Background & AimsDiligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.MethodsMouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors.ResultsIn contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures.ConclusionOur study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.
BACKGROUND:
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastro-intestinal tract for which the key elements in disease initiation and perpetuation are defects in epithelial barrier integrity and repair. Inducing and maintaining mucosal healing has the potential to address unmet medical need in IBD of achieving greater efficacy and more durable disease remission than current therapies. Recent studies suggest that IL-22 regulates epithelial homeostasis and promotes repair of epithelial damage, thus making IL-22 a promising target for IBD therapy. In an effort to better understand IL-22 biology we have used human colon organoids to strengthen our understanding of the mechanisms by which IL-22 regulates mucosal healing.
METHODS:
Human colon organoids derived from healthy and ulcerative colitis (UC) donor biopsies were cultured using a standard protocol and treated with recombinant IL-22 protein alone or in combination with microbial-derived metabolites at multiple concentrations and time points. Cell viability, organoid forming capacity, epithelial cell function, as well as gene expression analysis of markers of epithelial cell types and signaling pathways were then assessed. To recapitulate the inflammatory environments associated with IBD, organoids were also co-cultured with anti-CD3/CD28 activated T cells. In-vivo studies were also performed in naïve mice dosed with IL-22-Fc to evaluate the in-vitro/in-vivo translatability.
RESULTS:
IL-22 treatment led to stem cell survival, cell proliferation and production of anti-microbial peptides in organoids. IL-22 treatment also altered the mucus barrier by inducing an increase in membrane mucus but a decrease in both secreted mucus and goblet cell number. Interestingly, most of the IL-22 effects on the organoids were also observed in-vivo thus confirming the in vitro/in vivo translatability. In addition, IL-22 had the same inducing effect on anti-microbial peptides and the membrane mucus barrier in both healthy (n = 3) and UC (n = 3) organoids. In contrast, this IL-22-associated epithelial phenotype was different when organoids were co-cultured with activated T cells. Finally, IL-22 showed synergistic and additive effects with microbial-derived metabolites in inducing expression of antimicrobial peptides and membrane mucins.
CONCLUSION(S):
Our data indicate that IL-22 acts directly on the gut epithelium of both healthy and UC individuals to promote epithelial regeneration, innate defense and production of membrane mucus. In addition, our findings indicate that these IL-22 effects on epithelial cell function and mucosal healing mechanisms are differentially regulated in presence of either microbial-derived metabolites or activated T cells. Overall, this data strongly supports clinical relevance of IL-22 as a mucosal healing therapy in IBD and suggests that the efficacy may be impacted by microbial-derived metabolites and inflammatory environments.
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