Suspension culture promotes serosal mesothelial development in human intestinal organoids.

Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.