Tubb3

TS formation from human GBM specimens followed previous methods (Kong et al. 2013b (link)), and their expression of stemness markers, CD133, nestin, musashi, and podoplanin (Abcam, Cambridge, UK), was tested by immunocytochemistry. Neuroglial differentiation in GBM TSs was evaluated by monitoring the expression of GFAP (Dako, Carpinteria, CA, USA), MBP, NeuN, and TUBB3 (Chemicon, Temecula, CA, USA).

Immunohistochemistry (IHC) and immunofluorescence (IF) of the selected tissue was performed using a polyclonal antibody against CB1R (Abcam Cat# ab23703, RRID: AB_447623, dilution 1:200 IHC, 1:20 IF), the immunogen corresponding to C terminal amino acids 461 ± 472 of human cannabinoid receptor. For double immunofluorescent labeling, the following monoclonal antibodies were additionally used: anti-glial fibrillary acidic protein (GFAP, Sigma-Aldrich Cat# G-A-5, RRID: AB_2314539, dilution 1:500), anti-beta-tubulin III (TUBB3, Sigma-Aldrich Cat# T8660, RRID: AB_477590, dilution 1:200), anti-neuronal nuclei (NeuN, Millipore Cat# MAB377, RRID: AB_2298772; dilution 1:800), anti-synaptophysin (SYP, Dako Cat# M0776, RRID: AB_2199013; dilution 1:250).

NCCs were plated onto matrigel‐coated coverslips and cultured in N2 medium supplemented with 10 ng/ml bFGF and 20 ng/ml EGF. The cells were incubated for 48 hr for characterisation studies and 5 days for spontaneous differentiation analysis. Immunocytochemistry was performed as previously described (Kingham, Kolar, Novikova, Novikov, & Wiberg, 2014). The following primary antibodies were used: transcription factor AP‐2 alpha (TFAP2A; DHSB, mouse monoclonal, 1:100), glial fibrillary acidic protein (Dako, rabbit polyclonal, 1:400), β‐1,3‐glucuronyltransferase 1 (B3GAT1; Sigma‐Aldrich, mouse monoclonal, 1:100), microtubule‐associated protein 2 (Chemicon, rabbit polyclonal, 1:100), TUBB3 (Sigma‐Aldrich, rabbit polyclonal, 1:500), NEFH (Abcam, mouse monoclonal, 1:500), NGFR (Advanced Targeting Systems, mouse monoclonal, 1:100), S100 calcium‐binding protein B (S100B; Dako, rabbit polyclonal, 1:2000), and SRY‐box 10 (SOX10; R&D Systems, mouse monoclonal, 1:100). The specificity of NGFR antibody was validated by immunocytochemical staining of both hESCs‐ and MACS‐enriched cells. NGFR positive cells were only observed in the enriched population, thus confirming the specificity of the NGFR antibody for NCCs as has been previously documented (Lee et al., 2007). No NGFR positive cells were observed in the hESCs cultures.

Proteins (30–40 μg) were separated using SDS-PAGE, transferred to nitrocellulose membranes, and stained with Ponceau Red. According to the MW markers and Ponceau patterns, membranes were cropped to detect different proteins and were separately blocked with blocking buffer (PBS/0.05% Tween/5% non-fat milk). Membranes were incubated at 4 °C overnight with primary antibodies, followed by incubation for 1 h with a secondary HRP-conjugated anti-IgG antibody (Jackson). The primary antibodies were Flag (Cell Signaling D6W5B), Snail (Abcam 135708), Twist (Abcam 49254), E-cadherin (Abcam 76055), β-actin (Santa Cruz Biotechnology 47778), MMP9 (Santa Cruz Biotechnology 4778), Nestin (Abcam 22035), CD44 (Cell Signaling 3570S), GFAP (Cell Signaling 3670S), TUBB3 (Sigma Aldrich 4700544), and Sox2 (Cell Signaling 3579S). Bands were revealed using the West Dura chemiluminescence system (Thermo-Fisher), and imaging was performed on a Syngene G:Box instrument (Synoptics, Cambridge, UK).

Histological and immunohistochemical staining was performed on conduit cryosections (16 μm) as previously described (Jones et al., 2015). The following primary antibodies were used: human nuclear antigen (HNA; Millipore, mouse monoclonal, 1:100), TUBB3 (Sigma‐Aldrich, rabbit polyclonal, 1:500), and S100B (Dako, rabbit polyclonal, 1:2000). The specificity of the HNA antibody was validated by immunohistochemical staining of conduit cryosections harvested from all PNI treatment groups. HNA positive cells were only observed in the hESC and NCC transplanted animals, thus confirming the specificity of the HNA antibody to cells of human origin. No HNA positive cells were observed in the rat Schwann cell treatment group (Figure S1).