Cell culture

Human fetal brain tissue from a cadaver at 13 weeks of gestation was obtained with full parental written consent and approval of the Research Ethics Committee of Yonsei University College of Medicine, Seoul, Korea (Permit Number: 4-2003-0078) [25]. In this study, huNSPCs for transplantation were derived from such a single donated fetal tissue. The culture of NSPCs was previously described in detail [22]. Briefly, after dissociation of telencephalic tissue in trypsin (0.1% for 30 min; Sigma, St. Louis, MO), cells were plated at 4×10⁵ cells/mL in serum-free culture medium (DMEM/F12; Gibco, Grand Island, NY), N2 formulation (1% v/v; Gibco), and 8 µg/mL heparin (Sigma) supplemented with 20 ng/mL fibroblast growth factor-2 (FGF-2; R&D Systems, Minneapolis, MN) and 10 ng/mL leukemia inhibitory factor (LIF; Sigma). All cultures were maintained in a humidified incubator at 37°C and 5% CO₂ in air, and half of the growth medium was changed every 3–4 days. Proliferating single cells in culture generated free-floating neurospheres during the first 2–5 days of growth. They were passaged every 7–8 days by dissociation of bulk neurospheres with 0.05% trypsin/EDTA (T/E; Gibco).

For proliferation conditions, 8×10⁵ cells/well were maintained with mitogens in 6-well plates for quantitative RT-PCR, Western blot and HPLC analysis. For differentiation conditions, neurospheres were trypsinized and dissociated into single cells, and cells were then placed on poly-L-lysine (PLL; 10 µg/mL; Sigma)-coated 8-well chamber slides (Nunc, Roskilde, Denmark) at 8×10⁴ cells/well for immunocytochemical analysis or on PLL-coated 6-well plates (Sigma) at 1×10⁶ cells/well for quantitative RT-PCR, Western blot and HPLC analysis in the growth medium without mitogens. The medium was replaced every 2 days, and cells were analyzed at day 7 of differentiation.

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was isolated from cell cultures using the TRI reagent (MRC, Inc., Cincinnati, OH), and 2 µg of RNA was reverse-transcribed into cDNA using random hexamer primers (Bioneer, Daejeon, Korea) and Superscript III reverse transcriptase (Invitrogen, Grand Island, NY) in a thermal cycler (Eppendorf, Happauge, NY) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in a total volume of 10 µL containing 5 µL of LightCycler® 480 SYBR Green I Master (Roche Diagnostics Ltd., Rotkreuz, Switzerland), 0.5 µM of each primer and 2.5 µL of 1∶10 diluted cDNA using LightCycler® 480 instrument (Roche Diagnostics Ltd). The cycling conditions were: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. All the samples were carried out in triplicates. The expression levels of each mRNA expression were normalized to the housekeeping gene GAPDH using LightCycler® 480 Software, Version 1.5 (Roche Diagnostics Ltd). Primer sequences for FOXG1, DLX2, NKX2.1, GAD1, calbindin2 (CALB2) and somatostatin (SST) have been previously described [26]. Other primer sequences were retrieved from PrimerBank Database http://pga.mgh.harvard.edu/primerbank/ [27], with the exception of SLC32A1 which was designed using ProbeFinder software Version 2.49 from Roche Applied Science. Sequences of primer sets were listed in Table S1.

Western blots

At the protein level, the expression of GAD65 (GAD2) and GAD67 (GAD1) was quantified by Western blotting. Cells were lysed in tissue protein extraction reagent (T-PER reagent; Pierce, Rockford, IL) with a protease inhibitor cocktail (Sigma), and lysates were centrifuged (16,000×g, 30 min, 4°C). The supernatant was collected and stored at –80°C. The samples were assayed for total protein using a bicinchoninic acid assay (BCA kit; Pierce), and 20 µg of protein was mixed with 2% β-mercaptoethanol in tricine sample buffer (Bio-Rad, Hercules, CA) and denatured by heating to 95°C. Then, samples (20 µL) were loaded onto 10% Tris–glycine gels, and the proteins were transferred from the gel onto a 0.45-µm nitrocellulose membrane (Thermo Scientific, Suwanee, GA) over 4 h at 4°C. The protein blots were blocked with 5% skimmed milk in TBST and then incubated with 0.25% bovine serum albumin in TBST overnight at 4°C with the following primary antibodies: anti-GAD65 (1∶1,000; Sigma), anti-GAD67 (1∶1,000; Chemicon, Temecula, CA), or β-actin (1∶2,000; Sigma). Immunoblots were rinsed with TBST, incubated with a horseradish peroxidase-conjugated secondary antibody (1∶20,000; Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature, and developed using SuperSignal West Pico Chemiluminescent substrate (1∶20,000; Thermo Scientific). The images were scanned with a Fujifilm LAS-4000 mini imager and analyzed with the MultiGauge software (Fujifilm, Tokyo, Japan).

High-performance liquid chromatography (HPLC)

Intracellular GABA content was measured 7 days after plating as follows. The cells under proliferation or differentiation conditions were washed twice with ice-cold Hank's balanced salt solution-HEPES (H-H) buffer (Gibco), treated with 0.4 M perchloric acid, and scraped from each well. The cell lysates were centrifuged (16,000×g, 4°C, 30 min), and the supernatant was collected and adjusted to pH 7–8. The samples were filtered and stored at −80°C. To measure the level of GABA release, the cells were incubated in basal medium (144 mM NaCl, 1 mM MgCl₂, 4 mM KCl, 1.8 mM CaCl₂, 5 mM glucose, and 10 mM HEPES) and high K⁺ medium (same as basal medium except 94 mM NaCl and 53 mM KCl) for 60 min. After incubation, the supernatant was collected, filtered, and stored at −80°C. The GABA levels in the cell lysates and supernatants from basal and high K⁺ media were analyzed by reverse-phase HPLC using o-phthalaldehyde derivatization with a fluorescence detector [28], [29]. All experiments were done in triplicate. Values were normalized to total cellular protein, measured using the BCA assay.

Immunostaining

For immunocytochemical analysis, cultured cells were fixed with 4% paraformaldehyde (PF) in PIPES buffer (Sigma), rinsed with phosphate-buffered saline (PBS) solution, blocked with 3% bovine serum albumin (Sigma), 10% normal horse serum, and 0.3% Triton X-100 (Sigma) in PBS. For immunohistochemical analysis, animals were deeply anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.) and perfused with 4% PF in 0.1 M PIPES buffer. Brains were then removed, post-fixed, transferred in 30% sucrose in PBS for cryoprotection, and frozen in O.C.T compound (Sakura Finetek, Torrance, CA, USA). The brains were coronally sliced into 16-µm sections using a cryostat. Sections were washed in PBS and blocked as mentioned above. Cultures or Sections were incubated with following primary antibodies: anti-human specific nestin (anti-hNestin; 1∶200; Chemicon), anti-human specific nuclear pore (hNP; 1∶20; Oncogene, Cambridge, MA), anti-human specific cytoplasm SC121 (1∶500; Stem Cells, Inc., Cambridge, UK), anti-human specific GFAP SC123 (1∶500; Stem Cells, Inc., Cambridge, UK), anti-glial fibrillary acidic protein (GFAP; 1∶1,000; Dako, Glostrup, Denmark), anti-neuronal class β-tubulin III (TUJ1; 1∶1,000, Covance, Princeton, NJ), anti-GABA (1∶500; Sigma), anti-calretinin (1∶2,000; Chemicon), anti-GAD65 (1∶250; Sigma), anti-GAD67 (1∶500; Chemicon), anti-platelet-derived growth factor receptor alpha (PDGFR-α; 1∶100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-adenomatous polyposis coli-CC1 (APC-CC1; 1∶30; Abcam, Cambridge, MA), anti-Olig2 (1∶500; Millipore, Billerica, MA), anti-glial cell-derived neurotrophic factor (GDNF; 1∶50; Santa Cruz Biotechnology), anti-S100β (1∶1000; Sigma), and anti-human specific nuclei (hNuc; 1∶100; Chemicon) antibodies. Species-specific secondary antibodies, conjugated with fluorescein (FITC; 1∶180; Vector, Burlingame, CA) or Texas Red (TR; 1∶180; Vector) were used to detect the binding of primary antibodies. Specimens were mounted using Vectashield mounting medium with 4,6-diamino-2-phenylindole (DAPI; Vector), and were analyzed by an immunofluorescence microscopy (BX51; Olympus, Center Valley, PA) or a confocal laser scanning microscopy (LSM 700; Carl Zeiss, Oberkochen, Germany).

Quantification

For in vitro quantification, the total number of TUJ1-, PDGFR-α-, GFAP-, GABA-, CALB2-, and GAD2-expressing cells were counted in three to five randomly selected fields. The percentage of each marker-positive cell among total DAPI-positive cells was calculated. This sampling was replicated three times.

To quantify the survival and migration of engrafted huNSPCs, the number of BrdU⁺ cells were quantified in every fifth section through the entire anteroposterior extent of grafts at 4 and 8 weeks after grafting in the kindling model. Estimates of total BrdU⁺ cell numbers from raw cell counts were corrected according to the Abercrombie formula [30]. The mean yield of surviving cells in each rat was depicted as the percentage of transplanted cells. Cell numbers versus distance to the injection site were quantified and values were expressed as a percentage of the total number of surviving cells. To determine differentiation patterns of grafted cells, sets of 50–100 BrdU⁺ or hNuc⁺ cells from every fifth section were used to calculate the percentages of TUJ1⁺, GFAP⁺, APC-CC1⁺, PDGFR⁺, or GABA⁺ cells at 8 weeks following transplantation.

Kindling process

Adult male Sprague–Dawley rats (∼300 g) were kept on a 12/12-h light/dark cycle (lights on at 07:00 h) with free access to food and water. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Yonsei University College of Medicine (Permit Number: 2010-0201). The rats were anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.) and implanted with a bipolar electrode-cannula guide (Plastics One, Roanoke, VA) into the right hippocampal CA3 using the following stereotaxic coordinates: 4.2 mm lateral, 4.52 mm caudal, and 4.5 mm ventral to bregma. The electrode-cannula consisted of a cylindrical plastic pedestal molded around a piece of stainless steel tubing, which guided an infusion cannula, and two individually insulated wire electrodes that attached to the sides of the stainless steel tubing [31]. Via the stainless steel tubing, the infusion cannula penetrated to a consistent depth and was used to transfer vehicle or dissociated NSPCs. Two steel screws served as ground electrodes. All electrodes were anchored in place with dental cement. After a recovery period of 1 week, afterdischarge threshold (ADT) was determined by passing constant pulse trains (1 ms, biphasic rectangular wave, 50 Hz for 1 s). The initial current of 10 µA was delivered, increasing the current intensity in steps of 10 µA (to a maximum of 150 µA), with intervals of 1 min between current deliveries until an afterdischarge (AD) was seen, defined as poststimulus electroencephalogram (EEG) spikes with a frequency of greater than 1 Hz and an amplitude at least two times greater that of the pre-stimulus recording [32]. The rats were stimulated at the ADT intensity twice daily until five consecutive stage 5 or higher seizures (according to the additional Racine scale [33]) were elicited: Stage 1, facial movements only; Stage 2, facial movements and head nodding; Stage 3, facial movements, head nodding, and forelimb clonus; Stage 4, facial movements, head nodding, forelimb clonus, and rearing; Stage 5, facial movements, head nodding, forelimb clonus, rearing, and falling; Stage 6, cluster of multiple Stage 5 seizures; Stage 7, jumping and running seizures; and Stage 8, Stage 7 plus tonic hindlimb extension and tail rigidity, sometimes culminating in death. In total, 25 fully hippocampal-kindled rats were randomly allocated to the two groups: the vehicle-injected control group (n = 13) or the NSPC-transplanted group (n = 12). The average ADT between the groups was not significantly different (47.7±11.4 and 44.1±7.0 µA in the vehicle and NSPCs groups, respectively; P = 0.80).

For fully hippocampal-kindled animals, electrical responses generally consisted of three epochs: primary AD, silent period, and subsequent secondary AD [34]. AD duration (ADD) refers to the cumulative duration of primary and secondary ADs recorded within ∼2–3 min after stimulation at the intensity of ADT. In addition to ADD, behavior seizure stage and seizure duration were measured. Seizure stage was classified according to the additional Racine scale. Seizure duration was defined as the period of limbic seizure (stages 1–2) and subsequent motor seizure (stages 3–7), excluding limbic seizure after motor seizure [35].

Induction of status epilepticus by pilocarpine

Adult male Sprague–Dawley rats (200–220 g) were infused with lithium chloride (127 mg/kg, i.p.; Sigma) 24 h prior to the administration of pilocarpine. On the next day, the rats were injected with methylscopolamine bromide (1 mg/kg, i.p.; Sigma) to limit the peripheral effects of pilocarpine, and 30 min later were injected with pilocarpine hydrochloride (45 mg/kg, i.p.; Sigma) to induce SE. Seizure events in pilocarpine-treated rats were scored according to the additional Racine scale [33], and rats that did not develop stage 5 or higher seizure were excluded from this study. Diazepam (Samjin, 10 mg/kg, i.p.) was injected 1 h after SE onset to cease seizure activity. The rats that went into SE were injected with 2.5 mL of 5% dextrose intraperitoneally twice a day and were given a moistened rat diet during the following 2–3 days. For 14–20 d after SE, pilocarpine-treated rats were monitored to confirm the emergence of SRMSs by video recording. The frequency of SRMSs (stages 3–7 seizure) was scored for 12 h/day. After monitoring, epileptic rats were randomly allocated to two groups: the vehicle-injected control group (n = 18) or the NSPC-transplanted group (n = 21). The frequency of SRMSs before transplantation between two groups was not significantly different (0.18±0.04 and 0.15±0.02 seizures/day in the vehicle and NSPCs groups, respectively; P = 0.87).

Preparation and transplantation of NSPCs

huNSPCs were maintained by passaging through the dissociation of bulk neurospheres and cryopreserved at each passage in the Good Manufacturing Practice facility. For transplantation, NSPCs taken at between passage number 10 (P10) and P20 were selected and prepared. Cells were labeled with bromodeoxyuridine (BrdU; 3 µM; Sigma) for 5 days before grafts. At the time of grafting, cells were harvested by trypsinization after which the enzymatic activity was halted by soybean trypsin inhibitor (Sigma). The cells were centrifuged (900×g, 3 min), the cell pellet was washed three times with H-H buffer, and the entire cell pellet was then resuspended in H-H buffer at a density of 1.0×10⁵ cells/µL. The concentrated NSPCs in a sterile freezing tube (Nunc) were then delivered to the animal operation room. For the kindling model, 1 week after reaching the kindling criteria, rats were anesthetized and injected with 4 µL of vehicle (H-H buffer only) or NSPC suspension into the stimulated site (CA3 region of the right hippocampus) using an infusion cannula and tubing (Plastics One). For the pilocarpine model, at 3 weeks after SE, epileptic rats were anesthetized and injected with 4 µL of vehicle or NSPC suspension into the CA3 regions of bilateral hippocampus using the following stereotaxic coordinates: 4.2 mm left and right lateral, 4.52 mm caudal, and 5.0 mm ventral to bregma. Vehicle or NSPC suspension was infused at a flow rate of 1 µL/min using a 10-µL Hamilton syringe placed on an infusion pump (KD Scientific, Holliston, MA) controlled by a microprocessor. All animals in both groups received daily injections of cyclosporine (10 mg/kg, i.p.) from a day before transplantation to the end of the experiment.

Evaluation of NSPC grafts on kindled seizures and SRMS

For the kindling model, after a recovery period of 1 week following transplantation, the hippocampus of each animal was stimulated weekly at ADT intensity. Regular determinations of seizure parameters—ADD, seizure duration, and seizure stage—in both vehicle-injected and NSPC-transplanted groups were performed weekly for 8 weeks with video and EEG recordings. For the pilocarpine model, the behavior of epileptic rats was observed at 1–3 months following transplantation to analyze the frequency, severity and duration of SRMSs in both the vehicle-injected and NSPC-transplanted groups. Starting at 2 weeks following transplantation, the rats were video-monitored for 60 h per week (12 h/day, 5 days/week, 2 weeks/month, and 360 h in total). Rats were given free access to water and food in individual cages and video-monitored during the daylight period. The video recordings were analyzed by observers blinded to group allocation. Rats were scored according to the same additional Racine scale used in the establishment of the kindled model [33]. The frequency and severity of seizure, and the total time spent in seizure were assessed in each rat. At the end of experiments, all rats were sacrificed by CO₂ inhalation, in accordance with institutional and AVMA guidelines for the euthanasia [36].

Morris water maze

The Morris water maze test was performed to assess the effect of huNSPCs grafts on learning and memory function in epileptic rats. The detailed method has been previously described [37]. Briefly, a circular polypropylene pool (200 cm in diameter and 40 cm in height) was filled with water (22±1°C) made opaque with a food coloring agent, rendering it impossible for rats to see through it. On the pool rim, four points were designated (north, east, south, west), dividing the pool into four quadrants (NE, NW, SW, SE). A circular platform (15 cm in diameter and 30 cm in height) was positioned at the center of the SE quadrant and hidden 1 cm below the water surface, and the position of the platform was kept unchanged throughout the training days. A trial started when the rat was positioned in the water from a quasirandom start points and ended when the rat reached and escaped onto the hidden platform. The swim paths of rats were recorded by a video tracking system (SMART; Panlab, Barcelona, Spain) and analyzed thereafter. For the pilocarpine model, the water maze test was performed 3 months following transplantation in an age-matched intact group (n = 6), the vehicle-injected group (n = 11), and the NSPC-transplanted group (n = 12). Rats were trained for 4 days, and the training day consisted of six trials with an interval of 5–15 min. If the rat was not able to find the platform in 120 s, the rat was guided to it by the investigator. Mean escape latency was calculated for each day during the training days. At the probe trial, on day 5, the rats were positioned in the opposite quadrant where the platform was previously located and allowed to swim in the pool without the platform for 60 s. All parameters of memory retention were measured. At the end of experiments, all rats were sacrificed by CO₂ inhalation as described above.

Determination of GDNF-positive astrocytes in the hippocampus

To investigate whether huNSPC grafting affects the level of GDNF expression in host hippocampal astrocytes, double immunofluorescence for GDNF and the astrocytic marker, S100β was done 12 weeks after transplantation in age-matched intact controls and pilocarpine-treated rats that received vehicle or NSPCs. The ratio of double-labeled GDNF- and S100β-positive cells among total S100β-positive cells in the bilateral CA3 regions of the hippocampus was determined by a confocal laser scanning microscopy in every 10^th section throughout the dorsal hippocampus. At least 80 S100β-positive cells per animal were analyzed at 400x magnification.

Timm staining

After completion of the Morris water maze, pilocarpine-induced epileptic rats were anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.) and perfused transcardially for 10 s with 0.1% sodium sulfide, 3 min with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and 7 min with 0.1% sodium sulfide [38]. The brains were postfixed for 24 h at 4°C and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer. The brains were embedded in O.C.T compound (Sakura Finetek), coronally sectioned at 16-µm, and stored at −20°C. A 1-in-10 series of sections from dorsal hippocampus was dried, and developed for 60 min in a solution of 50% gum Arabic (120 mL), 20 ml of 2 M citrate buffer, 3.4 g of hydroquinone in 60 mL of H₂O, and 1 mL of 17% silver nitrate [39]. After rinsing, sections were dehydrated in graded alcohol and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). Sections from age-matched intact control rats were stained at the same time as sections from pilocarpine-induced epileptic rats. Mossy fiber sprouting was assessed by scoring a five-point scale, as previously reported [40]. The Timm score of each section was calculated by an observer blinded to group allocation, and averaged from all the stained sections of bilateral hippocampi.

Statistical analysis

All data are shown as means ± standard error of the mean (SEM). Differences in GABA content or GABA release from huNSPCs were evaluated with the Mann–Whitney U-test. Significant differences in average yield of viable huNSPCs and between 4 and 8 weeks after transplantation in the kindling model were analyzed with Student's t-test. To compare the mean rostrocaudal distance to injection site of grafted NSPCs between 4 and 8 weeks post-transplantation, we used Student's t-test. The Timm scores were analyzed using one-way analysis of variance (ANOVA) followed by the post hoc Bonferroni test. Comparison of percentages of S100β⁺ astrocytes expressing GDNF in the CA3 region of the hippocampus among the three groups was performed by one-way ANOVA with LSD post hoc test. In the kindling model, Student's t-test was used to compare the effects of NSPC transplantation with vehicle injection on ADD, seizure duration, and seizure stage. Escape latency in the Morris water maze was compared among groups using repeated-measures ANOVA with Tukey's HSD post hoc procedure. Dwell time in the target quadrant, dwell time in the platform area, latency to the platform area, and platform area crossings in the Morris water maze were compared among groups using one-way ANOVA followed by the post hoc LSD test. In the pilocarpine model, Student's t-test was used to compare the effects of NSPC transplantation with vehicle injection on the frequency, severity and duration of SRMSs. Differences were considered statistically significant for P<0.05. Group sizes were calculated based on power analysis (PASS program). We used a power of 0.8 and significance levels of 0.05.

Multipotent human NSPCs and differentiation into GABAergic neurons in culture

huNSPCs have the ability to differentiate into all three neural cell types in vitro: neurons, oligodendrocytes, and astrocytes (Fig. 1A–C). At 7 days after plating of neurosphere-derived single cells under differentiation conditions, ∼61% of NSPCs had differentiated into TUJ1⁺ neurons, ∼2% into PDGFR-α⁺ oligodendrocyte progenitors, and ∼5% into GFAP⁺ astrocytes (Fig. 1J). To investigate whether NSPCs could differentiate into GABAergic interneurons, we examined the expression of GABAergic neuronal markers in NSPCs using Western blots and immunocytochemistry (Fig. 1D-J). Histological analysis showed that ∼26% of NSPC-derived differentiated cells expressed GABA (Fig. 1E, J), and ∼37% of NSPC-derived TUJ1⁺ neurons expressed GABA with small bipolar processes (Fig. 1D–F, J). Additionally, ∼30% of cells expressed interneuron subtype marker CALB2, also known as calretinin [26] (Fig. 1G, J), and ∼11% of cells expressed GABA-synthesizing enzyme, GAD2 (Fig. 1H, J). Western blot showed that protein levels of GAD1 and GAD2, the two isoforms of the GABA-synthesizing enzyme, were elevated markedly in cells under differentiation (Diff) conditions, versus proliferation (Prol) conditions (Fig. 1I).

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Figure 1. Multipotent human NSPCs and differentiation into GABAergic neurons in culture.

(A–C) Under differentiation conditions, differentiation of fractions of NSPCs into TUJ1⁺ neurons visualized by Texas Red (A), PDGFR-α⁺ oligodendrocyte progenitors identified by fluorescein (B), and GFAP⁺ astrocytes imaged using fluorescein (C) could be observed. Nuclei were counterstained with DAPI. (D–F) Many NSPC-derived differentiated neurons were co-labeled with TUJ1 (red) and GABA (green) with small bipolar processes. (G, H) Fractions of NSPC-derived cells also expressed CALB2 (calbindin2, green) and GAD2 (GAD65, green). Scale bar, 50 µm. (I) Western blot analysis shows that GAD1 and GAD2 were expressed highly in NSPCs under differentiation conditions. (J) The bar chart illustrates percentages of NSPCs that exhibit differentiation into TUJ1⁺ neurons, PDGFR-α⁺ oligodendrocyte progenitors, GFAP⁺ astrocytes, GABA⁺ neurons, CALB2⁺ neurons, and GAD2⁺ neurons. Quantification of the data presented in (J); mean ± SEM (n = 3).

https://doi.org/10.1371/journal.pone.0104092.g001

We analyzed transcript expression for region-specific and GABAergic interneuron lineage markers in huNSPCs using qRT-PCR (Fig. 2). Telencephalic marker FOXG1 and ventral telencephalic markers (OLIG2, ASCL1, and DLX2) were expressed in cells under both Prol and Diff conditions [26], [41], and the expression levels of makers were significantly elevated under Diff condition compared to under Prol condition except OLIG2 (Fig. 2A, B). The MGE, marked by NKX2.1 and LHX6 expression, is known the primary source of cortical interneurons both in rodents and in humans, and the caudal ganglionic eminence (CGE), marked by NR2F2 (COUP-TFII) expression, give rise to a greater proportion of cortical interneurons in humans than in rodents [26], [42]-[45]. We found that NSPCs not only expressed MGE markers, but also abundantly the CGE marker (Fig. 2C, D). In addition, GABAergic markers (GAD1, SLC32A1, and SLC6A1) and interneuron subtype markers (CALB2, SST, and NPY) were robustly expressed under differentiation conditions (Fig. 2E, F). These data demonstrate that huNSPCs not only give rise to all three CNS neural cell types, but also that many of them differentiate into the MGE and CGE-derived GABAergic interneurons.

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Figure 2. Transcript expression profiling of human NSPCs.

(A–F) Expression patterns of genes associated with development and function of GABAergic neurons were analyzed in NSPCs under proliferation (Prol) and differentiation conditions (Diff) using quantitative RT-PCR. The expression levels of each mRNA expression were normalized to levels of GAPDH. Panels show: telencephalic (A), ventral telencephalic GABAergic neuronal lineage (B), medial ganglionic eminence (MGE) (C), caudal ganglionic eminence (CGE) (D), GABAergic neuron (E), and interneuron (IN) subtype markers (F). The expression levels of makers were significantly elevated under Diff condition compared to under Prol condition except OLIG2. Data represented as mean ± SEM (n = 3).

https://doi.org/10.1371/journal.pone.0104092.g002

HPLC analysis of GABA release from human NSPCs in culture

After finding evidence that huNSPCs could differentiate into GABAergic neurons, we examined whether NSPC-derived cells actually released GABA under basal and high K⁺ conditions in culture using HPLC. Intracellular GABA content of NSPCs under differentiation conditions (8,018.4±514.9 pmol/mg, n = 3) was about twice higher than that under proliferation conditions (4,288.4±118.4 pmol/mg) (P = 0.010; Fig. 3A). When NSPCs under differentiation conditions were incubated in basal or high K⁺ medium, the intracellular GABA content of cells incubated in basal medium (11,119.8±871.8 pmol/mg, n = 3) was slightly higher than that in the high K⁺ medium (8,494.0±682.0 pmol/mg) (P = 0.20; Fig. 3B). However, the amount of GABA released from differentiated NSPCs into the medium was significantly higher in the high K⁺ medium (507.69±11.39 pmol/mg) than that in the basal medium (293.20±23.78 pmol/mg) (P = 0.029; Fig. 3C), suggesting that huNSPC-derived differentiated cells can increase efflux of GABA in response to depolarization induced by elevated K⁺.

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Figure 3. HPLC analysis for GABA in human NSPCs.

(A) huNSPCs contain GABA under both proliferation (Prol) and differentiation (Diff) conditions in culture. Note that the total intracellular GABA content of NSPCs was significantly higher under Diff conditions than under Prol conditions. (B, C) NSPCs under Diff conditions were incubated in basal (4 mM KCl) or high K⁺ (53 mM KCl) medium, and intracellular GABA content (B) and GABA release into the medium (C) were quantified. * Significantly different from that under Prol conditions at P<0.05; † significantly different from that in the basal medium at P<0.05; error bars indicate ±SEM.

https://doi.org/10.1371/journal.pone.0104092.g003

Engraftment and distribution of human NSPCs following transplantation

In the kindling model, we transplanted NSPCs into the CA3 region of the right hippocampus of fully kindled rats. To evaluate the grafted cells, animals were killed at 4 and 8 weeks following transplantation, and brain tissues were processed for immunohistochemistry. BrdU⁺ grafted cells had migrated away from the injection site and dispersed throughout the hippocampus. Many grafted cells were predominantly located in the radiatum layer of the CA3 region, molecular and granular layer of the dentate gyrus (DG), and hilus of the hippocampus (Fig. 4A-F).

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Figure 4. Engraftment and distribution of human NSPCs into the hippocampus of kindled rats.

(A–E) Serial sections (320 µm apart) throughout the hippocampus were used to determine the location of BrdU⁺ grafted cells, visualized with fluorescein at 4 weeks following transplantation. A–E panels are representative images of serial coronal sections of the hippocampus which are ordered from anterior to posterior. Grafted cells had migrated apart from the injection site (arrowhead in C) and dispersed throughout the adjacent hippocampal subfields. Many grafted cells were predominantly placed in the radiatum layer (Rad) of the CA3 region, molecular (ML) and granular cell layer (GCL) of the dentate gyrus (DG), and hilus (HI) of the hippocampus. Dotted lines denote the boundary between HI and GCL of the DG. In panel C, the boxed regions of the Rad (1), and ML, GCL and HI (2) are magnified in panel C-1 and C-2, respectively. Scale bar, 500 µm (A–E) and 100 µm (C-1, C-2). (F) The bar chart illustrates the distribution of grafted cells versus rostrocaudal distances to injection site at 4 and 8 weeks after transplantation. The number of grafted cells/distance of serial sections was expressed as a percentage of the total number of surviving cells. Values are represented as mean ± SEM.

https://doi.org/10.1371/journal.pone.0104092.g004

Quantification of the total number of BrdU⁺ cells demonstrated that the survival rate of grafted cells was 57.4±16.6% (229,469.1±66,242.0, n = 4 rats) and 29.3±10.0% (117,127.8±39,809.3, n = 4) at 4 and 8 weeks following transplantation, respectively. To evaluate the migratory ability of NSPCs, we quantified the number of grafted cells versus rostrocaudal distances to injection site at 4 and 8 weeks after transplantation (n = 4, respectively). Grafted cells showed to migrate from the injection site in both rostral and caudal directions. There was a significant difference between the mean distance of migration at 8 weeks and that at 4 weeks (1.44±0.18 vs. 0.96±0.07 mm, P = 0.041), suggesting that rostrocaudal migration is continuously progressing until 8 weeks post-transplantation. In contrast, when NSPCs were transplanted into the CA3 region of the right hippocampus of age-matched non-kindled rats, there was no apparent grafted cell migration from CA3 to the DG (Fig. S1). These results suggest that transplanted huNSPCs engraft robustly, migrate extensively, and survive for a long time after grafting into epileptic hippocampus. In the pilocarpine model, grafted cells also showed robust engraftment and long-term survival, and extensive distribution around the injection site when brains were analyzed 12 weeks following transplantation (Fig. S2).

Differentiation of human NSPCs in epileptic rats following transplantation

To study differentiation patterns of donor-derived cells, the whole hippocampal area of kindled rats was analyzed at 8 weeks following transplantation. BrdU⁺ cells were co-labeled with the early neuronal marker TUJ1 in the CA3 region of the hippocampus (Fig. 5A–C) and fimbria (Fig. 5D–F). Confocal microscopic images of hNP⁺-grafted cells demonstrated dual labeling with the TUJ1 antibody (Fig. 5G–J). Quantification revealed that donor-derived cells differentiated into TUJ1⁺ neurons (33.8±4.0%, n = 4), APC-CC1-expressing oligodendrocytes (27.7±6.2%, n = 4), and GFAP-expressing astrocytes (8.2±1.8%, n = 4) in the hippocampus (Fig. 5K, M). About 40% of GFAP-expressing astrocytes expressed GDNF (Fig. S3). Additionally, ∼24% of BrdU⁺ cells expressed GABA in the hippocampus and surrounding structures (n = 4; Fig. 6A–I). Confocal microscopy images showed that BrdU⁺ cells co-localized with GABA⁺ cells (Fig. 6J), and hNP⁺ cells dual-labeled with GABA had small bipolar neuronal processes (Fig. 6K–N). Most grafted cells, however, appeared not to show the morphological features of mature interneurons resembling host inhibitiory hippocampal interneurons. A few BrdU⁺ cells (∼3%) were found to express CALB2, a GABAergic interneuron subtype (Fig. 6O–Q). In the pilocarpine model, grafted NSPCs also differentiated into TUJ1⁺ neurons, OLIG2⁺ oligodendrocyte progenitors, and GFAP⁺ astrocytes, although a large number of cells expressed an undifferentiated cell marker, nestin (n = 4). Moreover, ∼21% of donor-derived cells expressed GABA and few of them expressed GDNF (Fig. S4). Most grafted cells also appeared not to show the morphological features of mature interneurons. These findings suggest that huNSPCs can give rise to neurons including GABAeric neurons albeit still immature, oligodendrocytes, and astrocytes in the epileptic hippocampus and adjacent structures in TLE models.

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Figure 5. Differentiation of human NSPCs following transplantation into the hippocampus of kindled rats.

(A–F) BrdU⁺ grafted cells (green) were co-stained with TUJ1 (red, arrowheads in B, C, E, F) in the CA3 region of the hippocampus (A–C) and fimbria (D–F) in rats. (G–J) hNP⁺ grafted cells (red, arrowheads in G, I, J) were co-localized with TUJ1 (green, arrowheads in H–J) in the hilus of the hippocampus. (J) Orthogonal view from confocal z-series showed that hNP (red) in nuclei and TUJ1 (green) in cytoplasm were expressed in the same cell. (K, M) Under the dual-filter microscope, BrdU⁺ grafted cells co-expressed APC-CC1 in the fimbria (arrows in K), and hNuc⁺ grafted cells co-expressed GFAP in the CA3 region (an arrow in M). Scale bar; 200 µm (A), 20 µm (D), 10 µm (G, J), 20 µm (K).

https://doi.org/10.1371/journal.pone.0104092.g005

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Figure 6. Differentiation of human NSPCs into GABAergic neurons in the hippocampus of kindled rats.

(A–J) BrdU⁺ grafted cells (green, A, C, D, F, G, I, J), were co-labeled with GABA (red, B, C, E, F, H–J), in the molecular and granular layer of the dentate gyrus, hilus of the hippocampus (A–C), and the radiatum layer of the CA3 region (D–J) of rats. (G–I) A large fraction of BrdU⁺ grafted cells co-expressed GABA, as viewed under the dual-filter microscope. (J) Orthogonal view from confocal z-series visualized co-expression of BrdU (green) and GABA (red) in a grafted cell. (K–N) Orthogonal images show that hNP or hNuc⁺ grafted cells (red) co-expressed GABA (green) in different layers of the CA3: the stratum lucidum (K), stratum pyramidale (L and M), and stratum oriens (N). (O–Q) A BrdU⁺ grafted cells (green) were also co-labeled with calbindin2 (CALB2, red) in the stratum oriens around the CA3 area. Scale bar, 200 µm (A), 100 µm (D), 50 µm (G), 10 µm (J), 20 µm (K), 10 µm (O).

https://doi.org/10.1371/journal.pone.0104092.g006

Effect of human NSPC transplantation on kindled seizures and SRMS

The effect of huNSPCs transplantation on kindled seizure activity was evaluated weekly for 8 weeks following transplantation. Compared with the vehicle-injected group (n = 13), the transplanted group (n = 12) showed substantial decreases in three seizure parameters—ADD on EEG examination, behavioral seizure duration, and seizure stage—although no significant difference was observed in seizure parameters between pre-grafts in both groups (Fig. 7A–C). In the transplanted group, ADD decreased steadily until 4 weeks after grafting and significantly decreased compared with the vehicle group at the third week (24% reduction, P = 0.047) and fourth week (39% reductions, P = 0.005) (Fig. 7A).

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Figure 7. Effect of human NSPC grafting on hippocampal-kindled seizures in rats.

The mean values of afterdischarge duration (ADD) in electroencephalograms (A), behavioral seizure duration (B), and seizure stage (C) between vehicle-injected and NSPC-transplanted groups were compared before (pre-Tx) and after NSPC grafting (post-Tx). Error bars indicate ±SEM. huNSPC grafting significantly reduced all three seizure parameters—ADD, behavioral seizure duration, and seizure stage—although this seizure-suppressing effect was not permanent. * Significantly different from the vehicle-injected group at P<0.05; ** significantly different from the vehicle-injected group at P<0.01.

https://doi.org/10.1371/journal.pone.0104092.g007

Seizure duration declined significantly during the first, second, and third weeks after grafting (27%, 22%, and 25% reductions, respectively; P<0.05; Fig. 7B). Seizure stage also displayed a significant decrease from the second to sixth week (P<0.05; Fig. 7C). Furthermore, seizure parameters in NSPC-transplanted rats did not exceed those of vehicle-injected rats at almost every time point. Thus, huNSPCs grafting resulted in significant reductions in all seizure parameters in the kindling model. However, the significant anticonvulsant effect was not permanent, but disappeared slowly by the seventh week following transplantation (Fig. 7).

To examine the effect of huNSPCs transplantation on spontaneous seizures, we performed video monitoring in the pilocarpine model. Rats were video-monitored to document the emergence of SRMSs for 14–20 d after pilocarpine-induced SE. Previously, adult rats that underwent pilocarpine-induced SE for 1 h were reported to exhibit spontaneous seizures before 20 days after SE [24]. We also observed the emergence of at least one spontaneous seizure in all experimental animals. After confirmation of the occurrence of SRMSs, we injected NSPCs into both right and left hippocampus at 3 weeks after SE, and the rats were then video-monitored from 2 weeks to 3 months following transplantation. During the video monitoring period, we measured the frequency and severity of SRMS, and the total time spent in SRMS (Fig. 8). The mean frequencies of SRMSs in transplanted group (0.10±0.03 and 0.12±0.06 seizures/day at 2 and 3 months after grafting, respectively; n = 21) were significantly reduced compared with those in the vehicle group (0.26±0.06 and 0.61±0.15, respectively; n = 18; Fig. 8A). Thus, epileptic rats that received NSPC grafts into the hippocampus had significantly lower seizure frequencies as compared to vehicle-injected epileptic rats at 2 months (62% reduction, P = 0.029) and 3 months (80% reduction, P = 0.004) following transplantation. Seizure severity was usually stage 4 or 5. The mean seizure stage of SRMS was not different between both transplanted and vehicle groups (P>0.05; Fig. 8B). There were significant decreases in the average total time spent in SRMS at 2 and 3 months after grafting in the transplanted group (28.0±8.0 and 47.6±24.0 s, respectively) as compared to the corresponding time points in the vehicle group (69.0±16.9 and 213.5±51.5 s, respectively) (P = 0.033 and P = 0.007, respectively; Fig. 8C). The mean duration of individual SRMSs was not different in the transplanted group than in the vehicle group at any time point (P>0.05). The data suggest that huNSPCs grafting resulted in long-term significant attenuation of SRMS in a representative TLE model.

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Figure 8. Effect of human NSPC grafting on spontaneous recurrent motor seizures (SRMSs) in pilocarpine-treated rats.

The mean seizure frequencies (A), seizure stages (B) and total time spent in seizures (C) were calculated during 1, 2, and 3 months following transplantation in vehicle-injected and NSPC-transplanted groups. * Significantly different from the vehicle-injected group at P<0.05; error bars indicate ±SEM.

https://doi.org/10.1371/journal.pone.0104092.g008

Effect of human NSPC transplantation on learning and memory function

Epilepsy in patients is often accompanied by cognitive decline [46]. Thus, we examined whether huNSPC transplantation could affect performance in a Morris water maze test, which assesses hippocampal-dependent learning and memory function. To estimate the effect of NSPC transplantation on learning and memory function in the pilocarpine model, we performed water maze testing at 3 months post-grafting. Age-matched intact controls (n = 6) showed the marked decreases in the escape latencies over the sessions, indicating normal spatial learning ability. However, epileptic rats in the vehicle group (n = 11) did not improve significantly in locating the submerged platform over the trials (Fig. 9A), consistent with other reports, demonstrating impaired spatial learning [47], [48]. During the probe test to evaluate the memory function, epileptic rats in the vehicle group spent significantly shorter times in the target quadrant and in the platform area (Fig. 9B, C), took longer to reach the platform area (Fig. 9D), and barely crossed the platform area compared with intact controls (Fig. 9E). In the transplantation group (n = 12), rats did not show amelioration in overall spatial learning or memory function, and were not considerably different from the vehicle-injected group.

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Figure 9. Effect of human NSPC grafting on spatial learning and memory function in the pilocarpine-treated rats.

(A) Latency to locate the hidden platform (escape latency) was recorded. In the age-matched intact controls, escape latency decreased gradually and significantly over the 4 days of testing, indicating excellent spatial learning. In contrast, NSPC- and vehicle-injected epileptic rats showed markedly longer latency to escape the maze than intact controls (P = 0.024 and P = 0.006, respectively). (B–E) During probe testing on day 5, parameters of memory retention were measured in the three groups: dwell time in the target quadrant (B), dwell time in the platform area (C), latency to the platform (D), and platform area crossings (E). Vehicle-injected epileptic rats exhibited significant deficits in memory retention in terms of all parameters. NSPC-transplanted epileptic rats also showed significant memory deficits, except dwell time in the platform area. * Significantly different from age-matched intact controls at P<0.05; error bars indicate ±SEM.

https://doi.org/10.1371/journal.pone.0104092.g009

We performed water maze testing at 9 weeks post-grafting in fully kindled rats. Kindled rats of the vehicle-injected and NSPC-transplanted groups (n = 11 and 8, respectively) showed gradual decline in escape latency across training days. The probe test was performed at 24 h after the last training day to evaluate memory function. NSPC-grafted group were indistinguishable in all parameters of reference memory (latency to reach the platform area, platform area crossings, dwell time in the target quadrant, and dwell time in the platform area) compared to vehicle-injected group. No significant difference was observed in the learning and memory function between both groups (data not shown). Thus, huNSPCs transplantation did not interfere with the ability of spatial learning and memory retention in kindled rats.

Effect of human NSPC grafting on the expression of GDNF in host hippocampal astrocytes

Increased GDNF levels in hippocampal astrocytes of the epileptic brain are known to suppress seizures [49], [50]. In this study, few huNSPC-derived cells after grafting differentiated into GDNF-expressing astrocytes in the hippocampus in either TLE model. However, NSPC transplantation induced GDNF expression in a large part (>80%, n = 6) of host hippocampal astrocytes in the pilocarpine-treated TLE model, whereas the level of GDNF expression of host astrocytes was ∼72% (n = 4) in age-matched intact controls and ∼46% (n = 6) in vehicle-injected epileptic rats (Fig. 10). This suggested that the level of GDNF expression was restored to closer to that of the intact controls after NSPCs grafting. Thus, the induction of GDNF expression in host hippocampal astrocytes by huNSPCs transplantation may be involved in suppressing seizures.

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Figure 10. Effect of human NSPC grafting on the expression of GDNF in host hippocampal astrocytes.

GDNF expression in S100β⁺ hippocampal astrocyte was observed in an age-matched intact control (A–D), vehicle-injected pilocarpine-treated (E–H), and NSPC-transplanted pilocarpine-treated rats (I–L). Nuclei were counterstained with DAPI (C, G, and K). Arrowheads in A–L indicated S100β/GDNF double-labeled cells. Arrows in E, G and H denoted S100β⁺ host hippocampal astrocytes that were devoid of GDNF immunoreactivity in vehicle-injected epileptic rats. Scale bar, 50 µm. (M) The bar chart represents percentages of S100β⁺ astrocytes expressing GDNF in the CA3 region of the hippocampus in the three groups. There was a significant difference between intact controls and vehicle-injected epileptic rats (P = 0.022) and between vehicle-injected and NSPC-transplanted epileptic rats (P = 0.038). * Significantly different from the age-matched intact control group at P<0.05; † significantly different from vehicle-injected group at P<0.05; error bars indicate ±SEM.

https://doi.org/10.1371/journal.pone.0104092.g010

Effect of human NSPC transplantation on aberrant mossy fiber sprouting

Aberrant sprouting of mossy fibers into the inner molecular layer of the DG of the hippocampus is one of the best-known structural changes in TLE models [2]–[5]. Because mossy fiber sprouting (MFS) is known to be linked to increased seizure susceptibility in TLE [51], we examined whether huNSPCs grafting could reduce MFS in the pilocarpine model. To visualize mossy fibers, we performed Timm staining that selectively labeled zinc-containing mossy fibers and recorded the Timm score to evaluate the extent of MFS. In age-matched intact rats (n = 4), Timm staining was nearly absent in the supragranular region of the DG (Fig. 11A, D). However, compared with intact rats, prominent Timm granules were present in the supragranular region in pilocarpine-treated rats, indicating aberrant MFS (Fig. 11B, C, E, F). The statistical analysis showed that the Timm score for MFS was not significantly different between vehicle-injected and NSPC-transplanted rats (n = 4 and 4, respectively; P = 1.0; Fig. 11G). This finding indicates that the significant reduction in seizure frequency seen with huNSPCs transplantation was not the result of a change of aberrant mossy fiber sprouting in the dentate gyrus.

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Figure 11. Effect of human NSPC grafting on aberrant mossy fiber sprouting in pilocarpine-treated animals.

(A–F) Timm staining exhibits mossy fiber staining from age-matched intact control (A, D), vehicle-injected pilocarpine-treated (B, E) and huNSPCs-transplanted pilocarpine-treated rats (C, F) 3 months following transplantation. D–F are high-magnification views of areas indicated by arrows in A–C, respectively. Arrowheads in D–F indicate the supragranular region of the dentate gyrus. Pilocarpine-treated rats reveal mossy fiber sprouting with an increased density of Timm granules in the supragranular region (B, C, E, F), whereas control rats did not (A, D). Scale bar, 500 µm (C), 100 µm (F). (G) NSPC-transplanted and vehicle injected rats had higher Timm scores than the control group (*P<0.05). Error bars, SEM. No significant difference regarding Timm score was found between NSPC- and vehicle-injected groups.

https://doi.org/10.1371/journal.pone.0104092.g011