The pathological analysis and establishment of patient-derived cells were approved by the Ethics Review Committee of the National Hospital Organization, Nagara Medical Center (Approval number: 26-15), and informed consents were obtained from the parents of our pediatric patients. Informed consents were obtained from the subjects after explanation of the nature and possible consequences of the study.

Fibroblasts taken from two SMA type III patients (SMA01 and SMA05) and three SMA type I patients (SMA06, SMA08 and SMA12) were used in the present study. The control human fibroblast cell line TIG-119 was obtained from the National Institutes of Biomedical Innovation, Health and Nutrition (Osaka, Japan). TIG-119 and fibroblasts derived from SMA patients were maintained in Dulbecco’s modified Eagle medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, biowest, Nuaillé, France) and 500 U/mL penicillin/streptomycin (PS, Life Technologies) under a humidfied atmosphere of 5% CO₂ at 37˚C. These cells were passaged by trypsinization every 2 - 3 days.

Fibroblasts were seeded at a density of 100,000 cells per well into 6 well plate, and incubated in 5% CO₂ at 37˚C. After 24 h incubation, cells were treated by rufinamide (Tokyo chemical industry, Tokyo, Japan) for 24 h.

The iPSCs derived from an SMA type III patient (SMA01) and an SMA type I patient (SMA06) were used in this study. The iPSCs were maintained in primate ES cell medium (ReproCELL, Kanagawa, Japan) supplemented with 4 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan) and 500 U/mL PS (Life Technologies). The iPSC colonies were cultured in 5% CO₂ at 37˚C and passaged every 7 days.

For spinal motor neuron differentiation from iPSCs, 9000 single iPSCs were seeded into a primary differentiation medium containing Dulbecco’s modified Eagle medium (DMEM)/F12 (Life Technologies), 5% Knockout Serum Replacement (KSR, Life Technologies), and 500 U/mL PS with 2 μM dorsomorphin (Sigma-Aldrich, St. Louis, MO, USA), 10 μM SB431542 (SB, Cayman, San Diego, CA, USA), and 10 μM Rho-associated coiled-coil forming kinase inhibitor, Y-27632 (Wako). The medium was changed the medium supplemented with 2 μM dorsomorphin and 10 μM SB431542 after 3 days. Spheres were then plated in Matrigel-coated 24-well plates (Becton, Dickinson and Company, NJ, USA) in medium supplemented with 2 μM dorsomorphin and 10 μM SB431542 for another 7 days. The neural precursor cells were then cultured in the second differentiation medium containing DMEM/F12, 1% N2 supplement (Life Technologies), and 500 U/mL PS with 0.1 μM retinoic acid (RA, Sigma Aldrich). The cells were then cultured in the third differentiation medium with 1 μM purmorphamine (PMN, MiltenyiBiotec, Bergisch, Gladbach, Germany) and 0.1 μM RA for 7 days. Finally, the generated spinal motor neurons were cultured in the supplemented medium containing 10 ng/mL brain-derived neurotrophic factor (BDNF, R & D Systems Inc., Minneapolis, MN, USA), 10 ng/mL glial cell line-derived neurotrophic factor (GDNF, R&D), 1 μM cyclic adenosine monophosphate (cAMP, Wako) and 200 ng/ml ascorbic acid (AA, Sigma-Aldrich). During all differentiation stages, medium was changed every 2 or 3 days. The differentiated spinal motor neurons were exposed to rufinamide for 42 to 56 days of our induction protocol by changing the medium every 3 days.

RNA purification was performed using RNeasy Mini Kit (QIAGEN, Hilden, Germany). The concentration of RNA extracted was determined spectrophotometrically in a NanoView Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). First-strand cDNA was synthesized from 1 µg of total RNA using ReverTra Ace (Toyobo, Osaka, Japan). The cDNA samples were subjected to PCR amplification using a thermal cycler 2720 (Applied Biosystems, Carlsbad, CA, USA).

The PCR assay was performed using power SYBR green PCR master mix (Applied Biosystems) containing 250 nM of SMN primer. The SMN primer sequences were described in the previous report [13] . Amplification of SMN mRNA was performed as follows; 50˚C for 2 min, 95˚C for 10 min, followed by 45 cycles of 95˚C for 15 s and 60˚C for 1 min. The amplification of HB9 was performed as follows; 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 s, and 60˚C for 1 min. The samples were run using the comparative CT (ΔΔCT) method. The GAPDH transcript levels were determined as reference genes for qPCR.

Plated cells were washed with PBS three times. Cells were fixed in 4% paraformaldehyde (NacalaiTesque, Kyoto, Japan) for 20 min at 4˚C and then washed again with PBS three times. Cells were blocked with 5% donkey serum in PBS for 30 min at room temperature, and incubated with primary antibodies for 24 h at 4˚C. After washing two times with PBS, the cells were labelled with the appropriate secondary antibody-tagged fluorescent dye for 1 h. Nuclear staining was performed using Hoechst 33342 (diluted 1:1000; Life Technologies). The following primary antibodies were used: rabbit anti-TUJ1 (βIII-tubulin) antibody (diluted 1:1000; BioLegend, San Diego, CA, USA), the secondary Alexa Fluor-labeled antibodies were 488 donkey anti-rabbit IgG (diluted 1:1000; Life Technologies). All images were taken by BIOREVO BZ-9000 (Keyence, Osaka, Japan).

Data are presented as mean ± S.E.M. Statistical comparisons were performed using Student’s t-test. P values less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS Statistics (IBM, Armonk, NY, USA).

In SMA pathology, systemic deletion of SMN protein is observed. Fibroblasts derived from SMA patients have often been used for drug discovery research for SMA. To investigate the effect of rufinamide on SMN mRNA expression, we used type III SMA patient-derived fibroblasts (SMA01 and SMA05). Each patient has 3 copies of SMN2 gene. TIG-119 was used as the control human fibroblasts. Type III SMA patients-derived fibroblasts were treated by 5 µM rufinamide for 24 h. SMN mRNA was significantly increased by addition of rufinamide in type III SMA patient-derived fibroblasts (Figure 1).

We then determined the effect of rufinamide on SMN mRNA in more severe disease class, type I SMA patient-derived fibroblasts (SMA06, SMA08 and SMA12). Each patient has two copies of SMN2 gene. In contrast of the result using type III SMA patient-derived fibroblasts, the expression level of SMN mRNA was not changed after rufinamide treatment in type I SMA patient-derived fibroblasts (Figure 2).

Degeneration of spinal motor neuron is the most distinctive feature of SMA. To evaluate the potential efficacy of rufinamide on spinal motor neuron, we used spinal motor neurons differentiated from SMA patients-derived iPSCs. SMA-iPSCs derived-spinal motor neuron (SMA-iPSCs-MNs) was treated by rufinamide for 2 weeks, then neurite outgrowth was evaluated to determine the effect of rufinamide. To evaluate neurite length, we performed immunostaining using anti-TUJ1 antibody. In type III SMA patient derived-iPSCs-MNs (SMA 01), rufinamide stimulated neurite elongation (Figure 3(a) and Figure 3(b)). On the other hand, rufinamide did not affect neurite outgrowth in type I SMA patients derived-iPSCs-MNs (SMA 06) (Figure 3(c)).