To confirm whether the NSE was suitable with NSCs for this combined treatment strategy, both a neuronal cell-type-inducible luciferase expression system (NSE::Luci) and a general luciferase expression system (SV::Luci) were made by pGL3 luciferase reporter vectors. SV::Luci was purchased from Promega (Madison, WI, USA, Cat.no E1761). Neuron-specific enolase (NSE) promoter was provided by Prof. Lee at Hanyang University, Seoul, Korea. The NSE region was inserted between NheI and BglII sites of the pGL3-basic vector (Promega, Cat.no E1751). The construction designs of the plasmid are shown in Fig. 1.

We used mouse NSCs in this study. NSCs (CRL-2925, ATCC, Manassas, VA, USA) were cultured in dulbecco's modified eagle medium (DMEM)/F12 and supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). Approximately 4×10⁵ cells were plated in six-well culture dishes (4×10⁴ cells/cm²) and incubated at 37℃ in a humidified atmosphere containing 5% CO₂.

For the other experiment, we used the human embryonic kidney 293 cell (293FT) provided from Prof. Kang at Yonsei University. Cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, 1X non-essential amino acid (NEAA; Invitrogen), and 1X sodium pyruvate (Invitrogen). Approximately 6×10⁵ cells were plated in six-well culture dishes (6×10⁴ cells/cm²) and incubated at 37℃ in a humidified atmosphere containing 5% CO₂.

Transfection was performed using Lipofectamine 2000 (Invitrogen) as previously described.9 Briefly, NSE::Luci, SV::Luci, or SV::DsRed (Control) plasmid (each 4 µg) were individually mixed with serum-free and antibiotic-free DMEM/F12 medium. Lipofectamine (10 µL) also was mixed with serum-free and antibiotic-free DMEM/F12 medium. Plasmid DNA solution was mixed with the lipofectamine-DMEM mixture and then incubated for 20 min at room temperature (RT). The DNA-lipofectamine mixture was added to NSCs or 293FT cells, and the cells were incubated at 37℃ in a humidified atmosphere containing 5% CO₂.

For neural differentiation, NSCs were cultured in presence of retinoic acid (RA; Sigma, St. Louis, MO, USA) for 7 days. RA was dissolved in dimethyl sulfoxide (DMSO) (working solution, 10 mM) and diluted with culture media (final concentration, 0.01 µM, 0.1 µM, and 1 µM). Briefly, NSCs were seeded on poly-l-lysine (2 µg/cm², Sigma)-coated glass coverslips at a density of 2×10⁴ cells/cm². After 24-h incubation, the medium was replaced with a complete medium with or without RA and then changed every 2 days. Neural differentiation was carried out for 7 days.

All protocols were approved by the Animal Care and Use Committee of the Medical Research Institute at Yonsei University College of Medicine. All experiments were conducted in accordance with international guidelines on the ethical use of animals, and the number of animals used was minimized. For anesthesia, ICR mice (male, 6 weeks, 30 g, OrientBio, Seongnam, Gyeonggi-do, Korea) were intraperitoneally injected with ketamine (100 mg/kg, Yuhan, Seoul, Korea) and rompun (10 mg/kg, Bayer Korea, Seoul, Korea). Laminectomy was performed at thoracic level 11. SCI was carried out using self-closing forceps (compression injury for 10 sec). Animals were divided into two groups, those treated with SV::Luci-NSCs and those treated with NSE::Luci-NSCs. NSCs (5×10⁵ cells/2 µL) were directly injected into the spinal cord using a microinjection pump (injection rate, 1 µL/min). After transplantation, the exposure site was closed using suture wound clips. Cyclosporin A (10 mg/kg, Chong Kun Dang, Seoul, Korea) and cefazolin (20 mg/kg, Yuhan) were intraperitoneally injected daily.

The In Vivo Imaging System (IVIS) Spectrum (Xenogen, Alameda, CA, USA) was used to detect luciferase expression. For in vivo imaging, mice were anesthetized with ketamine (100 mg/kg) and rompun (10 mg/kg), and then the substrate D-luciferin (150 mg/kg) was injected intraperitoneally. Twenty minutes after injection, bioluminescence images were captured for 10 min. Regions of interest (ROIs) were analyzed, and total quantification of bioluminescence was quantified using Living Image® (Xenogen) software. For in vitro imaging, the substrate D-luciferin (150 µg/mL) was added to the SV::DsRed-NSCs (Control), SV::Luci-NSCs, or NSE::Luci-NSCs in medium, followed by mounting and bioluminescence detection for 2-3 min.

A luciferase assay was performed 24-48 hrs after gene transfection or cell transplantation, and each sample (cells or tissues) were lysed with Pro-prep™ (INTRON Biotechnology, Gyeonggi, Korea). The concentration of protein in lysates was measured using a bicinchoninic acid protein assay kit (Pierce, Iselin, NJ, USA), following the manufacturer's protocol. Luciferase expression was measured using a luminometer for 10 s. The level of luciferase expression was expressed in relative light units (RLU) per mg of protein.

Differentiated cells were fixed in 4% paraformaldehyde, pH 7.4 (Merck, Darmstadt, Germany), for 10-15 min at RT. Samples were washed with phosphate buffered saline (PBS, Thermo) three times. Blocking was performed with 10% normal donkey serum in PBS containing 0.3% Triton X-100 for 60 min at RT. Primary antibodies against beta-III-Tubulin (TUJ1), microtubule-associated protein 2 (MAP2), neural cell adhesion molecule (NCAM), and neurofilament (NF) were incubated overnight at 4℃. Samples were washed three times with PBS, and secondary antibodies were added and incubated for 1 h at RT. Samples were washed three times with PBS, and 4',6'-diamino-2-phenylinodole (DAPI; Vector Labs, Burlingame, CA, USA) was added. Samples were covered with glass coverslips. A confocal laser scanning microscope (LSM 700, Zeiss, Oberkochen, Germany) was used for analysis.

Statistical analysis was performed using Prism 6 (Graphpad, San Diego, CA, USA). All data were evaluated using Student's t-test and a one-way analysis of variance (ANOVA). All data are expressed as the mean±standard error of the mean. All p-values<0.05 were considered statistically significant.

NSE::Luci was constructed using NSE promoter and pGL3-basic plasmid (Fig. 1). To confirm whether this neuronal cell-type-inducible gene overexpression system is suitable for NSCs, we investigated the expression level of luciferase in NSCs transfected with SV::DsRed (Control), SV::Luci plasmid, and NSE::Luci plasmid. Based on the results of IVIS and the luciferase assay, we confirmed that luciferase expression significantly increased in NSE::Luci-NSCs, compared with SV::Luci-NSCs (Fig. 2). These results indicate that the neuronal cell type-inducible transgene overexpression system is suitable for NSCs. To confirm whether the transgene overexpression pattern can be sustained after hypoxic injury (mimicking tissue ischemia after SCI), we investigated the luciferase expression level of Control, SV::Luci-NSCs, and NSE::Luci-NSCs after hypoxic injury for 24 h. We confirmed that the luciferase expression level significantly increased in NSE::Luci-NSCs, compared with that in SV::Luci-NSCs (Fig. 2). These results indicate that combined therapy with NSCs and a neuronal cell type-inducible gene overexpression system can be applied to spinal cord injuries that cause tissue ischemia.

To confirm whether the NSE was selectively working in neuronal lineage cells, we examined the luciferase expression level in 293FT cells, non-neuronal cell, transfected with SV::DsRed (Control), SV::Luci, or NSE::Luci plasmid. We confirmed that the luciferase expression level in 293FT transfected with NSE::Luci plasmid was significantly lower than 293FT transfected with SV::Luci plasmid (Fig. 3). This result means that the efficacy of the neuronal cell type-inducible gene expression system depends on neuronal lineage cell type.

To confirm the differentiation potency of NSCs, neural differentiation was induced by RA treatment. Differentiated cells cultured in the presence of 1 µM RA for 7 d were stained for neuronal specific markers, such as TUJ1, MAP2, NCAM, and NF. We confirmed that differentiated cells were positive for all neuronal markers (Fig. 4), meaning that NSCs used in this experiment were efficiently differentiated into neurons.

To confirm whether the luciferase expression pattern of the NSE::Luci-NSCs can be increased after neuronal differentiation (1 µM RA), compared with NSC stage (0 µM RA), we investigated the luciferase expression level at the stage of NSCs and differentiated neurons after transfection with NSE::Luci or SV::Luci plasmids. We confirmed that the luciferase expression level of NSCs transfected with NSE::Luci plasmid was much higher after neuronal differentiation (Fig. 5A). At the neuronal differentiation condition (1 µM RA), the luciferase expression level of NSE::Luci-NSCs was significantly higher than that of SV::Luci-NSCs (Fig. 5B).

These results mean that the transgene expression by NSE promoter was induced by neuronal differentiation. Thus, these findings indicate that the neuronal cell-type-inducible transgene overexpression system appropriates with NSCs, which have a neuronal differentiation potency for combined treatment strategies.

To confirm whether NSE::Luci-NSCs show a consistent high expression pattern in vivo, both SV::Luci-NSCs and NSE::Luci-NSCs were transplanted into the spinal cord, and an IVIS analysis and luciferase assay was carried out 24-48 h after transplantation. Similar to our results in vitro, the luciferase expression level was significantly higher in NSE::Luci-NSCs than in SV::Luci-NSCs (Fig. 6). These results indicate that a combined treatment strategy based on a neuronal cell type-inducible transgene expression system and NSCs can be applied to SCI.