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Simultaneous and quantitative monitoring transcription factors in human embryonic stem cell differentiation using mass spectrometry–based targeted proteomics

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Abstract

Human embryonic stem cells (hESCs) can be self-propagated indefinitely in culture while holding the capacity to generate almost all cell types. Although this powerful differentiation ability of hESCs has become a potential source of cell replacement therapies, application of stem cells in clinical practice relies heavily on the exquisite control of their developmental fate. In general, an essential first step in differentiation is to exit the pluripotent state, which is precariously balanced and depends on a variety of factors, mainly centering on the core transcriptional mechanism. To date, much evidence has indicated that transcription factors such as Sox2, Oct4, and Nanog control the self-renewal and pluripotency of hESCs. Their expression displays a restricted spatial-temporal pattern and their small changes in level can significantly affect directed differentiation and the cell type derived. So far, few assays have been developed to monitor this process. Herein, we provided a mass spectrometry (MS)–based approach for simultaneous and quantitative monitoring of these transcription factors, in an attempt to provide insight into their contributions in hESC differentiation.

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Funding

The authors are grateful for the financial support from Natural Science Foundation of China (21722504, 21675089), SEU-NJMU cooperation project (2242017K3DN12), SEU-NJMU-CPU cooperation project (2242019K3DNZ2), Primary Research & Development Plan of Jiangsu Province (BE2018725), and Open Foundation of State Key Laboratory of Reproductive Medicine [SKLRM-GA201804] awarded to Dr. Chen, and Natural Science Foundation of China (21605086), Natural Science Fund Project of Colleges in Jiangsu Province (16KJB150028) and Fundamental Research Funds for the Central Universities(2042019kf0129) awarded to Dr. Xu.

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Author notes

  1. Mengying Xu, Lei Xu and Jianxiang Cao contributed equally to this work.

Authors and Affiliations

  1. School of Pharmacy, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 211166, China

    Mengying Xu, Lei Xu, Jianxiang Cao, Yechen Hu, Feifei Xu, Yan Liu & Yun Chen

  2. Key Laboratory of Cardiovascular & Cerebrovascular Medicine, 211166, Nanjing, China

    Yan Liu & Yun Chen

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  1. Mengying Xu
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  2. Lei Xu
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Contributions

Mengying Xu and Jianxiang Cao did the MS experiments and performed the statistical analysis. Lei Xu prepared the cells for the study. Yechen Hu and Feifei Xu drafted the manuscript. Yan Liu and Yun Chen designed the study and provide technological supports. All authors read and approved the final manuscript.

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Correspondence to Yan Liu or Yun Chen.

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This study was approved by the Institutional Review Board of Nanjing Medical University, Nanjing, China.

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Xu, M., Xu, L., Cao, J. et al. Simultaneous and quantitative monitoring transcription factors in human embryonic stem cell differentiation using mass spectrometry–based targeted proteomics. Anal Bioanal Chem 413, 2081–2089 (2021). https://doi.org/10.1007/s00216-021-03160-7

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