Abstract

Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. 4-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestine, organoids (epithelial "miniguts"), and nontransformed (MSIE) and transformed (Caco-2) intestinal epithelial cells. Here we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice display robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids are similarly robust, synchronize with serum shock, and persist beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronize rhythms within 12 hours of recording. The onset of this autonomous synchronization is slowed by >2 hours in the presence of glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of organoid growth factors EGF, Noggin, and R-spondin enhances PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects. Growth factor pulses induce modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence align with Per2 mRNA expression by qPCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis, and disease.