Abstract
Over the past decade, significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation, with organoids emerging as key experimental models. These three-dimensional in vitro cellular structures mimic the organization and functions of various gut regions, providing a powerful tool for research. By replicating critical stages of gut development, we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol, we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First, we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three-dimensional spheroids form spontaneously, can be collected, embedded in an extracellular matrix, and cultured over time. During this phase, the organoid epithelium develops, supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture, HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development, model diseases, and test therapeutic agents.