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 I_Na by TTX was quantified according to the following equation: 100-(200*I_Na^TTX/[I_Na^control+I_Na^wash]) = 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.