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


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.

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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.

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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



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.

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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)

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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).

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