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Purification of Specific Cell Populations Differentiated from Stem Cells Using MicroRNA-Responsive Synthetic Messenger RNAs

Part of the Methods in Molecular Biology book series (MIMB,volume 2312)


Pluripotent stem cells have the potential to differentiate into various cell types that can be used for basic biological studies, drug discovery, and regenerative medicine. To obtain reliable results using the differentiated cells, the contamination of nontarget cells should be avoided. microRNAs (miRNAs) can serve as indicators to distinguish target and nontarget cells, because the activities of miRNAs are different among cell types.

In this chapter, we introduce a method to purify target cells using synthetic messenger RNAs (mRNAs) that respond to cell-specific miRNAs. The method is composed of five steps: mRNA sequence design, template DNA preparation by PCR, in vitro mRNA transcription, mRNA transfection into cells, and fluorescence-activated cell sorting. This synthetic mRNA-based cell purification method will advance various applications of pluripotent stem cells.

Key words

  • Cell sorting
  • microRNA
  • Stem cell
  • Differentiation
  • Synthetic mRNA
  • Synthetic biology
  • Regenerative medicine

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  • DOI: 10.1007/978-1-0716-1441-9_5
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  1. Kozomara A, Griffiths-Jones S (2011) MiRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157

    CrossRef  CAS  Google Scholar 

  2. Parr CJC, Katayama S, Miki K, Kuang Y, Yoshida Y, Morizane A, Takahashi J, Yamanaka S, Saito H (2016) MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells. Sci Rep 6:32532

    CrossRef  CAS  Google Scholar 

  3. Hirosawa M, Fujita Y, Parr CJC, Hayashi K, Kashida S, Hotta A, Woltjen K, Saito H (2017) Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch. Nucleic Acids Res 45:e118

    CrossRef  CAS  Google Scholar 

  4. Miki K, Endo K, Takahashi S et al (2015) Efficient detection and purification of cell populations using synthetic MicroRNA switches. Cell Stem Cell 16:699–711

    CrossRef  CAS  Google Scholar 

  5. Lambert TJ (2019) FPbase: a community-editable fluorescent protein database. Nat Methods 16:277–278

    CrossRef  CAS  Google Scholar 

  6. Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T (2015) N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release 217:337–344

    CrossRef  CAS  Google Scholar 

  7. Svitkin YV, Cheng YM, Chakraborty T, Presnyak V, John M, Sonenberg N (2017) N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density. Nucleic Acids Res 45:6023–6036

    CrossRef  CAS  Google Scholar 

  8. Parr CJC, Wada S, Kotake K, Kameda S, Matsuura S, Sakashita S, Park S, Sugiyama H, Kuang Y, Saito H (2020) N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells. Nucleic Acids Res 48:1–7

    CrossRef  Google Scholar 

  9. Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16:1833–1840

    CrossRef  Google Scholar 

  10. Nakanishi H, Miki K, Komatsu KR, Umeda M, Mochizuki M, Inagaki A, Yoshida Y, Saito H (2017) Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors. Biomaterials 128:121–135

    CrossRef  CAS  Google Scholar 

  11. Matsuura S, Ono H, Kawasaki S, Kuang Y, Fujita Y, Saito H (2018) Synthetic RNA-based logic computation in mammalian cells. Nat Commun 9:4847

    CrossRef  Google Scholar 

  12. Endo K, Hayashi K, Saito H (2019) Numerical operations in living cells by programmable RNA devices. Sci Adv 5:eaax0835

    CrossRef  CAS  Google Scholar 

  13. Endo K, Hayashi K, Saito H (2016) High-resolution identification and separation of living cell types by multiple microRNA-responsive synthetic mRNAs. Sci Rep 6:21991

    CrossRef  CAS  Google Scholar 

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We would like to thank Dr. Peter Karagiannis and Ms. Miho Nishimura (Kyoto University) for English proofreading and administrative support, respectively.

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Correspondence to Hirohide Saito .

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Nakanishi, H., Saito, H. (2021). Purification of Specific Cell Populations Differentiated from Stem Cells Using MicroRNA-Responsive Synthetic Messenger RNAs. In: Kojima, R. (eds) Mammalian Cell Engineering. Methods in Molecular Biology, vol 2312. Humana, New York, NY.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1440-2

  • Online ISBN: 978-1-0716-1441-9

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