Combined Method of Neuronal Cell-Inducible Vector and Valproic Acid for Enhanced Gene Expression under Hypoxic Conditions

Abstract

Background:

Gene therapy shows the ability to restore neuronal dysfunction via therapeutic gene expression. The efficiency of gene expression and delivery to hypoxic injury sites is important for successful gene therapy. Therefore, we established a gene/stem cell therapy system using neuron-specific enolase promoter and induced neural stem cells in combination with valproic acid to increase therapeutic gene expression in hypoxic spinal cord injury.

Methods:

To examine the effect of combined method on enhancing gene expression, we compared neuronal cell-inducible luciferase levels under normoxia or hypoxia conditions in induced neural stem cells with valproic acid. Therapeutic gene, vascular endothelial growth factor, expression with combined method was investigated in hypoxic spinal cord injury model. We verified gene expression levels and the effect of different methods of valproic acid administration in vivo.

Results:

The results showed that neuron-specific enolase promoter enhanced gene expression levels in induced neural stem cells compared to Simian Virus 40 promoter under hypoxic conditions. Valproic acid treatment showed higher gene expression of neuron-specific enolase promoter than without treatment. In addition, gene expression levels and cell viability were different depending on the various concentration of valproic acid. The gene expression levels were increased significantly when valproic acid was directly injected with induced neural stem cells in vivo.

Conclusion:

In this study, we demonstrated that the combination of neuron-specific enolase promoter and valproic acid induced gene overexpression in induced neural stem cells under hypoxic conditions and also in spinal cord injury depending on valproic acid administration in vivo. Combination of valproic acid and neuron-specific enolase promoter in induced neural stem cells could be an effective gene therapy system for hypoxic spinal cord injury.

Introduction

Spinal cord injury (SCI) causes serious neurologic dysfunction in both the short and long term due to various features, including degeneration of nerve tissue, demyelination, neuropathic pain, infection, and blood vessel destruction [1–4]. It is important to focus on the fundamental problems in SCI, such as interruption of blood vessels, which results in an insufficient blood supply to the spinal cord and hypoxic conditions. Stem cell transplantation or therapeutic gene expression for SCI treatment has become a promising therapy. However, it is difficult to achieve complete recovery of SCI with stem cell or gene therapy alone because they have a lack of gene delivery and regeneration efficiency. Thus, we developed a combined therapy system to increase the expression of therapeutic genes based on neural stem cells (NSCs) for SCI recovery. This system consists of neuron-specific enolase promoter (pNSE) for neuronal cell-inducible gene expression under hypoxic conditions in NSCs and valproic acid (VPA) to enhance the gene expression system.

Recently, many studies have been reported that NSCs were used as a vehicle for gene delivery in SCI model [5–10]. Induced neural stem cells (iNSCs) were developed by pluripotency factors, OCT4, SOX2, KLF4 and microRNAs. Recently, three of the four reprogramming factors were replaced to chemical cocktail, resulting in OCT4 alone was found to induce direct conversion of iNSCs from human adult fibroblasts [11]. iNSCs produced by direct conversion are very similar to NSCs in the aspects of gene expression, action potential, and cell morphology [12, 13]. iNSCs are not only suitable for human studies because they are human-derived cells, but can also be used for both gene and cell therapy when a therapeutic gene is loaded into the cells. In our previous studies, we showed that pNSE induced neuronal cell-inducible gene expression in NSCs but not in non-neuronal cell such as Human embryonic kidney 293 (HEK293) and undifferentiated Pheochromocytoma 12(PC12) [14, 15]. Also, pNSE was used to establish a neuronal cell-inducible gene expression system in NSCs by introducing granulocyte–macrophage colony-stimulating factor (GMCSF) [16] and vascular endothelial growth factor (VEGF) [15] to treat SCI by promoting the survival of neuronal cells. VEGF has been used as a therapeutic gene to improve the ischemic environment by promoting angiogenesis [17] and neuronal regeneration [18]. Meanwhile, VPA is a histone deacetylase (HDAC) inhibitor molecule that can increase gene expression levels [19–24]. We hypothesized that VPA would have a synergic effect on the expression of therapeutic genes in iNSCs with pNSE.

Here, we investigated whether the combination of VPA and neuronal cell-inducible gene expression controlled by pNSE in iNSCs leads to gene overexpression under hypoxic conditions in vitro and in vivo. Also, we examined the concentration and administration method of VPA in a SCI model.

Materials and methods

Plasmid construction

The plasmids were referred to our previous study [15]. In order to construct promoter Simian Virus 40-VEGF (pSV-VEGF) and pNSE-VEGF, the luciferase gene was removed from the promoter Simian Virus 40-Luciferase (pSV-Luc) and pNSE-Luc, and the pSV-VEGF-pGL3-promoter including poly-A was introduced to the luciferase location at HindIII and BamHI sites. Plasmids were amplified in E. coli DH5α, and a Plasmid Maxiprep kit (Qiagen, Hilden, Germany) was used for purification. NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA) was used to measure the quantity of plasmids based on absorbance at 260 nm. To measure the purity of DNA, the ratio of absorbance at 260 nm and 280 nm was used.

Cell culture and neural differentiation

iNSCs were kindly provided by Janghwan Kim of Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. iNSCs were directly reprogrammed from human neonatal fibroblasts (ATCC, CRL-2097) with OCT4 (Fig. 1) [11]. iNSCs were cultured with advanced Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (advanced DMEM/F12, Gibco, Grand Island, NY, USA) and neurobasal medium (Gibco, Grand Island, NY, USA) with the ratio of 1:1 supplemented with 1 × N2 supplement (Gibco, Grand Island, NY, USA), 1 × B27 supplement (Gibco, Grand Island, NY, USA), 5% bovine serum albumin (Gibco, Grand Island, NY, USA), Glutamax™-I(Gibco, Grand Island, NY, USA), 1000 × 2-mercaptoethanol (Gibco, Grand Island, NY, USA), 1% penicillin/streptomycin (P/S, Gibco, Grand Island, NY, USA), leukemia inhibitory factor (LIF, Millipore, Burlington, MA, USA), 0.1 μM A83-01 (Tocris Bioscience, Bristol, UK), and 3 μM CHIR99021 (Tocris Bioscience, Bristol, UK). HEK293 cells were cultured in DMEM containing 90% FBS and 1% P/S (All from Gibco, Grand Island, NY, USA).

Fig. 1.

Illustration of direct reprogramming. iPS induced pluripotent stem cells, NSC neural stem cells

For hypoxic conditions, cells were cultured at 37 °C in 5% CO₂ and hypoxic (1% O₂) conditions for 48-h. CoCl₂ (Sigma-Aldrich, St Louis, MO, USA) was used to mimic hypoxic conditions.

For neuronal differentiation, the culture media was changed to differentiation media containing 10 ng/ml BDNF, GDNF, and NT3 (All from Peprotech, Seoul, Korea) with removal of LIF, A83-01, and CHIR99021.

Whole-cell patch clamp

Whole-cell patch clamp experiments were conducted as described in a previous study [25]. Cells were seeded in a 24well plate at a density of 2 × 10⁴ cells/well and differentiated for 4 weeks. A cover slip with cultured cell was transferred to a recording chamber (Warner instrument, Hamden, CT, USA) and placed under a microscope (Olympus, Tokyo, Japan). Artificial CSF, containing 124 mM NaCl, 3 mM KCl, 1.3 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 2.4 mM CaCl₂–2H₂O, and 10 mM glucose (Sigma-Aldrich, St Louis, MO, USA-Aldrich, St Louis, MO, USA Aldrich) was continuously perfused over the cells and aerated with O₂ 95%/CO₂ 5% mixed gas at RT. A glass capillary pipette was directed towards the cell surface. Negative pressure was provided to elicit a giga seal for whole-cell recording. The internal pipette solution contained 115 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, 5 mM Mg-ATP, and 0.5 mM Na²⁺-GTP (Sigma-Aldrich, St Louis, MO, USA Aldrich), with pH 7.3 and 280–285 mOsm. Sodium current was recorded in voltage clamp mode, and electrical stimulation was provided in the range − 10 to + 30 mV. Thereafter, current clamp mode was utilized to evaluate action potential generation. To verify that sodium current and action potential were mediated by voltage gated sodium channels and potassium channels, the bath was treated with tetrodotoxin (TTX, 0.5 μM, Sigma-Aldrich, St Louis, MO, USA Aldrich) and tetraethylammonium (TEA, 10 mM, Sigma-Aldrich, St Louis, MO, USA Aldrich) prior to recording. Data acquisition was performed using Digitizer 1440A (Molecular Devices, Sunnyvale, CA, USA) and Clampex 10.3 (Molecular Devices, Sunnyvale, CA, USA, Sunnyvale, CA, USA). Analysis of data was conducted by using Clampfit 10.3 (Molecular Devices, Sunnyvale, CA, USA).

Gene transfection

Cells were seeded in a 6-well plate at 3 × 10⁵ cells/well and cultured at 37 °C in 5% CO₂ for 24-h. The plasmid and Viafect Transfection Reagent (Promega, Madison, WI, USA) were mixed with serum-free medium and incubated at room temperature. After the 15-min, the serum-free medium mixture was added to the cells and incubated at 37 °C in a 5% CO₂. After 24-h, change the media with or without VPA and cells were further incubated under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 48-h.

Valproic acid treatment

Twenty-four hours after transfection, change the media with 1 mM VPA (Sigma-Aldrich, St Louis, MO, USA Aldrich) and incubated for 48-h.

Animals were divided into six groups including control group to examine the effect of VPA. In the ‘i.p. injection’ group, 1 mM VPA was injected into the abdominal cavity twice a day after cell transplantation until sacrifice for tissue sampling. The ‘Pre-treatment’ group was cell transplantation with VPA-treated cells 24-h after plasmid transfection. The ‘0.1 mM VPA’ and ‘1 mM VPA’ groups were cell transplantation with media containing 0.1 mM and 1 mM VPA, respectively.

MTT assay

Cells were plated in 96-well plates at 1 × 10⁴ cells/well and incubated at 37 °C in 5% CO₂. The MTT assay with 0.5 mg/ml of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and incubated at 37 °C for 4-h. The MTT solution was then removed, and DMSO (Sigma-Aldrich, St Louis, MO, USA Aldrich) was added to dissolve the formazan crystals. Absorbance of the MTT reaction product was measured by a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 490 nm.

Immunofluorescence staining

Cells were fixed in 4% paraformaldehyde for 10-min at room temperature, and then washed with 0.3% Tween 20 in 1X phosphate buffered saline (PBS, Thermo Fisher Scientific, Wilmington, DE, USA). Cells were blocked with 10% NDS and 0.3% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA Aldrich) in 1X PBS at room temperature for 1-h. Immunostaining was performed with anti-Nestin (Millipore, Burlington, MA, USA), anti-Microtubule-associated protein 2 (MAP2, Abcam, Cambridge, UK), anti-vGLUT2 (Millipore, Burlington, MA, USA), anti-GAD67 (Abcam, Cambridge, UK), anti-Neural cell adhesion molecule (NCAM, Abcam, Cambridge, UK), anti-Neuronal Nuclei (NeuN, Millipore, Burlington, MA, USA), anti-beta III Tubulin (Tuj1, Abcam, Cambridge, UK), and anti-Glial fibrillary acidic protein (GFAP, Abcam, Cambridge, UK).

Luciferase assay

Cell samples were lysed with 300 μl of reporter lysis buffer for 1-h on ice. Tissue samples were homogenized with Pro-prep (iNtRON Biotechnology, Gyeonggi-do, Korea) and lysed for 1-h on ice. The lysates were added to a 1.5-ml tube and centrifuged at 13,000 rpm for 5-min. The supernatant was transferred to a fresh 1.5-ml tube. The BCA kit (Pierce, Rockford, IL, USA) was used to measure protein concentrations. Luciferase activity was examined by a microplate luminometer (Berthold, Bad Wildbad, Germany) through relative light units per milligram of total protein.

IVIS (in vitro imaging system) imaging

To measure luciferase activity, an in vitro imaging system (IVIS; Xenogen, Alameda, CA, USA) was used. Living Image Software (Xenogen, Alameda, CA, USA) was used to perform IVIS. Bioluminescence was examined 2–3-min after D-luciferin (150 μg/ml, Promega, Madison, WI, USA) substrate was added.

Enzyme-linked immunosorbent assay (ELISA)

Cells were transfected with pSV-VEGF or pNSE-VEGF and incubated under normoxic or hypoxic conditions. The supernatant sampling was performed each 2, 3, 5 and 7 days after transfection. Tissue samples were prepared same as luciferase assay protocol above. An ELISA kit (Abfrontier, Seoul, Korea) was used to measure the amounts of VEGF secreted from the cells in accordance with the manufacturer’s protocol. Samples were added to the wells of the pre-coated with VEGF monoclonal antibodies plate and incubated at 37 °C for 90-min. After the cell culture media samples were discarded, 0.1 ml of biotinylated VEGF antibody was added to each well and incubated at 37 °C for 60-min. Plates were washed three times in 0.01 M PBS. An Avidin–Biotin–Peroxidase Complex (ABC) working solution was added to each well and incubated at 37 °C for 30-min. The ABC solution was removed, wells were washed five times, and 90 μl of tetramethylbenzidine (TMB) color-developing agent was added and incubated at 25 °C in the dark for 20-min. After incubation, TMB stop solution was added, and catalyzed TMB had changed to yellow. The density of yellow was measured at an absorbance–wavelength of 450 nm. The relative amount of VEGF in each sample was determined with human VEGF standard curve.

Histone acetylation detection

Cells were plated in 6-well plates at 3 × 10⁵ cells/well and incubated at 37 °C in 5% CO₂. After 24-h transfection, 1 mM VPA was treated for 6-h. Histone extracts were prepared with Histone Extract kit (Abcam, Cambridge, UK). Briefly, cells were harvested and pellet the cells by centrifugation. Pellets were resuspended in the Pre-lysis buffer and lyse cells on ice for 10-min. Cell lysates were centrifuged at 10,000 rpm for 1-min and remove supernatant. Pellets were resuspended in Lysis buffer and incubated on ice. After 30-min, cell lysates were centrifuged and supernatant fraction was transferred into a new tube. Balance-DTT buffer was added to the supernatant immediately and protein concentration was quantified with the BCA kit (Pierce, Rockford, IL, USA).

Histone H3 total acetylation detection kit (Abcam, Cambridge, UK) was used to measure histone acetylation. 1 μg of the histone extract was added into sample wells with Antibody buffer and incubated at room temperature for 2-h. After washing and aspiration, Detection antibody was added and incubated for 1-h. Color developer and Stop solution was added following washing and aspiration, absorbance of yellow color was read on a microplate reader at 450 nm.

Spinal cord injury and cell transplantation

The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) in Yonsei University (IACUC approval No. 2017-0114). All procedures were performed according to international guidelines on the ethical use of animals, and the number of animals used was minimized.

Laminectomy was conducted at the T11 level in adult male C57BL/6 mice (20–25 g; OrientBio, Gyeonggi-do, Korea). Anesthesia was performed using ketamine (100 mg/kg, Yu-han, Seoul, Korea) and Rompun (10 mg/kg, Bayer Korea, Seoul, Korea), and the opened spinal cord was injured for 1 s with forceps.

One week after the SCI models were constructed, a Hamilton syringe (Hamilton, Reno, NV, USA) and microinjection pump (injection rate, 1 μl/min) were used to directly inject cells (1 × 10⁵/2 μl) that were transfected with pNSE or phosphate-buffered saline (PBS). Heart perfusion with saline (pH 7.5) was performed to obtain spinal cord tissues 2-day after transplantation. Obtained tissues were homogenized with Pro-prep (iNtRON Biotechnology, Gyeonggi-do, Korea) and incubated for 1-h on ice for tissue lysis.

Statistical analysis

Student’s t test and one-way ANOVA were used to evaluate all data. A p value *< 0.05, **< 0.01 and ***< 0.001 was considered statistically significant.

Results

Induced neural stem cells (iNSCs) properties

iNSCs directly reprogrammed from human fibroblast were identified as positive for the neuronal precursor cell marker Nestin and merged with originally labeled GFP [11]. Neuronal differentiation of iNSCs was confirmed by immunocytochemistry and electrophysiology. iNSCs-derived neural cells were positive for the neuronal markers glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), microtubule-associated protein 2 (MAP2), beta III tubulin (Tuj1), neuronal nuclei (NeuN), and astrocyte marker glial fibrillary acidic protein (GFAP) (Fig. 2A).

Fig. 2.

Induced neural stem cells derived from human fibroblasts are differentiated into neurons. A Immunostained with neural stem cell marker, Nestin was stably labeled in iNSCs. iNSCs was differentiated into neurons. Immunostaining with neuronal specific markers, such as GAD67, NCAM, MAP2, NeuN, Tuj1 and GFAP. Nuclei were stained with DAPI. Scale bar, 50 μM. B Representative traces of sodium current before (left) and after neural induction by 0.5 μM TTX and 10 mM TEA bath application (middle, right). C Representative traces of action potential before (left) and after neural induction by 0.5 μM TTX and 10 mM TEA bath application (middle, right). MAP2, microtubule-associated protein 2; GAD67, glutamate decarboxylase 67; NCAM, neural cell adhesion molecule; NeuN, neuronal nuclei; Tuj1, beta III Tubulin; GFAP, glial fibrillary acidic protein; TTX, Tetrodotoxin; TEA, Tetraethylammonium

Electrophysiological properties of differentiated iNSCs were examined by whole-cell patch clamp recording. In the voltage clamp mode, Na⁺ channels were found in differentiated iNSCs at sequential stimulations from − 10 mM to + 30 mV (Fig. 2B). Evoked action potentials in the current clamp mode were used to confirm the excitability of the differentiated iNSCs. Action potential spikes appeared at 40 pA and 80 pA (Fig. 2C).

These results indicated that iNSCs directly reprogrammed from human fibroblasts were characterized as self-renewing and multipotent stem cells that can differentiate to neurons.

NSE promoter activity was enhanced under hypoxic conditions

pSV-Luc and pNSE-Luc was transfected into iNSCs to confirm that pNSE was overexpressed than pSV in iNSCs. Transfection with pNSE-Luc showed a fivefold higher expression levels than transfection with pSV-Luc in iNSCs by luciferase assay (Fig. 3A) and IVIS imaging (Fig. 3B). We compared the expression levels of pNSE under hypoxic conditions to normoxic conditions. pNSE activity was eightfold higher, while pSV activity was threefold higher under hypoxic conditions, compared to normoxic conditions (Fig. 3C). IVIS imaging also measured luciferase levels (Fig. 3D). In order to mimic hypoxic conditions using a chemical reagent, various concentrations of CoCl₂ were added. The addition of 200 μM CoCl₂ showed threefold higher pNSE activity, compared to untreated cells (Fig. 3E). The luciferase gene was replaced with the VEGF gene downstream of the pSV or pNSE. pNSE-VEGF under hypoxic conditions showed higher levels of gene expression, compared to pSV-VEGF and/or normoxic conditions (Fig. 3F).

Fig. 3.

Increased expression of neuronal inducible gene expression system in iNSCs under hypoxic conditions. A Luciferase expression activity of pSV-Luc and pNSE-Luc was confirmed by luciferase assay at 48-h transfection. Control cells were transfected with carrier alone. n = 5. B Luciferase expression levels of pSV-Luc and pNSE-Luc with IVIS imaging. The color scale bar indicates the luciferase levels. C Luciferase expression activity of pSV-Luc and pNSE-Luc under normoxic or hypoxic conditions was confirmed by luciferase assay at 48-h transfection. n = 5. D Luciferase expression levels of pSV-Luc and pNSE-Luc under normoxic or hypoxic conditions on IVIS imaging. The color scale bar indicates the luciferase levels. E Luciferase expression levels were confirmed by quantification of IVIS imaging with ROI value. iNSCs were transfected with pNSE-Luc. Hypoxic conditions mimicking with various concentrations of CoCl₂. n = 2. F VEGF expression of pSV-VEGF and pNSE-VEGF under normoxic or hypoxic conditions. n = 3. Data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01. RLU relative light units

Valproic acid increases gene expression under the NSE promoter

We examined the effect of VPA treatment on the gene expression levels of pSV and pNSE by a luciferase assay. After 24-h plasmids were transfected, iNSCs were treated with VPA for 48-h (Fig. 4A). Gene expression levels were increased by twofold in pNSE-Luc-transfected cells. Both pNSE-Luc and pSV-Luc gene expression levels were increased when cells were treated with 1 mM VPA (Fig. 4B). Gene expression was also measured by IVIS imaging (Fig. 4C) and ROI (Region of Interest) values (Fig. 4D). Total histone H3 was more acetylated in VPA treatment following pNSE-Luc transfection in iNSCs compared to that without VPA (Fig. 4E). After transfection of pNSE-Luc into HEK293 cells, treatment with VPA showed an increase in gene expression compared to control (Fig. 4F). Therefore, we concluded that VPA does not increase gene expression specifically in iNSCs with the NSE promoter, but increases gene expression due to its function as an HDAC inhibitor. We examined differences in the degree of gene expression when cells were treated with VPA at various concentrations. The highest gene expression was observed when 5 mM VPA was applied (Fig. 4F), however, 5 mM VPA showed low viability of iNSCs (Fig. 4G). These results indicate that 1 mM VPA is the optimal concentration at which to increase gene expression levels, and a gene expression system combined with iNSCs, pNSE, and VPA can significantly increase the expression of a therapeutic gene.

Fig. 4.

VPA increases neuronal gene expression system in iNSC. A Time table of VPA treatment on iNSCs transfected with pSV-Luc or pNSE-Luc. iNSCs were seeded at day 0 and pSV-Luc or pNSE-Luc was transfected at day 1. Cells were treated with VPA at day 2 for 48-h. At day 4, cells were harvested to conduct assay. B Luciferase expression activity of iNSCs transfected with pSV-Luc or pNSE-Luc. n = 3 (pSV-Luc Control), n = 3 (pSV-Luc VPA), n = 9 (pNSE-Luc Control), n = 11 (pNSC-Luc VPA). C Luciferase expression levels of pSV-Luc and pNSE-Luc on IVIS imaging. The color scale bar indicates the luciferase levels. D Quantification of IVIS imaging in (C) with ROI value. n = 3. E Histone acetylation detection in iNSCs transfected with pNSE-Luc and treated with 1 mM VPA. n = 4 (Control), n = 5 (VPA). F Luciferase expression activity test of HEK293 cells transfected with pNSE-Luc and treated with 1 mM VPA. n = 3. G Luciferase expression activity of iNSCs transfected with pNSE-Luc. iNSCs were treated with 0, 1, 5, 10 mM concentrations of VPA for 48-h. n = 9 (0 mM), n = 11 (1 mM), n = 6 (5 mM), n = 6 (10 mM). H Cell viability test using MTT assay. iNSCs were transfected with pNSE-Luc and treated with 0, 1, 5, 10 mM concentrations of VPA. 490 nm was used to measure the absorbance. n = 6 (0 mM), n = 6 (1 mM), n = 6 (5 mM), n = 3 (10 mM). Data are represented ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001

Up-regulation of gene expression under hypoxic conditions with the NSE promoter and valproic acid

We tested whether the effect of increasing the gene expression levels through the addition of VPA is reenacted under hypoxic conditions. pNSE-Luc was transfected into iNSCs and incubated under normoxic or hypoxic conditions with VPA. The level of gene expression with VPA under hypoxic conditions was increased twofold, compared to hypoxic conditions without VPA (Fig. 5A). We also mimicked hypoxic conditions by treatment with 200 μM CoCl₂, and the results were measured by IVIS imaging (Fig. 5B) and ROI values (Fig. 5C). iNSCs treated with 200 μM CoCl₂ and 1 mM VPA showed twofold higher gene expression levels than those without VPA. We performed the same experiment with the therapeutic gene VEGF, and it was measured by ELISA. The highest level of VEGF expression was observed with VPA treatment under hypoxic conditions (Fig. 5D). We next examined the total VEGF expression for 7 days. VPA treatment under hypoxia showed a high level of expression compared to other conditions (Fig. 5E). As shown the results that 1 mM VPA is an optimal concentration in normoxic conditions, iNSCs treated with 1 mM VPA under hypoxia showed the highest level of cell viability (Fig. 5F).

Fig. 5.

VPA increases pNSE/iNSCs expression levels under hypoxia in vitro. A Luciferase expression levels of iNSCs transfected with pNSE-Luc. Cells were incubated under normoxic or hypoxic conditions and treated with or without 1 mM VPA. n = 9 (Normoxia Control), n = 11 (Normoxia VPA), n = 5 (Hypoxia Control), n = 4 (Hypoxia VPA). B Luciferase expression levels of pNSE-Luc under hypoxic conditions with the addition of CoCl2 on IVIS imaging. The color scale bar indicates the luciferase levels. C Quantification of IVIS imaging in (B) with ROI values. n = 3. D VEGF expression of pNSE-VEGF under normoxic or hypoxic conditions with or without VPA. n = 11. E VEGF expression of pNSE-VEGF under normoxic or hypoxic conditions with or without VPA for 3, 5 and 7 days. n = 3. FIn vitro cell viability assay. iNSCs were transfected with pNSE-Luc and treated 0, 1, 5, 10 mM concentrations of VPA under hypoxic conditions. 490 nm was used to measure the absorbance. n = 6 (0 mM), n = 6 (1 mM), n = 6 (5 mM), n = 3 (10 mM). Data are represented as mean ± S.E.M.. *p < 0.05, **p < 0.01, ***p < 0.001

A SCI mouse model was constructed to examine the effect of the pNSE and VPA in iNSCs on increasing gene expression levels in vivo. We split mice into six groups and treated each group with different concentrations or administrations of VPA. The Control group did not receive VPA treatment and cell transplantation. The ‘Cell’ group was transplanted with iNSCs transfected with pNSE (pNSE/iNSCs), but did not receive VPA treatment. The ‘i.p. injection (intraperitoneally injection)’group refers to mice injected with 1 mM VPA into the abdomen twice a day after pNSE/iNSCs transplantation until sacrifice. The ‘Pre-treatment’ group comprised mice transplanted with cells treated with 1 mM VPA in vitro. ‘0.1 mM VPA’ or ‘1 mM VPA’ comprised respective mice groups to which 0.1 or 1 mM VPA was added simultaneously with cell transplantation (Fig. 6A). Two days after cell transplantation, spinal cord tissue was obtained, and gene expression levels were measured by luciferase activity. The i.p. injection of VPA treatment and Pre-treatment groups did not show any increase in gene expression, compared to the ‘Cell’ group (Fig. 6B). These results imply that i.p VPA injection and VPA pre-treatment of in vitro do not have any effect on raising gene expression levels. However, transplantation of iNSCs with 0.1 mM or 1 mM VPA led to a significant increase in gene expression, compared to other groups (Fig. 6C). This indicates that intraperitoneal administration of VPA does not affect increasing gene expression and that treatment with VPA during iNSCs transplantation increases gene expression in vivo. According to the results of VPA administration methods, pNSE-VEGF loaded iNSCs was transplanted with 0.1 mM VPA and VEGF expression was increased in injured spinal cord site (Fig. 6D).

Fig. 6.

VPA increases pNSE/iNSCs expression levels in vivo, hypoxic spinal cord injury. A Schematic overview showing VPA treatment or administrations of VPA in vivo. SCI models were constructed 1 week prior to cell transplantation. iNSCs were seeded at day 0 and pNSE-Luc transfection was performed at day 1. iNSCs transfected with pNSE-Luc was transplanted at day 2 with various conditions of VPA treatment. Spinal cord tissues were obtained 48-h after transplantation. The ‘Cell’ group, iNSCs were transplanted only. The ‘i.p. injection’ group, 1 mM VPA was injected into the abdominal cavity twice a day after cell transplantation. The ‘Pre-treatment’ group, iNSCs were treated with 1 mM VPA at day 1 for 6-h and then cells were transplanted at day 2. The ‘0.1 mM VPA’ group, cells were transplanted with 0.1 mM VPA. The ‘1 mM VPA’ group, cells were transplanted with 1 mM VPA. B Luciferase expression levels of obtained mouse spinal cords in ‘Cell’, ‘i.p. injection’, and ‘Pre-treatment’ group. Control was transplanted with PBS only. n = 6 (Control), n = 6 (Cell), n = 10 (i.p. injection), n = 4 (Pre-treatment). C Luciferase expression levels of obtained mouse spinal cords in all groups. Direct injection of VPA with pNSE/iNSCs showed increased gene expression significantly. n = 6 (Control), n = 6 (Cell), n = 10 (i.p. injection), n = 4 (Pre-treatment), n = 6 (0.1 mM VPA), n = 4 (1 mM VPA). D VEGF expression levels of obtained mouse spinal cords in ‘Cell’ and ‘0.1 mM VPA’ group. n = 3 (Control), n = 5 (Cell), n = 5 (0.1 mM VPA). Data are represented as mean ± S.E.M.. *p < 0.05, **p < 0.01

Discussion

Spinal cord injury exhibits degeneration of tissue and blood vessels resulting hypoxic conditions, such that genes are difficult to deliver and express at the injury site. In this study, neuronal cell-inducible gene therapy was combined with VPA and iNSCs generated from human fibroblasts. This combined method was directly transplanted at injury sites to deliver therapeutic genes and resulted in stable gene expression at the injured region, and VPA induced increases in gene expression. Combined strategy may be an effective therapy system for SCI and a platform for patient-individualized gene therapy.

We previously demonstrated that genes are specifically overexpressed in mouse neural stem cells under the NSE promoter [14, 15]. In another our study, microarray analysis showed that therapeutic gene expression under NSE promoter (pNSE-VEGF) has no sign of a toxic effect on neural stem cells in regards to molecular and biological functions [15]. Building on these previous studies, we demonstrated in the present study that therapeutic gene expression was increased in human iNSCs using NSE promoter and that gene expression levels showed the highest under hypoxic conditions with the addition of VPA. VPA, known as histone deacetylase (HDAC) inhibitor molecules [23], is widely used in treatments for bipolar disorder and epilepsy [23], approved by Food and Drug administration (FDA) as an anticancer reagent [24], and also known to induce neural differentiation [19, 26]. In our expression vector system, VPA enhanced gene expression of neuronal cell-inducible vector using NSE promoter. NSE promoter showed fivefold higher gene expression levels than SV promoter in iNSCs and further eightfold higher under hypoxia than normoxia conditions. When VPA was added, the gene expression levels were twofold higher under hypoxia than without VPA. However, VPA also enhanced gene expression in SV promoter, HEK293 and under normoxia not specific to NSE promoter, iNSCs and under hypoxia. Total histone H3 acetylation was increased after VPA treatment, indicating VPA induced gene overexpression in overall due to its function as HDAC inhibition. Collectively, the combined method of neuronal cell-inducible vector and VPA induced gene overexpression to the maximum under hypoxic conditions.

In an in vitro model, treatment with 5 mM VPA showed the highest levels of gene expression. However, the viability of the cells significantly decreased, so this seemed to be a toxic concentration of VPA for the cells. Therefore, 1 mM VPA was determined to be the optimal concentration at which to increase gene expression without negatively affecting cell viability. In the in vivo study, our results demonstrated that gene expression was increased when VPA was added to cells to be transplanted at concentrations of 1 mM and 0.1 mM. However, anorexia and hypothermia were observed in 1 mM VPA-treated mice. Given this finding, 1 mM VPA was considered to be toxic, and 0.1 mM VPA was determined to be the optimal concentration for in vivo studies. Overall, we found VPA treatment to increase gene expression to be dependent on the dose and route of administration.

Our experiments showed that the NSE promoter is inducible in both iNSCs and hypoxic conditions. In combination with VPA, it induced gene overexpression of pNSE/iNSCs under hypoxic conditions. As a result, this combined method maximizes gene expression in hypoxic injury site compared to SV promoter or normoxic conditions. in vivo study showed direct injection of VPA with pNSE/iNSCs increases gene expression at the SCI injury site. In future studies, it will be necessary to investigate the effect of therapeutic genes and replacement of degenerative neural tissue with differentiated iNSCs in order to determine the efficiency of this therapy in regards to SCI outcomes.

Authors’ contribution

JSO and YH organized and supervised the study; YY and DB performed the experiments and wrote the manuscript; DL and EC performed electrophysiological studies; JK contributed to material preparation.

Compliance with ethical standards

Conflict of interest

The authors declare no financial conflicts of interest.

Ethical statement

The Animal Care and Use Committee of the Medical Research Institute of Yonsei University College of Medicine approved all protocols (IACUC approval 291 No. 2017-0114) in the current study.