Priming with FGF2 stimulates human dental pulp cells to promote axonal regeneration and locomotor function recovery after spinal cord injury

Human dental pulp cells (DPCs), adherent cells derived from dental pulp tissues, are potential tools for cell transplantation therapy. However, little work has been done to optimize such transplantation. In this study, DPCs were treated with fibroblast growth factor-2 (FGF2) for 5–6 consecutive serial passages and were transplanted into the injury site immediately after complete transection of the rat spinal cord. FGF2 priming facilitated the DPCs to promote axonal regeneration and to improve locomotor function in the rat with spinal cord injury (SCI). Additional analyses revealed that FGF2 priming protected cultured DPCs from hydrogen-peroxide–induced cell death and increased the number of DPCs in the SCI rat spinal cord even 7 weeks after transplantation. The production of major neurotrophic factors was equivalent in FGF2-treated and untreated DPCs. These observations suggest that FGF2 priming might protect DPCs from the post-trauma microenvironment in which DPCs infiltrate and resident immune cells generate cytotoxic reactive oxygen species. Surviving DPCs could increase the availability of neurotrophic factors in the lesion site, thereby promoting axonal regeneration and locomotor function recovery.


Preparation of the medium conditioned by DPCs
At the 6th passage, the DPC-S and the DPC-FS were seeded onto 6 cm dishes coated with Cellmatrix Type IP (Nitta Gelatin Inc), at a density of 5 × 10 5 cells per dish and cultured in the 10% FBS-±MEM supplemented without or with 10 ng/mL of FGF2, respectively. After 2 DIV, the cells were rinsed twice with PBS and the medium was replaced with neuronal culture medium without B27 supplement (Neurobasal medium [Invitrogen] containing 0.5 mM L-glutamine, and 100 U/ml penicillin). After 2 DIV, the medium was collected and filtered through a sterile 0.45 µm pore-size membrane filter Advantec,Tokyo,Japan). The medium was used as conditioned medium for neuron morphological analysis.

Isolation and culture of mouse cortical neurons and morphological analysis
Cortical neurons were prepared from cerebral cortices of embryonic day 15.5 mice and cultured as described previously. 37 In brief, embryos were collected removed from timed-pregnant ddY mice after killed and their cortices were dissected in L-15 medium (Sigma-Aldrich). The cortices were incubated in L-15 medium containing 0.25% trypsin and 0.1% DNase for 15 min at 37°C. After incubation, the digestion was stopped with FBS (HyClone, Thermo Fisher Scientific, Logan, UT, USA), and mechanically dissociated by pipette trituration in DMEM/F-12 (Sigma-Aldrich) containing 10% FBS. The cells were seeded onto round 13 mm coverslips (Matsunami Glass) coated with 0.05 mg/mL poly-L-ornithin (Sigma-Aldrich) in culture 24-wells plates at density of 5 × 10 4 cells per well. After cell attachment, the medium was replaced by culture medium (Neurobasal medium [Invitrogen] with 2% B27 supplement [Invitrogen], 0.5 mM L-glutamine, and 100 U/ml penicillin) with lenti-GFP (2.0 × 10 5 transduction units) under humidified conditions in 95% air and 5% CO 2 at 37°C. After 2 DIV, the medium was replaced with the DPC-cultured medium. The cortical neurons were incubated from 2 to 4 DIV and were processed for immunostaining and morphological analysis.
For morphological analysis, the cultured cortical neurons were immunostained and then processed using a confocal laser microscope (LSM 710; Carl Zeiss) as described previously. 37 Sholl analysis 2 with slight modification was performed for evaluating neuron morphology. In brief, circles with radii of 50 and 100 µm were centered on the cell body, respectively. Then, the number of intersections with both MAP2 and GFP double-positive neurites was recorded. All the measured neurites started at the cell body.

Membrane-based human growth factor antibody array and ELISA
At the sixth passage, the DPC-S and the DPC-FS cells were seeded onto Cellmatrix Type IP-coated 6-cm dishes at a density of 1 × 10 6 cells per dish and cultured in 10% FBS-±MEM without FGF2. The next day, cells were rinsed twice with PBS and cultured in 2 mL of fresh 10% FBS-±MEM for 2 days. For the microarray, a human growth factor antibody array was purchased from Abcam (ab134002). The cells were harvested with 500 µL of lysis buffer, and cell lysates were prepared for use in the microarray assay, which was performed according to the manufacturer's instructions.
Briefly, membranes were incubated with 1× blocking buffer at r.t. for 30 min, followed by overnight individual incubation at 4°C in 1 mL of the lysates from the DPC-S and DPC-FS cells. After washing and incubation with biotin-conjugated anti-cytokine antibodies at 4°C overnight, the membranes were again washed and then incubated with horseradish peroxidase-streptavidin at 4°C overnight. After washing, the membranes were incubated with the provided detection buffer, and signals were imaged using an LAS3000 mini-CCD camera (Fuji Film, Tokyo Japan).
The amount of BDNF in the cell culture medium and the DPC lysates was measured by a commercial ELISA kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, after the cells (1 × 10 6 cells per 6-cm dish) were cultured in 2 ml of fresh 10% FBS-±MEM for 2 days, cell lysates were harvested with 150 µl of lysis buffer. The cell lysates (diluted 1/4) and the undiluted culture medium were used as samples, which were tested at least in duplicate (n = 4-5).

Exposure to H 2 O 2 , western blot analysis, and immunocytochemistry
The DPCs were plated at 4 × 10 5 cells/6-cm dish for western blotting, and at 3 × 10 4 cells/coverslip coated with Cellmatrix Type IP for immunocytochemistry, in 24-well dishes. They were cultured for 2 days, and the medium was changed daily. The DPCs were then washed twice with PBS and exposed to fresh 10% FBS-±MEM with 0.3-0. determined by one-way ANOVA followed by Tukey' s test; *p < 0.05, **p < 0.01, and ***p < 0.001, respectively. and without FGF2, respectively (DPC-S/FS, and DPC-S/FS). a. At the indicated days after cultivation, the DPCs were exposed to 0.5 mM H 2 O 2 for 24 hrs, followed by MTT assay. b. On days 1 and 17 after cultivation, the DPCs were exposed to H 2 O 2 at various concentrations (0, 0.4, 0.5, and 0.6 mM) for 24 hrs, followed by MTT assay.  that the cell density of DPC-S was reduced at 24 h after exposure to H 2 O 2 ; however, the production of FGF2 and HGF was not altered after H 2 O 2 treatment. Scale bar, 100 μm.