H1138

Purified iMuSCs were plated onto a 1:100 Matrigel growth factor reduced (Corning 354230) in a StemFit media-coated 96-well microplate (IWAKI 3860–096). The microplate was pre-coated with Matrigel diluted by StemFit media at 1:100 for 1 h at 37°C. Then, 5,000 purified iMuSCs were seeded onto the plate and cultured in StemFit media containing 10 μM Y-27632 for expansion.
iMuSCs started to proliferate and reach confluency after approximately 5 days of culture. In this process, the medium was changed every day until day 5. After the day of seeding, the medium was replaced to Stemfit media without Y-27632. Differentiation was induced by switching to differentiation media composed of DMEM (Invitrogen 11885084) supplemented with 1% N2 supplement (Gibco 17502048) or 2% HS (Sigma H1138). Recombinant rat Agrin (R&D Systems 550-AG) was used for better differentiation.

All medium was based on Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM-HG cat no. D5796, Sigma-Aldrich). Components added to the medium were sterile filtered with syringe filters with a 0.45 µm pore size (GE Healthcare, Whatman). The myoblast cell line C2C12 (DSMZ, ACC 565) was maintained at 37°C in 5% CO₂ at 95% humidity in growth medium (GM) containing DMEM-HG (Sigma-Aldrich), 10% fetal bovine serum (F6178, Sigma-Aldrich), and 1% penicillin/streptomycin (15140122, Thermo Fisher Scientific). Differentiation medium (DM) was prepared from DMEM-HG with 2% horse serum (H1138, Sigma-Aldrich) and 1% penicillin/streptomycin.

For immunofluorescent staining, 20-μm serial sections were captured on superfrost+ glasses (Thermo Scientific, 10149870) using a cryostar NX70 (Thermo Scientific). Slides were dried 1 h at RT and stored at −20 °C before staining. Slides were washed in PBS/0.05% Tween-20 (Sigma) and incubated in PBS containing 10% NHS (Sigma, H1138) and 0.2% NP-40 (Sigma) at RT for 30 min to block non-specific binding, followed by incubation in a moist chamber with primary antibodies (for list of antibodies and dilutions used, see Supplementary Table 4) in PBS containing 1% NHS and 0.02% NP-40 at 4 °C overnight. After washing with PBS/0.05% Tween-20 (Sigma, P9416-100 ML), sections were incubated with the appropriate secondary Alexa Fluor 488, 594, or 647 (Invitrogen) antibodies in PBS/0.05% Tween-20 with DAPI (Sigma, D9542-10 mg) at RT for 1 h. After washing, the slides were mounted in fluorescent mounting solution (Dako, S302380-2).

Hippocampal slice cultures from Wistar rats of either sex were prepared at postnatal day 4–7^{54 (link)}. Briefly, rats were anesthetized with 80% CO₂ 20% O₂ and decapitated. Hippocampi were dissected in cold slice culture dissection medium containing (in mM): 248 sucrose, 26 NaHCO₃, 10 glucose, 4 KCl, 5 MgCl₂, 1 CaCl₂, 2 kynurenic acid, 0.001% phenol red (310–320 mOsm kg⁻¹, saturated with 95% O₂, 5% CO₂, pH 7.4). Tissue was cut into 400 µM thick sections on a tissue chopper and cultured at the medium/air interface on membranes (Millipore PICMORG50) at 37 °C in 5% CO₂. No antibiotics were added to the slice culture medium which was partially exchanged (60–70%) twice per week and contained (for 500 ml): 394 ml Minimal Essential Medium (Sigma M7278), 100 ml heat inactivated donor horse serum (H1138 Sigma), 1 mM l-glutamine (Gibco 25030-024), 0.01 mg ml⁻¹ insulin (Sigma I6634), 1.45 ml 5 M NaCl (S5150 Sigma)), 2 mM MgSO₄ (Fluka 63126), 1.44 mM CaCl₂ (Fluka 21114), 0.00125% ascorbic acid (Fluka 11140), 13 mM D-glucose (Fluka 49152). Wistar rats were housed and bred at the University Medical Center Hamburg-Eppendorf. All procedures were performed in compliance with German law and according to the guidelines of Directive 2010/63/EU. Protocols were approved by the Behörde für Gesundheit und Verbraucherschutz of the City of Hamburg.

TS603, TS667, TS543 were obtained from Memorial Sloan Kettering Cancer Center (MSKCC). SU-AO3, NCH612 glioma lines were described previously [23 (link)]. The patient-derived IDH1-mutant glioma lines were routinely subjected to panel sequencing analysis [26 (link)] and immunoblotting to confirm the expression of mutant IDH1 (R132H). Glioma lines were maintained as neurospheres in NeuroCult (StemCell Technologies) with 2 µg/ml heparin sulfate (Sigma), 20 ng/ml of EGF (StemCell Technologies), and FGF2 (StemCell Technologies). PC12 cells (provided by Dr. Agarwal, Heidelberg University; Dr. Chang, Gachon University) were maintained in DMEM (D5796, Sigma) supplemented with 10% horse serum (H1138, Sigma) and 1% penicillin–streptomycin. Human neural progenitor cells (SCC008, Sigma) were maintained in DMEM (D5796, Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin. All cells were grown in a 5% CO₂ humidified incubator at 37 °C.

After the dissection, the retina was washed 3 times with PBS at room temperature for 5 min each round. Then the retina was incubated with a blocking solution (5% horse serum (H1138, Sigma Aldrich) + 0.3% Triton X-100 (BP151–500, Fisher Scientific) in PBS) at room temperature for 1 h and then incubated with primary antibodies goat anti-VAChT (1:800, Millipore, ABN100), goat anti-ChAT (1:200, AB144P, Millipore), rabbit anti-RFP (1:1000, ab62341, Abcam) at 4 °C for 5 days. Next, the retina was washed 3 times with PBS at room temperature for 30 min each round and then incubated with secondary antibodies Donkey anti-goat-Alexa Fluor 488 (1:500, A32814, Invitrogen) and Donkey anti-rabbit-Alexa Fluor 594 (1:1000, A32754, Invitrogen) at room temperature for 2 h. Then the retina was washed 3 times with PBS at room temperature for 5 min each round and mounted in a homemade mounting medium (80% glycerol (BDH1172, VWR), 1% DABCO (D27802, Sigma Aldrich) in PBS, pH 8.6). The above commercial antibodies have been previously validated by their manufactures and were well characterized in the literature.

Mixed glial cultures generated from P0 to P3 neonatal wild-type mice were maintained in full DMEM for 10 to 14 days until a monolayer of astrocytes on the bottom and primary oligodendroglial progenitor cells (OPCs) with loosely attached microglia on the top, were apparent. The separation of OPCs was achieved initially with the removal of microglia, by shaking in 200 rpm for 1 h in 37 °C and then with continuous shaking under the same conditions for 18 h, as previously described^{56 (link)}. Afterwards, isolated cells were platted on poly-D-lysine-coated coverslips (P7405, Sigma-Aldrich, USA) with a density of 80,000 cells/mm² and maintained in SATO medium (284369) supplemented with Insulin-Transferrin-Selenium solution (41400045, ITS- Gibco, Invitrogen, Carlsbad, CA, USA), 1% penicillin/streptomycin and 1% horse serum (H1138; Sigma-Aldrich, St. Louis, MO, USA) for 4 days. αSyn fibrils (final concentration 0.5 µg/mL culture medium/well) amplified from human MSA and PD brains were added to TPPP/p25α-positive mature differentiated oligodendrocytes for 48 h and then cells were fixed and preceded for immunofluorescence analysis. All experimental procedures were approved by the Ethics Committee for the Use of Laboratory Animals in the Biomedical Research Foundation of Athens.