Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.

Chao Lu,Yang Yang,Ran Zhao,Bingxuan Hua,Zuoqin Yan,Chen Xu,Ning Sun,Ruizhe Qian

doi:10.1007/s13238-016-0319-9

Abstract

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0319-9) contains supplementary material, which is available to authorized users.

Highlights

Circadian rhythm is a fundamental biological system in humans, animals and plants that regulates various physiological functions such as sleep–wake cycle, energy metabolism, cell division, post-transcriptional regulation (Koike et al, 2012; Cardinal-Aucoin and Steel, 2016; Lopez-Minguez et al, 2016; McAlpine and Swirski, 2016; Nitschke et al, 2016), and endocrine system (Gachon et al, 2004; Lowrey and Takahashi, 2004)
We found that Clock knockout mouse embryonic stem cells (mESCs) still exhibited normal clonal morphology and pluripotency, which indicated that Clock gene might not be required for regular maintenance of mESCs
The results showed that both alleles were edited at exon 2 of the Clock gene in all the 3 homozygous mESC clones (Fig. 1D and Fig. S1)

Summary

Introduction

Circadian rhythm is a fundamental biological system in humans, animals and plants that regulates various physiological functions such as sleep–wake cycle, energy metabolism, cell division, post-transcriptional regulation (Koike et al, 2012; Cardinal-Aucoin and Steel, 2016; Lopez-Minguez et al, 2016; McAlpine and Swirski, 2016; Nitschke et al, 2016), and endocrine system (Gachon et al, 2004; Lowrey and Takahashi, 2004). Role of Clock during differentiation of stem cells the generation of circadian rhythms (Dunlap, 1999). The master clock is regulated by four major groups of genes: Clock, Bmal, Cry (Cryptochrome 1 and 2), and Per (Period 1 and 2). These core circadian genes are regulated in a transcription-translation feedback loop (TTFL) (Xue et al, 2016). Some recent studies have shown associations between clock genes and chronic inflammation, blood pressure and energy intake in humans, supporting the importance of the circadian rhythm in cardiac physiology (Dashti et al, 2016; Johnston and Ordovas, 2016). Adult stem cell functions are regulated by circadian oscillations (Casanova-Acebes et al, 2013; Janich et al, 2013; Karpowicz et al, 2013)

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Results

Conclusion