Abstract
Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-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 intestinal organoids (epithelial ‘miniguts’), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is 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.
Highlights
We show that: (1) jejunal explants and organoids derived from PERIOD2::LUCIFERASE fusion protein (PER2::LUC) mice (Yoo et al, 2004) display robust circadian rhythms, (2) PER2::LUC organoids synchronize their circadian clock in response to serum shock, (3) unshocked organoids autonomously synchronize their circadian clock within 12 hours of monitoring and (4) organoid growth factors and MatrigelTM enhance circadian rhythms
Bioluminescent PER2::LUC organoids showed structural features typical of the intestinal epithelium and exquisite circadian rhythms; these rhythms synchronized in response to a high concentration of serum, a typical external cue designed to align the clocks of mammalian cells in culture
Even in the absence of serum shock, PER2::LUC organoids demonstrated the unusual ability to self-synchronize circadian rhythms after 12 hours in culture. In this tissue culture model, the circadian patterns of Per2 gene expression over 48 hours were more robust as compared with serum-shocked intestinal epithelial cell lines from mice and humans (Caco-2 colorectal adenocarcinoma cells)
Summary
Received September 2013; Accepted June 2014 spectrum of organisms (Panda et al, 2002) These processes include the digestive, immune and regenerative functions of the gastrointestinal (GI) epithelium, which are necessary for organismal growth and survival (Potten et al, 1977; Hoogerwerf, 2006; Hussain and Pan, 2009; Karpowicz et al, 2013; Yu et al, 2013). Progress in understanding the mechanisms by which circadian rhythms influence GI health and disease has lagged, in part, due to a paucity of in vitro models with appropriate phenotypes and robust circadian rhythms (Hughes et al, 2009). Four-dimensional tissue culture models with organotypic features and the ability to reproduce the circadian rhythms of the GI epithelium would help advance our understanding of how circadian rhythms regulate GI health and disease
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