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Review
. 2024 Feb 8;25(4):2063.
doi: 10.3390/ijms25042063.

"Time Is out of Joint" in Pluripotent Stem Cells: How and Why

Francesca Agriesti  1 , Olga Cela  1 , Nazzareno Capitanio  1
Affiliations
Review

"Time Is out of Joint" in Pluripotent Stem Cells: How and Why

Francesca Agriesti et al. Int J Mol Sci. .

Abstract

The circadian rhythm is necessary for the homeostasis and health of living organisms. Molecular clocks interconnected by transcription/translation feedback loops exist in most cells of the body. A puzzling exemption to this, otherwise, general biological hallmark is given by the cell physiology of pluripotent stem cells (PSCs) that lack circadian oscillations gradually acquired following their in vivo programmed differentiation. This process can be nicely phenocopied following in vitro commitment and reversed during the reprogramming of somatic cells to induce PSCs. The current understanding of how and why pluripotency is "time-uncoupled" is largely incomplete. A complex picture is emerging where the circadian core clockwork is negatively regulated in PSCs at the post-transcriptional/translational, epigenetic, and other-clock-interaction levels. Moreover, non-canonical functions of circadian core-work components in the balance between pluripotency identity and metabolic-driven cell reprogramming are emerging. This review selects and discusses results of relevant recent investigations providing major insights into this context.

Keywords: cellular differentiation; circadian rhythm; clock genes; pluripotent stem cells; reprogramming.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
The circadian clock function is coupled to cellular differentiation. (a) Schematic representation of the transcriptional/translational feedback loops (TFFLs) of the circadian clock pathway. The transcription factors BMAL1 and CLOCK binds to E-boxes and drive the expression of clock-controlled genes (CCG) and their own inhibitors, PER1 and CRY1, which, if not degraded, block BMAL1::CLOCK transcriptional activity in a primary feedback loop. The ROR and REV-ERVB transcription factors govern the second feedback loop dependent on BMAL1::CLOCK. Through competitive binding to the ROR/REV-ERB-response element (RORE) in regulatory sequences, their proteins activate or repress Bmal1 transcription. The pathway’s robustness is further influenced by post-transcriptional, translational, and epigenetic modifications, ensuring the establishment of approximately 24 h rhythmic cycles of BMAL1::CLOCK-mediated transcriptional activation in CGCs. (b) Emergence of the circadian clock during differentiation. The core TTFLs of the circadian molecular oscillation in PSCs are not detectable but exit from pluripotency, and subsequent commitment of PSCs induces a cell-autonomous robust circadian oscillation that disappears after reprogramming differentiated cells into induced pluripotent cells (iPSCs). The diagram of the proteins shown is from the respective PDB depository code and created with BioRender.com. Ub-ligase, ubiquitin ligase; ncRNA, non-coding RNA.
Figure 2
Figure 2
Generation of pluripotent stem cells. The diagram compares the derivation of embryonic stem cell lines (ESCs) from the inner cell mass of the blastocyst and how iPSCs are derived from somatic cells following induction of the “Yamanaka factors” (Oct4, Kfl4, Sox2, cMyc) by different reprogramming methods. These PSCs can be expanded indefinitely and then be directed to differentiate in vitro into clinically relevant cell types. Cells differentiated from PSCs are expected to contribute to disease modelling in vitro and to regenerative medicine as cell therapies. The icons of the clock without and with hands imply the absence or presence of circadian oscillators, respectively, in the different cell types shown. SeV, Sendai virus; AdVs, adenovirus.
Figure 3
Figure 3
Differential cytosol–nucleus trafficking of circadian factors. Cytoplasmic retention of PER proteins and differentiation factors due to up-expression of importin α2 in PSCs prevents the proper nuclear function of the negative feedback loop required for cyclic circadian regulation and commitment induction (upper panel). Alternative expression of KPNB1 and importin α1 induces nuclear localization of core clock proteins along with commitment factors, resulting in establishment of circadian oscillations (lower panel). The look of the proteins shown is from the respective PDB depository code and created with BioRender.com. TTFL, transcriptional/translational feedback loops; PSC, pluripotent stem cell. See text for further details.
Figure 4
Figure 4
Mechanisms underlying the inhibition of circadian oscillation in pluripotent stem cells. (a) Though being expressed at the mRNA level, CLOCK protein translation is almost absent in PSCs because of microRNA (Dicer/DGCR8)-mediated post-transcriptional repression. (b) Hypermethylation of histone H3 lysine 27 trimethylation (H3K27me3) in the Per1 promoter, catalyzed by the histone methyl-transferase EZH2 (enhancer of zest homologue) of polycomb repressive complex 2 (PRC2), is responsible for circadian rhythm repression in PSCs. The image of the proteins shown is from the respective PDB depository code and created with BioRender.com. Green and red arrows stand for up- and down-regulation of EZH2/PRCs, respectively. See text for further details.
Figure 5
Figure 5
Biological significance of the delayed emergence of circadian clock oscillation in mammalian development. The upper panel shows the early- to middle-developmental stages where the cell-autonomous ultradian rhythm of the somitogenic segmentation clock, driven by a negative feedback loop involving Hes7 oscillation and NOTCH signaling, is essential for a proper developmental process. Expression of the key circadian components CLOCK/BMAL1 (shown in the lower panel) interferes with the Hes7 oscillations-mediated segmentation clock underlying the need of suppressing a functional CLOCK/BMAL1-mediated circadian clockwork for an unharmed process of mammalian embryogenesis to develop. NICD, Notch intracellular domain; LFNG, Beta-1,3-N-acetylglucosaminyltransferase lunatic fringe. The image of the proteins shown is from the respective PDB depository code and created with BioRender.com. See text for further details.
Figure 6
Figure 6
Clock-independent function for circadian clock genes in pluripotent stem cells. The upper panel shows the non-canonical function of CRY1 in controlling the capacity of self-renewal, stemness maintenance, and metabolic programs unique to PSCs. Nuclear accumulation of CRY1 is driven by down-regulation of its AMPK-mediated proteolytic degradation. The low expression of BMAL1, known to positively modulate mitochondrial respiratory functions, contributes to the PSCs’ metabolic signature. The lower panel illustrates the reversal of the above-mentioned PSCs features following induction of their differentiation consequently to down- and up-regulation of CRY1 and BMAL1, respectively. It is highlighted that the transcriptional activity of CRY1 and BMAL1 is exerted on gene responsive elements unrelated to the circadian clockwork. The diagram of the proteins shown is from the respective PDB depository code and created with BioRender.com. Green and red arrows stand for up- and down-regulation of mitochondrial and glycolytic functions, respectively. Question mark indicates unknown co-transcription factor. See text for further details.
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Grants and funding

This research was financed by local funds from the University of Foggia, Progetti di Ricerca d’Ateneo (PRA-2022).

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