Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.
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.
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.|Research Center on Aging and Medicine, Fudan University, Shanghai, 200032, China.
Chao
http://orcid.org/0000-0002-9484-6415
C
Lu
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Yang
http://orcid.org/0000-0002-3007-6861
Y
Yang
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Ran
R
Zhao
Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Bingxuan
B
Hua
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Chen
C
Xu
Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
Zuoqin
Z
Yan
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China. sunning@fudan.edu.cn.|Research Center on Aging and Medicine, Fudan University, Shanghai, 200032, China. sunning@fudan.edu.cn.|State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, 200032, China. sunning@fudan.edu.cn.
Ning
N
Sun
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China. rzqian@shmu.edu.cn.|Research Center on Aging and Medicine, Fudan University, Shanghai, 200032, China. rzqian@shmu.edu.cn.
Ruizhe
R
Qian
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D056926:CLOCK Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D049109:Cell Proliferation; D065150:Cellular Reprogramming; D057906:Circadian Clocks / Q000235:genetics*; D000072669:Gene Editing; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
C496503:Foxa2 protein, mouse; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D051542:Hepatocyte Nuclear Factor 3-beta; D056926:CLOCK Proteins; C538944:Clock protein, mouse
Circadian gene Clock; cell apoptosis; cell differentiation; cell proliferation; gene knockout; mouse embryonic stem cells; pluripotency
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