Nerve growth factor (ngf)

Neuronal MEMbranes were prepared from DRG of rat embryos at 15 gestational days as described in ref. 56 (link). Briefly, 20 DRGs were dissected and plated on 9.5-cm² dishes in C media: MEM (Gibco), 10% foetal bovine serum, 4 g l⁻¹D-Glucose, 2 mM L-Glutamine, 50 ng ml⁻¹ Nerve Growth Factor (Harlan). After 12 h cells were switched for 2 weeks in NB medium (Gibco), 1 × B27 supplement (Gibco), 4 g l⁻¹D-Glucose, 2 mM L-Glutamine, 50 ng ml⁻¹ Nerve Growth Factor (Harlan). To obtain neuron-only cultures, the cells were treated with FUDR (10 μM fluorodeoxyuridine (FdU) and 10 μM uridine) in NB medium for three cycles (2 days with FUDR, 2 days without FUDR). Neurons were collected by scraping the network with forceps and then they were homogenized in PBS with a Dounce homogenizer. The homogenized solution was centrifuged at 300 g for 20 min to remove debris and collagen. The supernatant was centrifuged at 30,000 g for 1 h. The pellets were resuspended in DMEM (67 μl for each 9.5-cm² dish) and stored at −80 °C for up to 6 months. To test their activity, MEMbranes were centrifuged onto serum starved, rat primary Schwann cells, incubated at 37 °C for an additional 20 min, lysed and analysed by western blotting. CHO and COS-7 MEMbranes were prepared as above using two 10-cm² dishes of confluent cells as starting material.

HEK293T, Hela, and Daoy cells were cultured on poly-D-lysine-coated dishes or coverslips with DMEM (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and 1% penicillin/streptomycin. Cells were transfected with plasmid DNA using PEI according to the manufacturer’s protocol. Cells were transfected with siRNA using Lipofectamine RNAimax according to the manufacturer’s protocol. The following siRNA target sequences were used: si-PHF5A-CDS, 5′-CCAUCGAAGACUGUGUGAAA-3′; si-PHF5A-UTR, 5′-GCCUACUACUACCAGCAGAAA-3′; si-CEP250-1#, 5′-GCTGACTCTATTCGACAACAA-3′; si-CEP250-2#, 5′-CCCTGACTCAAAGTCTGACAT-3′; si-Nek2-UTR, 5′-GCCATGCCTTTCTGTATAGTA-3′.
Before NGF (PeproTech) treatment, the cells were serum-starved for 16 h. The proper final concentration of NGF (50 or 100 ng/ml) was added to the cell culture medium still without serum.

Spheroids from oral mucosa-, skin-, and CBDC-derived cells were transferred into a new regular culture dish. After these cultures reached 50–60% confluence, the medium was changed to neurogenic induction medium. The medium used for neural cell differentiation was αMEM supplemented with l-glutamine, phenol red (Wako), 50 ng/ml nerve growth factor, 50 ng/ml brain-derived neurotrophic factor, 10 ng/ml NT-3 (all from Peprotech), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Biological Industries). For Schwann cell differentiation, the medium used was αMEM supplemented with l-glutamine, phenol red (Wako), 5 μM forskolin (Sigma), 50 ng/ml heregulin-1β (Peprotech), 2% v/v N2 supplement (Invitrogen), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Biological Industries). The cells were differentiated for 1 or 2 weeks, and 50% of the medium was changed every 2 days.

F11 cells (ECACC, UK), a hybrid of rat neuroblastoma and primary DRG neurons commonly used as model peripheral sensory neurons (Prucha et al., 2018), were chosen due to their ease of culture and well characterized electrophysiological properties including calcium (Kusano & Gainer, 1993), potassium (Fan, Shen, & Scheideler d, M. A., and Crain a, S. M., 1992) and TTX‐sensitive sodium (Gaudioso, Hao, Martin‐Eauclaire, Gabriac, & Delmas, 2012) ion channels. F11s were cultured on custom‐built deformable silicon substrates and differentiated in high glucose dulbecco’s modified eagle medium (ThermoFisher, UK), supplemented with 1% fetal bovine serum (ThermoFisher, UK), 1% penicillin/streptomycin, 0.5% insulin transferrin selenium (ThermoFisher, UK), 10 µM 3‐isobutyl‐1‐methylxanthine (IBMX, Sigma‐Aldrich, UK), 50 ng/ml nerve growth factor (Peprotech, UK), 2 µM retinoic acid (Sigma‐Aldrich, UK) and 0.5 mM bromoadenosine 3,5‐cyclic monophosphate. Cells were differentiated for 5 days prior to experiments and were confirmed to adhere to the deformable substrates by microscopy inspection.