Deriving human intestinal organoids with functional tissue-resident macrophages all from pluripotent stem cells

Background & Aims: Organs of the gastrointestinal tract contain tissue-resident immune cells that function during tissue development, homeostasis, and disease. However, the majority of published human organoid model systems lack resident immune cells, thus limiting their potential as disease avatars. For example, human intestinal organoids (HIOs) derived from pluripotent stem cells contain epithelial and various mesenchymal cell types but lack immune cells. In this study, we aimed to develop an HIO model with functional tissue-resident macrophages. Methods: HIOs and macrophages were generated separately through the directed differentiation of human pluripotent stem cells and combined in vitro. Following two weeks of co-culture, the organoids were used for transcriptional profiling, functional analysis of macrophages, or transplanted into immunocompromised mice and matured in vivo for an additional 10–12 weeks. Results: Macrophages were incorporated into developing HIOs and persisted in the 2-week in vitro HIOs and for at least 12 week in HIOs in vivo. These macrophages expressed CD163 and had a transcriptional signature that resembled those of the human fetal intestine, indicating that they were acquiring the features of tissue-resident macrophages. HIO macrophages could phagocytose bacteria and produced inflammatory cytokines in response to proinflammatory signals such as lipopolysaccharide, which could be reversed with IL-10. Conclusions: We generated an HIO system containing functional tissue-resident macrophages for an extended period. This new organoid system can be used to investigate the molecular mechanisms involved in inflammatory bowel disease.