Controlled aggregative assembly to form self-organizing macroscopic human intestine from induced pluripotent stem cells

Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) are promising resources for intestinal regenerative therapy as they recapitulate both endodermal and mesodermal components of the intestine. However, due to their hPSC-line-dependent mesenchymal development and spherical morphology, HIOs have limited applicability beyond basic research and development. Here, we demonstrate the incorporation of separately differentiated mesodermal and mid/hindgut cells into assembled spheroids to stabilize mesenchymal growth in HIOs. In parallel, we generate tubular intestinal constructs (assembled human intestinal tubules [a-HITs]) by leveraging the high aggregative property of assembled spheroids. Through rotational culture in a bioreactor, a-HITs self-organize to develop epithelium and supportive mesenchyme. Upon mesenteric transplantation, a-HITs mature into centimeter-scale tubular intestinal tissue with complex architectures. Our aggregation- and suspension-based approach offers basic technology for engineering tubular intestinal tissue from hPSCs, which could be ultimately applied to the generation of the human intestine for clinical application.