The circadian clock ticks in organoids

The circadian clock is an evolutionarily conserved molecular system possessed by organisms ranging from bacteria to humans. The role of the clock system is fundamental as it coordinates the internal rhythm of organisms with the daily changes in environments such as temperature, light, and ecological niche. In humans, many physiological functions, including body temperature, hormone secretion, and sleep and wake cycle, are under the control of the circadian clock. Notably, some pathological conditions are more apparent at a specific time of day (Durrington et al, 2014). Nearly half of the genes in the mouse genome show circadian rhythm somewhere in the body. Each organ in the body has its own set of genes that oscillate in their transcription (Zhang et al, 2014; Ruben et al, 2018). Furthermore, a majority of the most commonly used drugs in the world target pathways involving circadian genes. Many of these drugs have relatively short half‐lives, suggesting some medications are more effective when dosed in a time‐controlled manner (Zhang et al, 2014). Also, timed dosing of drugs may lower the untoward effects on organs without diseases. The integration of knowledge from circadian biology and medicine is forming a new research field of so‐called chronotherapy, chronopharmacology, or circadian medicine (Panda, 2016; Ruben et al, 2019). One apparent promising approach to facilitate the growth of circadian medicine is to develop efficient systems where researchers can evaluate the effects of drugs on multiple organs in vitro.

Classical cell lines and primary cells from model animals have been successful since the late twentieth century. The studies of molecular mechanisms of the circadian clock quickly advanced by using cultured fibroblast cells (Ukai & Ueda, 2010). Nevertheless, extrapolating results from 2D‐cultured cells to organs or even to humans has become a major bottleneck in medical studies today. Recent research has identified biological processes specific to cell types and unable to be well recapitulated in conventional cell cultures. For example, human noroviruses, the most common cause of epidemic and sporadic acute gastroenteritis worldwide, only recently became possible to be cultured using human intestinal enteroid (HIE) (Ettayebi et al, 2016). HIE is an artificially induced, multicellular in vitro culture mirroring the human intestinal epithelium. The advent of this reproducible model system for the analysis of viruses leads to the mechanistic understanding of the infection, better treatment of the illness, and prevention. These next generation of in vitro systems, i.e., enteroids or, more generally, organoids, serve to study medication in a human cell context and will be used as screening platforms to identify novel drug candidates (Kim et al, 2020).

Organoids are a promising technology also for achieving circadian medicine. But, the pluripotent stem cells or embryonic stem cells used to induce the organoids do not possess the circadian clock, in contrast to the differentiated cells showing the circadian oscillation in the core clock genes (Yagita et al, 2010). It has been largely unknown whether organoids possess circadian clocks similar to in vivo organs. One breakthrough recently occurred in this question. The current study by Rosselot et al (2021) constitutes a breakthrough, as it investigated the circadian clock in several types of artificially induced, multicellular structures that mimic the intestinal organ. The authors generated organoids that were different in terms of their maturity, cell origin, or induction process and reported that human intestinal organoids (HIO) induced from the pluripotent stem cells did not show circadian rhythm. On the other hand, the more mature structures such as human intestinal enteroids (HIEs) showed robust circadian oscillation of cardinal clock‐controlled genes (Fig 1). Importantly, they demonstrated that the HIE possessed the circadian phase‐dependent necrotic cell death response to Clostridium difficile toxin B (TcdB). They also showed that the mouse intestinal enteroids' response was anti‐phasic to that of the human intestinal enteroids. Strikingly, the authors revealed that the expression of the Rac1 gene, one of the target genes of TcdB, was oscillating in a circadian manner. Furthermore, the oscillation of Rac1 expression in mice was anti‐phasic to humans, consolidating the potential role of Rac1 in the circadian phase‐dependent sensitivity to TcdB. The work by Rosselot and colleagues provides a first detailed view of the circadian clock system and its function in organoids, demonstrating its potential for circadian medicine.

Organoids also offer a unique opportunity to understand the circadian clock in the context of development and evolution. There are just a handful of genes that are commonly oscillating in different organs. The transcription of these core clock genes is largely in phase in various organs. It is an attractive challenge to reveal the design principle of the circadian clock by investigating how the robust ~24 h oscillation emerges from the core clock genes and how each tissue builds its unique set of oscillating genes by observing organoids developing into different organs. This approach may also highlight some evolutionary differences of peripheral clocks, such as the molecular differences between nocturnal and diurnal animals.

One major hurdle to overcome in achieving circadian medicine is to measure the precise time (the oscillation phase) of the subject organ (Anafi et al, 2017; Wu et al, 2018). The molecular details of each organ, to be provided by the analysis of organoids, should shed new light on the search for molecular markers that are useful to determine the time of the organ. As organoids are promising technology in the interdisciplinary fields of medicine and biology, the circadian clock research in organoids should serve as a foundation for future studies among different research fields, including regenerative medicine, circadian medicine, and developmental biology in human cell context.