Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kozomara A, Griffiths-Jones S (2011) MiRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157
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
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
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
Lambert TJ (2019) FPbase: a community-editable fluorescent protein database. Nat Methods 16:277–278
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
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
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
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
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
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
Endo K, Hayashi K, Saito H (2019) Numerical operations in living cells by programmable RNA devices. Sci Adv 5:eaax0835
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
Acknowledgments
We would like to thank Dr. Peter Karagiannis and Ms. Miho Nishimura (Kyoto University) for English proofreading and administrative support, respectively.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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. https://doi.org/10.1007/978-1-0716-1441-9_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1441-9_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1440-2
Online ISBN: 978-1-0716-1441-9
eBook Packages: Springer Protocols