Preparation and purification of mouse cortical astrocytes
Mouse cortices were isolated from postnatal day 1 mouse pups, all experimental procedures were carried out in accordance with institutional guidelines for the care and use of laboratory animals, and the protocol was approved by the Animal Ethics Committee of Hangzhou Normal University, China. Cortical tissues were diced into ~ 1 mm3 pieces, then digested with 0.05% Trypsin/EDTA (w/v) in a 37°C incubator for 5 minutes, followed by adding D/F20S medium (DMEM/F-12 supplemented with 20% v/v FBS, all from Gibco) to terminate digestion. Tissues were pipetted up and down until homogenized, and then transferred into a 15 ml centrifuge tube and centrifuged at 200 g for 5 minutes at room temperature. After removing the supernatant, D/F20S medium was added to resuspend the pellet, the suspension was transferred into flasks (one brain to one T25 flask) (Corning) and incubated with 5% CO2 at 37°C for 72 hours. Culture medium was replaced every other day. Around the 10th day after plating, mixed glial cultures became confluent, and floating cells were removed by gently rinsing twice with 1 × PBS, before fresh D/F20S medium was added. Culture flasks were placed in incubator to equilibrate the medium for 2 hours, followed by shaking for 15–18 h (37°C, 250 rpm) with tightened caps on an orbital shaker model 420, Orbital Size 1.0 (Thermofisher, MA). During this process, microglias and OPCs that grew on the astrocyte layer detached gradually, while astrocytes adhered to the dish firmly.
For astrocyte purification, the remaining cells were digested with 0.05% Trypsin/EDTA (w/v) and plated into uncoated petri dishes, followed by incubating in a 5% CO2, 37°C incubator for 30 minutes. The floating cells were removed and rinsed twice with 1 × PBS. The remaining cells were cultured in D/F20S medium to confluency. The purity of astrocytes was determined by immunostaining.
Generation of lentiviral particles.
For viral production, pCDH-Sox10 and pCDH-Sox10-GFP plasmids were obtained by inserting Sox10 ORF into pCDH vectors after the CMV promoter. Plasmids were prepared as previously described [15]. Lentiviral particles were generated by transfecting 293T cells. Supernatants were collected after 48 hrs, filtered through an 0.45 µm polyvinylidene difluoride filter and concentrated overnight. The viral titer was determined as previously described [16], and stored at -80°C.
Induction of the transdifferentiation of astrocytes to oligodendroglias in vitro
Purified astrocytes were placed into a 12-well plate at a density of 2 × 104 cell/cm2, and infected with the reprogramming virus on the next day. 6 hours after the viral infection, the original medium was discarded and cells were washed twice with 1 × PBS and replaced with fresh glial basal media (DMEM/F12 supplemented with 1 × N2, 1 × B27 and 1 × P/S) containing 10 ng/ml PDGFaa. Medium was replaced every other day until d15.
iOPC differentiation
The directed differentiation of iOPCs into mature oligodendrocytes was performed as previously described [17]. Briefly, iOPCs were cultured in PDL/Laminin-coated chamber slides (Millipore, CA) with glial basal media supplemented with 30 ng/ml Triiodothyronine (T3) for 3 days, then examined for MBP antigen expression by immunostaining.
Preparation of O4+ iOPCs by immunopanning
Immunopanned Petri dishes were prepared as previously described [18, 19]. For obtaining more O4+ iOPCs, we used 100 mm petri dishes for transdifferentiation culture and extended the transdifferentiation culture time to 21 days. Then cells were digested with 0.05% Trypsin/EDTA (w/v), and were subsequently seeded at 2.5 × 106 per 100 mm uncoated dish and cultured for 30 min to enable astrocytes to attach to the dishes. After the incubation, the non-adherent cells were collected, and seeded into dishes coated with the O4 antibody and incubated for 30 min at room temperature. The adherent cells were then removed by treatment with 0.05% trypsin/EDTA (w/v) and replated onto the PDL/Laminin-coated dishes in glial basal media plus 10 ng/ml PDGFaa.
DRG co-culture for myelination assays
IOPCs that are used to test myelination ability were induced with pCDH-Sox10 lentivirus that does not express GFP, so that the iOPCs obtained were all GFP negative. Dorsal root ganglion neurons (DRGs) were isolated from E15 Sprague-Dawley rat embryos as previously described [20], and cultured for neurite outgrowth as described by Dincman et al., 2012 [21]. iOPCs/DRGs co-cultures were maintained for 8–12 days in glial basal media supplemented with 30 ng/ml Triiodothyronine (T3) (Sigma), and fixed for immunostaining with anti-MBP and anti-neurofilament (axons) antibodies.
qRT-PCR
For quantitative real-time reverse transcription (qRT) PCR, cells were lysed, and RNA was isolated using RNAiso Plus (9109, Takara) according to the manufacturer’s manual. Total RNA samples were subjected to complementary DNA (cDNA) generation using the cDNA Synthesis kit (6210A, Takara). Samples were then subjected to quantitative PCR analysis on a Bio-Rad CFX96 Real Time PCR System. The following primer pairs were used: 5’-TTTCTCCAACCTCCAGATCC-3’ and 5’-CCGCATCTCCACAGTCTTTA-3’ for GFAP; 5’-GCAGCCACCTATCTCCT CATC-3’ and 5’-CGAGTAGGGTAGGATAACTTCGC-3’ for Olig1; 5’-GGCGGTGG
CTTCAAGTCAT-3’ and 5’-CATGGCGATGTTGAGGTCG-3’ for Olig2; 5’-ACCAA
AAGCAACGGAGAAGAG-3’ and 5’-GGCATTCCGAAACAGGTAACTC-3’ for Glast; 5’-GAAGCGCATGTCGAAAGAAGA-3’ and 5’-GGCGGAGGCAGTCAATT
CTC-3’ for NFIA; 5’- AGTACCCGCATCTGCACAAC-3’ and 5’-ACGAAGGGTCT CTTCTCGCT-3’ for Sox9. Relative mRNA level was normalized against the house keeping gene GAPDH.
Surgical procedures
All experimental procedures were carried out in accordance with institutional guidelines for the care and use of laboratory animals, and the protocol was approved by the Animal Ethics Committee of Hangzhou Normal University, China. After intraperitoneal injection of tamoxifen for 5 days, adult hGFAPCre−ER:Rosa26-tdTomato transgenic mice were anesthetized with Nembutal (50 mg/kg, i.p.) and received a dorsal laminectomy at the tenth thoracic vertebral level (T10) to expose the spinal cord, and then a 75 kdyn contusive SCI using the IH-0400 Impactor (Infinite Horizons). At 2 d after injury, mice were randomly assigned to four groups, which received PBS, Sox10 virus, Sox10 virus + EGF, or Sox10 virus + EGF + Gefitinib, respectively. Animals were re-anesthetized as above, and the laminectomy site was re-exposed. Virus or vehicle injections were made at the center of the lesion at depth 0.7 mm. 2 µl of Sox10 lentivirus (1 × 108 IU/µl) or vehicle was injected through a glass micropipette with an outer diameter of 50–70 µm at rate of 0.5 µl/min. And then, Alzet® Osmotic Pumps (Model 1004) were installed according to the manufacturer’s manual, and PBS, EGF (10 ng/µl), or EGF (10 ng/µl) plus Gefitinib (1 µM) were continuously delivered (0.11 µl/hr) in the lesion site for 21 days.
Immunostaining
Immunochemical analysis was carried out as previously described [22]. Anti-mouse O4 IgM (50%, v/v) were produced by hybridoma culture. Anti-mouse Olig2 (1:1000), anti-mouse GFAP (1:1000), and anti-rabbit neurofilament (NF-1) (1:1000) were purchased from Millpore. Anti-rat MBP (1:500) and anti-mouse CC1 (1:500) was obtained from Abcam, and anti-rabbit PDGFRα (1:500) from Santa Cruz. Anti-mouse Nestin (1:1000) and the Alexa-488 or Alexa-594 conjugated secondary antibodies were purchased from Invitrogen. The nucleic acid dye 4’,6-diamidino-2-phenylindole (DAPI) was obtained from Roche.
Western immunoblotting
Western blotting was carried out as previously described [23]. Primary antibodies were used as follows: anti-Olig2 (1:3000, AB9610, Millpore), anti-GFAP (1:1000, MAB360, Millpore), anti-Nestin (1:1000, MA1-110, Invitrogen), anti-Erk1/2 (1:5000, ab184699, Abcam), and anti-p-Erk1/2 (1:5000, ab76299, Abcam). Horseradish peroxidase (HRP)-conjugated secondary antibody (Promega) was used at 1:2500. Chemiluminescent signals were detected by autoradiography using the ECL System (Amersham, Piscataway, NJ, USA).
Statistical analysis
All quantitative data are presented as Means ± SD. Statistical significance of the difference was evaluated by Student’s t-test. P-value < 0.05 was considered statistically significant.