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Microfluidic reprogramming to pluripotency of human somatic cells

Nature Protocolsvolume 14pages722737 (2019) | Download Citation

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 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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Key references using this protocol

Luni, C. et al. Nat. Methods 13, 446–452 (2016): www.nature.com/articles/nmeth.3832

Giobbe, G. G. et al. Nat. Methods 12, 637–640 (2015): www.nature.com/articles/nmeth.3411

Giulitti, S. et al. Nat. Cell Biol 21, 275–286 (2019): www.nature.com/articles/nmeth.3832

References

  1. 1.

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  2. 2.

    Luni, C. et al. High-efficiency cellular reprogramming with microfluidics. Nat. Methods 13, 446–452 (2016).

  3. 3.

    Banito, A. et al. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23, 2134–2139 (2009).

  4. 4.

    Luni, C., Serena, E. & Elvassore, N. Human-on-chip for therapy development and fundamental science. Curr. Opin. Biotechnol. 25, 45–50 (2014).

  5. 5.

    Giobbe, G. G. et al. Functional differentiation of human pluripotent stem cells on a chip. Nat. Methods 12, 1–7 (2015).

  6. 6.

    Gagliano, O., Elvassore, N. & Luni, C. Microfluidic technology enhances the potential of human pluripotent stem cells. Biochem. Biophys. Res. Commun. 473, 683–687 (2016).

  7. 7.

    Poleganov, M. A. et al. Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using nonmodified RNA for reprogramming and immune evasion. Hum. Gene Ther. 26, 751–766 (2015).

  8. 8.

    Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T. & Yamanaka, S. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5, 237–241 (2009).

  9. 9.

    Beers, J. et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029–2040 (2012).

  10. 10.

    Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendaivirus, an RNA virus that does not integrate into the host genome. Proc. Japan Acad. Ser. B 85, 348–362 (2009).

  11. 11.

    Schlaeger, T. M. et al. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63 (2015).

  12. 12.

    Churko, J. M. et al. Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods. Nat. Biomed. Eng. 1, 826–837 (2017).

  13. 13.

    Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409–412 (2011).

  14. 14.

    Yoshioka, N. et al. Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell 13, 246–254 (2013).

  15. 15.

    Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

  16. 16.

    Mandal, P. K. & Rossi, D. J. Reprogramming human fibroblasts to pluripotency using modified mRNA. Nat. Protoc. 8, 568–582 (2013).

  17. 17.

    Kogut, I. et al. High-efficiency RNA-based reprogramming of human primary fibroblasts. Nat. Commun. 9, 745 (2018).

  18. 18.

    Melin, J. & Quake, S. R. Microfluidic large-scale integration: the evolution of design rules for biological automation. Annu. Rev. Biophys. Biomol. Struct. 36, 213–231 (2007).

  19. 19.

    Qin, D., Xia, Y. & Whitesides, G. M. Soft lithography for micro- and nanoscale patterning. Nat. Protoc. 5, 491–502 (2010).

  20. 20.

    Martí, M. et al. Characterization of pluripotent stem cells. Nat. Protoc. 8, 223–253 (2013).

  21. 21.

    Cacchiarelli, D. et al. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency. Cell 162, 412–424 (2015).

  22. 22.

    Mali, P. et al. Butyrate greatly enhances derivationÿof human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 28, 713–720 (2010).

  23. 23.

    Zhu, S. et al. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7, 651–655 (2010).

  24. 24.

    Ichida, J. K. et al. A small-molecule inhibitor of Tgf-β signaling replaces Sox2 in reprogramming by inducing Nanog. Cell Stem Cell 5, 491–503 (2009).

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Acknowledgements

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).

Author information

Author notes

  1. These authors contributed equally: Onelia Gagliano, Camilla Luni.

Affiliations

  1. Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China

    • Onelia Gagliano
    • , Camilla Luni
    • , Wei Qin
    • , Erika Torchio
    • , Silvia Galvanin
    •  & Nicola Elvassore
  2. Department of Industrial Engineering, University of Padova, Padova, Italy

    • Onelia Gagliano
    • , Erika Torchio
    • , Silvia Galvanin
    • , Anna Urciuolo
    •  & Nicola Elvassore
  3. Venetian Institute of Molecular Medicine, Padova, Italy

    • Onelia Gagliano
    • , Enrica Bertin
    • , Erika Torchio
    • , Silvia Galvanin
    • , Anna Urciuolo
    •  & Nicola Elvassore
  4. Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    • Wei Qin
  5. University of Chinese Academy of Sciences, Beijing, China

    • Wei Qin
  6. Stem Cells & Regenerative Medicine Section, UCL Great Ormond Street Institute of Child Health, London, UK

    • Nicola Elvassore

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Contributions

O.G., C.L., and N.E. designed the study and wrote the manuscript. O.G. and C.L. optimized and performed the reprogramming experiments. W.Q. and S.G. produced the microfluidic devices. W.Q., E.B., and E.T. helped with reprogramming experiments. E.B., E.T., and A.U. helped with cell characterization. N.E. oversaw the project.

Competing interests

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.

Corresponding author

Correspondence to Nicola Elvassore.

Integrated supplementary information

  1. Supplementary Figure 1 Human fibroblast reprogramming by self-replicative RNA.

    (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.

  2. Supplementary Figure 2 Design of the photomask used for microfluidic chip production.

    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.

  3. Supplementary Figure 3 Expression of reprogramming transcription factors at day 4 of reprogramming.

    (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.

  4. Supplementary Figure 4 Fibroblast reprogramming troubleshooting.

    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.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Manual

  2. Reporting Summary

  3. Supplementary Video 1

    How to change medium in the microfluidic chambers.

  4. Supplementary Video 2

    How to carry out mechanical picking of hiPSC colonies.

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DOI

https://doi.org/10.1038/s41596-018-0108-4

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