Culture of human neural stem cells
Primary dissociated cell cultures of fetal human telencephalon tissues of 14 weeks gestation were prepared as described previously [23, 50, 51]. The cells were grown in T25 flasks in Dulbecco's modified Eagle medium (DMEM; HyClone, Logan, UT), supplemented with high glucose, 5% fetal bovine serum (FBS), 20 μg/ml gentamicin (Sigma, St Louis, MO), and 2.5 μg/ml amphotericin B (Sigma). The medium was changed twice a week. The permission to use the fetal tissues was granted by the Clinical Research Screening Committee involving Human Subjects of the University of British Columbia, and the fetal tissues were obtained from the Anatomical Pathology Department of Vancouver General Hospital.
PA317 amphotropic packaging cell line was infected with the recombinant replication-incompetent retroviral vector pLNX.v-myc, and the supernatants from the packaging cells were used to infect NSCs in human fetal telencephalon cultures. Stably transfected colonies were selected by neomycine resistance. Several stable clones of human NSCs were isolated, and one of them, HB1.F3 (F3 hereafter), was expanded for the present study. F3 human NSCs express ABCG2, nestin, and Musashi1, which are cell type specific markers for NSCs [28, 52, 53].
To generate Olig2 overexpressing human NSC line (F3.Olig2), Olig2 cDNA (a generous gift from Dr. Takebayashi, Okazaki, Japan) was ligated into multiple cloning sites of the retroviral vector pLPCX. PA317 amphotropic packaging cell line was infected with the recombinant retroviral vector, and the supernatants from the packaging cells were added to the F3 cells. Stably transfected colonies were selected by puromycine resistance.
RT-PCR and immunocytochemistry
For RT-PCR analysis, NSCs were grown on poly-L-lysine coated Petri dishes in DMEM with 2% FBS for 3 days. Total RNA was extracted from cultured cells using Trizol (GIBCO-BRL). One μg of total RNA was reverse-transcribed into first-strand cDNA using oligo-dT primer (Promega, Madison, WI). For PCR amplification, specific primer pairs were incubated with 1 μl of cDNA in a 20 μl reaction mixture containing Taq polymerase. The sequences of the primers were as follows; AAATCGCATCCAGATTTTC/CACTGCCTCCTAGCTTGTC for Olig2, TCTACGACAGCAGCGACAAC/CTTGGAGCTTGAGTCCTGAG for Nkx2.2, CATGACCACAGTCCATGCCATCACT/TGAGGTCCACCACCCTGTTGCTGTA for GAPDH.
To examine phenotypic differentiation, human NSCs were grown on poly-L-lysine coated 9 mm Aclar fluorocarbon plastic coverslips in DMEM with 2% FBS for 5 days. Cells were washed three times with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde for 10 minutes. After blocking with 5% normal goat serum, cells were incubated with primary antibodies for 2 hours at room temperature (RT). The following primary antibodies were used to examine the expression of neural cell phenotypic markers; anti-O4 (1:5; mouse monoclonal; Kim Lab), anti-galactocerebrosidase (GalC) (1:5; mouse monoclonal; Kim Lab), anti-myelin basic protein (MBP) (1:500; rabbit polyclonal; Chemicon, Temecula, CA), anti-glial fibrillary acidic protein (GFAP) (1:500; rabbit polyclonal; Chemicon), and anti-MAP2 (1:200; rabbit polyclonal; Chemicon). The coverslips were then incubated with appropriate secondary antibodies tagged with Alex-488 or Alexa-594 fluorophores (Molecular probes, Eugene, OR) for an hour. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI; Sigma) before mounting on the slides.
Growth rate of NSCs was determined by measuring viable cell numbers at different time points after plating. The number of cells was measured by Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, cells were dispensed as 100 μl of cell suspension (1000 cells/well) in a 96-well plate. Ten μl of CCK-8 solutions were added to each well at different time points (0, 6, 12, 18, 24, 48, and 72 hours), and the plate was incubated at 37°C for 4 h. The absorbance value at 450 nm wavelength was measured with a dual-beam microtiter plate reader. The experiment was replicated four times with triplicate samples included in each experiment.
For 5-bromo-2'-deoxyuridine (BrdU) incorporation experiment, 100 μl of cell suspension (1000 cells/well) was plated on a 9 mm coverslip and incubated for 36 hours. BrdU (final concentration 2 μM, Sigma) was added to each well for 2 hours followed by immunocytochemistry as described above using anti-BrdU (1:500; rat monoclonal; Oxford, UK). Counting was performed in three randomly selected fields (×200) and the average value was obtained from three coverslips for each condition. The percent of BrdU positive cells out of all DAPI stained nuclei was obtained as BrdU incorporation index.
Animals and surgery
Adult female Sprague Dawley rats (250 - 300 gram, Orient Bio Inc. Seongnam, Korea) were used in this study. All rats were maintained on a 12:12 hour dark and light cycle with food and water provided ad libitum. After being anesthetized with 4% chloral hydrate (10 ml/kg, injected intraperitoneally), rats were subjected to a dorsal laminectomy at the 9th thoracic vertebral level (T9-10) to expose the dorsal surface of the spinal cord. The NYU spinal cord impactor was used to inflict a standardized contusion; a 10 g impactor head was dropped from a height of 12.5 mm onto the exposed T9-10 spinal cord. Muscles and subcutaneous tissues were sutured in layer, and the skin was stapled. Sham operation involved only laminectomy without contusion on the spinal cord. The bladder was manually expressed twice daily until the animals resumed self voiding. Seven days after spinal injury, the spinal cord was reexposed for cellular transplantation. Animals were randomly divided into three groups: 1) vehicle (PBS) injected (Vehicle group), 2) transplantation of F3 cells (F3 group), and 3) transplantation of F3.Olig2 cells (F3.Olig2 group). Two injections were made at 2 mm rostral and caudal to the epicenter using a glass micropipette (tip diameter < 70 μm) configured with Hamilton syringe. The pipette pierced the dorsal spinal cord slightly off the dorsal median sulcus, avoiding blood vessels at the midline. We advanced the pipette with a depth of 1.2 mm from the dorsal surface and kept it for 3 minutes during the injection which was controlled by Nanoliter syringe pump (KD scientific; Holliston, MA, USA). Each injection consisted of 1 × 105 of the dissociated cells in 2 μl of PBS. Thus, a total of 2 × 105 cells were transplanted for each animal. To prevent leakage from the injection site, the pipette was maintained for three more minutes and then withdrawn slowly. Animals were assigned with new identification codes after transplantation to ensure blind evaluation of behavioral performance. All animals received daily intraperitoneal injection of cyclosporine (Sandimmun; Novartis, Bern, Switzerland) at a dosage of 10 mg/kg, beginning from one day prior to transplantation to three weeks after transplantation. After that, cyclosporine (50 μg/ml) was administered through drinking water until animals were sacrificed. Prophylactic antibiotics were intraperitoneally injected on the next day after each surgery.
Assessment of locomotor recovery
A total of 35 animals (N = 11, 12, and 12 for Vehicle, F3, and F3.Olig2 groups, respectively), divided in three series, underwent behavioral tests to assess locomotor recovery. The BBB (Basso, Beattie, and Bresnahan) locomotor rating scale was used to assess the extent of locomotor recovery during open field locomotion. Two experimenters who were blind to experimental conditions scored BBB scale separately and the average of the two scores was obtained. The grid walk test was conducted at 4 and 7 weeks after initial injury. We performed grid walk test for the second and third series of animals, so the animal in the first series were excluded (total 5; Vehicle = 1, F3 = 3, F3.Olig2 = 2). For grid walk, rats were trained to cross a grid runway (30 cm × 140 cm with 50 × 50 mm holes) for a water reward. Two animals in Vehicle group and one animal in F3.Olig2 group could not be trained adequately enough to undergo grid walk test. Therefore, grid walk data were obtained from 27 rats (Vehicle = 8, F3 = 9, F3.Olig2 = 9). On the day of test, four runs were recorded using a three-CCD digital video camera (NV-GS250, Panasonic, Japan), and later analyzed frame by frame in slow motion. The average number of limb placement errors per run was obtained for each animal. For footprint analysis, rats were trained to walk on a runway with a narrow alley made of transparent Plexiglas (7 cm × 170 cm) on top for a water reward during the same training session for grid walk. The animals' hindpaws were inked and footprints were obtained on white paper covering the floor of the runway. The base of support, which is a distance between the two hindlimbs, was determined by measuring the distance between the central pads of both hindpaws. Right and left stride lengths were measured between two consecutive prints on each side. Footprint analysis was performed only in the third series of animals (total 14; Vehicle = 4, F3 = 5, F3.Olig2 = 5). Normal foot print data were obtained from 5 rats with sham operation.
Histological processing and immunohistochemistry
For histological analysis, animals were anesthetized with an overdose of chloral hydrate and perfused with heparinized saline (0.9%), followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The spinal cord was dissected and post-fixed in 4% paraformaldehyde at RT for 2 hours, followed by cryoprotection in a graded series of sucrose solutions (10%-30%) in 0.1 M phosphate buffer at 4°C. Transverse sections (20 μm) of the spinal cord were cut using cryostat (Leica CM 1900; Wetzlar, Germany) in a 1:10 series and thaw-mounted onto silane-coated glass slides. To quantify the area of myelinated white matter, transverse spinal cord sections were stained with eriochrome cyanine that stains myelinated white matter . The transverse sections were immersed for 8 minutes in the staining solution consisting of 240 ml of 0.2% eriochrome cyanine RC (Sigma) and 10 ml of 10% FeCl3·6H2O (Sigma) in 3% HCl. The sections were then washed with running tap water, followed by differentiation in 1% aqueous NH4OH.
For immunohistochemistry, transverse spinal cord sections were incubated overnight at 4°C with anti-human mitochondria (1:400; mouse monoclonal; Chemicon), anti-Ki-67 (1:500; rabbit polyclonal; Novocastra, Newcastle, UK), anti-myelin basic protein (MBP) (1:400; rabbit polyclonal; Chemicon), anti-APC-CC1 (1:200; mouse monoclonal; Calbiochem, La Jolla, CA), anti-GFAP (1:500; rabbit polyclonal; Chemicon), anti-nestin (1:500; rabbit polyclonal; Chemicon), and anti-MAP2 (1:200: rabbit polyclonal; Chemicon). The spinal cord sections were washed and then incubated with biotinylated or fluorophore-tagged secondary antibodies. For chromogenic detection of antigen-antibody reaction, preformed avidin-biotinylated peroxidase complexes were applied for 30 minutes, followed by incubation with peroxidase substrate (DAB) until desired intensity developed. For fluorescence staining, coverslips were mounted onto glass slides using Gelvatol and examined under Olympus confocal laser scanning microscope (Model FV 300, Tokyo, Japan).
To analyze the thickness of myelin sheath wrapping individual axons, a separate series of animals (16 rats; Vehicle = 3, F3 = 6, F3.Olig2 = 7) were perfused with modified Karnovsky's fixative solution (2% glutaraldehyde and 1% paraformaldehyde in 0.1 M cacodylate buffer). Dissected spinal cord was divided into smaller blocks with about 5 mm in length. The tissue block containing caudal regions to the epicenter was transversely sectioned into 200 μm slices with a vibratome (model Vibratome Series 1000; Technical Products Int'l. Inc., O'Fallon, MO). The slices containing the regions around 1 mm caudal to the epicenter were immersed in 1% OsO4 solution, and then embedded in Poly/Bed 812 embedding media (Polysciences Inc., Warrington, PA). Transverse semithin (1 μm) sections were cut from the rostral surface with glass knife and then stained with toluidine blue.
Quantitative cell counting and image analysis
Human mitochondria positive cells (developed with DAB substrates) were counted using unbiased stereology. Two transverse sections 200 μm apart from each other at the epicenter, ± 1 mm, ± 2 mm, ± 3 mm, and ± 4 mm rostral and caudal from the epicenter were chosen. Cell counting was performed on an Olympus BX51 Microscope with a MAC 6000 Motorized Stage Encoder System that was coupled with a computer running Stereo Investigator 8 software (MBF Bioscience, Williston, VT). Human mitochondria positive cells within optical dissectors randomly placed in regions of interest were counted using standard stereological criteria for inclusion. Stereological estimates were done in a systematic way using the formula in the software. The average of the two sections at each level was obtained for each animal. The percentage of the cell number in the spared ventrolateral white matter was calculated by dividing the number of cells in the ventrolateral white matter by the total number in the entire section. To determine the percentage of grafted human NSCs that were colocalized with proliferation marker Ki67 or neural cell specific markers, two transverse sections with the highest graft survival were chosen for each animal. The number of human NSCs colocalized with these markers was counted in the entire section and divided by the total number of human mitochondria positive cells. The average from the two sections was calculated as the final percentage value for each animal.
The volume of myelinated white matter and cystic cavities were determined for the animals that completed behavioral tests. Every 10th eriochrome-stained transverse spinal cord sections were viewed on an optical microscope (Olympus BX41). The section containing injury epicenter was defined visually as the one with a smallest visible rim of spared myelin. Then, serial sections with an equal distance (400 μm) spanning ± 2 mm from epicenter were imaged at a 40× magnification and captured with CCD camera (Olympus DP11). Myelinated spared ventrolateral white matter anterior to the dorsal horn was drawn with the Pen Tablet input device (Bamboo MTE-450K; Wacom Co., Tokyo, Japan) on each image and the cross-sectional areas were measured using publicly available Image J software (NIH, Bethesada, MD). The total estimated volume was calculated using the Cavalieri's Principle. The individual subvolumes were obtained by multiplying the cross-sectional area by the distance between sections, and the subvolumes were summed to generate the total volume of spared white matter (∑n [cross sectional areas × intersection distance], n = number of sections analyzed). The volume of cystic cavities was calculated by the same equation.
Thickness of myelin sheath in individual axons was determined on toluidine blue stained transverse semithin sections. Two sections with adequate preparation were selected for each animal. In order to have corresponding regions imaged across all the animals, a single image field (120 μm × 90 μm) per section was located in the ventral white matter just anterior to the ventral gray horn by an independent experimenter who was blind to the experimental conditions. Images were taken at 1000× magnification using a Zeiss Axiophot upright microscopy equipped with Axiocam HR digital camera. Horizontal grid lines with a 10 μm interval were drawn using Photoshop software and the same grid lines were applied to all images. Only the axons that were intersected by the grid lines were included for quantification. The thickness of myelin sheath and the shortest axon diameter were measured using the Image J software. According to the previously published report by Karimi-Abdolrezaee et al. (2006) , the myelin ratio was obtained by dividing the total axonal diameter including thickness of myelin sheath by the diameter of axonal fiber excluding myelin sheath.
Statistical analysis was performed with SPSS version 12.0 (Chicago, IL, USA) or GraphPad Prism software version 4.0 (San Diego, CA, USA). The unpaired Student T test or one-way ANOVA followed by Tukey's post hoc test was used for statistical comparison of group means. Repeated measures two-way ANOVA was used to compare the number of cells or the percent cells in the white matter at different regions and BBB locomotor scores at multiple time points.