Intestinal organoids (IOs) are versatile, physiologically relevant models of the human gut. In cystic fibrosis research, IOs help evaluate CFTR correctors, potentiators, and translational readthrough compounds. Here, we present a protocol for generating induced pluripotent stem cell (iPSC)-derived IOs with high phenotypic similarity to primary material. We describe steps for establishing iPSC culture, generating intestinal progenitors via directed differentiation, and maturing these intestinal progenitors using three-dimensional culture. We then detail procedures for establishing intestinal organoid cultures from these progenitors using dissection-based techniques. • Steps for preparing iPSC cultures for directed differentiation into intestinal progenitors • Instructions for generating intestinal progenitors from iPSCs • Directions for maturing intestinal progenitors toward DiO lineages • Guidance on the culture and maintenance of DiO lineages Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Intestinal organoids (IOs) are versatile, physiologically relevant models of the human gut. In cystic fibrosis research, IOs help evaluate CFTR correctors, potentiators, and translational readthrough compounds. Here, we present a protocol for generating induced pluripotent stem cell (iPSC)-derived IOs with high phenotypic similarity to primary material. We describe steps for establishing iPSC culture, generating intestinal progenitors via directed differentiation, and maturing these intestinal progenitors using three-dimensional culture. We then detail procedures for establishing intestinal organoid cultures from these progenitors using dissection-based techniques.
Stuffer et al. (Mon,) studied this question.