Sensory Neuron Differentiation Protocol

For sensory neuron differentiation, a previously reported method was used with modifications [19 (link)]. iPSCs were maintained in Stem Fit medium containing 0.3 μM LDN193189 (Stemgent), 2 μM A83-01 (Wako), 6 μM CHIR99021, 2 μM RO4929097 (Cellagen Technology), 3 μM SU5402 (TOCRIS), and 0.3 μM retinoic acid for 8 days from the day after passage. The cells were dissociated into single cells with 0.5× TrypLE Select Solution and replated on plates coated with poly-l-ornithine and laminin at a density of approximately 0.5 × 10⁴ cells/cm² to induce differentiation into sensory neurons. After seeding, the sensory neurons were cultured with neuron medium consisting of KBM neural stem cell medium containing 2% B27, 10 ng/ml neurotrophin-3 (R&D Systems), 20 ng/ml NGF (R&D Systems), 20 ng/ml BDNF, and 20 ng/ml GDNF. The medium was changed every 2–3 days.