Serum free neurobasal medium

Purified rat RGCs were isolated on postnatal day 5 from retinas that were dissociated with MACS dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The RGCs were isolated with the MACS RGC isolation kit (Miltenyi Biotec). The dissociation of the retinas and isolating the RGCs were performed following the manufacturer’s instructions. Cells were plated at about 2,500 cells/well in 96-well plates and cultured in serum-free neurobasal medium (Invitrogen) supplemented with 2% B-27 supplement (Invitrogen), 1 mM pyruvate acids (Sigma-Aldrich), 60 ng/ml N-acetylcysteine (Wako), 10 µM forskolin (Wako), 2mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 40 ng/ml triiodothyronin (Sigma-Aldrich), 5 µg/ml insulin (Sigma-Aldrich), 100 U/ml penicillin (Meiji Seika Pharma, Tokyo, Japan), and 50 mg/ml streptomycin (Meiji Seika Pharma). The plates had been coated with 0.05 mg/ml poly-D-lysine (Sigma-Aldrich) overnight, rinsed three times with PBS, and then coated for 2 hours with 1 µg/ml of laminin (Corning, Corning, NY, USA).
After incubating for 24 h, the RGCs were exposed to 3 µM AQEE-30 or a combination of 50 ng/ml recombinant human BDNF (Miltenyi Biotec) and 10 ng/ml recombinant human CNTF (Miltenyi Biotec) for 48 h. Then, 2 mM calcein-AM (Dojindo Laboratories, Kumamoto, Japan) was added to label the surviving RGCs.

PD-MSCs from 6 pd (corresponding to passage 4) were plated in nonadherent conditions: serum-free neurobasal medium (Gibco), supplemented with 20 ng/ml epidermal growth factor (EGF) (Sigma), 40 ng/ml bFGF (Sigma) and 1% neuronal supplements N2 (Gibco) and B27 (Gibco), and 2 μg/ml heparin using 60 mm low-attachment culture dishes at a density of 1.9 × 10⁶ cells/dish. After four days from seeding, the cells formed primary floating neurosphere-like structures. These structures grew rapidly until day 7. At this time, before the obtained neurosphere-like structures became necrotic, we harvested them and resuspended them in Accutase enzymatic solution (Gibco) for five minutes at 37°C and then mechanically dissociated them into a single cell suspension. The cells were re-seeded in the same non-adherent conditions as above, and the secondary spheres were allowed to form. This protocol was applied for more rounds of spheres formation. As positive or negative controls we used NB LAN-5 (kindly provided by Dr. Doriana Fruci) and SK-N-SH (purchased from American Type Culture Collection, Manassas, VA, USA) cell lines, respectively.

Rabbits were anesthetized with pentobarbital sodium and 1 mL of bone marrow was harvested from the left iliac crest. The bone marrow cells were washed and cultured in D-MEM/F-12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Sigma). Epidermal growth factor (EGF, Invitrogen) and basic fibroblast growth factor (bFGF, Invitrogen) were added when cells became adherent. Upon reaching 80% confluence, the cells were cultured in serum-free neurobasal medium (Gibco) supplemented with 1% N2 (Gibco) and 2% B27 (Gibco). Following the formation of cell clusters, retinoic acid (RA, 2000 nM, Sigma) and sonic hedgehog (SHH, 500 ng/mL, ProSpec-tany) were added, then the cells were used 48 hours after addition.

Primary neuronal culture was performed as described previously (10 (link),21 (link),22 (link)). Freshly dissociated (trypsin) hippocampi were plated (10⁵ cells/well in a 12-well dish containing an 18-mm coverslip) in neuronal attachment media consisting of 10% horse serum (Eurobio), 1 mM sodium pyruvate (Thermo Fisher Scientific), 2 mM Glutamax-100X (Thermo Fisher Scientific), and penicillin/streptomycin (Thermo Fisher Scientific) in MEM (Thermo Fisher Scientific) for 3 h. The attachment medium was replaced, and cells were maintained in serum-free neurobasal medium (Thermo Fisher Scientific) supplemented with B27 (Gibco, Gaithersburg, MD) and 2 mM Glutamax-100X. Exposure to fibrillar α-Syn polymorphs was performed at days in vitro 14 (DIV 14). Fibrillar α-Syn polymorphs were diluted in fresh neurobasal medium. The “cell-conditioned neurobasal medium” was replaced with fibrillar α-Syn containing neurobasal medium for 15 min, the former kept aside at 37°C. After 15 min exposure, fibrillar-α-Syn-containing medium was removed, and the well was washed thrice. Lastly, the cells were replenished with “cell-conditioned neurobasal medium” and transferred back to the incubator until DIV 21.

Muse‐AT cells were seeded onto adherent dishes for induction into the three germline cell lineages. For myocyte induction, Muse‐AT cells were incubated with 5% N‐hydroxysuccinimide (NHS), 50 µM hydrocortisone, and antibiotics in DMEM supplemented with 20% FBS. Hepatocyte differentiation medium consisted of DMEM plus 20% FBS, 10 µg/ml insulin, 5 µg/ml transferrin, 7 ng/ml Na₂SeO₃, 10 nM dexamethasone, and 100 ng/ml hepatocyte growth factor‐4. For myocyte and hepatocyte induction, Muse‐AT cells were cultured in their respective differentiating media for 7 days, and their identity was revealed by immunofluorescence microscopy of smooth muscle actin (SMA) expression and cytokeratin‐7, respectively. For neuron formation, serum‐free neurobasal medium (Thermo Fisher) supplemented with B‐27, 2 mM glutamine, 30 ng/ml basic fibroblast growth factor (bFGF), and 30 ng/ml endothelial growth factor (both from Peprotech, Rocky Hill, NJ,
http://www.peprotech.com) was used for 7 days. Next, the cells were cultured for an additional 7 days in DMEM plus 2% FBS, 25 ng/ml bFGF, and 25 ng/ml brain‐derived growth factor (Peprotech).