Abstract

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

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References

  1. 1.

    & Pluripotent stem cells and disease modeling. Cell Stem Cell 5, 244–247 (2009).

  2. 2.

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

  3. 3.

    & The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295–305 (2012).

  4. 4.

    et al. Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes. Nat. Genet. 44, 981–990 (2012).

  5. 5.

    et al. A practical guide to induced pluripotent stem cell research using patient samples. Lab. Invest. 95, 4–13 (2015).

  6. 6.

    & Origins and implications of pluripotent stem cell variability and heterogeneity. Nat. Rev. Mol. Cell Biol. 14, 357–368 (2013).

  7. 7.

    et al. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell 9, 588–598 (2011).

  8. 8.

    & Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell 13, 149–159 (2013).

  9. 9.

    , , & Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell 14, 13–26 (2014).

  10. 10.

    et al. Automated, scalable culture of human embryonic stem cells in feeder-free conditions. Biotechnol. Bioeng. 102, 1636–1644 (2009).

  11. 11.

    et al. Automated maintenance of embryonic stem cell cultures. Biotechnol. Bioeng. 96, 195–201 (2007).

  12. 12.

    et al. Scalable 96-well plate based iPSC culture and production using a robotic liquid handling system. J. Vis. Exp. 99, e52755 (2015).

  13. 13.

    et al. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci. Rep. 2, 213 (2012).

  14. 14.

    et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460, 1145–1148 (2009).

  15. 15.

    et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009).

  16. 16.

    , , , & Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 85, 348–362 (2009).

  17. 17.

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

  18. 18.

    , , & Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Sci. Rep. 2, 657 (2012).

  19. 19.

    et al. Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting. PLoS ONE 8, e59867 (2013).

  20. 20.

    et al. Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011).

  21. 21.

    et al. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7, 521–531 (2010).

  22. 22.

    et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438–442 (2012).

  23. 23.

    et al. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Stem Cell 10, 337–344 (2012).

  24. 24.

    et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013).

  25. 25.

    et al. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease. Cell Reports 9, 1173–1182 (2014).

  26. 26.

    , , & Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc. 8, 430–437 (2013).

  27. 27.

    et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 14, 53–67 (2014).

  28. 28.

    et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Reports 3, 250–259 (2014).

  29. 29.

    et al. A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture. Sci. Rep. 5, 11319 (2015).

  30. 30.

    et al. Rapid and efficient generation of transgene-free iPSC from a small volume of cryopreserved blood. Stem Cell Rev. 11, 652–665 (2015).

  31. 31.

    & What is the point of large-scale collections of human induced pluripotent stem cells? Nat. Biotechnol. 31, 875–877 (2013).

  32. 32.

    , , & The aging signature: a hallmark of induced pluripotent stem cells? Aging Cell 13, 2–7 (2014).

  33. 33.

    et al. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 109, 12538–12543 (2012).

  34. 34.

    et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

  35. 35.

    et al. Genetic heterogeneity of induced pluripotent stem cells: results from 24 clones derived from a single C57BL/6 mouse. PLoS ONE 10, e0120585 (2015).

  36. 36.

    et al. Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin. Science (New York, N.Y.) 348, 880–886 (2015).

  37. 37.

    et al. Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell 10, 595–609 (2012).

  38. 38.

    et al. Erosion of X chromosome inactivation in human pluripotent cells initiates with XACT coating and depends on a specific heterochromatin landscape. Cell Stem Cell 16, 533–546 (2015).

  39. 39.

    et al. Research electronic data capture (REDCap)—a metadata-driven methodology and workflow process for providing translational research informatics support. J. Biomed. Inform. 42, 377–381 (2009).

  40. 40.

    et al. Expansion of a 12-kb VNTR containing the REXO1L1 gene cluster underlies the microscopically visible euchromatic variant of 8q21.2. Eur. J. Hum. Genet. 22, 458–463 (2014).

  41. 41.

    R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2012).

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Acknowledgements

We thank L. Rubin, Z. Hall and S. Lipnick for critical reading of the manuscript. This work would not have been possible without S. Solomon's leadership, vision, continual encouragement and unstinting support. The authors also thank The Genomics Core, National Human Genome Research Institute, for performing the SNP arrays and the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health, Bethesda, USA for their contributions. A.M. receives support as a New York Stem Cell Foundation Robertson Investigator, with additional funding through US National Institutes of Health grant P01GM099117.

Author information

Author notes

    • Keren A Weiss
    •  & David J Kahler

    Present addresses: New York University School of Medicine, RNAi High Throughput Screening Core, New York, New York, USA (D.J.K.); Department of Cell & Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia (K.A.W.).

    • Daniel Paull
    • , Ana Sevilla
    • , Hongyan Zhou
    • , Aana Kim Hahn
    • , Hesed Kim
    •  & Christopher Napolitano

    These authors contributed equally to this work.

Affiliations

  1. The New York Stem Cell Foundation Research Institute, New York, New York, USA.

    • Daniel Paull
    • , Ana Sevilla
    • , Hongyan Zhou
    • , Aana Kim Hahn
    • , Hesed Kim
    • , Christopher Napolitano
    • , Linshan Shang
    • , Katie Krumholz
    • , Premlatha Jagadeesan
    • , Chris M Woodard
    • , Bruce Sun
    • , Matthew Zimmer
    • , Eliana Forero
    • , Dorota N Moroziewicz
    • , Hector Martinez
    • , Keren A Weiss
    • , Lauren B Vensand
    • , Carmen R Dusenberry
    • , Hannah Polus
    • , Karla Therese L Sy
    • , David J Kahler
    • , Susan L Solomon
    • , Stephen Chang
    •  & Scott A Noggle
  2. The Broad Institute, Cambridge, Massachusetts, USA.

    • Alexander Tsankov
    • , Alexander Meissner
    •  & Kevin Eggan
  3. The Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Alexander Tsankov
    • , Alexander Meissner
    •  & Kevin Eggan
  4. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Alexander Tsankov
    • , Alexander Meissner
    •  & Kevin Eggan
  5. Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.

    • Thierry Vilboux
    • , May Christine V Malicdan
    •  & William A Gahl
  6. Division of Medical Genomics, Inova Translational Medicine Institute, Inova Health System, Falls Church, Virginia, USA.

    • Thierry Vilboux
  7. NIH Undiagnosed Diseases Program, Common Fund, Office of the Director, National Institute of Health and National Human Genome Research Institute, National Institute of Health, Bethesda, Maryland, USA.

    • William A Gahl
  8. The Howard Hughes Medical Institute, Cambridge, Massachusetts, USA.

    • Kevin Eggan

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Contributions

D.P. designed and performed iPSC reprogramming, expansion and QC assays. A.S. designed and performed iPSC expansion and RNA QC assays. H.Z. designed and performed iPSC reprogramming, selection and passaging biology. A.K.H. engineered methods for iPSC expansion and EB and fibroblast QC methods. H.K. engineered methods for fibroblast derivation, iPSC reprogramming, selection and passaging. C.N. designed the integration of the robotic platform and sample tracking systems, and contributed to engineering methods. A.T. performed statistical analysis. K.K. and P.J. performed fibroblast derivation. D.P., A.S., L.S., B.S., C.M.W., D.N.M., H.M., M.Z., K.A.W and S.A.N., performed iPSC reprogramming, expansion, QC and differentiation experiments. E.F., H.P., K.T.L.S., C.R.D. and L.B.V. were involved in the collection of fibroblast samples. T.V., M.C.V.M. and W.A.G. performed SNP genotyping and analysis. K.K., D.J.K. and S.A.N. were involved in system protocol development. S.L.S., S.C., K.E. and S.A.N. designed and supervised the project. A.M. provided statistical tools and supervised statistical analysis. D.P., K.E. and S.A.N. wrote the manuscript with contributions from other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Daniel Paull or Scott A Noggle.

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DOI

https://doi.org/10.1038/nmeth.3507

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