Abstract

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20–80 individuals to detect the effects of regulatory variants with moderately large effect sizes.

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Acknowledgements

The iPSC lines were generated under the Human Induced Pluripotent Stem Cell Initiative (HIPSCI) funded by a grant from the Wellcome Trust and Medical Research Council (WT098503), supported by the Wellcome Trust (WT098051) and the NIHR/Wellcome Trust Clinical Research Facility. HIPSCI funding was used for sensory neuron RNA-sequencing. We acknowledge Life Science Technologies Corporation as the provider of Cytotune. Pfizer Neuroscience (Pfizer Ltd.) funded neuronal differentiation, functional assays, single-cell RNA-sequencing, and collection and sequencing of dorsal root ganglion samples. The authors gratefully acknowledge N. Kumasaka for help with RASQUAL. We thank F. Merkle for comments on the manuscript. J.S. gratefully acknowledges support from the Wellcome Trust for his PhD studentship. We also thank three anonymous reviewers whose feedback greatly improved this manuscript.

Author information

Author notes

  1. A list of members and affiliations appears in Supplementary Note 1.

Affiliations

  1. Wellcome Trust Sanger Institute, Hinxton, UK

    • Jeremy Schwartzentruber
    • , Julia Rodrigues
    • , Kaur Alasoo
    • , Andrew J. Knights
    • , Minal Patel
    • , Angela Goncalves
    •  & Daniel J. Gaffney
  2. Pfizer Neuroscience and Pain Research Unit, Pfizer Ltd., Cambridge, UK

    • Stefanie Foskolou
    • , Rita Ferreira
    • , Caroline Louise Benn
    • , Anna Wilbrey
    • , Magda Bictash
    • , Emma Impey
    • , Lishuang Cao
    • , Sergio Lainez
    • , Alexandre Julien Loucif
    • , Paul John Whiting
    •  & Alex Gutteridge
  3. European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, UK

    • Helena Kilpinen
  4. AR-UK Drug Discovery Institute, Institute of Neurology, University College London, London, UK

    • Paul John Whiting
  5. European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, UK

    • Jeremy Schwartzentruber

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Consortia

  1. HIPSCI Consortium

    Contributions

    J.S. analyzed data, and J.S. and D.J.G. wrote the manuscript. S.F. performed all differentiations. A. Gutteridge analyzed data. A. Gutteridge, D.J.G., and P.J.W. conceived and supervised the project. H.K. compared eQTLs with GTEx and identified tissue-specific eQTLs. J.R. and M.P. cultured iPSC samples. A.J.K. performed all ATAC-seq. K.A. and A. Goncalves assisted with data analysis. A.W. performed single-cell RNA work and assisted with data analysis. R.F. and C.L.B. performed RNA extraction and quantification. E.I. performed cell culture and Ca2+ flux assays. M.B. assisted with experimental design and Ca2+ flux assays. L.C., S.L., and A.J.L. performed electrophysiology measurements. All authors reviewed the manuscript.

    Competing interests

    S.F., R.F., C.L.B., A.W., M.B., E.I., L.C., S.L., A.J.L., P.J.W., and A. Gutteridge were all employees of Pfizer at the time the experiments were performed.

    Corresponding authors

    Correspondence to Jeremy Schwartzentruber or Alex Gutteridge or Daniel J. Gaffney.

    Supplementary information

    1. Combined Supplementary Figures and Supplementary Note

      Supplementary Figures 1–25 and Supplementary Note 1.

    2. Life Sciences Reporting Summary

    3. Supplementary Tables

      Supplementary Tables 1–11.

    4. Supplementary Tables

      Supplementary Tables 12–22.

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    DOI

    https://doi.org/10.1038/s41588-017-0005-8

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