N2 supplement

After transduction with scrambled-shRNA, shRNA-LSD1-927-treated hiPSCs were treated with puromycin for 72 h. A modified four-step induction protocol to direct pancreatic differentiation from hiPSCs was employed as described previously [5 (link)]. In stage 1 of the procedure, the hiPSCs were cultured for 4 days to form definitive endoderm in DMEM/F12 containing 0.2% BSA (Sigma, USA), 0.5× N2-supplement (Gibco, USA), 0.5× B-27 supplement (Gibco, USA), 100 ng/mL Activin A (Peprotech, USA), and 1 μM Wortmannin (Sigma, USA) with medium changes every day. In stage 2, the differentiated cells were cultured for 4 days in F12/IMDM (1:1) mixed with 0.5% BSA, 0.5× ITS-X supplement (Gibco, USA), 0.5× B-27 supplement, 2 μM RA (Sigma, USA), 20 ng/mL FGF7 (Peprotech, USA), and 50 ng/mL Noggin (Peprotech, USA) with medium changes every day. In stage 3, the cells were cultured for 5 days in DMEM/H (high glucose) containing 0.5% BSA, 1× ITS-X supplement, 1× N2-supplement, and 50 ng/mL EGF (Sigma, USA). The medium was refreshed every day. For maturation, cells were incubated another 7–9 days in DMEM/F12 containing 1× ITS-X supplement, 10 ng/mL bFGF (Peprotech, USA), 10 mM nicotinamide (Sigma, USA), 50 ng/mL Exendin-4 (Sigma, USA), and 10 ng/mL BMP4 (Peprotech, USA). All media and supplements were purchased from Gibco, and growth factors were from Peprotech, unless indicated.

Embryonic mouse oligodendrocyte precursor cells (Oli-neu) were grown in DMEM (Gibco-Life Technologies 41965062) containing N2 supplement (Life Technologies), penicillin–streptomycin–glutamine (Life Technologies 10378016), T3 (Sigma-Aldrich; 340 ng ml⁻¹), T4 (Sigma-Aldrich 89430; 400 ng ml⁻¹), bFGF (PeproTech; 10 ng ml⁻¹) and PDGF-ββ (R&D Systems; 1 ng ml⁻¹) on plates coated with poly-l-lysine (Sigma-Aldrich; 0.01%). Cells were differentiated with differentiation medium (DMEM containing N2 supplement; penicillin–streptomycin–glutamine; T3 (Sigma-Aldrich; 340 ng ml⁻¹); T4 (Sigma-Aldrich; 400 ng ml⁻¹); ErbB inhibitor (Santa Cruz sc-204170; 1 µM)) for 1 day.

GMSC‐derived NPC‐like cells at passages 3–4 were seeded on poly‐d‐lysine‐ and laminin‐coated plastic coverslips (Nunc) and cultured in neurobasal medium supplemented with 1% N‐2 Supplement, 5% fetal bovine serum, 0.5 µM all‐trans‐retinoic acid (Sigma‐Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), and 10 ng/ml brain‐derived neurotrophic factor (PeproTech) for neuronal differentiation 33. For Schwann cell differentiation, cells were cultured in regular MSC culture medium supplemented with 35 ng/ml all trans‐retinoic acid for 72 hours. The medium was changed to regular culture medium supplemented with 5 µM forskolin (Sigma‐Aldrich), 10 ng/ml bFGF, 5 ng/ml platelet‐derived growth factor AA, and 200 ng/ml heregulin‐β‐1 (PeproTech) 14, 19. The medium was replenished every 3 days. After 2 weeks, cells were prepared for immunocytofluorescence studies to examine the expression of neuronal or glial cell markers.

Primary human nasal epithelial cells (hNEPCs) were maintained in Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 (Cat.No.11320033, Gibco), which contained human epithelial growth factor (Cat.No.100-15-500, Peprotech), insulin (Cat.No.I5500-50MG, Sigma), cholera toxin (Cat.No.C9903-0.5MG, Sigma), hydrocortisone (Cat.No.H0888-1G, Sigma), 3,3’,5-triiodo-l-thyronine (Cat.No.T6397-100MG, Sigma), and N-2 supplement (Cat.No.17502001,Gibco-Invitrogen). Further, a 1% Antibiotic-Antimycotic solution (Cat. No.MIR 5970, Mirusbio) was added. hNEPCs were cultured at 37°C in a 5% CO2 cell incubator.
The NIH/3T3 cell line purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured in DMEM (Cat.No.C11995500BT, Gibco) containing 10% fetal bovine serum (FBS) (Cat. No. 10099-141, Gibco-Invitrogen) and 1% penicillin-streptomycin (Cat. No.15140-122, Gibco). When the cell density reached about 80%, mitomycin C (Cat.No.M4287-2MG, Sigma) was added, treated in a 37°C cell incubator for 2 h, washed three times with PBS, and then co-cultured with the isolated primary cells.
Madin-Darby canine kidney (MDCK) cells were cultured in the following manner: 5% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/ml streptomycin, and 2 mM glutamine were added to the DMEM. Further, the cells were cultured at 5% CO2 and 37°C.

Prior to differentiation, iPSC lines were adapted to feeder-free conditions using Matrigel (BD) and EBs were formed. After 4 days, neural induction was initiated as previously described (32 (link),40 ). Briefly, EBs were plated onto Geltrex-coated plates in Neural Induction medium 1 (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, bovine serum albumin (BSA) (1 mg/ml), Y27632 (10 μM; Tocris), SB431542 (10 μM, Tocris), Noggin (200 ng/ml) and antibiotic/antimycotic (1% v/v)). After 4 days, medium was changed to Neural Induction medium 2 (as NI1, without SB431542 and Noggin and with addition of SHH C24II (200 ng/ml; SHH C24II; R&D Systems). After 6 days, medium was supplemented with FGF8a (100 ng/ml; R&D Systems), Heparin (5 μg/ml; Sigma), BDNF (20 ng/ml) and Ascorbic Acid (200 μM; Sigma)) and incubated for 7 days, until the appearance of dense neural rosette structures. Neural progenitor cells were manually selected and re-plated onto Poly-D-Lysine/Laminin-coated plates in final differentiation medium (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, BDNF (20 μg/ml), GDNF (20 μg/ml), N6, 2′ -O-dibutyryladenosine 3′,5′ -cyclic monophosphate sodium salt (dcAMP, 0.5 mM; Sigma), Laminin (1 μg/ml) and antibiotic/antimycotic (1% (v/v)). Neurons were matured for 2 weeks in this medium before experimental procedures were carried out.