37 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition Impact of amino acid starvation on structure and function of liver organoids Liver Progenitor Organoids were matured into hepatocyte-like cells according to an established protocol35. Maturation was confirmed by the reduction in expression of stem cell markers and the increase in hepatocyte differentiation markers (e. g. Albumin, Hnf-4α, Cyp1a2 and Cyp3a11) in organoids grown in differentiation medium compared to progenitors, as previously described in the literature2. Maturation of the organoids was also confirmed by increased albumin production, measured in the supernatant. Peroxisomal enzymes acylCoA oxidase 1 and catalase were significantly upregulated in the differentiated organoids, indicating the suitability of these organoids for the study of peroxisomes. (Supplementary Figure 1). Deprivation of amino acids up to 96 hours did not significantly affect the growth nor the morphology of mature hepatic organoids (Figure 1a, b). In contrast, when liver progenitor organoids received the starvation medium, organoids remained smaller than control organoids (Supplementary Figure 2). The different response to removal of amino acids reflects the lower proliferative state of mature hepatic organoids as indicated by lower gene expression levels of stem cells markers Axin2 and Lgr5 (Supplementary Figure 1b). Starvation of mature hepatic organoids for 48 hours substantially reduced levels of albumin released in the supernatant (Figure 1c) and increased fat deposition as indicated by an accumulation of intracellular triglycerides as well as an increase in lipid droplets (Figure 1d, e). Re-supplementation of amino acids after 48 hours of starvation completely restored albumin production and significantly decreased lipid accumulation (Figure 1d, e).
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