Reconstruction of the Human Colon Epithelium In Vivo

Abstract

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.

In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium

“…These results suggest that local transplantation of organoids in combination with scaffolds does not induce any side effects inside the body. Studies have shown that tumorigenesis is not caused by human colonoid transplantation in mice, even after 10 mo of tracking (18). As shown here, locally-transplanted organoids from tissue-specific stem cells are safer and more effective than treatment of pluripotent and mesenchymal stem cells in tissue regeneration.…”

“…Transplantation into recipient mouse colons occurred as previously described by Sugimoto et al (18). Y-27632 is used to prevent cell apoptosis to increase cell survival rate of colonoids as previously described (17,36,37).…”

“…In studies that have transplanted organoids into recipient mice, organoids were dissociated into small fragments, suspended in Matrigel, and injected by enema (17)(18)(19)(20). Use of Matrigel could significantly increase the efficiency of colonoid engraftment into injured epithelia (17).…”

Brief summary of the current protocols for generating intestinal organoids

“…Unlike stem cells derived from the small intestine, colonic stem cells form spherical organoids (SOs) without crypt-like structures and give rise to their progenies expressing colonic epithelial cell markers, similar to epithelial tissues in the colon Yui et al, 2012). Moreover, a recent study showed that epithelial tissues in the injured mouse colon could be reconstituted by human colonic epithelial tissues after transplantation of adult human-derived colonic organoids (Sugimoto et al, 2018). Scale bars, 100 μm F I G U R E 2 Generation of mouse and human intestinal organoids.…”

“…Also, the other group has shown that human colonic stem cells can expand by forming organoids in 3D culture with EGF, noggin, R-spondin1, Wnt3a, nicotinamide, gastrin, LY2157299 (an inhibitor of transforming growth factor beta receptor type 1 kinase), SB202190, and prostaglandin E2 (PGE-2) (Jung et al, 2011). Moreover, a recent study showed that epithelial tissues in the injured mouse colon could be reconstituted by human colonic epithelial tissues after transplantation of adult human-derived colonic organoids (Sugimoto et al, 2018). Meanwhile, human FIPCs are able to form SOs in culture medium containing EGF, noggin, R-spondin1, and PGE-2, although, unlike mouse FIPC-derived SOs, a culture method capable of developing human FIPCs-derived SOs into BOs remains to be established ( Figure 2b; Fordham et al, 2013).…”