Figure 1.

Differentiation of hPSCs into Human Nociceptors

(A) Overview of differentiation of human ESCs/iPSCs to nociceptors by LSB + 3i. L = LDN193189; SB = SB431542; 3i = DAPT, SU5402, CHIR99021.

(B) Bright-field image of a ganglion-like cluster. A nociceptor suitable for patch-clamp experiments is indicated by arrowhead.

(C) Ganglion-like clusters stain for BRN3A (green) and PERIPHERIN (red).

(D) Most cells in clusters co-express β3-TUBULIN (TUJ1, red), PERIPHERIN (green), and BRN3A (blue).

(E) Eighty-three percent of TUJ1-positive cells in clusters also express TRPV1.

(B–E) Scale bars represent 100 μm (B and C), 25 μm (D), and 20 μm (E).

(F and G) mRNA expression levels of neuronal NAVs (F) (n ≥ 4 independent experiments, measured as duplicates) and of typical nociceptor ion channels (G) (n ≥ 4 independent experiments, measured as duplicates). Error bars indicate the SEM.

(H–J) Functional characterization of hiPSC-derived nociceptors. (H) Current clamp recordings of representative phasically firing (top) and repetitively firing (bottom) nociceptors, elicited by injection of 100 pA for 500 ms. (I) Representative traces recorded with the indicated protocol (top) show sodium and potassium currents (bottom). (J) Example trace of voltage clamp recordings before and after addition of 500 nM TTX to the recording solution.