Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells [RNA-seq]

Definitive endoderm (DE) derived from human pluripotent stem cells (hPSCs) holds great promise for cell-based therapies and drug discovery. However, current DE differentiation methods required undefined components and/or expensive recombinant proteins, limiting their scalable manufacture and clinical use. Homogeneous DE differentiation in defined and recombinant protein-free conditions remains a major challenge. Here, by systematic optimization and high-throughput screening, we report a fully synthetic, small molecule-based defined system that contains only four components (4C), enabling highly efficient and cost-effective DE specification of hPSCs in the absence of recombinant proteins. 4C-induced DE can differentiate into functional hepatocytes, lung epithelium, and pancreatic ß cells in vitro and multiple DE-derivatives in vivo. Genomic accessibility analysis reveal that 4C reconfigures chromatin architecture to allow key DE transcription-factor binding and identify TEAD3 as a novel key DE regulator. This system may facilitate mass production of mature DE-derivatives for drug discovery, disease modelling, and cell therapy. Overall design: Total RNA of cells was isolated using RNAzol. Sequencing libraries were constructed using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-1) according to the manufacturer's instruction.ATAC-seq sequencing libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to the manufacturer's instruction. Quality control of the libraries was performed using an Agilent 2100 Bioanalyzer and sequencing was performed using an Illumina NovaSeq 6000 Sequencer.