Abstract
Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naïve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations. To overcome such limitations, we developed protocols for high-efficiency generation of either conventional or naïve iPSCs by delivery of messenger RNAs (mRNAs) using a microfluidic system. In this protocol we describe how to produce microfluidic devices, and how to reprogram human somatic cells into naïve and primed iPSCs using these devices. We also describe how to transfer the iPSC colonies from the microfluidic devices over to standard multiwell plates for subsequent expansion of the cultures. Our approach does not require stable genetic modifications, is reproducible and cost-effective, allowing to produce patient-specific iPSCs for cell therapy, disease modeling, and in vitro developmental studies.
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Acknowledgments
We thank the Martello and Elvassore laboratories for comments and suggestions. GM is supported by the Giovanni Armenise-Harvard Foundation, the Telethon Foundation (TCP13013) and The ERC Starting Grant (MetEpiStem). OG is supported by 2019 STARS Starting Grant of University of Padova. NE is supported by 2017 STARS-WiC Grant of University of Padova and Progetti di Eccellenza Cariparo.
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Zorzan, I., Gagliano, O., Elvassore, N., Martello, G. (2022). Using Microfluidics to Generate Human Naïve and Primed Pluripotent Stem Cells. In: Rugg-Gunn, P. (eds) Human Naïve Pluripotent Stem Cells. Methods in Molecular Biology, vol 2416. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1908-7_5
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DOI: https://doi.org/10.1007/978-1-0716-1908-7_5
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1907-0
Online ISBN: 978-1-0716-1908-7
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