Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.

Human organoid model systems lack important cell types that, in the embryo, are incorporated into organ tissues during development. We developed an organoid assembly approach starting with cells from the three primary germ layers-enteric neuroglial, mesenchymal, and epithelial precursors-that were derived separately from human pluripotent stem cells (PSCs). From these three cell types, we generated human antral and fundic gastric tissue containing differentiated glands surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered antral tissue. Using this experimental system, we show that human enteric neural crest cells (ENCCs) promote mesenchyme development and glandular morphogenesis of antral stomach organoids. Moreover, ENCCs can act directly on the foregut to promote a posterior fate, resulting in organoids with a Brunner's gland phenotype. Thus, germ layer components that are derived separately from PSCs can be used for tissue engineering to generate complex human organoids.