Pdgf aa

A human NPC line, established from human-induced pluripotent stem cells (hiPSCs), was obtained from Royan Institute, Tehran, Iran [23 (link)], and used. NPCs were passaged in a 1:3 ratio for expansion on poly-d-lysine (PDL)-coated plates and cultured in neurobasal medium (Gibco) supplemented with 1× penicillin/streptomycin, 25 ng/ml bFGF, 20 ng/ml epidermal growth factor (EGF), and 2 mM L-glutamine (all from Invitrogen).
At about 70% confluency, OPC differentiation was induced according to a previously published protocol with minor modifications [24 ]. Briefly, NPCs were grown for 3 weeks in the oligo medium containing serum-free DMEM/HAMS F12 medium (Gibco) supplemented with 1% bovine serum albumin, 2 mM L-glutamine, 50 μg/ml gentamicin, 1× N2 supplement, 3 nM T3 (SIGMA), 2 ng/mL Shh (SIGMA), 2 ng/mL NT-3 (SIGMA), 20 ng/mL bFGF, and 10 ng/mL PDGF-AA (SIGMA). Differentiation of OPCs to OLs was initiated by growth factors withdrawn for 2 days.
Human embryonic kidney cells (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin sulfate). Cells were grown in a humidified atmosphere containing 5% CO² at 37 °C.

New born Sprague-Dawley rats within 48 hours were used to isolate primary OPC as described 51 (link). Briefly, the cerebral cortex was gently dissected from the rat pup brain and washed with PBS three times. Then the cerebral cortex was chemically digested and mechanically pipetted to single-cell suspension. After filtering through a 70 μm filter, approximately 1X10⁷ cells were plated on a T75 poly-D-lysine (PDL, Sigma-Aldrich) coated flask and grown for 7-10 days in DMEM with 10% FBS and 1% penicillin-streptomycin antibiotic. When the mixed glial cultures were observed to be confluent, the flasks were first shaken at 180 rpm at 37 °C for 45 to 60 minutes to remove microglia, then the medium was changed and cells were shaken for another 16-20 hours to collect OPCs. The obtained OPCs were further purified by 30-minute culture dish panning, then seeded on PDL coated plates in OPC proliferation medium: Neurobasal-A(Gibco, CA) with 2% B27 (Gibco), 10 ng/mL platelet-derived growth factor AA (PDGF-AA, Gibco), 10 ng/mL basic fibroblast growth factor (bFGF, Peprotech, NJ) and 2 mM glutamine (Gibco).

The brain cortex from P1 Sprague-Dawley rat pups was dissociated into a single-cell suspension by trypsinize at 37°C for 10 min and the suspension was filtered with a 70-um filter. The cells were seeded on poly-d-lysine (PDL, Sigma-Aldrich, San Louis, MO) coated culture flasks in DMEM (Corning, New York, NY) with 10% fatal calf serum (Gibco, Carlsbad, CA). After 8 days, the microglia was separated from glia cell mixtures after 30 min of culture by a 220 rpm shake. Then cells were collected by 20 h of culture by a 200 rpm shake to inject into the mice or seed on a PDL coated culture dish in Neurobasal-A (Gibco) containing 2% B27 (Gibco), 10 ng/ml PDGF-AA (Gibco), 10 ng/ml bFGF (Peprotech, NJ, United States) and 2 mmol/l glutamine (Gibco) (Yuan et al., 2018 (link); Wang et al., 2020 (link)).
Cells were fixed with 4% paraformaldehyde for 5 min at room temperature and blocked by 10% bovine serum albumin. Then OPCs were incubated with primary antibodies against PDGFR-α (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) and NG2 (1:200, Millipore) at 4°C overnight. Cells were incubated with the fluorescence conjugated second antibodies for 1 h at room temperature.

The brain cortex was dissected from P1 Sprague-Dawley rat pups as described^{25 (link)}. Brain tissue was dissociated into a single-cell suspension by trypsin at 37 °C for 10 min. After the suspension was filtered with a 70-µm filter, cells were seeded on poly-d-lysine (PDL, Sigma, St. Louis, MO) coated culture flasks in DMEM (Gibco, Carlsbad, CA) with 10% fatal calf serum (Gibco). Eight to ten days later, the microglia were separated from glia cell mixtures after 30 min of culture by a 220 rpm shake and then OPCs were collected by 20 h of culture by a 200 rpm shake. Collected cells were injected into the mouse striatum or seeded on a PDL-coated culture dish in Neurobasal-A (Gibco) containing 2% B27 (Gibco), 10 ng/ml PDGF-AA (Gibco), 10 ng/ml bFGF (Peprotech, Rocky Hill, NJ), and 2 mmol/l glutamine (Gibco).
For identification, cells were fixed with 4% paraformaldehyde and blocked by 10% bovine serum albumin. Then OPCs were incubated with primary antibodies against PDGFR-α (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), NG2 (1:200, Millipore, Bedford, MA), GAFP (1:200, Millipore), MBP (1:200, Abcam, Cambridge, UK), NeuN (1:200, Millipore), and Iba-1 (1:200, WAKO, Osaka, Japan) at 4 °C overnight. Cells were incubated with the fluorescence conjugated second antibodies 37 °C for 1 h.