Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating, often dose limiting side effect of radiation treatment. A mouse model of mucositis, induced by gamma irradiation, leads to weight loss and tissue damage, similar to that observed in patients. This model reflects the human ailment as it responds to keratinocyte growth factor (KGF), the standard of care treatment. Culturing of intestinal crypt organoids derived from primary cells allowed the development of a 3D assay to monitor the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay closely resembles the mouse model as KGF and Roof Plate-Specific Spondin-1 (RSPO1) enhanced the recovery of crypt organoids following radiation. Screening identified tool compounds that increased the survival of organoids post radiation. Repeated testing of these compounds revealed that the organoids changed their response over time. To investigate this adaptive behavior, intestinal organoid cultures were studied over time. Samples of organoids at various time points were used to prepare mRNA for unbiased transcriptome analyses. This expression profiling revealed a number of genes and pathways that were modulated over time, providing a rationale for the altered sensitivity of the intestinal crypt organoid cultures. This report describes the development of an in vitro assay that reflects the response of disease to therapeutic treatment. The assay was miniaturized and used to identify bioactive tool compounds, which served as probes to interrogate the patho-physiology of organoids over prolonged culture conditions. In vitro disease models based on primary 3D cell cultures represent valuable tools to identify potential drug targets and bioactive hits

Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/β-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD