Generating Myelinating Co-Cultures from iPSC-derived Sensory Neurons

Myelinating co-cultures were prepared as previously described.²⁴ (link)^,25 (link) In brief, 30,000 rat Schwann cells were added to iPSCdSNs in Schwann cell basal medium [DMEM/F12 (ThermoFisher), 5 mg/ml insulin (Sigma), 100 mg/ml transferrin (Millipore), 25 ng/ml recombinant-human NGF (Peprotech), 25 ng/ml Selenium (Sigma), 25 ng/ml thyroxine (Sigma), 30 ng/ml progesterone (Sigma), 25 ng/ml triiodothyronine (Sigma) and 8 mg/ml putrescine (Sigma)]. Cells were maintained in this medium for one week to allow Schwann cell proliferation and alignment. Myelination was subsequently induced by exposing the cells to myelination medium ((N2 medium, 1:300 phenol-free Matrigel (Corning), 5% charcoal-stripped FBS (ThermoFisher), 25 ng/ml recombinant- human NGF (Peprotech), 50mg/ml ascorbic acid (Sigma)). Myelinating co-cultures were maintained for 8 or 26 weeks with twice weekly medium changes before fixation and immunocytochemistry.