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.

PIEZO2 is required for mechanotransduction in human stem cell–derived touch receptors

Nature Neuroscience volume 18, pages 10–16 (2015)Cite this article

Subjects

Abstract

Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell–derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor–specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Generation of hES cell–derived sensory neurons.
Figure 2: Expression of TRK receptors in hES cell–derived sensory neurons.
Figure 3: Molecular characterization of hES cell–derived neurons.
Figure 4: Functional characterization of hES cell–derived LTMRs.
Figure 5: Generation and characterization of PIEZO2-knockout LTMRs.
Figure 6: Functional analysis of PIEZO2-knockout LTMRs.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Abraira, V.E. & Ginty, D.D. The sensory neurons of touch. Neuron 79, 618–639 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. 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 

  3. Lallemend, F. & Ernfors, P. Molecular interactions underlying the specification of sensory neurons. Trends Neurosci. 35, 373–381 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. 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 

  5. Young, G.T. et al. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research. Mol. Ther. 22, 1530–1543 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Coste, B. et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Coste, B. et al. Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483, 176–181 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Maksimovic, S. et al. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature 509, 617–621 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Woo, S.H. et al. Piezo2 is required for Merkel-cell mechanotransduction. Nature 509, 622–626 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ikeda, R. et al. Merkel cells transduce and encode tactile stimuli to drive Abeta-afferent impulses. Cell 157, 664–675 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Marmigère, F. & Ernfors, P. Specification and connectivity of neuronal subtypes in the sensory lineage. Nat. Rev. Neurosci. 8, 114–127 (2007).

    Article  PubMed  Google Scholar 

  12. 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 

  13. Schwartz, P.H., Brick, D.J., Stover, A.E., Loring, J.F. & Muller, F.J. Differentiation of neural lineage cells from human pluripotent stem cells. Methods 45, 142–158 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bajpai, R. et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463, 958–962 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Airaksinen, M.S. et al. Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation. Neuron 16, 287–295 (1996).

    Article  CAS  PubMed  Google Scholar 

  16. Bourane, S. et al. Low-threshold mechanoreceptor subtypes selectively express MafA and are specified by Ret signaling. Neuron 64, 857–870 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Carroll, P., Lewin, G.R., Koltzenburg, M., Toyka, K.V. & Thoenen, H. A role for BDNF in mechanosensation. Nat. Neurosci. 1, 42–46 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Luo, W., Enomoto, H., Rice, F.L., Milbrandt, J. & Ginty, D.D. Molecular identification of rapidly adapting mechanoreceptors and their developmental dependence on ret signaling. Neuron 64, 841–856 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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 

  20. Lechner, S.G., Frenzel, H., Wang, R. & Lewin, G.R. Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development. EMBO J. 28, 1479–1491 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wende, H. et al. The transcription factor c-Maf controls touch receptor development and function. Science 335, 1373–1376 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Heidenreich, M. et al. KCNQ4 K(+) channels tune mechanoreceptors for normal touch sensation in mouse and man. Nat. Neurosci. 15, 138–145 (2012).

    Article  CAS  Google Scholar 

  23. Zhang, Y. et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78, 785–798 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Somers, A. et al. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28, 1728–1740 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rose, R.D., Koerber, H.R., Sedivec, M.J. & Mendell, L.M. Somal action potential duration differs in identified primary afferents. Neurosci. Lett. 63, 259–264 (1986).

    Article  CAS  PubMed  Google Scholar 

  27. Ho, C. & O'Leary, M.E. Single-cell analysis of sodium channel expression in dorsal root ganglion neurons. Mol. Cell. Neurosci. 46, 159–166 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Akopian, A.N. et al. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat. Neurosci. 2, 541–548 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Amaya, F. et al. The voltage-gated sodium channel Nav1.9 is an effector of peripheral inflammatory pain hypersensitivity. J. Neurosci. 26, 12852–12860 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Poole, K., Herget, R., Lapatsina, L., Ngo, H.D. & Lewin, G.R. Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch. Nat. Commun. 5, 3520 (2014).

    Article  PubMed  Google Scholar 

  31. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Coste, B. et al. Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of distal arthrogryposis. Proc. Natl. Acad. Sci. USA 110, 4667–4672 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. McMillin, M.J. et al. Mutations in piezo2 cause Gordon syndrome, Marden-Walker syndrome, and distal arthrogryposis type 5. Am. J. Hum. Genet. 94, 734–744 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  36. Anders, S., Pyl, P.T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics doi:10.1093/bioinformatics/btu638 (2014).

  37. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J. Rossius for help with generating the targeting constructs, C. Birchmeier, T.J. Jentsch and T.C. Südhof for providing antisera and viral expression constructs, V. Benes and J. Blake from the Genomics Core Facility, EMBL Heidelberg, for advice concerning design and analysis of the deep sequencing experiment, and A. Littlewood-Evans for critical reading of the manuscript. This work was supported by the European Research Council (ERC grant agreement 280565), the Alexander von Humboldt Foundation (AvH), the Human Frontiers Science Program (HFSP) (to J.S.) and the German Research Foundation (grant LE3210/2-1 to S.G.L.). Additional support was provided by the German Research Foundation (SFB665 to G.R.L. and SFB873 to J.U.).

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of Heidelberg, Heidelberg, Germany

    Katrin Schrenk-Siemens, Hagen Wende, Vincenzo Prato, Kun Song, Charlotte Rostock, Stefan G Lechner & Jan Siemens

  2. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Alexander Loewer & Gary R Lewin

  3. Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and University Medical Center Mannheim, Mannheim, Germany

    Jochen Utikal

Authors
  1. Katrin Schrenk-Siemens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hagen Wende
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vincenzo Prato
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kun Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Charlotte Rostock
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexander Loewer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jochen Utikal
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gary R Lewin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Stefan G Lechner
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jan Siemens
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.S.-S. designed and conducted the differentiation protocol, performed all cell culture work, generated the PIEZO2 knockout cell line and performed immunocytochemistry and qPCR experiments. H.W. analyzed the deep-sequencing and qPCR data, carried out in situ hybridization and helped with the targeting strategy. V.P. conducted the electrophysiological recordings. K.S. prepared the samples for deep-sequencing analysis and performed Ca2+ imaging. C.R. performed viral infections and Southern blot analysis. A.L. generated Supplementary Video 1. J.U. established the iPS cell line. S.G.L. designed and analyzed the electrophysiological recordings. G.R.L., S.G.L., K.S.-S. and H.W. contributed to the editing of the manuscript. J.S. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Jan Siemens.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of hES cell–derived neural crest (NC) cells.

(a) qRT-PCR for known NC markers SOX9, SOX10, PAX3 and P75 and (b) early sensory fate markers BRN3A, NGN1 and NGN2. Lysates prepared from one culture dish of hESC-derived NC cells and processed in triplicates. Fold increase is shown relative to undifferentiated hESCs. Mean ± s.d. (c) Immunocytochmistry of hES-cell-derived neural crest cells with anti-sera against the indicated proteins P75, HNK1, BRN3A. Scale bar, 20μm.

Supplementary Figure 2 Transcriptional profiling of hES cell–derived NC cells and sensory neurons.

Transcript abundance of selected genes are indicated as reads per kilobase transcript per million total reads (RPKM) obtained from dissociated hES-cell-derived NC cells and manually picked sensory neurons as indicated (N=3 for each cell type; mean ± s.e.m.).

Supplementary Figure 3 Time course of virally induced NGN2 expression in hES cell–derived NC cells.

(a-c) Phase contrast and (a’-c’) EGFP expression in hESC-derived NC cells infected with TetO-NGN2-EGFP virus. (a’) EGFP signal 24h after doxycycline (DOX) treatment. Infection efficiency 54.5±2.5%; mean ± s.e.m. (b’) EGFP signal decreases 10h after DOX withdrawal and is almost absent 48h after DOX withdrawal (c’). Scale bar 50μm.

Supplementary Figure 4 Directed generation of sensory neurons after virally induced NGN2 expression in hES cell–derived NC cells.

(a-b) Immunocytochemistry for ISL1 (upper panel) and ISL1 and DAPI (lower panel) in hESC-derived NC cells with or without NGN2 virus treatment. Stainings were performed after 4div and 2 days after NGN2 induction. (c-d) NGN2 infected hNC cultures showed an increase in the number of MAFA (upper panel) and NF200 (lower panel) positive neurons after 21 days of differentiation (d), compared to non-infected cells (c). Scale bar 50μm (b, d).

Supplementary Figure 5 Characterization of hiPS cells.

(a-c) Hematoxylin and eosin staining of human iPS cells subjected to a teratoma formation assay to verify pluripotency. Generated human iPS cells have the capability to differentiate into all 3 germ layers: (a) mesoderm (cartilage), (b) endoderm (non-keratinized epithelium) and (c) ectoderm (pigmented cells). (d-i) Immunocytochemistry of differentiating human iPS-cell-derived sensory neurons, showing the expression of the pan-sensory marker ISL1 (d) after 1div (e) and f) show the overlay with DAPI) and expression of MAF and NF200 after 21div (g-i). Scale bar 50μm (f, i).

Supplementary Figure 6 hES cell–derived LTMRs lack nociceptor-specific ion channels.

(a) Whole-cell sodium currents (bottom traces) were elicited at an interval of 20s using the illustrated voltage-pulses (top trace, red). Cells were hyperpolarized to -120mV for 120ms to completely remove inactivation from Na+-channels followed by a 40ms step depolarization to -20mV (top trace, red). To assess TTX-sensitivity of Na+-currents, increasing concentrations of TTX (1nM to 10µM) were applied. After each TTX application the toxin was washed out for 40s to ensure full recovery of the Na+-currents. Inhibition of INa by TTX was quantified according to the following equation: 100-(200*INaTTX/[INacontrol+INawash]) = percent inhibition; and plotted as a function of TTX concentration. The EC50 value (8.38nM) of TTX-inhibition was determined with a sigmoidal dose-response fit-function (hill-slope = 1); n=7. (b) Mouse DRG neurons (red, blue and yellow traces) and hES-cell-derived LTMRs (green traces) were loaded with Fura-2 and challenged with hypotonic solution (45%), Menthol (500 μM), Mustard Oil (200 μM), Capsaicin (1 μM) and high Potassium using the indicated application schema. Traces are shown as normalized fluorescence ratios (F340/F380) for Capsaicin-only (n=19; red trace), Mustard Oil+Capsaicin (n=8; yellow trace) and Menthol-only responsive neurons (n=3; blue trace) of mouse DRG cultures and human ES-cell-derived sensory neurons (n=17; green traces), upper panel. F340/F380 for hypotonic solution responsive mouse DRG neurons (n=10; red traces) together with human ESC-derived neurons (n=3; green traces) are shown in the lower panel. Note that the derived human sensory neurons only respond to the depolarizing (high potassium) stimulus but not to any of the pungent chemicals that activate TRP channels in mouse nociceptors, serving as positive control.

Supplementary Figure 7 Functional characterization of hES cell– and hiPS cell–derived LTMRs.

(a) Examples of mechanotransduction currents (bottom traces) elicited by mechanical stimuli of increasing magnitude (top traces) in hESC-derived LTMRs. Note that the mechanoreceptors do not show any response upon removal of the mechanical stimulus. (b) Representative example of an action potential (AP) recorded from human iPS-cell-derived neurons. Half peak duration (HPD) was 1.81±0.81ms (n=8), after-hyperpolarization (AHP) was 12.79±8,10ms (n=8) and time constant (Tau) was 7.96±1.75ms (n=6). (c) Example traces of mechanotransduction currents (bottom traces) elicited by mechanical stimuli of increasing magnitude (top traces) in human iPS-cell-derived LTMRs.

Supplementary Figure 8 Uncropped Southern blot of Figure 5b.

Uncropped Southern blot showing the band for the hPIEZO2 wild-type locus (10.1 kb), the hPIEZO2 Neo allele (8.5kb) and the hPIEZO2 Puro allele (12.2kb) after PvuII digest.

Supplementary Figure 9 Half-maximal activation of PIEZO2+/+, PIEZO2+/– and PIEZO2–/– neurons using Boltzmann fitting.

Half maximal activation is reached at a displacement of 3.43±0.86μm and 3.58±0.74μm for hPIEZO2+/+ and hPIEZO2+/– neurons, respectively as deduced by Boltzmann-fitting of the current-displacement curve. hPIEZO2+/+, n=15; hPIEZO2+/–, n=15; hPIEZO2–/–, n=46 (for hPIEZO2–/–, three independent cell clones were analyzed: clone 1: n=18, clone 2: n=16, clone 3: n=12), mean ± s.e.m.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 4077 kb)

Supplementary Table 1: Expression profile of TRP channels and other selected sensory neuron markers in hES cell–derived LTMRs.

Expression profiling of hNC cells and hLTMRs by deep sequencing. Average expression (RPKM) and s.e.m. for all known TRP channels and selected sensory neuron markers (NTRK1-3, MAF, MAFA, RET, PIEZO1-2) in hLTMRs and hNC cells are shown. Transcriptome analysis was performed on three biological replicates of ∼100 hNC cells and three manually isolated hES-cell-derived LTMR clusters (each cluster containing 10 to 20 hLTMRs). (XLSX 11 kb)

Delamination of hES cell–derived neural crest cells from a neuroectodermal sphere.

The movie shows the migration of human ES-cell-derived neural crest cells from a neuroectodermal sphere. The sphere was imaged under controlled environmental conditions. Pictures were taken every 15 min. For further details see Methods. (MOV 5429 kb)

Mechanical stimulation of hES cell–derived LTMRs.

The movie shows the indentation of the cell membrane of a human ES-cell-derived LTMR by a nanomotor-driven probe. (MOV 273 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schrenk-Siemens, K., Wende, H., Prato, V. et al. PIEZO2 is required for mechanotransduction in human stem cell–derived touch receptors. Nat Neurosci 18, 10–16 (2015). https://doi.org/10.1038/nn.3894

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3894

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