39 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition 2c, d and Supplementary Figure 4b). Amino-acid deprivation upregulated the expression of markers of Paneth cells (lysozyme), goblet cells (mucin-2) and enteroendocrine cells (chromogranin A), but did not affect the expression of VIL1 (enterocytes) or stem cell and proliferation markers (Supplementary Figure 5). FITC-dextran (4 and 10kDa) leakage into the lumen of starved organoids was increased, as indicated by increased number of FITC-dextran positive organoids as well as higher luminal fluorescence, showing that aminoacid starvation compromised the intestinal barrier function (Figure 3a-c, Supplementary figure 5). This is consistent with in vivo findings36 and was supported by reduced levels of the tight-junction protein claudin-3 in aminoacid deprived intestinal organoids (Figure 3d). Importantly, re-supplementation of amino acids restored organoid size, crypt number, claudin-3 protein level, and expression of mucin-2 and chromogranin A, and increased stem cell markers LGR5 and Axin2 (Figure 2a-d, Supplementary Figure 4). Restoration of intestinal barrier function by amino-acid re-supplementation could not be properly assessed with FITC-dextran due to the high amount of content (e.g. dead cells) in the organoids lumen after 96 hours.
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