PDGF AA

We used a multiplex Xmap technology that uses magnetic microspheres internally coded with two fluorescent dyes to measure markers of neurodegeneration (Millipore, Cat#: HNABTMAG-68K). All samples including placebo and resveratrol at baseline and 52 weeks were analyzed in parallel using the same reagents. Through precise combinations of these two dyes, multiple proteins are measured within the sample. Each of these spheres is coated with a specific capture antibody. The capture antibody binds to the detection antibody and a reporter molecule, completing the reaction on the surface of the bead. CSF or plasma (25 μl) was incubated overnight at 4 °C with 25 μl of a mixed bead solution, containing human total tau, p-tau181, Aβ42, and Aβ40 (CSF Aβ40 is diluted 1:10). After washing, samples were incubated with 25 μl detection antibody solution for 1.5 h at room temperature. Streptavidin-phycoerythrin (25 μl) was added to each well containing the 25 μl of detection antibody solution. Samples were then washed and suspended in 100 μl of sheath fluid. Samples were then run on MAGPIX with Xponent software. The median fluorescent intensity (MFI) data was analyzed using a 5-parameter logistic or spline curve-fitting method for calculating analyte concentrations in samples. We also performed multiplex ELISA (Millipore, CAT#: HCYTOMAG-60K) to profile a panel of plasma and CSF markers that are indicative of inflammation, including human EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFNγ, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα, TNFβ, and VEGF.

Murine glioma cell lines were generated from retrovirus induced orthotopic murine glioma models as previously described^{21 (link)}. Briefly, C57Bl/6 neonatal (p4) mice harboring floxed p53 and stop-flox mCherry-luciferase were anesthetized using hypothermia and orthotopically injected with a PDGFA–internal ribosomal entry site (IRES)–cyclization recombination (Cre) retrovirus (stereotaxic coordinates relative to bregma: 1 mm anterior, 1 mm lateral, 1 mm deep), resulting in tumor cells that overexpress PDGFA and mCherry-Luciferase, and have deleted p53^{36 (link)}. End-stage tumors were harvested and tumor cells isolated and cultured in basal media (BFP), containing DMEM (Gibco™ 11965092) with 0.5% FBS (Gibco™ 16000044), antibiotic-antimycotic (Thermo Scientific 15240096), N2 supplement (Thermo Fisher Scientific, 17502-048), and 10 ng/ml each of recombinant human PDGF-AA (Peprotech, 100-13 A) and FGFb (Peprotech, 10018B50UG). Three biological replicates of PDGFA driven cells made from three independent tumors with the same genetic background were used for this study. A Pten^−/− P53^−/− PDGFB⁺ cell line was also used^{21 (link)}. All cells were grown at 37 °C with 5% CO2. For all murine glioma cell lines cysteine methionine deprived media was made from basal DMEM without cysteine, methionine and glutamine (Thermo Fisher Scientific, 21013024) that was supplemented with L-glutamine to a final concentration of 4 mM. All other components of the media were the same for control and CMD media. Human glioma cells were cultured as previously described^{37 (link),38 (link)}. TS543 cell neurosphere cell lines were cultured in Neurocult media as previously described^{37 (link)}, but were dissociated and plated in a single cell monolayer in 96 well plates in either BFP or cysteine methionine deprived BFP for dose response experiments. KNS42 cell lines were cultured in DMEM + 10%FBS or cysteine methionine deprived DMEM + 10%FBS. Thus, for all experiments the only difference between CMD media and control media used for each cell line was the concentration of cysteine and methionine.

Several in vitro assays were performed to assess the cellular activities of MSCs. An MTT assay (Sigma-Aldrich) was used to assess cell proliferation, and a colony forming unit-fibroblast (CFU-F) assay was used to assess self-renewal. Multipotency (in vitro differentiation into chondrogenic, osteogenic, or adipogenic lineages) and transwell migration in response to platelet-derived growth factor (PDGF; 10 ng/mL PDGF-AA, R&D Systems, Minneapolis, MN, USA) were also assessed. Angiogenesis was quantified using Matrigel, and in vitro anti-inflammation was analyzed as described previously^{8 (link)–10 (link),20 (link),21 (link)}. The digital images generated in these assays were assessed quantitatively using Image-Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).