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 system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.
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The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files.
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This work was supported by the Natural Science Foundation of China (31601178), ShanghaiTech University, the University of Padova (TRANSAC and PRAT), the CaRiPaRo Foundation, the Telethon Foundation (GGP15275), and an Oak Foundation Award (W1095/OCAY-14-191).
O.G., C.L., and N.E. are co-inventors on patent applications describing the reprogramming and differentiation processes in microfluidics: application nos. PD2013A000220, IT UA20162645 and 102016000039189, and PCT/IB2017/052167. O.G. and N.E. are cofounders of uSTEM Srl and Onyel Biotech Srl.
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Integrated supplementary information
(a) Time lapse of cell morphology changes along the process of cell reprogramming of BJ fibroblasts, seeded at 60 cell/mm2, and reprogrammed using the commercial kit Simplicon™ RNA Reprogramming Technology (Merck Millipore), containing self-replicative RNA for OCT4, SOX2, KLF4, and GLIS1. At day 27, phase contrast, TRA-1-60 live staining, and the two merged figures are shown (scale bar = 100 µm). (b) Dot plot indicating the number of hiPSC colonies obtained in each independent microfluidic culture chamber from BJ fibroblast under the conditions described in (a). The superimposed red error bar represents mean ± standard deviation of the data. The number of replicates is 20.
The design of one chip includes 10 microfluidic culture chambers. Two 10-chamber devices can fit on a 4-inch silicon wafer for the production of a master mold. On the left of each chamber, 5 markers identify the channel number and section area (highlighted in the inset), to assist in positioning during microscopy imaging. Crosses drawn on the left and on the right of the design are useful if alignment is required when attaching the PDMS at specific positions on glass. The full resolution design to be used for master mold fabrication is reported in the supplementary file Photomask.pdf.
(a) Immunofluorescence analysis of the indicated proteins in BJ cells undergoing reprogramming. Overlay figures contain enlargements in the insets to show the nuclear localization of the transcription factors assayed. (scale bar = 100 µm). (b) Western blot of the indicated proteins (SOX2, OCT4, KLF4, NANOG, VIM, and GAPDH) in H9, BJ fibroblasts and the sample treated as in (a). GAPDH was assessed for loading. The data are representative of three independent experiments.
Images of unfavorable conditions during fibroblast reprogramming: (a) cells are almost confluent at Day 3. (b) Cell morphology does not show mesenchymal-to-epithelial transition at Day 5.
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Gagliano, O., Luni, C., Qin, W. et al. Microfluidic reprogramming to pluripotency of human somatic cells. Nat Protoc 14, 722–737 (2019). https://doi.org/10.1038/s41596-018-0108-4
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