Circadian clocks: from stem cells to tissue homeostasis and regeneration

Abstract

The circadian clock is an evolutionarily conserved timekeeper that adapts body physiology to diurnal cycles of around 24 h by influencing a wide variety of processes such as sleep‐to‐wake transitions, feeding and fasting patterns, body temperature, and hormone regulation. The molecular clock machinery comprises a pathway that is driven by rhythmic docking of the transcription factors BMAL1 and CLOCK on clock‐controlled output genes, which results in tissue‐specific oscillatory gene expression programs. Genetic as well as environmental perturbation of the circadian clock has been implicated in various diseases ranging from sleep to metabolic disorders and cancer development. Here, we review the origination of circadian rhythms in stem cells and their function in differentiated cells and organs. We describe how clocks influence stem cell maintenance and organ physiology, as well as how rhythmicity affects lineage commitment, tissue regeneration, and aging.

Glossary

Adpn

adiponutrin

Arnt

aryl hydrocarbon receptor nuclear translocator

Arntl

aryl hydrocarbon receptor nuclear translocator‐like

BAT

brown adipose tissue

Bmal1

brain and muscle ARNT‐like 1

bNGF

β‐nerve growth factor

Cav1

caveolin 1

CCG

clock‐controlled gene

CCM

cardiomyocyte‐specific CLOCK mutant

Cdk1

cyclin‐dependent kinase 1

CKIδ

casein kinase 1, delta

CKIε

casein kinase 1, epsilon

CLOCK

Circadian Locomoter Output Cycles Protein Kaput

Cry1

cryptochrome 1

Cry2

cryptochrome 2

Dbp

D site albumin promoter binding protein

Dgat2

diacylglycerol O‐acyltransferase 2

E4bp4

E4 promoter binding protein

E‐box

enhancer box

ES

embryonic stem

GLUT1/8

glucose transporter 1/8

HDAC3

histone deacetylase 3

Hlf

hepatic leukemia factor

HNF6

hepatocyte nuclear factor 6

KChIP2

Kv channel interacting protein 1

LIF

leukemia inhibitor factor

Lipe

lipase, hormone sensitive

NCoR

nuclear receptor co‐repressor

Npas2

neuronal PAS domain protein 2

Nr1d1/2

nuclear receptor subfamily 1, group D, member1/2

Per1

period circadian clock 1

Per2

period circadian clock 2

PGIF‐1

human placental growth factor

Pnpla2

patatin‐like phospholipase domain containing 2

Ppp1cc

protein phosphatase 1, catalytic subunit, gamma isoform

REV‐ERBα/β

reverse ERB

RGS2

regulator of G‐protein signaling 2

Rorα/β

RAR‐related orphan receptor alpha/beta

RRE

Rev‐ErbA/ROR response elements

SCA1

stem cell antigen 1

SCF

stem cell factor

SCN

suprachiasmatic nucleus

Slc2a1

solute carrier family 2 member 1

Slc2a8

solute carrier family 2 member 8

Tcap

titin‐cap

Tef

thyrotroph embryonic factor

TGFβ

transforming growth factor beta

TSPO

translocator protein

VEGF‐A

vascular endothelial growth factor A

WAT

white adipose tissue

ZT

Zeitgeber

Introduction

The fact that planet Earth spins around its axis every 24 h results in day and night cycles that influence organismal functioning, which is illustrated by circadian phenotypes in almost every form of life. The circadian clock, an evolutionarily conserved time‐keeping mechanism, allows organisms to anticipate the fluctuating environment, as illustrated by, for instance, the opening and closing of plant leaves, and to turn it into their advantage.

In mammals, the central or “master clock” consists of around 20,000 neurons that are located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light that is sensed by the retina is transmitted to the SCN, and via neural and humoral factors such as glucocorticoids and melatonin, “peripheral clocks” in all organs are synchronized to entrain the whole body to diurnal cycles. This rhythm persists in the absence of physiological clues (e.g., in complete darkness), has a “free‐running” period of around 24 h, and is even apparent in cells cultured in vitro 1, 2.

The molecular machinery that underlies the circadian clock consists of a transcriptional/translational feedback loop that is coordinated by the rhythmic binding of a heterodimer of two core clock transcription factors, BMAL1 and CLOCK, to enhancer boxes (E‐boxes) in the promoters of the negative regulators Period (Per) and Cryptochrome (Cry). Subsequent expression of Per and Cry suppresses the transcription of Bmal1 and Clock, thereby establishing an oscillating negative feedback loop (Fig 1). While this core pathway is shared among tissues, the resulting rhythmic transcription of clock‐controlled output genes (CCGs) is highly tissue‐specific. This is necessary to meet the physiological needs of every organ, and it has been shown that 43% of all protein‐coding genes display circadian expression in at least one organ 3. The liver, for instance, has up to 15% of its transcripts expressed in a diurnal manner 3, 4.

Figure 1. The molecular clock pathway.

Schematic of the transcriptional/translational feedback loop. The BMAL1:CLOCK heterodimer binds enhancer (E)‐Box elements within regulatory sequences of its target genes, such as Per, Cry, Rev‐erb, Ror, Dbp, Tef, Hlf, E4bp4, and clock‐controlled genes (CCGs). Upon transcriptional induction of Per and Cry, PER and CRY proteins accumulate and dimerize in the cytosol, where they either get degraded by the 26S proteasome upon CK1ε/δ‐mediated phosphorylation or from where they migrate to the nucleus to inhibit BMAL1:CLOCK transcriptional activity and therefore their own transcription. Upon gradual phosphorylation and ubiquitination by FBXL3, they are degraded in the nucleus, completing the first feedback loop. The second BMAL1:CLOCK‐dependent feedback loop is driven by rhythmic Ror and Rev‐erb transcription. Upon accumulation of their respective proteins in the cytosol, ROR and REV‐ERB shuttle to the nucleus where they activate/repress Bmal1 transcription via competitive binding to the REV‐ERB/ROR response (RRE) element in its regulatory sequences. Additional post‐transcriptional/translational/epigenetic modifications mediate robustness of the pathway, thereby establishing cycles of around 24 h of rhythmic BMAL1:CLOCK‐mediated transcriptional activation of CCGs.

The importance of maintaining proper clock function is illustrated by the fact that its disturbance is implicated in multiple pathological conditions, such as impaired metabolism, cardiovascular diseases, sleep disorders, cancer, and even hampered regenerative capacities 5. Therefore, the circadian clock is under intense investigation in differentiated cells, adult stem cells, and even embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells, derived from the inner cell mass of the blastocyst and can form all cells of the embryo proper 6. In vitro, these cells proliferate indefinitely and can give rise to derivatives of all three germ layers (ecto‐, meso‐, and endoderm). Indeed, directed differentiation toward clinically relevant cell types such as neurons 7, hepatocytes 8, and cardiac cells 9 is common practice nowadays. Therefore, these cells hold great promise for disease modeling, drug development as well as cell‐based regenerative therapies.

In contrast to ES cells, adult stem cells are multipotent cells that can proliferate and differentiate into specific lineages only. This type of stem cells is now known to be present in several organs, such as the bone marrow (hematopoietic stem cells), the intestine (intestinal stem cells), the skin (epidermal stem cells), the heart (cardiac stem cells), and many more organs 10. These cells provide a pool for cell renewal, thereby ensuring tissue homeostasis, but can also be activated specifically upon damage.

In this review, we describe how circadian rhythms evolve during embryonic stem cell differentiation, elaborate on the role of rhythmicity in adult stem cell maintenance, and discuss how the clock controls tissue physiology, regeneration, and aging by driving tissue‐specific gene expression programs.

The molecular clock comprises a transcriptional/translational feedback loop

Practically, all cells of the human body possess a functional circadian clock. The underlying molecular machinery involves a mechanism of transcriptional and translational feedback loops (Fig 1). The central players in this pathway are BMAL1 (brain and muscle ARNT‐like1) and CLOCK (Circadian Locomotor Output Cycles Kaput) that are encoded by the genes Arntl and Clock, respectively. Upon heterodimerization, these bHLH‐PAS (basic‐helix‐loop‐helix Per‐Arnt‐Single‐minded) proteins activate the transcription of other clock genes, such as Period (Per1‐2‐3) and Cryptochrome (Cry1‐2), by binding to E‐boxes near their respective promoters. Protein levels of PER and CRY are tightly regulated by stabilization and proteasome‐based degradation upon (de)phosphorylation 11, 12. After accumulation and dimerization of PER and CRY in the cytoplasm, they translocate to the nucleus in which they inhibit BMAL1:CLOCK‐mediated transcription 13 and therefore also their own transcription 14, 15, 16. Disturbing this molecular negative feedback loop results in an affected period or amplitude of circadian rhythmicity 5, 17.

A second feedback loop consists of two sets of nuclear receptors: the transcriptional activator ROR (RAR‐related orphan receptor, RORα/β) and transcriptional repressor REV‐ERB (REV‐ERBα/β, encoded by Nr1d1/2) that are all activated by the BMAL1:CLOCK heterodimer 18, 19. RORs and REV‐ERBs compete for Rev‐ErbA/ROR response elements (RRE) within the regulatory sequence of core clock genes such as Bmal1, Cry1, E4bp4, and Npas2 to fine‐tune their transcription 20, 21. In addition to transcriptional‐based circadian rhythms, non‐transcriptional oscillatory patterns in post‐transcriptional/translational modulation 22, chromatin modifications 23, binding of RNA binding factors 24, redox 25, and metabolic 26 fluxes also occur. They mainly stabilize the precise regulation of the well‐conserved clock pathway and contribute to its robustness (summarized in detail in 5).

Establishment of the clock through tissue‐specific transcription factors

The core pathway, present in every organ, ultimately results in a set of tissue‐specific clock‐controlled genes (CCGs) that are rhythmically expressed. With up to 15% of all mRNAs in a given tissue oscillating in a diurnal manner, these output genes reflect the specific temporal control of cellular physiology that is unique to each tissue 3. Intriguingly, different groups of genes peak at different times during the day (Fig 2). This is partially established by rhythmic binding of the BMAL1:CLOCK heterodimer onto E‐boxes in proximal and distal cis‐regulatory elements of CCGs. BMAL1:CLOCK abundance, and therefore, binding occurs within a specific and short time window (ZT (Zeitgeber) 6‐ZT9), but the transcription of other genes can peak at other time points in the 24‐h cycle, which suggests that additional regulatory mechanisms regulate the expression of different subsets of CCGs within one organ 27. Among the BMAL1:CLOCK‐controlled transcripts are a number of PAR‐bZIP genes, such as DBP, TEF, HLF, and E4BP4 that on their turn recognize D‐box motifs in the regulatory sequences of other CCGs. Circadian enhancers phasing in ZT9‐ZT12 were found to be enriched for this D‐box motif, while REV‐DR2/ROR motifs were found enriched in regulatory sequences of a distinct set of CCGs that peak around ZT18‐ZT24 27. The rhythmic binding of these respective binding factors (BMAL1/CLOCK, E4BP4, REV‐ERB/ROR) hints toward a molecular mechanism in which phase‐specific oscillators rhythmically influence circadian enhancers 27, 28.

Figure 2. Organ‐specific clock‐controlled genes peak at different times during the circadian cycle.

The central clock, located in the suprachiasmatic nucleus in the brain, synchronizes the clocks of peripheral clocks, which on their turn drive rhythmic expression of clock‐controlled genes (CCGs) that are often tissue‐specific (depicted as differentially colored heatmaps). This is mediated by tissue‐specific transcription factors that bind regulatory elements of CCGs, which results in peaks/phases of transcription at different ZTs (zeitgeber times).

Core clock factors are expressed in most organs, but CCGs are often organ‐specific. This can be explained by tissue‐specific chromatin conformations that are established by pioneering factors during development 29. These are able to open up nucleosome‐bound DNA, thereby opening up certain genomic regions to co‐factors that influence gene transcription. Once their binding motifs are accessible, core clock transcription factors can bind and drive rhythmic expression of CCGs by the recruitment of other regulatory complexes. In addition to this, rather than by direct binding, core clock factors can also be tethered to regulatory sequences of CCGs by cell type‐specific transcription factors. For instance, the liver‐specific transcription factor HNF6 (hepatocyte nuclear factor 6) recruits REV‐ERBα to HNF6 binding motifs in the regulatory sequences of liver metabolism genes. Subsequent recruitment of NCoR/HDAC3 (nuclear receptor co‐repressor‐histone deacetylase 3) by REV‐ERBα leads to rhythmic transcriptional repression, which results in oscillatory expression of liver metabolism genes 21, 30. For the heart, Jain and colleagues showed that the transcription factor KLF15 is responsible for REV‐ERBα‐mediated NCoR/HDAC3 facilitated oscillatory target gene expression 31. While this shows how tissue specificity is established, the tethering co‐factors that direct rhythmic target gene expression remain to be determined for many organs.

Tissue‐specific circadian clocks influence organ physiology

In a recent study, Zhang and colleagues profiled CCGs in 12 murine organs 3. These data, combined with other circadian experiments on tissues and cell lines, have been implemented in an online database (http://circadb.hogeneschlab.org) that facilitates a straightforward search for oscillators in any organ of interest. Mining this database can help to understand or explain tissue‐specific circadian physiology. For instance, CCGs in the murine heart are involved in cardiac glucose and fatty acid metabolism (e.g., Dgat2, Adpn, Ppp1cc 32), but also electrophysiology which results in oscillatory contractile properties 33 (e.g., Tcap 34 , Kv4.2, and KChIP2 35). Surprisingly, the cardiac‐specific clock not only drives rhythmic output under normal physiological conditions, but also under pathophysiological conditions as noted by its oscillating responsiveness to myocardial infarction mediated by oscillating phosphorylated Akt and GSK‐3 levels 36. In adipose tissue, the clock drives rhythmicity of lipogenesis and lipolysis, and even lipid release 37, via output genes such as Pnpla2 and Lipe 38. In the pancreas, the clock mainly regulates insulin secretion, the importance of which is indicated by the development of β‐cell failure and diabetes mellitus upon genetic clock ablation 39. In brown adipose tissue (BAT), REV‐ERBα modulates the response to cold tolerance via transcriptional repression of Ucp1 40, while the lung clock steers a phasic response to pro‐inflammatory stimuli 41, highlighting how the circadian clock can drive anticipation to stress.

The skin consists of numerous cell types, such as keratinocytes, melanocytes, and dermal fibroblasts, most of which have been shown to possess circadian clocks (expertly reviewed by Plikus et al 42). These compartmented dermal clocks mediate several circadian responses, such as cycles of DNA replication and repair in keratinocytes 42, 43, 44, 45, 46 as well as oscillatory waves of hair follicle regeneration via hair growth cycles 47. Indeed, BMAL^−/− mice show accelerated skin aging 48, suggesting an underlying oscillating pattern of CCGs driving skin homeostasis.

In conclusion, organ‐specific peripheral clocks influence tissue physiology in many ways by driving rhythmic and phased CCG mRNA expression, which entrains organs to deal with diurnal fluctuations of the environment.

The circadian clock in stem cell‐derived cells

In‐depth studies of the molecular clock and its CCGs in different murine organs have significantly increased our understanding of circadian rhythmicity. Nonetheless, the time resolution as well as a necessity of multiple replicates that are needed for these types of in vivo studies results in the requirement of large number of animals. This, in combination with limited options to study transcriptional rhythmicity in humans, has driven the investigation of use of in vitro stem cell‐derived cell types to investigate the circadian clock. This has led to the understanding that pluripotent embryonic stem (ES) cells do not possess a functional clock system (further discussed in the next section), but that a clock emerges in a spontaneous manner upon in vitro differentiation (Fig 3).

Figure 3. The circadian clock during in vitro (de)differentiation.

(A) Random differentiation of mouse embryonic stem (ES) cells leads to gradual activation of the molecular circadian clock, while reprogramming decreases rhythmicity of the expression of clock genes. (B) Directed differentiation of human ES cells toward the cardiac lineage leads to activation of the circadian clock that drives oscillatory gene expression of a set of clock‐controlled genes.

In murine pluripotent stem cells, circadian rhythms were shown to be established when differentiation is induced upon withdrawal of leukemia inhibitor factor (LIF) (passive) or by the addition of retinoic acid (active) 49, 50, 51. When reversing differentiation through reprogramming 52, the clock is switched off again 49 (Fig 3A), which indicates that the (in)activation of the diurnal clock is a reversible process that is intensively linked with the differentiation state of a cell.

We have recently shown that human ES cells also lack a circadian clock 53 (Fig 3B) and that a functional circadian clock emerges after directed differentiation toward cardiac cells by the use of growth factors (BMP4 and activin A) 53. In these human ES cell‐derived cardiac cultures, ~7.5% of the transcriptome was expressed with significant diurnal rhythmicity, 20% of which had been shown to oscillate in the murine heart 3. This suggests conserved mechanisms between both species, but still the overlap is modest. Differences might, however, be explained by factors such as cellular heterogeneity. Most circadian transcriptomic experiments are done on whole murine hearts, which include atrial, ventricular and nodal cardiomyocytes, smooth muscle cells, endothelial cells, epicardium cells, pacemaker cells, Purkinje cells, and cardiac fibroblasts 54. Our human ES cell‐derived cardiac cultures are mixed cultures as well, but with a different constitution of cells compared to the murine heart. In addition, in vitro differentiated cardiac cells are known to remain embryonic‐like. Importantly, however, in vitro‐derived cardiac cells are the best proxy for transcriptional oscillations in human hearts. We uncovered several genes, known for their role in human heart physiology, that were expressed in an oscillatory manner. For instance, factors such as TSPO (translocator protein), a therapeutic target, CAV1 (caveolin‐1), an ion channel regulator, PLN (phospholamban), a cardiac contractility regulator, RGS2 (regulator of G‐protein signaling 2), an inhibitor of cardiac hypertrophy, showed rhythmic transcription.

This highlights that unraveling the oscillating transcriptome or proteome of in vitro‐derived cells can lead to the discovery of previously unidentified oscillators that might influence organ physiology as well as therapeutic efficacy. We thus believe that studying phenotypical oscillations of in vitro differentiated human ES cells can serve as a proxy for circadian organ parameters in vivo.

Absence of a functional circadian clock in embryonic stem cells

Conclusions on the absence of a functional circadian clock in murine and human pluripotent ES cells were based on qRT–PCR experiments as well as reporter assays, and while no rhythmicity of clock genes could be observed, most of the core clock genes were found to be expressed in these cells 49, 53, 55. It is evident that the stoichiometry of their transcription is different compared to differentiated cells. Most of the core clock factors are expressed at lower levels in pluripotent stem cells compared to differentiated cells, but Cry1 is an exception as expression levels are higher in stem cells 49, 53, 55 (Fig 4). Since expression ratio of the core genes and availability of clock proteins are the main mechanism by which the diurnal oscillatory network is established and maintained, it is to be expected that such changes result in the absence of a functional clock.

Figure 4. Altered clock factor stoichiometry might shift the functional role of core clock genes toward modulation of proliferation.

ES cells express core clock factors (ARNTL, PER2, CLOCK, CRY1, and NR1D1) at a different level than differentiated cells. These factors may exert non‐circadian roles in proliferative ES cells that is distinct from their circadian and cell division clock‐related role in differentiated cells.

Aberrant localization of core clock proteins might also contribute to a non‐functional clock in ES cells. It has been proposed that importin α2, encoded by Kpna2, plays a role in this. Importin α2 is a nuclear transporter that shuttles specific pluripotency factors, such as OCT3/4 and differentiation‐related factors like OCT6, out of the nucleus in mouse ES cells in order to retain a pluripotent state 56. This nuclear transporter regulates the cellular differentiation to maintain an undifferentiated state, which induces the retention of core clock factors PER1 and PER2 out of the nucleus, thereby preventing a functional clock pathway through the disturbance of the PER‐based negative feedback loop 57. It is not known yet whether the same holds true for human ES cells, but importin α2 levels gradually decreased upon differentiation toward cardiomyocytes 53, which suggests that similar regulatory mechanisms are involved.

Both stoichiometry and localization differences may contribute to the absence of a clock in ES cells. It would be interesting to study the (transcriptional) effect of overexpression of certain (sets of) core clock factors to see whether this will speed up transcriptional circadian clock formation during differentiation or even whether a functional clock could be established in pluripotent ES cells without them differentiating.

Why are clock genes expressed in embryonic stem cells?

As they are expressed, the question remains whether clock factors do play a role in ES cells even though they do not participate in the functional clock wheelwork that drives the well‐known circadian rhythmicity of CCG expression in differentiated cells. It is known that clock factors control essential cell cycle genes. PER2, for example, can activate p16 (Ink4a), which inhibits the G1‐S transition, in murine fibroblasts 58. Per2 levels in ES cells are lower compared to differentiated cells 49, 53, which might possibly allow stem cells to proliferate faster than differentiated cells. The finding that mice devoid of Per2 are cancer prone is in line with this 59. CRY1 levels on the other hand are upregulated in colorectal cancer cells, and overexpression of Cry1 accelerates the proliferation of HCT116 cells 60. Increased Cry1 levels in ES cells compared to differentiated cells 49, 53 might therefore also influence the difference in proliferation rates between these cell types. CLOCK⁻/⁻ mouse embryonic fibroblasts display hampered proliferation with concomitant overexpression of p21 and p27, two inhibitors of cellular proliferation 61. In line with this, a recent report demonstrated that CLOCK KO mouse ES cells exhibit decreased proliferation (associated with lower mRNA levels of c‐Myc, Cdk1, and cyclin D1) and increased apoptosis, while pluripotency was unaffected 62. This suggests a strong interaction between cell cycle regulation and clock factors and indicates that altered stoichiometry of clock factors in pluripotent stem cells might shift the role of these factors from regulators of diurnal rhythmicity to key regulators of the cell cycle (Fig 4).

While this hints toward similar consequences of clock factors in proliferation in stem, cancer as well as differentiated cells, other findings report that the regulation of clock genes in stem cells is different or even opposite from the one in differentiated cells. For example, overexpression of a dominant‐negative (DN) form of Bmal1, a positive regulator of Per, Cry, and Dbp in NIH3T3 cells, leads to broad suppression of their mRNA levels. In contrast, overexpression of DN‐Bmal1 in mouse ES cells had no effect on Cry and Dbp expression and induced (rather than reduced) Per2 mRNA levels 49. Remarkably, no attempts to knockout Bmal1 or Per2 have been reported for in vitro‐cultured ES cells. Other (combinatorial) knockout ES cells have been generated for Cry1, Cry2, CKIδ, CKIε, and while these cells show no apparent phenotype when pluripotent, defects in circadian rhythmicity become apparent upon in vitro differentiation 63.

The latter is in line with the observation of impairments later in life rather than at embryonic stages in clock gene knockout mice. Bmal1, Clock, and Per2 knockout mice are not embryonic lethal 48, 64, 65, but a phenotype of premature aging and age‐related pathologies were noted in Bmal1 knockouts 48. That the basis for this is possibly laid during development is indicated by the fact that deletion of both Bmal1 alleles in adult mice (deleted at 3 months of age) does not result in the reduced lifespan, defects in hair regrowth, arthropathy, atherogenesis, and reduced body weight as seen in Bmal1 knockout mice 66. This likely means that BMAL1 exerts a non‐clock‐related role during embryonic development that is important for proper organismal functioning at later stages in life. One could speculate that embryonic BMAL1 depletion leads to epigenetically modified regulatory regions, the result of which only becomes prevalent upon adulthood. Therefore, it would be commendable to study the BMAL1 cistrome (and that of other clock factors) during development and in ES cells, to analyze the putative transcriptional program that is established and which may explain dominant phenotypes later in life.

The role of the clock in adult stem cells

Adult stem cells have the capacity to proliferate as well as to differentiate and, in contrast to embryonic stem cells, contain a functional circadian clock (reviewed by Weger et al 67). Rhythmicity is confirmed to contribute to the properties of these multipotent cells that assure a constant renewal of cells in (some) human organs and can become activated upon injury.

The intestinal epithelium is a good example of this as its stem cells constantly renew the epithelial layer that lines the gut and facilitate regeneration via proliferation and differentiation upon damage 68. The latter has been shown to depend on rhythmic Per and Cycle expression which influences transcription of more than 100 CCGs that are involved in stress response and regeneration 69. Similarly, intestinal stem cell proliferation exhibits a diurnal rhythm in mice that suffer from gastrointestinal syndrome. Here, BMAL1 controls the inflammatory cytokine TNF that drives both p21 and a jnk expression‐mediated stress response, thereby coordinating intestinal regeneration 70.

Similar observations were made in the skin. A robust clock is present in stem and progenitor cells of the basal epidermis, and circadian rhythmicity establishes diurnal cycles of DNA replication and regeneration 43, 44, 45, 46, 71, 72. Interestingly, patchy expression of clock genes creates heterogeneity in the dormant hair follicle stem cells, which results in a subset of cells being more prone to activation. BMAL1 (clock) high subpopulations show more WNT activity and less TGFβ signaling in comparison with clock low cells. Clock ablation leads to the disruption of this equilibrium and results in accumulation of dormant stem cells in Bmal1 knockouts and depletion of the stem cell population upon loss of Per2, which results in premature epidermal aging and increased tumor formation 71. These different phenotypes hint toward a factor‐specific, clock‐unrelated role for these proteins in aging. Another two recent studies show that epidermal as well as skeletal muscle stem cells retain robust circadian rhythms upon aging, but that the set of oscillatory output gene changes from genes involved in homeostasis to tissue‐specific stress‐associated genes involved in DNA damage and inefficient autophagy 73, 74. The authors could prevent age‐associated rewiring of the circadian transcriptome by long‐term caloric restriction in aged mice, which is line with an earlier found correlation between low‐fat diet and reduced aging 73, 75. This means that stem cells can adapt their circadian transcriptome to different conditions of metabolic states and organ‐specific stresses. The question remains whether additional adult stem cell types show this transcriptomic rewiring. Understanding the true mechanism behind this age‐associated switch could lead to therapies ensuring healthy aging.

The circadian clock in (heart) disease and regeneration

The clock has not only been shown to influence healthy (and/or aged) stem cells, but also cancerous cells. Leukemic stem cells have the capacity to self‐renew and are able to propagate disease upon serial transplantation. They display circadian rhythms, and disruption of the clock genes BMAL1 and CLOCK hampers their proliferation, induces myeloid differentiation, and depletes the leukemic stem cell pool 76. This suggests that core clock genes affect disease and that this system could possibly be targeted for (cancer) therapy.

Circadian rhythms have also been shown to play a major role in heart pathophysiology. This is illustrated by increased incidence of myocardial infarction in the morning compared to the evening 77, 78, 79, and a correlation between infarct size and the time of the day at which it occurs is noted 80, 81, 82. Mice that overexpress a dominant‐negative form of the CLOCK protein in cardiomyocytes specifically (CCM) show abolished rhythmicity in cardiac CCG expression, which results in an abrogated circadian response in infarct size after ischemia/reperfusion 36. This shows that the cardiac‐specific clock is important for the heart's response to external stress and that it contributes to the organ its regenerative potential. This is most likely driven by the oscillatory expression of a set of transcripts, but the identity of these CCGs is extremely difficult to uncover in human hearts. Nonetheless, a circadian network of stress‐associated genes was discovered in human ES cell‐derived cardiomyocytes 53. This was found to result in a rhythmic response to doxorubicin, a widely used anti‐cancer drug that causes severe cardiotoxic side effects 83.

We now know that this is not restricted to in vitro ES cell‐derived cardiac cultures as neonatal rat cardiomyocytes 84 and SCA1⁺ stem cells derived from human hearts 85 also show an oscillatory response to external stressors. Next to that, human SCA1⁺ cardiac stem cells showed circadian rhythmicity in proliferation and the secretion of a number of paracrine factors, such as β‐nerve growth factor (bNGF), stem cell factor (SCF), human placental growth factor (PGIF‐1), and vascular endothelial growth factor A (VEGF‐A) 85, was found to be rhythmic as well. Strikingly, VEGF‐A, a well‐known growth factor involved in angiogenesis, also oscillated in human ES cell‐derived cardiomyocytes 53 and murine hearts 3. This suggests that the rhythmic nature of this pro‐angiogenic factor may contribute to the oscillatory reaction of the heart to stress. In light of cell‐based cardiac regeneration, one could speculate whether the contribution of human ES cell‐derived cardiomyocytes, or human cardiac stem cells to repair, could be increased by timed administration after cardiac injury.

Future perspectives

In the mammalian body, nearly all cells possess a functional clock to anticipate diurnal variations. Over the last decade, the tissue specificity of the clock has become apparent, which is now known to be established by the interplay between transcription factors and regulatory elements within or close to CCGs. These insights were gained from bulk tissue samples that consist of a heterogeneous population of cells. While the circadian clock is principally organized to synchronize cells, differences in levels of clock genes (and more importantly output genes) have been described. The presence and consequences of this remain to be determined for many (single) cell types within one organ.

Focusing on defined cell types may also help to unravel the mechanisms behind the birth of the clock. There is no functional clock at the earliest steps of development (the oocyte and early zygote), but clock factor levels increase after fertilization, which allows for the gradual development of diurnal clock gene rhythmicity during and after gestation 86, 87, 88. This is in line with the emergence of circadian rhythmicity during differentiation of embryonic stem cells that do not possess a functional circadian clock 49, 53. Clock genes are expressed in ES cells, however, and the emergence of a functional clock system is closely linked to differentiation 49, 50, 51, 53, but these factors may have additional functions in those cells. The intimate link between clock proteins and the cell cycle was discussed above, and stem cell proliferation may be regulated by these factors. Future studies using inducible knockout systems could possibly shed light on the clock‐unrelated roles of clock proteins in pluripotent ES cells (see also Box 1).

Box 1: In need of answers.

1. Do clock factors exert non‐circadian roles in embryonic stem cells?

   Although ES cells do not possess a classical transcriptional/translational feedback loop‐based circadian clock, they do express most of the clock factors. Whether they exert specific roles in stem cell maintenance is unknown so far, but knockout during development can lead to premature aging later in life.

2. Which machinery drives metabolic rhythms in embryonic stem cells?

   ES cells do not possess a functional circadian core clock machinery, but they do show rhythmic glucose uptake. Metabolic oscillations have also been observed in other cell types such as red blood cells that show oxidation–reduction cycles of peroxiredoxins. Understanding how metabolic ES cell rhythms are established could help to uncover additional non‐traditional oscillatory systems.

3. Do organ‐specific clocks consist of cell type‐specific clocks?

   Clock‐controlled genes are organ‐specific and mostly identified in bulk tissue. Deconvoluting this into cell type‐specific output genes may help to understand how subclocks are synchronized to retain proper organ physiology.

4. Can we apply knowledge of circadian rhythms in human embryonic stem cell‐derived cells for regenerative purposes?

   Differentiating human ES cells toward specific lineages provides an unlimited source of cells that could be used for therapy. In addition, they can be used to identify clock‐controlled genes of (specific) cell types that cannot be investigated for humans otherwise. Uncovering oscillatory networks involved in cell survival as well as factor secretion could ultimately be used to ameliorate cell‐based therapy after injury.

5. Are small‐molecule modulators of the circadian clock specific and safe?

   The generation of synthetic clock modulators has gained attention over the last years. While some have been shown to have beneficial effects in multiple organs/diseases, the specificity and safety of these compounds are underexplored. Thorough investigation will be needed before taking these modulators of the circadian clock to the clinic.

A functional clock is present in adult stem cells, in which circadian oscillations have been shown to drive proliferation and differentiation, thereby facilitating tissue homeostasis and regeneration 89. Targeting the clock in adult stem cells in vivo by the use of drugs might enhance regeneration after damage. Another route for treatment could be ex vivo culturing of stem cells to synchronize their clocks and administering them in the time window in which the patient is most receptive and in which the cells are most likely to engraft. Additional knowledge on the circadian clock in adult stem cells could thus allow for or improve stem cell‐based therapies.

A set of novel synthetic ligands that target components of the clock pathway are currently being investigated for different purposes 90, 91. Compounds, such as SR9009 and SR9011, have been shown to activate the clock factors REV‐ERBα/β, the role of which has been studied extensively in oscillatory metabolic processes of the liver. While these REV‐ERB agonists have been shown to be (beneficial) for energy expenditure and to reverse diet‐induced obesity 92, 93, the effects of these drugs should be closely examined for ramifications on other cellular processes. For instance, SR9009 and SR9011 were shown to induce an anti‐proliferative response in leukemic stem cells 76, therefore providing potential new therapeutic avenues, but their consequences in healthy adult stem cells are unknown. In‐depth characterization of small‐molecule modulators of circadian factors is needed, as it may provide powerful tools for tissue regeneration as well as anti‐cancer therapies.

In conclusion, the circadian clock plays a major role in facilitating tissue homeostasis and regeneration via a number of processes. Circadian rhythms drive stem cell metabolism, self‐renewal, and differentiation, and can even create stem cell heterogeneity in one tissue to protect the organism from stem cell depletion upon activation. Finding methods to preserve the circadian rhythm in stem cells will unequivocally lead to proper tissue homeostasis and healthy aging.

Conflict of interest

The authors declare that they have no conflict of interest.

See the Glossary for abbreviations used in this article.