Isolation and cultivation of NPCs
Neural precursor cells were prepared according to the method of Hu [29], with appreciable modifications. Briefly, NPCs were isolated from the embryonic brain of the E16 Sprague-Dawley rat. After removal of the amnion and dura, the brain was dissected out in a sterile dish containing ice-cold Leibovitz's L15 medium (Invitrogen, Grand Island, NY, USA), and dissociated by gentle mechanical pipeting through fire-polished Pasteur pipettes to achieve single-cell suspensions. The dissociated cells were filtered through a nylon mesh of 70 μm (Falcon, NJ, USA), seeded into a T25 Corning tissue culture flask (Corning Inc., Corning, NY, USA) containing growth medium at a density of 1 × 105cells/ml, and incubated in a humid atmosphere containing 5% CO2 at 37°C. The culture medium for NPCs contained: 1 × Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12, Invitrogen), 1 × N2 (Invitrogen), 2% B27 (Invitrogen), 0.06% glucose, and 2 mM glutamine (Invitrogen), 1.34 mM bicarbonate sodium, 0.5 mM HEPES, 2 μg/ml heparin (all from Sigma, St.Louis, MO, USA), and freshly added 20 ng/ml epidermal growth factor (EGF, Sigma) and 20 ng/ml bFGF (Invitrogen). After 3 or 4 days, the cells grew and developed into visible neurospheres of 50-200 cells/sphere; then they were collected and mechanically pipetted into single cells for passage. The NPCs of passage 2 were collected for the following assay.
To identify the expression of CSPGs on the NPCs, 50 μl dissociated cells in single-cell suspension were seeded onto laminin (10 μg/ml, Gibco) coated coverslips in 35-mm dishes at a density of 3 × 104 cells/coverslip, and cultured in NPC growth medium supplemented with EGF and bFGF. 48 hours after culture, the cells were fixed for immunostaining.
All embryonic rats were obtained from female pregnant SD rats bred in the Animal Care Facility at Shanghai Jiaotong University School of Medicine. All animal care was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23; revised 1996).
Differentiation of NPCs
To induce the differentiation of NPCs, 65 μl dissociated cells in single-cell suspension were seeded onto poly-L-lysine (200 μg/ml, Sigma) coated coverslips in 35-mm dishes at a density of 5 × 104 cells/coverslip. Then, growth factors were removed from the growth medium, and 1% fetal bovine serum (FBS, Invitrogen) was added. The cultures were allowed to differentiate for 5 days in vitro before being fixed for immunostaining.
[3H] Thymidine Incorporation Assay
NPCs of passage 2 were dissociated and transferred to 96-well plates (1.0 × 104cells/100 μl/well), after that, another 100 μl of different concentrations (0.05-50 mU/mL) of Chondroitinase ABC (Chase ABC, Seikagaku, Tokyo, Japan) or 100 μl medium alone (control) were added to the wells, then cultured in medium with EGF and bFGF for 24 hours at 37°C containing 5% CO2. And 1 μCi/well [3H] thymidine (Amersham Piscataway, NJ, USA) was added to the cultures for the last 16 hours. The cells were harvested on fiberglass filters (Whatman, Kent, UK) using a cell harvester (Tomtec, CT, USA). Incorporated radioactivity was measured using standard scintillation techniques.
Immunocytochemistry (ICH)
For floating neurospheres, they were harvested and fixed using 4% paraformaldehyde (PFA) in 0.01 M PBS (pH 7.4) at 4°C overnight and cryoprotected in PBS containing 30% sucrose, then embedded in OCT (Sakura FineTec Inc., Torrance, CA, USA) and sectioned with a cryostat. For cell cultures on the coverslips, they needed to be fixed with 4% PFA for 10 min at room temperature (RT) in advance, then washed and stored in 0.01 M PBS (PH7.4). Thereafter, the sections of neurospheres or cell cultures were blocked in 10% goat serum in PBS (for cell surface staining) or 0.3% Triton X-100-containing 10% goat serum in PBS (for intracellular staining) for 1 hour at RT, then sequentially incubated with the following primary antibodies overnight at 4°C: the monoclonal mouse antibodies IgM against CS-56 (1:200, Sigma) for CSPGs, the monoclonal mouse antibodies IgG against nestin (1:800, BD Pharmingen, San Jose, CA, USA) for NPCs, βIII-tubulin (1:800, Sigma) for neurons, glial fibrillary acidic protein (GFAP, 1:200, Sigma) for astrocytes, or the monoclonal mouse antibodies IgM against O4 (1:600, R & D, Minneapolis, MN, USA) and the monoclonal mouse antibodies IgG against myelin basic protein (MBP, 1:40, Siemens Inc, Oncogene Group, Cambridge, MA, USA) for oligodendrocytes. After being washed with PBS, sections and cell cultures were incubated for 60 min at 37°C with the appropriate secondary antibody: fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FITC-conjugated goat anti-mouse IgG (1:100, Sigma), rhodamine-conjugated goat anti-mouse IgM (1:200, Santa Cruz Biotechnology), and rhodamine-conjugated goat anti-mouse IgG (1:100, Sigma). After staining, the slides or coverslips were rinsed and mounted with Gel/Mount aqueous mounting media (Biomeda Corp., Foster City, CA, USA) containing Hoechst 33342 (1 μg/ml, Sigma), a fluorescent nuclear dye. The images were acquired using an Olympus BX60 microscope equipped with a digital camera and SPOT 4.0.1(G) software. For cell counts, at least five randomly selected fields with a total of more than 1000 cells were counted. For neurite outgrowth assay, the neurite lengths of a total of 50 neurons in each group from four independent experiments were measured with a Neurolucida system (MicroBrightField Inc., Colchester VT, USA). In all experiments, primary antibody omission controls were used to confirm the specificity of Immunocytochemistry.
Western blot analysis
Dissociated NPCs were seeded into 60-mm dishes (Corning Inc.) and cultured in D/F12 medium containing 1% FBS for 5 days, with or without Chase ABC administration. Then cells were washed and lysed with a lysis buffer (50 mM Tris-HCl, 150 mM NaCl buffer, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 4 μg/ml leupeptin, 1 μg/ml aprotinin and 100 μg/ml PMSF; all from Sigma). The supernatant was clarified by centrifugation at 16,000 g for 10 min at 4°C. The protein concentration of the lysate was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). For western blotting, Protein samples containing an equal amount of protein (15 μg) were electrophoresed on SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride filters (Millipore, Bedford, MA, USA). The filters were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 hour at RT and then incubated overnight at 4°C with primary antibodies (in TBST-5% BSA) including GFAP (1:1000, Sigma), βIII-tubulin (1:2000, Sigma), MBP (1:100, Oncogene) as markers for the differentiated neural cells, and GAPDH (1:8000, Kangchen Bio-Tech) as an internal control. After being rinsed with TBST, the membranes were incubated with the secondary antibody HRP-conjugated goat anti-mouse IgG (1:1000, R & D) for 1 hour at RT. To visualize the immunoreactive proteins, the ECL kit (Pierce) was used, following the manufacture's instructions.
FACS analysis
For flow cytometric analysis of DNA content, NPCs were cultured in medium alone or incubated with Chase ABC at 37°C for 48 hours, and approximately 106 cells were harvested, washed with ice-cold PBS, then fixed with 70% ethanol at 4°C overnight. After that, fixed cells were washed in PBS and re-suspended in a staining solution containing 50 μg/mL propidium iodide (BD Pharmingen) in 0.1% sodium citrate, 0.1% NP-40, and 12.5 μL ribonuclease (RNase) 1 mg/mL. Cells were incubated for 15 min at 37°C, then kept in the dark and analyzed using a FACSCalibur (BD Biosciences, Mountain View, CA, USA) with excitation at 480 nm and the fluorescent signal was collected at 585 nm. Cell cycle populations in the G1-, S-, and G2-phases were quantified with the ModFit LT v3.0 software.
Cell migration assay
Neurospheres of passage 2, grown for 3 days in growth medium supplemented with EGF and bFGF, were uniformly seeded on the well bottoms of a sterile 35-mm dish, which were in advance coated with Laminin (10 μg/mL, Invitrogen). Neurospheres were plated at low density to ensure large distance between individual spheres and incubated with Chase ABC at 37°C in NPCs growth medium containing EGF and bFGF. The control groups were grown under identical conditions except for Chase ABC. At three selected time point, i.e. 2, 4.5 and 8 hours, the cultures were observed and photographed by an inverted microscope (IX70, Olympus, Tokyo, Japan) equipped with a CCD camera (DP70, Olympus). Three samples were evaluated for each set of conditions, and a total of more than 30 neurospheres at each selected time point were randomly captured and imaged. The average migration distance of the neurospheres was expressed by the diameter of the spheres surrounded by the rim of migrating cells, based on previous reports [7, 15], and represented as the mean of the straight-line measurement from the center of the neurosphere to the farthest defined edge of the migratory cell boundary in four vertical directions using SPOT 4.0.1(G) software.
Integrin blocking test
For neurosphere growth test, NPCs were divided into 4 groups: control group in regular growth medium, Chase ABC treatment group, integrin blocking group which were incubated with Echistatin (Ech, Sigma), the most potent known inhibitor of integrin function, and combined group with Chase ABC and Ech. The NPCs cultured on the uncoated dishes in the growth medium containing EGF and bFGF, were observed and photographed 24 hours after culture, by an inverted microscope (IX70, Olympus) equipped with a CCD camera (DP70, Olympus). For differentiation experiments, there were 3 groups: control group, Chase ABC group, and Chase ABC+Ech group. The dissociated NPCs of each group were cultured on the poly-L-lysine coated coverslips in differentiation medium with 1% FCS for 5 days, then, fixed with 4% PFA for MBP immunostaining as described above.
Statistical analysis
Data were presented as mean ± standard error of the mean (SEM) values. Statistical analysis was performed by SPSS 10.0 and One-way analysis of variance (ANOVA) with post hoc Tukey LSD test or two-tailed independent sample T-Test was used to determine statistical significance. A P-value less than 0.05 was considered statistically significant.