Pdgf aa

Dulbecco’s modified Eagle Medium (DMEM) (10-017-CV), DMEM/F12 50:50 (10-090-CV), N2-supplement (17502-048), and penicillin/streptomycin (15140) were from Invitrogen (Carlsbad, CA, USA); Hank’s balanced salt solution (HBSS) (21-022-CV), 0.25% trypsin (25-050-CV) and sodium pyruvate (25-000-Cl) were from Mediatech (Manassas, VA, USA); fetal bovine serum (FBS) (SH30070.03) was from Hyclone (Waltham, MA, USA); FGF-2 (01–106) and PDGF-AA (01–309) was from Millipore (Temecula, CA, USA). Laminin was from R&D (Minneapolis, MN, USA). All other chemicals not specifically mentioned were purchased from Sigma (St. Louis, MO, USA).

Primary cerebellar cultures were prepared from C57BL/6J mice at postnatal day 10 as previously described [55 (link), 56 (link)]. Briefly, cerebella were embedded in 1% agarose and sagittally sectioned in ice-cold DPBS (Sigma‒Aldrich, G8769) supplemented with 5% glucose (Sigma‒Aldrich, G8769) at 300 μm using a Leica VT1000S vibrating microtome. Slices were immediately placed in 24-well plates on individual 0.4 μm 12 mm diameter Millicell-CM cell culture inserts (Millipore, PICM01250) and grown in 350 μL of cell culture medium containing 50% BME (Thermo Fisher, 21010046), 15% heat-inactivated horse serum (Thermo Fisher, 16050114), 25% Hank's solution (Sigma‒Aldrich, H4641), 0.5% glucose, 1% GlutaMAX™ Supplement (Thermo Fisher, 35050061), 1% penicillin‒streptomycin (Corning, 30-002-CI), N2, and 10 ng/mL PDGF-AA (Sigma‒Aldrich, P3076). The slices were incubated at 37 °C in 5% CO₂. Half-volume media changes were made two days after plating. After 4 days, mouse cerebellar slices were treated with 10 µL of Nef or Ctrl EVs once daily for 2 or 4 days. The slices were fixed with 4% PFA for 15 min, delipidated in ice-cold 2.5% acid methanol for 15 min at − 20 °C, and subsequently processed for immunohistochemistry as described below.

Based on the minimal requirements for NPC proliferation and differentiation, we created four distinct experimental groups with different growth factor concentrations/combinations for our in vitro experiment (Table 1): no growth factors (group 1; control group), 10 ng/ml EGF + 10 ng/ml bFGF (group 2; minimum concentration/normal combination group), 20 ng/ml EGF + 20 ng/ml bFGF (group 3; normal concentration/combination group), and 20 ng/ml EGF + 20 ng/ml bFGF + 6 ng/ml PDGF-AA (Sigma-Aldrich, USA; group 4; normal concentration/enhanced combination group). NPCs at the third passage (p3) with a density of 2 − 3 × 10⁵ cells/ml were incubated with the corresponding growth factor combination/concentration groups in DMEM/F12 with sodium bicarbonate and L-glutamine, 1% penicillin/streptomycin and 1x N2 supplement in a humidified incubator at 37°C with 5% CO₂ for seven days. Medium change (50%) was performed daily. After 7 days, the cells were fixed with 4% paraformaldehyde (PFA) (Santa Cruz, USA) for 20 minutes and washed with PBS before they were subjected to immunofluorescence staining. All experiments were performed in triplicate on 24-well plates.

Mixed glial cultures were generated from 1 to 2-day-old mice pups as described previously [17 (link)]. Briefly, the cerebra of mice pups were dissected, minced, and digested at 37 °C using a neural tissue dissociation kit (Miltenyi Biotec, catalog #130-092-628) to generate a single-cell suspension. Cells were plated into 75-cm² flasks and grown in DMEM with 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂ for the next 10 days. After 6–8 days, oligodendrocyte precursor cells (OPCs) can be visualized growing upon a mesh of astrocytes. On the 10th day, OPCs were purified from mixed glial cells by shaking. Cells were shaken initially for 1 h at 200 rpm to remove microglia, refed, and shaken again for 18–20 h at 37 °C at 200 rpm. OPCs were collected by centrifugation at 10 min at 100 × g. OPCs were then cultured in oligodendrocyte medium (Sciencell, catalog #1621). PDGF-AA (Sigma) was added at 10 ng/ml to Sato medium for allow for oligodendrocyte growth.

Primary cortical OPCs were prepared and maintained as described previously.
³⁵ Briefly, cells were dissociated from the cortex of neonatal mice after 1–3 days. Dissociated cells were plated in 75‐cm² flasks coated with 0.01% Poly‐D‐lysine (A3890401; Gibco) and maintained in DMEM containing 20% heat‐inactivated fetal bovine serum and 1% penicillin/streptomycin. After 10–12 days of inoculation, cells were placed on an orbital shaker at 37°C and shaken at 200 rpm for 1 h to remove microglia. The medium was then replaced with fresh medium and allowed to stand for 4 h before being shaken horizontally at 220 rpm for 16–18 h. The suspension was collected and left in the incubator for 30 min to remove astrocytes and microglia. The non‐adherent cells were collected and replanted in neurobasal medium containing glutamine, 1% penicillin/streptomycin, 10 ng/mL PDGF‐AA (100‐13A; Peprotech), 10 ng/mL FGF (AF‐100‐18B; Peprotech), 2% B27 (17504044; Gibco), and 1% N2 (17502048; Gibco) supplement on poly‐DL‐ornithine‐coated plates. Four to five days after plating, the OPCs were used for experiments. For cell differentiation, PDGF‐AA and FGF growth factor were excluded from the OPCs medium, and 3,3′,5‐triiodo‐L‐thyronine (T3; 40 ng/mL; Sigma‐Aldrich) and ciliary neurotrophic factor (CNTF; 10 ng/mL; MCE) were added to allow their differentiation.

Selective growth factors and cytokines, including TNF-α (20 ng/mL), FGF2 (25 ng/mL, Chiron, Emeryville, CA, USA), VEGF (25 ng/mL, Genetech, Plano, TX, USA), IGF-1 (50 ng/mL, Sigma), PDGF-AA (20 ng/mL, Sigma), PDGF-BB (20 ng/mL, Sigma), PDGF-AB (20 ng/mL, Sigma), HGF (20 ng/mL, Sigma) and IL1-β (5 ng/mL, Sigma), were added in BAECs or HAECs. The concentration of the growth factors was in accordance with doses used in a previous study [60 (link)]. After 4 h treatment, Northern blot analysis or Quantitative-PCR analysis was performed.

LSCs from all 13 testis biopsies were cultured in an expansion medium (EM) adapted for embryonic stem cell culture with little modification [44 (link)]. LSCs were maintained in this medium for at least 14 days. To induce differentiation, these cells were replated in a new medium containing differentiation-inducing factors. Differentiation-inducing medium (DIM) included DMEM containing 2 mM l-glutamine and 15 mM HEPES (Thermo Fisher Scientific), 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 2 ng/ml EGF (Sigma-Aldrich), 1 ng/ml LIF (Sigma-Aldrich), 10 ng/ml PDGF-AA (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), and the insulin transferrin selenium (ITS) supplement (Thermo Fisher Scientific).