Reconstruction of the Human Colon Epithelium In Vivo

Highlights

• Growing human normal colonic epithelium in orthotopically xenografted mouse colon

• Human colon crypt architectures are preserved in mouse colon environments

• Lineage tracing demonstrates stem cell functions of human LGR5⁺ colon cells in vivo

• Unlike mouse colon, human colon stem cells are found to be slow cycling in vivo

Summary

Genetic lineage tracing has revealed that Lgr5⁺ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5⁺ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5⁺ CoSCs, human LGR5⁺ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.