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Melatonin improves the reprogramming efficiency of murine-induced pluripotent stem cells using a secondary inducible system

Authors

  • Shuai Gao,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. National Institute of Biological Sciences, NIBS, Beijing, China
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    • These authors contributed equally to this work.
  • Zhi-Long Wang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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    • These authors contributed equally to this work.
  • Ke-Qian Di,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. Health Science Center, Hebei University, Baoding, Hebei, China
    3. Department of Animal Reproduction, Faculty of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei, China
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    • These authors contributed equally to this work.
  • Gang Chang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. National Institute of Biological Sciences, NIBS, Beijing, China
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  • Li Tao,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. College of Life Science, Jilin Agricultural University, Changchun, Jilin, China
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  • Lei An,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Fu-Jia Wu,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Ji-Qiang Xu,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. Department of Animal Reproduction, Faculty of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei, China
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  • Ying-Wei Liu,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
    2. College of Life Science, Jilin Agricultural University, Changchun, Jilin, China
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  • Zhong-Hong Wu,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Xiang-Yun Li,

    1. Department of Animal Reproduction, Faculty of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei, China
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  • Shaorong Gao,

    Corresponding author
    1. National Institute of Biological Sciences, NIBS, Beijing, China
    • Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Jian-Hui Tian

    Corresponding author
    • Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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Address reprint requests to Jian-Hui Tian, College of Animal Science and Technology, China Agricultural University, Yuanminyuan West Road 2#, Beijing 100193, China.

E-mail: tianjh@cau.edu.cn

or Shaorong Gao 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China.

E-mail: gaoshaorong@nibs.ac.cn

Abstract

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all-iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10−6, 10−7, 10−8, 10−9, or 10−10 m) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10−8 (0.81 ± 0.04%), and 10−9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (< 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis-related genes p53 and p21 was lower in the group treated with 10−9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53-mediated apoptotic pathway.

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