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