(P01-019-24) Astaxanthin Regulates Gene Expression in Mouse Small Intestinal Organoids in Culture

Objectives: To establish mouse intestinal organoid culture and to determine the effect of astaxanthin on gene expression profiles in organoids in culture.

Methods: The distal jejunum to proximal ileum were collected from eight-week-old C57BL/6J (WT) and beta-carotene oxygenase 2 (BCO2) gene knockout (KO) mice. After washing, tissues were incubated with cell dissociation reagent, followed by resuspension in ice-cold 0.1% BSA to release crypts. Approximately 6,000 crypts were isolated from individual animals. Freshly isolated crypts were suspended with complete growth media and subsequently embedded in Matrigel Matrix. After polymerization, complete growth medium was added to each well, and the plate was maintained in a CO2 incubator with 5 % CO₂ at 37 °C. Cultures were passed in a 1:3 split ratio on day 7 post-plating. Then, on day 4 after passage, organoids were treated with astaxanthin (at 0, 1, and 5μmol/L working concentrations) for three days. Organoid growth rates and crypt morphology were assessed. RNAs were extracted (n=4 samples/group) followed by RNA sequencing (RNA-seq) (Novogene Corporation Inc) and results were verified by qPCR. Data were analyzed by two-way ANOVA.

Results: Astaxanthin treatment did not show any significant changes in growth rates and crypt morphology of organoid cultures. RNA-seq results indicated that organoids isolated from BCO2 KO mice showed distinct gene expression patterns compared to WT. Consistently, qPCR results indicated that KO organoids presented markedly decreased expressions of Chromogranin A (CHGA) than those isolated from WT mice. Compared to WTs, KO organoids were more responsive to astaxanthin treatments as astaxanthin induced more differential genes in KO organoids than in WTs. Moreover, astaxanthin had the potential to regulate cell proliferation and differentiation in organoids, as indicated by Gene Ontology (GO) enrichment analyses and confirmed by qPCR results. For example, the astaxanthin-treated KO organoids exhibited enrichment of the Wnt signaling pathway, as well as increased gene transcription of hairy and enhancer of split 1 (Hes1), a key player in the Notch signaling pathway.

Conclusions: Our results suggest that astaxanthin can differentially regulate gene expression in intestinal epithelial organoid cultures, especially in those derived from BCO2 KO mice.

Funding Sources: USDA NIFA