NS/PC culture and analysis
NS/PCs were cultured and expanded, as previously reported. Briefly, the striata of transgenic mice ubiquitously expressing ffLuc-cp156, a fusion protein of firefly luciferase and a circularly permuted Venus protein, were dissociated using a fire-polished glass pipette on embryonic day 14. Venus is a fluorescent protein with fast and efficient maturation that was originally engineered from GFP, and therefore grafted cells can be detected as fluorescent Venus signals using anti-GFP antibody[17, 26]. Dissociated cells were collected by centrifugation and re-suspended in culture medium, which consisted of Dulbecco’s modified Eagle medium/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a previously described hormone mixture. Human recombinant FGF-2 (Peprotech, Rocky Hill, NJ, USA) and EGF (Peprotech) (20 ng/ml each) were added every 2 days. The cells formed floating cell clusters (neurospheres) within 2–3 days. After propagation for 3 passages, the neurospheres were used for in vitro BLI, differentiation, and proliferation assays, and for cell transplantation.
For differentiation analysis, the neurospheres were plated onto poly-L-ornithine/fibronectin-coated 8-well chamber slides (Iwaki; Asahi Glass Co Ltd., Tokyo, Japan) at a density of 1 × 105 cells/ml and cultured in culture medium without serum or growth factors at 37°C in 5% CO2 and 95% air for 7 days. The differentiated cells were then fixed with 4% paraformaldehyde in 0.1 M PBS and stained with the following primary antibodies for immunocytochemistry: anti-Tuj-1 (mouse IgG, 1:1000, Sigma-Aldrich), anti-GFAP (rat IgG, 1:1000, Invitrogen, Carlsbad, CA, USA), and anti-CNPase (mouse IgG1, 1:1000, Sigma-Aldrich). Nuclei were stained with Hoechst 33258 (10 μg/ml, Sigma-Aldrich). In vitro images were obtained using a fluorescence microscope (BZ-9000; Keyence Co., Osaka, Japan).
The proliferation assay was performed by measuring ATP, which indirectly reflects the number of viable cells[15, 17, 54]. In brief, NS/PCs were first incubated in culture medium without serum or growth factors in 48-well cell-culture plates (Corning Inc., Corning, NY, USA) at 37°C in 5% CO2 and 95% air for 24 or 72 h. D-luciferin was then added to each well, and the luminescent signal was detected immediately using a Xenogen-IVIS spectrum cooled charged-coupled device (CCD) optical macroscopic imaging system (Caliper Life Sciences, Hopkinton, MA, USA). To determine the population doubling time, the ATP assay was modified, as described elsewhere[15, 17, 54].
Adult female C57BL/6J mice (8–10 weeks old, 18–22 g, n = 52; Clea, Tokyo, Japan) were anesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After laminectomy at the 10th thoracic spinal vertebra (Th10), the dorsal surface of the dura mater was exposed. Contusive SCI was induced using a commercially available SCI device (IH Impactor, Precision Systems and Instrumentation, Lexington, KY, USA), as previously described. This device creates a reliable contusion injury by rapidly applying a force-defined impact (60 kdyn) with a stainless steel-tipped impactor. All experiments were approved by the ethics committee of Keio University and fully in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).
The injured mice were anesthetized and transcardially perfused with heparinized saline (5 U/ml) at 9 DPI or 42 DPI (n = 3 each). Dissected segments of spinal cord at the Th10 level were rapidly frozen and placed in TRIzol (Invitrogen). Total RNA was isolated using an RNeasy Mini Kit (Qiagen Inc., Hilgen, Germany), in accordance with the manufacturer’s instructions. As a control, samples of naïve spinal cord were harvested by the same protocol. For microarray analysis, RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and 100 ng of total RNA was reverse transcribed, biotin labeled, and hybridized to a GeneChip® Mouse Genome 430 2.0 Array (Affymetrix Inc., Santa Clara, CA, USA). The array was then washed and stained in a Fluidics Station 450, according to the manufacturer’s instructions[55, 56]. The microarrays were scanned using a GeneChip Scanner 3000 7G, and the raw image files were converted to normalized signal intensity values using the MAS 5.0 algorithm. PCA was carried out with Gene Spring GX software (Agilent Technologies Inc.), using the full set of normalized data. For the clustering analysis, the normalized data were narrowed down by the cut-off values of expression levels (>50) and by fold change (>1.5, versus the signal of intact spinal cord), and statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test (P < 0.05). The heat map was visualized with Gene Spring GX.
Various numbers of NS/PCs (approximate range 2.5 × 104 to 5 × 105 cells/2 μl) were transplanted into uninjured naïve mice (n = 3, each), and NS/PCs (5 × 105 cells/2 μl) were transplanted at 9 DPI (sub-acute TP group, n = 10) or 42 DPI (chronic TP group, n = 10) as previously reported[4, 16, 17, 26, 30, 57]. The NS/PCs were transplanted into the lesion epicenter with a glass micropipette at a rate of 1 μl/min using a Hamilton syringe (25 μl) and a stereotaxic microinjector (KDS 310, Muromachikikai Co. Ltd., Tokyo, Japan). PBS (2 μl) was similarly injected into the lesion epicenter of the control mice at each time point (sub-acute and chronic PBS groups, n = 10 each).
A Xenogen-IVIS spectrum CCD optical macroscopic imaging system was used for in vitro and in vivo BLI as previously reported[4, 16, 17]. In brief, the signal intensity of NS/PCs in vitro was assessed using plated cells at various cell numbers (approximate range 1 × 102 to 1 × 104 cells/well), and BLI was performed immediately after adding D-luciferin (150 μg/ml) (n = 3). The integration time was fixed at 1 min for each image.
In vivo imaging was performed 15 min after the i.p. injection of D-luciferin (0.3 mg/g body weight) with the field-of-view set at 13.2 cm, because the photon count was most stable during this period. The intensity peaked between 10 and 30 min after the i.p. injection of D-luciferin. The integration time was fixed at 5 min for each image. All images were analyzed with Living Image software (Caliper Life Sciences), and the optical signal intensity was expressed as photon flux (photon count) in units of photons/s/cm2/steradian. Each result was displayed as a pseudo-colored photon count image superimposed on a gray-scale anatomic image. To quantify the measured light, a region of interest was defined in the cell-implanted area, and all values at the same region of interest were examined.
The motor function of each mouse was evaluated weekly using the BMS up to 49 DPI in the sub-acute TP and PBS groups and up to 84 DPI in the chronic TP and PBS groups (n = 10 per group). This assessment was performed by two investigators blinded to the identity of the experimental mice.
Motor coordination was evaluated using a rotating rod apparatus (Rotarod, Muromachikikai Co., Ltd.), which consisted of a plastic rod (3 cm diameter, 8 cm length) with a gritted surface flanked by two large discs (40 cm diameter) (n = 10 per group). At 42 days after cell transplantation or PBS injection, each mouse was placed on the rod while it rotated at 20 rpm for 2 min sessions. Three trials were conducted, and the maximum number of seconds the mouse stayed on the rod was recorded.
Gait analysis was performed using the DigiGait system (Mouse Specifics, Quincy, MA, USA) (n = 10 per group)[17, 26, 60]. Each mouse demonstrated weight-supported hindlimb stepping at 42 days after cell transplantation or PBS injection. The stride length was determined on a treadmill set to a speed of 7 cm/s.
Electrophysiological experiments were performed using a Neuropack S1 MEB-9402 (Nihon Kohden, Tokyo, Japan) at 42 days after cell transplantation or PBS injection (n = 7 per group). The animals were anesthetized with an i.p. injection of ketamine (60 mg/kg) and xylazine (6 mg/kg), and the stimulation was applied through the occipitocervical area of the spinal cord and the hindlimb by needle electrodes. The active electrode was placed in the quadriceps muscle belly, and the reference electrode was placed near the distal quadriceps tendon. The ground electrode was placed on the tail. A stimulus of 0.4 mA intensity, 0.2 ms duration, and 1 Hz interstimulus interval were used. The latency was measured as the length of time from the stimulus to the onset of first response wave. The amplitude was measured from the initiation point of the first response wave to its highest point.
Injured animals were deeply anesthetized and transcardially perfused with 4% PFA in 0.1 M PBS at 9 DPI or 42 DPI (n = 3 each). The treated animals were similarly prepared 42 days after cell transplantation or PBS injection. The spinal cords were removed, postfixed overnight in 4% PFA, soaked overnight in 10% sucrose, followed by 30% sucrose, embedded in Optimal Cutting Temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), frozen, and sectioned in the sagittal or axial plane at 12 μm thickness on a cryostat (CM3050 S; Leica Microsystems, Wetzlar, Germany). The injured spinal cord sections were histologically evaluated by HE staining and immunohistochemistry, followed by quantitative analysis. The sections of transplanted spinal cord were subjected to HE staining, LFB staining, and immunohistochemistry followed by quantitative analyses.
For immunohistochemistry, tissue sections were stained with the following primary antibodies: anti-GFP (rabbit IgG, 1:200; Frontier Institute, Hokkaido, Japan), anti-Hu (human IgG, 1:1000, a gift from Dr. Robert Darnell, The Rockefeller University, New York, NY, USA), anti-GFAP (rat IgG, 1:200, Invitrogen), anti-APC CC-1 (mouse IgG, 1:200; Calbiochem, San Diego, CA, USA), anti-nestin (mouse IgG1, 1:500; BD Bioscience Pharmingen, San Jose, California, USA), anti-CS56 (a marker for CSPG, mouse IgM, 1:200; Sigma-Aldrich), anti-Iba1 (a marker for microglia/macrophages, rabbit IgG, 1:200; Wako Pure Chemical Industries, Osaka, Japan), anti-arginase-1 (a marker for M2 macrophages, goat IgG, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LAMP2 (a marker for endosomes or lysosomes, rat IgG, 1:200; Abcam, Cambridge, UK), anti-NF-H (mouse IgG1, 1:200; Chemicon, Millipore, Billerica, MA, USA), and anti-5HT (goat IgG, 1:200; Immunostar, Hudson, WI, USA). For immunohistochemistry with anti-GFP, -NF-H, and -5HT, a biotinylated secondary antibody (Jackson Immunoresearch Laboratory Inc., West Grove, PA, USA) was used after exposing the sections to 0.3% H2O2 for 30 min at room temperature to inactivate endogenous peroxidases. The signals were enhanced with the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). Nuclei were stained with Hoechst 33258 (10 μg/ml, Sigma-Aldrich). All images were obtained using a fluorescence microscope (BZ 9000; Keyence Co.) or a confocal laser scanning microscope (LSM 700; Carl Zeiss, Munich, Germany).
Quantitative analyses of the histological findings (HE, LFB staining, and immunostaining for CS56, Iba1, NF-H, 5HT, GFP/each phenotypic marker, and arginase-1/Iba1) were performed using a BZ 9000 microscope and Dynamic Cell Count BZ-HIC software (Keyence Co.). The threshold values were maintained at a constant level for all analyses. The GFP+ area was quantified using images of axial sections of the lesion epicenter and 0.5 mm, 1.0 mm, 2.0 mm, 3.0 mm, and 4.0 mm rostral and caudal to the epicenter at 100× magnification (n = 4 each). To determine the spinal cord area, HE-stained images of axial sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 100× magnification were used (n = 3 each). Quantitative analysis of the LFB+ area was similarly performed using axial sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 100× magnification (n = 3 each). CS56+ areas as well as Iba1+ areas were quantified in the midsagittal sections of injured spinal cords at 100× magnification (n = 3 each).
To quantify NF-H+ fibers, four regions were automatically captured within the midsagittal sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 400× magnification (n = 3 each). To assess the 5HT+ fibers, five automatically captured regions within the axial sections were analyzed at the lumbar intumescence, which was 6–8 mm caudal to the lesion epicenter (n = 3 each).
To quantify the proportion of each cell phenotype among the grafted cells in vivo, five regions were captured within sagittal sections at 200× magnification using an LSM 700 confocal laser scanning microscope. GFP and phenotypic marker double-positive cells were counted in each section (n = 3 each). To quantify the infiltrated M2 macrophages in the injured spinal cord, the number of Iba1 and arginase-1 double-positive cells was counted within five regions of sagittal sections of the lesion epicenter at 200× magnification with an LSM 700 confocal laser scanning microscope (n = 3 each).
All data are reported as the mean ± SEM. An unpaired two-tailed Student’s t test was used to evaluate the differences between groups with respect to microarray gene expression profile, in vivo BLI analysis, in vivo differentiation assays, and analyses of the CS56+, Iba1+ and GFP+ areas, and arginase-1+/Iba1+ cells. One-way ANOVA followed by the Tukey-Kramer test for multiple comparisons was used in the analyses of the HE, LFB, NF-H+, and 5HT+ areas and the Rotarod and DigiGait results. Repeated-measures two-way ANOVA followed by the Tukey-Kramer test was used for the BMS analysis. P values < 0.05 were considered statistically significant.