery by covalent phenotypic screening in a stem cell differentiation model

The phenotypic screen was designed to identify signalling pathway modulators and constituted a differentiation assay whereby primed human induced pluripotent cells (iPSCs) were differentiated to definitive endoderm over a period of 4 days in the presence of DMSO (control) or the individual covalent fragments from the library.

We discovered a hit (PP1071) that prevented differentiation to endoderm. To learn more about the biological effect of PP1071, we performed a bulk RNA-seq on pluripotent iPSCs and cells differentiated to endoderm treated for three days with DMSO or with 10 µM PP1071. We showed that PP1071 prevented differentiation to endoderm, but the resulting cells were transcriptionally distinct from the starting iPSCs, despite expressing markers characteristic of pluripotency.