Probes for LGR5

The turnover of the intestinal epithelium is driven by multipotent LGR5+ crypt-base columnar cells (CBCs) located at the bottom of crypt zones1. However, CBCs are lost following injury, such as irradiation2, but the intestinal epithelium is nevertheless able to recover3. Thus, a second population of quiescent '+4' cells, or reserve stem cells (RSCs), has previously been proposed to regenerate the damaged intestine4-7. Although CBCs and RSCs were thought to be mutually exclusive4,8, subsequent studies have found that LGR5+ CBCs express RSC markers9 and that RSCs were dispensable-whereas LGR5+ cells were essential-for repair of the damaged intestine3. In addition, progenitors of absorptive enterocytes10, secretory cells11-15 and slow cycling LGR5+ cells16 have been shown to contribute to regeneration whereas the transcriptional regulator YAP1, which is important for intestinal regeneration, was suggested to induce a pro-survival phenotype in LGR5+cells17. Thus, whether cellular plasticity or distinct cell populations are critical for intestinal regeneration remains unknown. Here we applied single-cell RNA sequencing to profile the regenerating mouse intestine and identified a distinct, damage-induced quiescent cell type that we term the revival stem cell (revSC). revSCs are marked by high clusterin expression and are extremely rare under homoeostatic conditions, yet give rise-in a temporal hierarchy-to all the major cell types of the intestine, including LGR5+ CBCs. After intestinal damage by irradiation, targeted ablation of LGR5+ CBCs, or treatment with dextran sodium sulfate, revSCs undergo a YAP1-dependent transient expansion, reconstitute the LGR5+ CBC compartment and are required to regenerate a functional intestine. These studies thus define a unique stem cell that is mobilized by damage to revive the homoeostatic stem cell compartment and regenerate the intestinal epithelium.

Background and Aims

Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium’s role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell–derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli.

Methods

Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate–dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function.

Results

The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability.

Conclusions

We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.