Abstract
Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. We successfully downscaled an 8-d protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160 ± 20 mean ± s.d (n = 48)) in a single 27-mm2 microfluidic chamber) 15 d after seeding ~1,500 cells per independent chamber and under xeno-free defined conditions. Only ~20 µL of medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization because of the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold reduction in costs of raw materials compared to those for standard multiwell culture conditions. This sys...Continue Reading
References
Feb 3, 2007·Annual Review of Biophysics and Biomolecular Structure·Jessica Melin, Stephen R Quake
Nov 24, 2007·Cell·Kazutoshi TakahashiShinya Yamanaka
Aug 22, 2009·Genes & Development·Ana BanitoJesús Gil
Sep 1, 2009·Cell Stem Cell·Yoshinori YoshidaShinya Yamanaka
Oct 13, 2009·Cell Stem Cell·Justin K IchidaKevin Eggan
Oct 20, 2009·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Noemi FusakiMamoru Hasegawa
Mar 5, 2010·Stem Cells·Prashant MaliLinzhao Cheng
Mar 6, 2010·Nature Protocols·Dong QinGeorge M Whitesides
Oct 5, 2010·Cell Stem Cell·Luigi WarrenDerrick J Rossi
Nov 30, 2010·Cell Stem Cell·Saiyong ZhuSheng Ding
Apr 5, 2011·Nature Methods·Keisuke OkitaShinya Yamanaka
Oct 27, 2012·Nature Protocols·Jeanette BeersGuokai Chen
Jan 12, 2013·Nature Protocols·Mercè MartíJuan Carlos Izpisua Belmonte
Feb 23, 2013·Nature Protocols·Pankaj K Mandal, Derrick J Rossi
Aug 6, 2013·Cell Stem Cell·Naohisa YoshiokaSteven F Dowdy
Feb 4, 2014·Current Opinion in Biotechnology·Camilla LuniNicola Elvassore
Dec 2, 2014·Nature Biotechnology·Thorsten M SchlaegerGeorge Q Daley
Jun 2, 2015·Nature Methods·Giovanni G GiobbeNicola Elvassore
Jul 18, 2015·Cell·Davide CacchiarelliTarjei S Mikkelsen
Sep 19, 2015·Human Gene Therapy·Marco Alexander PoleganovUgur Sahin
Jan 17, 2016·Biochemical and Biophysical Research Communications·Camilla LuniNicola Elvassore
Apr 19, 2016·Nature Methods·Camilla LuniNicola Elvassore
Feb 23, 2018·Nature Communications·Igor KogutGanna Bilousova
Sep 29, 2018·Nature Biomedical Engineering·Jared M ChurkoJoseph C Wu
Citations
May 14, 2020·Nature Communications·Irene ZorzanGraziano Martello
Mar 9, 2021·Frontiers in Cellular Neuroscience·Anna Maria TolomeoNicola Elvassore
May 1, 2021·International Journal of Molecular Sciences·Quentin AlleJean-Marc Lemaitre
Jun 2, 2021·Biomedical Microdevices·Anke R VollertsenMathieu Odijk
Jun 11, 2020·Nano Letters·Yuan WangPeng Shi
Nov 3, 2021·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Araceli Rivera-OrdazLorenza Lazzari