biostudies-literatureZhao ZNational Key Research and Development Program of China Stem Cell and Translational ResearchGuangdong Provincial Natural Science FoundationNational Natural Science Foundation of ChinaQingdao Municipal Science and Technology BureauNational Key Research and Development Program of ChinaTaishan Scholar Foundation of Shandong Province102382https://www.ebi.ac.uk/biostudies/studies/S-EPMC11784501biostudies-literatureUnknown20(1)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 chemically defined, 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 β cellsStem cell reportsChemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells.PMC117845012022A1515011819322006853247116032430031tsqn2023062719226810523-2-8-smjk-11-nsh2023YFA1801200En HChan WYCao NGu SLu GZeng FXu HZhao ZWang JNie YfalseChemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells.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 chemically defined, 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 β cells2025-01-01T00:00:00Z2025 Jan2026-06-01T08:59:30.98Z2025-04-03T23:26:45.653ZS-EPMC117845013972998910.1016/j.stemcr.2024.11.012