Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens.

PURPOSE: Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids. METHODS: Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 °C for various intervals post-resection. RESULTS: We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids. CONCLUSION: SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid.

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.