Protocol for differentiation of human pluripotent stem cells into definitive endoderm lineage in a chemically defined and growth factor-free system.

Here, we present a protocol for the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) lineage. We describe steps for the resuscitation, passaging, and plating of hPSCs. We then detail procedures for culturing cells followed by immunostaining, imaging, and analysis. This chemically defined, small-molecule-based, recombinant protein-free system offers a cost-effective and scalable platform for generating endodermal derivatives, demonstrating efficiency for applications in drug screening, disease modeling, and regenerative medicine. For complete details on the use and execution of this protocol, please refer to Zhao et al.(1).