The circadian clock ticks in organoids
Rikuhiro G Yamada1 & Hiroki R Ueda1,2,*
Organoids are self-organizing in vitro 3D
cultures that are histologically similar to avariety of human organs. A recent study
by Rosselot et al (2021) shows that matureintestinal organoids possess speciesspecific circadian clocks similar to theirrespective in vivo context, suggestingorganoids as promising platforms to study
circadian medicine.
The EMBO Journal (2022) 41: e110157

See also: AE Rosselot et al (January 2022)

The circadian clock is an evolutionarily
conserved molecular system possessed by organisms ranging frombacteria to humans. The role of the clock
system is fundamental as it coordinates theinternal rhythm of organisms with the dailychanges in environments such as temperature, light, and ecological niche. In humans,
many physiological functions, including
body temperature, hormone secretion, andsleep and wake cycle, are under the controlof 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 somewherein 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 mostcommonly used drugs in the world target
pathways involving circadian genes. Manyof these drugs have relatively short halflives, suggesting some medications are more
effective when dosed in a time-controlledmanner (Zhang et al, 2014). Also, timed dosing of drugs may lower the untoward effects
on organs without diseases. The integrationof knowledge from circadian biology andmedicine 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 ofdrugs on multiple organs in vitro.
Classical cell lines and primary cells frommodel animals have been successful since
the late twentieth century. The studies ofmolecular mechanisms of the circadianclock quickly advanced by using cultured
fibroblast cells (Ukai & Ueda, 2010). Nevertheless, extrapolating results from 2Dcultured cells to organs or even to humans
has become a major bottleneck in medicalstudies today. Recent research has identified
biological processes specific to cell types
and unable to be well recapitulated in conventional cell cultures. For example, humannoroviruses, the most common cause of epidemic and sporadic acute gastroenteritis
worldwide, only recently became possible tobe 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 themechanistic 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 ahuman cell context and will be used as
screening platforms to identify novel drugcandidates (Kim et al, 2020).
Organoids are a promising technology
also for achieving circadian medicine. But,
the pluripotent stem cells or embryonicstem 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 thisquestion. The current study by Rosselot etal (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 weredifferent in terms of their maturity, cell origin, or induction process and reported thathuman intestinal organoids (HIO) induced
from the pluripotent stem cells did notshow circadian rhythm. On the other hand,
the more mature structures such as humanintestinal enteroids (HIEs) showed robust
circadian oscillation of cardinal clockcontrolled genes (Fig 1). Importantly, they
demonstrated that the HIE possessed thecircadian phase-dependent necrotic cell
death response to Clostridium difficile toxin
B (TcdB). They also showed that the mouseintestinal enteroids’ response was antiphasic to that of the human intestinal enteroids. Strikingly, the authors revealed thatthe expression of the Rac1 gene, one of
the target genes of TcdB, was oscillating
in a circadian manner. Furthermore, theoscillation of Rac1 expression in mice wasanti-phasic to humans, consolidating thepotential role of Rac1 in the circadian phasedependent sensitivity to TcdB. The workby Rosselot and colleagues provides a firstdetailed 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. Thereare just a handful of genes that are commonly oscillating in different organs. The
transcription of these core clock genes islargely in phase in various organs. It is an

1 Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Suita, Japan
2 Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan*Correspondence. E-mail: uedah-tky@umin.ac.jp
DOI 10.15252/embj.2021110157 | The EMBO Journal (2022) 41: e110157 | Published online 22 November 2021ª 2021 The Authors

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41: e110157 | 2022

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Rikuhiro G Yamada & Hiroki R UedaATURAT I O N© EMBOM

Figure 1. Tissue-level oscillations of gene expression and drug responses in mouse and human intestinal organoids.
The recent methodological advances have realized self-organizing 3D culture systems that are histologically similar to a variety of human organs. Rosselot et al (2021)
report that mature organoids possess circadian clocks and are susceptible to time-controlled dosing of drugs.

attractive challenge to reveal the designprinciple of the circadian clock by investigating how the robust ~24 h oscillation
emerges from the core clock genes and howeach tissue builds its unique set of oscillating genes by observing organoids developing
into different organs. This approach may
also highlight some evolutionary differencesof peripheral clocks, such as the moleculardifferences between nocturnal and diurnal
animals.
One major hurdle to overcome in achieving circadian medicine is to measure theprecise time (the oscillation phase) of thesubject 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 formolecular markers that are useful to determine the time of the organ. As organoids
are promising technology in the interdisciplinary fields of medicine and biology, thecircadian 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.

2 of 2The EMBO Journal 41: e110157 | 2022

Conflict of interest
The author declares no conflict of interest.

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