Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts

Nature Neuroscience volume 18, pages 17–24 (2015)Cite this article

Subjects

This article has been updated

Abstract

Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus–detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Combinations of transcription factors result in nociceptor production.
Figure 2: Induced nociceptors (iNoc) express characteristic nociceptor genes.
Figure 3: Induced neurons respond to different Trp channel agonists.
Figure 4: Whole-cell patch-clamp recordings of tdTomato-positive induced nociceptors.
Figure 5: Sensitization of induced nociceptors treated with the inflammatory mediator PGE2 and the chemotherapeutic drug oxaliplatin.
Figure 6: Human fibroblast–derived neurons for human disease modeling.

Similar content being viewed by others

Change history

  • 10 December 2014

    In the version of this article initially published online, an affilation was missing for author Julia T. Oliveira: Laboratório de Neurodegeneração e Reparo, Departamento de Patologia, Faculdade de Medicina, Universidade Federal Do Rio De Janeiro, Rio de Janeiro, Brazil. The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. Qiang, L., Fujita, R. & Abeliovich, A. Remodeling neurodegeneration: somatic cell reprogramming-based models of adult neurological disorders. Neuron 78, 957–969 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Sandoe, J. & Eggan, K. Opportunities and challenges of pluripotent stem cell neurodegenerative disease models. Nat. Neurosci. 16, 780–789 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Liu, M.L. et al. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat. Commun. 4, 2183 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Son, E.Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Costigan, M., Scholz, J. & Woolf, C.J. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu. Rev. Neurosci. 32, 1–32 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Basbaum, A.I., Bautista, D.M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Blair, N.T. & Bean, B.P. Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J. Neurosci. 22, 10277–10290 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chambers, S.M. et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 30, 715–720 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Waxman, S.G. Channelopathic pain: a growing but still small list of model disorders. Neuron 66, 622–624 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Huang, J. et al. Small-fiber neuropathy Nav1.8 mutation shifts activation to hyperpolarized potentials and increases excitability of dorsal root ganglion neurons. J. Neurosci. 33, 14087–14097 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Han, C. et al. Nav1.7-related small fiber neuropathy: impaired slow-inactivation and DRG neuron hyperexcitability. Neurology 78, 1635–1643 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Sorge, R.E. et al. Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat. Med. 18, 595–599 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Reimann, F. et al. Pain perception is altered by a nucleotide polymorphism in SCN9A. Proc. Natl. Acad. Sci. USA 107, 5148–5153 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Pitchford, S. & Levine, J.D. Prostaglandins sensitize nociceptors in cell culture. Neurosci. Lett. 132, 105–108 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Lopshire, J.C. & Nicol, G.D. Activation and recovery of the PGE2-mediated sensitization of the capsaicin response in rat sensory neurons. J. Neurophysiol. 78, 3154–3164 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Anand, U., Otto, W.R. & Anand, P. Sensitization of capsaicin and icilin responses in oxaliplatin treated adult rat DRG neurons. Mol. Pain 6, 82 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lee, K.J. & Jessell, T.M. The specification of dorsal cell fates in the vertebrate central nervous system. Annu. Rev. Neurosci. 22, 261–294 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Knecht, A.K. & Bronner-Fraser, M. Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3, 453–461 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Woolf, C.J. & Ma, Q. Nociceptors–noxious stimulus detectors. Neuron 55, 353–364 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Ma, Q., Fode, C., Guillemot, F. & Anderson, D.J. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 13, 1717–1728 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dykes, I.M., Lanier, J., Eng, S.R. & Turner, E.E. Brn3a regulates neuronal subtype specification in the trigeminal ganglion by promoting Runx expression during sensory differentiation. Neural Dev. 5, 3 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chen, C.L. et al. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron 49, 365–377 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Sun, Y. et al. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nat. Neurosci. 11, 1283–1293 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lei, L. et al. The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons. Genes Dev. 19, 1354–1364 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cavanaugh, D.J. et al. Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons. J. Neurosci. 31, 10119–10127 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wu, C., Macleod, I. & Su, A.I. BioGPS and MyGene.info: organizing online, gene-centric information. Nucleic Acids Res. 41, D561–D565 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Allen Institute for Brain Science. Allen Spinal Cord Atlas http://mousespinal.brain-map.org/ (2012).

  29. Fornaro, M. . et al. Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: an in vivo and in vitro study. Neuroscience 153, 1153–1163 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Cavanaugh, D.J. et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J. Neurosci. 31, 5067–5077 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bautista, D.M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Chen, C.C. et al. A P2X purinoceptor expressed by a subset of sensory neurons. Nature 377, 428–431 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Jarvis, M.F. et al. A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc. Natl. Acad. Sci. USA 99, 17179–17184 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dib-Hajj, S.D., Cummins, T.R., Black, J.A. & Waxman, S.G. Sodium channels in normal and pathological pain. Annu. Rev. Neurosci. 33, 325–347 (2010).

    Article  CAS  PubMed  Google Scholar 

  35. Renganathan, M., Dib-Hajj, S. & Waxman, S.G. Na(v)1.5 underlies the 'third TTX-R sodium current' in rat small DRG neurons. Brain Res. Mol. Brain Res. 106, 70–82 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Dib-Hajj, S.D. et al. Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons. FEBS Lett. 462, 117–120 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Nagy, I., Urban, L. & Woolf, C.J. Morphological and membrane properties of young rat lumbar and thoracic dorsal root ganglion cells with unmyelinated axons. Brain Res. 609, 193–200 (1993).

    Article  CAS  PubMed  Google Scholar 

  38. Emery, E.C., Young, G.T., Berrocoso, E.M., Chen, L. & McNaughton, P.A. HCN2 ion channels play a central role in inflammatory and neuropathic pain. Science 333, 1462–1466 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Pang, Z.P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Slaugenhaupt, S.A. et al. Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am. J. Hum. Genet. 68, 598–605 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Cuajungco, M.P. et al. Tissue-specific reduction in splicing efficiency of IKBKAP due to the major mutation associated with familial dysautonomia. Am. J. Hum. Genet. 72, 749–758 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lee, G. et al. Modeling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Woolf, C.J. Overcoming obstacles to developing new analgesics. Nat. Med. 16, 1241–1247 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Davidson, S. et al. Human sensory neurons: membrane properties and sensitization by inflammatory mediators. Pain 155, 1861–1870 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li, Y. et al. Cloning and expression of a novel human gene, Isl-2, encoded a LIM-homeodomain protein. Mol. Biol. Rep. 34, 19–26 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Dietrich, P., Alli, S., Shanmugasundaram, R. & Dragatsis, I. IKAP expression levels modulate disease severity in a mouse model of familial dysautonomia. Hum. Mol. Genet. 21, 5078–5090 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. George, L. et al. Familial dysautonomia model reveals Ikbkap deletion causes apoptosis of Pax3+ progenitors and peripheral neurons. Proc. Natl. Acad. Sci. USA 110, 18698–18703 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Rebelo, S., Chen, Z.F., Anderson, D.J. & Lima, D. Involvement of DRG11 in the development of the primary afferent nociceptive system. Mol. Cell. Neurosci. 33, 236–246 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Anderson, D.J. Lineages and transcription factors in the specification of vertebrate primary sensory neurons. Curr. Opin. Neurobiol. 9, 517–524 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Cheng, L. et al. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7, 510–517 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Banerjee, A., Roach, M.C., Trcka, P. & Luduena, R.F. Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin. J. Biol. Chem. 265, 1794–1799 (1990).

    CAS  PubMed  Google Scholar 

  52. Lysakowski, A., Alonto, A. & Jacobson, L. Peripherin immunoreactivity labels small diameter vestibular 'bouton' afferents in rodents. Hear. Res. 133, 149–154 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Belghiti, M. et al. Potentiation of the transient receptor potential vanilloid 1 channel contributes to pruritogenesis in a rat model of liver disease. J. Biol. Chem. 288, 9675–9685 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Maatkamp, A. et al. Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 mice. Eur. J. Neurosci. 20, 14–28 (2004).

    Article  PubMed  Google Scholar 

  55. Ghilardi, J.R. et al. Sustained blockade of neurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skeletal pain but not the maintenance of sensory and sympathetic nerve fibers. Bone 48, 389–398 (2011).

    Article  CAS  PubMed  Google Scholar 

  56. Regad, T., Roth, M., Bredenkamp, N., Illing, N. & Papalopulu, N. The neural progenitor–specifying activity of FoxG1 is antagonistically regulated by CKI and FGF. Nat. Cell Biol. 9, 531–540 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Skalli, O. et al. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103, 2787–2796 (1986).

    Article  CAS  PubMed  Google Scholar 

  58. Panayi, H. et al. Sox1 is required for the specification of a novel p2-derived interneuron subtype in the mouse ventral spinal cord. J. Neurosci. 30, 12274–12280 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Hoffmann, S. et al. Retinoic acid inhibits angiogenesis and tumor growth of thyroid cancer cells. Mol. Cell. Endocrinol. 264, 74–81 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Costigan for assistance with RT-PCR, A. Yekkirala and J. Sprague for help with calcium imaging, Q. Ma (Dana-Farber Cancer Institute) and E. Turner (Seattle Children's Research Institute) for constructs, J. Gardner and J. McNeish for helpful advice and support, and K. Wainger for assistance with figure preparation. We also thank the Boston Children's Hospital IDDRC Molecular Genetics Core Facility for RNA Bioanalyzer analyses and the Harvard Medical School ICCB Screening Facility for assistance with ImageXpress and MetaXpress analyses. This research was supported by the National Institute of General Medical Sciences (T32 GM07592) and National Institute of Neurological Disorders and Stroke (1K08-NS082364) to B.J.W. Conselho Nacional de Desenvolvimento Científico e Tecnológico (J.T.O.), GlaxoSmithKline Regenerative Medicine DPU (C.J.W.), the National Institute of Neurological Disorders and Stroke (NS038253 to C.J.W.), and the Dr. Miriam and Sheldon G. Adelson Medical Foundation (C.J.W.).

Author information

Author notes

  1. Brian J Wainger and Elizabeth D Buttermore: These authors contributed equally to this work.

Authors and Affiliations

  1. F.M. Kirby Neurobiology Center, Boston Children's Hospital and Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Brian J Wainger, Elizabeth D Buttermore, Julia T Oliveira, Cassidy Mellin, Seungkyu Lee, Wardiya Afshar Saber, Amy J Wang, Isaac M Chiu, Lee Barrett, Eric A Huebner, Canan Bilgin, Christian Brenneis, Kush Kapur & Clifford J Woolf

  2. Department of Anesthesia, Critical Care and Pain Medicine and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Brian J Wainger

  3. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Brian J Wainger, Elizabeth D Buttermore, Seungkyu Lee, Isaac M Chiu, Eric A Huebner & Clifford J Woolf

  4. Departamento de Patologia, Laboratório de Neurodegeneração e Reparo, Faculdade de Medicina, Universidade Federal Do Rio De Janeiro, Rio de Janeiro, Brazil

    Julia T Oliveira

  5. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA

    Justin K Ichida, Naomi Tsujimoto, Lee L Rubin & Kevin Eggan

  6. Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, USA

    Justin K Ichida

  7. The Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Kevin Eggan

Authors
  1. Brian J Wainger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth D Buttermore
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julia T Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cassidy Mellin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seungkyu Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wardiya Afshar Saber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Amy J Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Justin K Ichida
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Isaac M Chiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lee Barrett
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Eric A Huebner
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Canan Bilgin
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Naomi Tsujimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Christian Brenneis
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Kush Kapur
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Lee L Rubin
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Kevin Eggan
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Clifford J Woolf
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.J.W. conceived, designed and performed the lineage reprogramming experiments and physiological experiments, analyzed data, and wrote the manuscript. E.D.B. designed, performed and analyzed reprogramming, quantitative PCR, single-cell RT-PCR, immunohistochemistry and CGRP ELISA experiments, and wrote the manuscript. J.T.O. performed and optimized the induced nociceptor technique. C.M. performed and analyzed physiological studies and edited the manuscript. S.L. performed CGRP ELISA and single-cell RT-PCR assays. W.A.S. performed reprogramming and immunohistochemistry experiments. A.J.W. performed initial cloning and transduction experiments. J.K.I. provided essential advice for nociceptor reprogramming strategy and edited the manuscript. I.M.C. gave critical advice regarding the genetic reporter, choice of transcription factors, performed cell sorting experiments and edited the manuscript. L.B. advised and performed molecular biology experiments. E.A.H. performed image quantification and analysis. C. Bilgin assisted with reprogramming and immunohistochemistry. N.T. assisted with human motor neuron culture and, together with C. Brenneis performed culture and characterization using initial approaches. K.K. performed statistical modeling of human nociceptor data. L.L.R. advised regarding reprogramming experiments and edited the manuscript. K.E. provided advice and reagents for reprogramming and edited the manuscript. C.J.W. designed experiments, interpreted findings and wrote the manuscript.

Corresponding author

Correspondence to Clifford J Woolf.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Lineage reprogramming schematic.

MEFs were isolated from TrpV1+/-::tdTomato+/- mice and transduced with candidate transcription factors. After 2 weeks, we detected tdTomato-positive induced neurons.

Supplementary Figure 2 Mouse embryonic fibroblasts (MEFs) do not contain neural precursors or neurons.

(a) Representative images of TrpV1-Cre::tdTomato MEFs reveal lack of staining for Nestin (upper), Sox1 (middle), and Ki67 (lower). Bright field images confirm the presence of cells in the field of view. (b) Positive-control rat neural stem cells stain positive for Nestin (upper), Sox1 (middle), and Ki67 (lower). (c) TrpV1-Cre::tdTomato MEFs reveal lack of staining for Tuj1. (d) Positive-control TrpV1-Cre::tdTomato induced nociceptors (iNoc) stain positive for Tuj1. Representative images of induced neurons were selected from n = 4 wells per antibody from 2 separate transductions and from rat neural stem cells from n = 4 wells from one plating. Scale bars: 100 µm.

Supplementary Figure 3 Single transcription factor dropouts from 12 factors identify inhibitory and essential factors.

(a) Retroviral transduction of MEFs with a combination of 12 transcription factors produces a small number of tdTomato-positive, Tuj1-positive neurons. (b,c) Removing Runx1 (b), or Brn3a (c) from the 12 factors increases the number of tdTomato-positive cells. (d-f) Removing Ascl1 (d), Myt1l (e), or Klf7 (f) results in a decrease of tdTomato-positive cells. Representative images for each transcription factor dropout were taken from n = 4 wells from 2 separate transductions. Scale bars: 100 µm.

Supplementary Figure 4 Alternative factor combinations generate low numbers of tdTomato-, Tuj1-positive neurons.

(a) Ngn1 alone produces a small number of tdTomato, Tuj1-positive cells. (b) The BAM factors produce large numbers of Tuj1-positive cells, a few of which are tdTomato-positive. (c) BAM factors and Ngn1 produce tdTomato, Tuj1-positive neurons, but much less efficiently than the 5 factors (Fig. 1). Representative images for each transcription factor combination study were taken from n = 4 wells from 2 separate transductions. Scale bars: 100 µm. (d) Quantification of the number of tdTomato-positive neurons per well.

Source data

Supplementary Figure 5 Removal of any of the five factors disrupts nociceptor formation.

(a) Transduction of MEFs with all 5 factors (Ascl1, Myt1l, Ngn1, Isl2, and Klf7) efficiently produces tdTomato, Tuj1-positive neurons. (b-f) Removal of Ascl1 (b), Myt1l (c), Ngn1 (d), Isl2 (e), or Klf7 (f) dramatically reduces the number of tdTomato, Tuj1-positive neurons. Representative images of each transcription factor drop out were selected from n = 4 wells from 2 separate transductions. Scale bars: 100 µm.

Supplementary Figure 6 Trpv1-Cre::tdTomato iNoc do not express a smooth muscle marker and BAM-derived neurons do not express nociceptor markers.

(a,b) Representative images of TrpV1-Cre::tdTomato iNoc (a) revealing lack of staining for smooth muscle actin (SMA), compared to positive-control C2C12 mouse myoblast cells (b). (c-g) BAM-factor derived, non-subtype specific induced neurons express the pan-neuronal marker Tuj1 (c), but not the nociceptor-specific markers TrpV1 (d), Prph (e), CGRP (f) or NF200 (g). Representative image for SMA in induced nociceptors was selected from n=4 wells from 2 independent transductions and representative image for SMA in myoblasts was selected from n = 4 wells from 1 plating of myoblasts. Representative images for BAM-derived neurons were selected from immunostaining of n = 6 wells from 3 independent transductions. Scale bars: 100 µm.

Supplementary Figure 7 Responses to Trp agonists in induced and adult primary nociceptors.

(a) Examples of tdTomato-negative induced neurons that responded to menthol (250 µM) but not mustard oil (100 µM) or capsaicin (1 µM). Calcium imaging was completed in 19 plates from 3 different transductions and examples of tdTomato-negative cells were found while imaging tdTomato-positive cells. (b) Calcium imaging responses from a single field of adult primary tdTomato-positive nociceptors (n = 249 cells from 12 plates of primary nociceptors from 2 mice). (c) Venn diagram showing subgroups of tdTomato-positive cells that responded to KCl (40 mM, grey), capsaicin (1 µM, Cap, red), mustard oil (100 µM, MO, lower small circle, green) and menthol (250 µM, ME, upper small circle, blue). No cells responded to mustard oil and menthol without responding to capsaicin (white).

Supplementary Figure 8 CGRP release from primary DRGs in response to KCl and capsaicin.

CGRP ELISA reveals a dose-dependent increase in CGRP release from in vitro primary DRGs in response to increasing concentrations of KCl compared to capsaicin (100 nM).

Source data

Supplementary Figure 9 NeuroD1 inhibits the transdifferentiation of human fibroblasts to nociceptors.

(a,b) Representative low-mag images of human iNoc derived with 5 factors (without NeuroD1), stained with Tuj1. (c,d) Representative low-mag images of human iNoc derived with 6 factors (including NeuroD1), stained with Tuj1. Representative images for human induced neurons with or without NeuroD1 were selected from immunostaining that was repeated in n = 6 wells from one transduction. Scale bars: 500 µm. The reprogramming efficiency was greater without NeuroD1 than with NeuroD1 (20.7 ± 1.4 cells per field without NeuroD1; 9.7 ± 1.1 cells per field with NeuroD1, n = 6 wells/group; t-test p = 1.0×10−4).

Source data

Supplementary Figure 10 Single-cell RT-PCR full gels from Figure 6f.

RT-PCR for IKBKAP and GAPDH from single human induced neurons (a) and single human fibroblasts (b) show normal (black arrow) and abnormally spliced (red arrowhead) transcripts.

Supplementary information

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wainger, B., Buttermore, E., Oliveira, J. et al. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nat Neurosci 18, 17–24 (2015). https://doi.org/10.1038/nn.3886

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3886

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing