B 27 serum free supplement

Tissue-culture-treated plates (12-well polystyrene plates, VWR, Radnor, PA, USA) were incubated with 5 µg/mL laminin-511-E8 solution (iMatrix-511) in sterile water for 3 h at room temperature. hiPS cells were dissociated from Matrigel-coated plates by treatment with enzyme-free cell dissociation buffer (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged (Avanti J15-R, Beckman-Coulter, Pasadena, CA, USA) at 200 g for 5 min in DMEM/F12. Then, the cells were resuspended in a mesoderm differentiation medium containing DMEM/F12 with GlutaMax (GIBCO) supplemented with 100 ng/mL activin A (Thermo Fisher Scientific, Waltham, MA, USA), 3 µM CHIR99021 (Stemgent, Cambridge, MA, USA), 10 µM Y27632 (TOCRIS, Minneapolis, MN, USA), and 1 × B27 serum-free supplement (GIBCO, Waltham, MA, USA), and 100,000 cells were plated onto each well of a 12-well plate coated with the laminin-511-E8 solution. After 2 days of differentiation, the cell culture medium was switched to an intermediate mesoderm induction medium containing DMEM/F12 with GlutaMax supplemented with 100 ng/mL BMP7 (Thermo Fisher Scientific, Waltham, MA, USA), 3 µM CHIR99021, and 1 × B27 serum-free supplement, and the cells were incubated for a minimum of 14 days with daily medium replenishment.

To obtain primary neuronal cells, male and female mice were mated. If a vaginal plug was observed the next morning, it was designated as embryonic day (E0.5). Embryos were collected at day E15.5 from a pregnant dam and brains were obtained. Meninges were discarded and forebrains were collected in Hibernate^®-E (Thermo Scientific, Waltham, MA, USA) supplemented with 2% B-27^® Serum-Free Supplement (Thermo Scientific) and 0.5 mM GlutaMAX™-I (Thermo Scientific) at 4 °C. Next, brains were washed two times with Hanks’ Balanced Salt Solution (HBSS, PAN-BioTech, Aidenbach, Germany). Afterwards, they were thoroughly resuspended in Neurobasal medium supplemented with 2% B-27^® Serum-Free Supplement (Thermo Scientific) and 0.5 mM GlutaMAX™-I (Thermo Scientific) and seeded on poly L-ornithine coated plates. Every three-four days half of the medium was aspirated and replaced with fresh Neurobasal medium. At days in vitro 7 (DIV7) neurons were treated with RCM/MCM or EGF as described below.

Five-thousand cells were dissociated as described above, plated in eight-well chamber slides coated with poly-L-ornithine and laminin, and incubated for 1 day in stem cell media, containing KnockOut D-MEM/F12, GlutaMax-I supplement [2 mM), bFGF (20 ng/ml), EGF (20 ng/ml), 2% StemPro Neural Supplement, penicillin/streptomycin (0.1%) and Fungizone (40 ng/ml)]. Media were then replaced by neuronal differentiation medium [Neurobasal medium, 2% B27 Serum-Free Supplement (ThermoFisher Scientific, 17504), GlutaMax-I supplement (2 mM)], Astrocyte differentiation medium [D-MEM, 1% N-2 Supplement (ThermoFisher Scientific, 17502), GlutaMax-I supplement (2 mM), 1% FBS) or oligodendrocyte differentiation medium [Neurobasal medium, 2% B-27 Serum-Free Supplement (ThermoFisher Scientific, 17504), GlutaMax-I supplement (2 mM), T3 (Sigma, cat. D6397)]. Media were replaced every 2 days for 10 DIV. Cells were fixed with 4% PFA for 15 min at room temperature and immunostained using mouse anti-Tuj1 antibody (neuronal III β-tubulin, 1/500, Covance, MMS-435P), goat anti-Pdgfrα antibody (1/500, R&D Systems, AF1062) or rabbit anti-S100b antibody (1/500, Dako, Z031129). Secondary antibodies were conjugated to Alexa fluor 568 (Molecular Probes) or Alexa fluor 488 (Molecular Probes).