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Using Microfluidics to Generate Human Naïve and Primed Pluripotent Stem Cells

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Part of the Methods in Molecular Biology book series (MIMB,volume 2416)

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.

Key words

  • Reprogramming
  • mRNAs
  • Microfluidics
  • Pluripotency
  • Induced pluripotent stem cells

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  • DOI: 10.1007/978-1-0716-1908-7_5
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References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    CAS  CrossRef  Google Scholar 

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

    CAS  CrossRef  Google Scholar 

  3. Yu J, Vodyanik MA, Smuga-Otto K et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920

    CAS  CrossRef  Google Scholar 

  4. Osafune K, Caron L, Borowiak M et al (2008) Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26:313–315

    CAS  CrossRef  Google Scholar 

  5. Tchieu J, Kuoy E, Chin MH et al (2010) Female human iPSCs retain an inactive X chromosome. Cell Stem Cell 7:329–342

    CAS  CrossRef  Google Scholar 

  6. Lister R, Pelizzola M, Kida YS et al (2011) Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471:68–73

    CAS  CrossRef  Google Scholar 

  7. Perrera V, Martello G (2019) How does reprogramming to pluripotency affect genomic imprinting? Front Cell Dev Biol 7:76

    CrossRef  Google Scholar 

  8. Takashima Y, Guo G, Loos R et al (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158:1254–1269

    CAS  CrossRef  Google Scholar 

  9. Theunissen TW, Friedli M, He Y et al (2016) Molecular criteria for defining the naïve human pluripotent state. Cell Stem Cell 19:502–515

    CAS  CrossRef  Google Scholar 

  10. Guo G, von Meyenn F, Santos F et al (2016) naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Reports 6:437–446

    CAS  CrossRef  Google Scholar 

  11. Giulitti S, Pellegrini M, Zorzan I et al (2019) Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics. Nat Cell Biol 21:275–286

    CAS  CrossRef  Google Scholar 

  12. Liu X, Nefzger CM, Rossello FJ et al (2017) Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming. Nat Methods 14:1055–1062

    CAS  CrossRef  Google Scholar 

  13. Wang Y, Zhao C, Hou Z et al (2018) Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state. elife 7:1–21

    Google Scholar 

  14. Kilens S, Meistermann D, Moreno D et al (2018) Parallel derivation of isogenic human primed and naive induced pluripotent stem cells. Nat Commun 9:360

    CrossRef  Google Scholar 

  15. Dong C, Beltcheva M, Gontarz P et al (2020) Derivation of trophoblast stem cells from naïve human pluripotent stem cells. elife 9:e52504

    CrossRef  Google Scholar 

  16. Cinkornpumin JK, Kwon SY, Guo Y et al (2020) naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and Methylome. Stem Cell Reports 15:198–213

    CAS  CrossRef  Google Scholar 

  17. Guo G, Stirparo GG, Strawride S et al (2021) Human naïve epiblast cells possess unrestricted lineage potential. Cell Stem Cell 28:1040–1056

    Google Scholar 

  18. Linneberg-Agerholm M, Wong YF, Herrera JAR et al (2019) Naïve human pluripotent stem cells respond to Wnt, nodal and LIF signalling to produce expandable naïve extra-embryonic endoderm. Development 146:dev180620

    CAS  CrossRef  Google Scholar 

  19. Sahakyan A, Kim R, Chronis C et al (2017) Human naive pluripotent stem cells model X chromosome dampening and X inactivation. Cell Stem Cell 20:87–101

    CAS  CrossRef  Google Scholar 

  20. Luni C, Giulitti S, Serena E et al (2016) High-efficiency cellular reprogramming with microfluidics. Nat Methods 13:446–452

    CAS  CrossRef  Google Scholar 

  21. Gagliano O, Luni C, Qin W et al (2019) Microfluidic reprogramming to pluripotency of human somatic cells. Nat Protoc 14:722–737

    CAS  CrossRef  Google Scholar 

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

    CAS  CrossRef  Google Scholar 

  23. Chen G, Gulbranson DR, Hou Z et al (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429

    CAS  CrossRef  Google Scholar 

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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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Correspondence to Graziano Martello .

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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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