Scientific Papers

Hepatocyte growth factor pretreatment boosts functional recovery after spinal cord injury through human iPSC-derived neural stem/progenitor cell transplantation | Inflammation and Regeneration


Adult (eight-week-old) female athymic nude rats (F344/NJcl-rnu/rnu, weight = 110–180 g, CLEA Japan, Inc., Tokyo, Japan) were used in these experiments. The rats were housed randomly in groups of three or four per cage (24 × 42 × 24 cm) regardless of the experimental group. The animals were kept on a 12/12 h light/dark cycle in an environment with controlled temperature and humidity and provided ad libitum access to food and water. Antibiotics (orbifloxacin; Sumitomo Dainippon Pharma Animal Health, Inc., Osaka, Japan) were injected for 3 days after SCI and other surgeries. All experimental procedures were approved by the Experimental Animal Care Committee of KEIO University, School of Medicine (assurance no. 13020) and performed per the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). In this study, all rats were anesthetized by subcutaneous injection of 0.4 mg/kg medetomidine hydrochloride, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol.

NS/PC-derived human iPSC culture

The YZWJs513 hiPSC line was used throughout this study. NS/PCs were prepared at a Good Manufacturing Practice (GMP)-grade cell processing facility at Osaka National Hospital, Japan. The clinical-grade human leukocyte antigen superdonor-derived, integration-free hiPSC line YZWJs513, established by the iPSC Stock Project and organized by the Kyoto University (CiRA) for iPS Cell Research and Application, was used. For in vivo analyses, the cells were cultured for 5 days in a floating culture, and the neurospheres were used for transplantation. Cells were treated with the small molecule GSI and N-[N-(3,5-difluorophenacetyl)-l-ananyl]-S-phenylglycine t-butyl ester (DAPT) (10 μM, Sigma-Aldrich, Inc., D5942) for 1 day before transplantation, as described in a previous study [4].

Preparation of osmotic mini-pump containing rhHGF

Osmotic mini pumps (Alzet model 2002; nominal pumping rate 0.5 μl/hr, nominal duration 2 weeks, nominal reservoir 200 μl, Alzet, CA, USA) were filled with PBS or with rhHGF (Kringle Pharma, Inc., Osaka, Japan) diluted with dilution medium (10 mM citrate buffer, 0.3 M NaCl, 0.01% polysorbate 80, pH 6.0) so that each pump contained 200 μg of rhHGF. Each pump was connected to an intrathecal catheter (rat Intrathecal Catheter short; Alzet, CA, USA), and the apparatuses were incubated in sterile PBS at 37 °C for 12 h before use.

Severe contusive SCI, intrathecal infusion of rhHGF and NS/PC transplantation

Severe contusive SCI was induced at the level of the tenth thoracic spinal vertebra using an Infinite Horizon impactor (240 kdyn; Precision Systems and Instrumentation, Fair-fax Station, VA, USA) with a 2 mm tip, as described previously [49]. This force was chosen because pilot studies revealed that milder (220 kdyn) injuries allowed the animals to walk via their hindlimbs over time, thus failing to recreate the recovery course of severe SCI. For SCI at the T10 level, a T12 laminectomy was carried out, and an intrathecal catheter was inserted from the T12 level to the T10 level. An osmotic mini pump filled with 200 μg rhHGF (HGF group) or PBS (control group) was connected to a catheter and placed in the subcutaneous space on the middle side of the animal’s back. The pump was left in place to deliver a 200 μg total dose of rhHGF for 2 weeks. The pump was removed 2 weeks after administration. The rats with SCI were randomly assigned to each group. For the sham group, the same surgical procedure was performed as for the SCI experimental groups; however, no SCI was inflicted.

For TP group rats (combination group: n = 16, 1 rat died; TP group: n = 14, 3 rats died), 9 days after injury, hiPSC-NS/PCs (1 × 106 cells in 2 μl of phosphate-buffered saline [PBS]) were transplanted into the lesion epicenter of each rat with a Hamilton syringe with a 27G metal needle using a micro stereotaxic injection system (KDS310; Muromachi-Kikai Co., Ltd.). An equal volume of PBS was similarly injected into PBS group rats (HGF group: n = 15, 2 rats died; control group: n = 15, 2 rats died) in place of the hiPSC-NS/PCs. The injected depth was 0.4 to 1.0 mm, and the injection speed was 1 μl/min.

Transcriptome analysis

Spinal cord samples collected 2 or 7 days after SCI were used for analysis, and samples were pooled in the control (PBS) and HGF (HGF-treated) groups (n = 4 each, total: n = 16). Then, 3 mm-long dissected spinal cords were rapidly frozen, and total RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions with slight modifications. Total RNA from each sample was purified, and mRNA libraries were prepared according to the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, United States) protocol and sequenced using NovaSeq 6000 (Illumina) to obtain paired-end reads. Raw reads were trimmed based on the read quality and read length using Trimmomatic 0.38. After read mapping to the reference genome (rn6), StringTie version 2.1.3b was used for transcript count. The read count of each sample was normalized to fragments per kilobase of transcript per million mapped reads (FPKM) and transcripts per kilobase million (TPM).

Differentially expressed gene (DEG) analysis was performed on 2 comparison pairs as requested using edgeR. The raw count data were normalized with the trimmed mean of M-values (TMM) method in the edgeR package. Statistical analysis was performed using edgeR with Fold Change, exactTest for each comparison pair. The significant results were selected under the conditions of |FC|≥ 2 and exact test raw p value < 0.05. An enrichment test based on the GO ( DB was conducted with a significant gene list using the g:Profiler tool ( For GO analysis, adjusted p values were calculated by Benjamini‒Hochberg tests (FDR: false discovery rate).

R software version 4.2.3 was utilized to generate gene expression heatmaps. The normalized expression values of the selected genes were used as input data. Z scores were calculated for each gene across the samples to compare gene expression profiles. The resulting Z score matrix was visualized as a heatmap. Color gradients were assigned to indicate relative gene expression levels, with warmer colors representing higher expression and cooler colors representing lower expression.

Behavioral analyses

Hindlimb locomotor function was evaluated weekly until 12 weeks after SCI using the BBB scale [50]. Two investigators blinded to the groups performed the behavioral analyses. The stride length of the hindlimbs on a treadmill was measured at a speed of 5 cm/s (Rat Specifics, Inc., Framingham, MA, USA). Briefly, the animal’s gait was video recorded with the treadmill system. The footsteps were manually marked in the video frames and then subjected to analysis to evaluate the gait of the rats. Kinematics analysis was performed using four cameras (Go Pro HERO5 Black CHDX-502) to record multiple movements of the shoulders and hindlimbs [2]. The bilateral shoulders, hips, knees, ankles, and toes were labeled, and the images were analyzed using KinemaTracer software (Kissei Comtec).

Tissue processing and histological analyses

The spinal cord was dissected and postfixed in 4% PFA and was then embedded in frozen section compound. The tissue sections were stained with the following primary antibodies: c-Met (mouse IgG1, 1:300, Cell Signaling Technology, Inc., 3127), phosphorylated c-Met (rabbit IgG, 1:100, Invitrogen, Inc., 44-882G), RECA1 (mouse IgG1, 1:200, Santa Cruz Biotechnology, Inc., sc-52665), Iba1 (rabbit IgG, 1:250, Wako, Inc., 01919741), GFAP (rabbit IgG, 1:500, Proteintech, Inc., 16,825–1-AP; mouse IgG2a, Thermo Fisher, Inc., 13–0300), SOX2 (goat IgG, 1:50, R&D Systems, Inc., AF2018), HNA (mouse IgG1, 1:100, Millipore, Inc., AB1281), STEM121 (mouse IgG1, 1:200, TaKaRa Bio, Inc., Y40410), ELAVL3/4 (mouse IgG2b, 1: 100, Molecular Probes, Inc., A-21271), APC (mouse IgG2b, 1:300, Abcam, Inc., ab16794), Ki67 (rabbit IgG, 1:1000, Leica, Inc., NCL-Ki67p), Nestin (rabbit IgG, 1: 500, IBL, Inc., 18,741), NF–H (200 kDa) (mouse IgG1, 1:200, Sigma-Aldrich, Inc., MAB5262), 5-HT (goat IgG, 1:500, Immunostar, Inc., 20,079). Then, the sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:1000) and Hoechst 33,258 (10 μg/ml, Sigma-Aldrich). H&E staining and MGB staining were performed with the Black Gold II Ready-to-Dilute (RTD) Staining Kit (Biosensis, SA, Australia). Images were acquired at room temperature using standard filter sets with a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan) and a confocal laser-scanning microscope (LSM 780; Carl Zeiss, Jena, Germany), and the acquisition settings were kept constant for all groups for each experiment. Quantitative analysis of all images was performed using Zen 2012 SP5 software (version; Carl Zeiss) or ImageJ software (version 13.0.6/1.53 k).

EdU labeling and staining

EdU labeling was performed with an EdU Click 647 Kit (Base Click, Munich, Germany) according to the manufacturer’s protocol. Nude rats were intraperitoneally injected with EdU (50 mg/kg/day) daily after SCI. The injections started the day after SCI and continued until 7 days, with the last injection occurring 4 h before sacrifice. Immunohistochemical staining for EdU was conducted according to the manufacturer’s protocol.

In vivo imaging (bioluminescence imaging) of transplanted cells

For confirmation of the survival of the transplanted hiPSC-NS/PCs, a Xenogen-IVIS spectrum-cooled charge-coupled device (CCD) optical macroscopic imaging system (Caliper Life-Science, Hopkinton, MA, USA) was used for bioluminescence imaging (BLI) [51]. Monitoring was performed once per week following cell transplantation. Eight rats in the TP group and nine rats in the Combination group were examined for in vivo imaging. D-Luciferin (VivoGlo Luciferin; Promega, Madison, WI) was intraperitoneally injected at a dose of 800 mg/kg body weight. Animals were placed in a light-tight chamber, and photons emitted from luciferase-expressing cells were collected with integration times of 5 s to 2 min, depending on the intensity of bioluminescence emission. BLI signals were quantified in maximum radiance units [photons per second per centimeter squared per steradian (p/s/cm2/sr)] and are presented as log10 (photons per second) values.

Immunoelectron microscopy analysis

Immunoelectron microscopy analysis was performed on spinal cord sections. The detailed immunoelectron microscopy procedure was performed as described in a previous study [52]. In brief, spinal cord tissues were perfused and postfixed with 4% PFA for 12 h, followed by cryoprotective treatment with 15% and 30% sucrose. Frozen tissue blocks in cryocompound were sectioned at 20 μm thicknesses using a cryostat (CM3050S, Leica Microsystems, Wetzlar, Germany). Sections were incubated with 5.0% Block Ace solution (DS Pharma Promo) containing 0.01% saponin in 0.1 M PB for 1 h, then incubated with primary mouse anti-human cytoplasm antibody (STEM121, 1:200, TaKaRa Bio, Inc., Y40410) for 72 h at 4 °C, followed by incubation with FluoroNanogold-conjugated goat anti-mouse secondary antibody (1:100, Thermo Fisher Scientific, Inc.) for 24 h at 4 °C. After 2.5% glutaraldehyde fixation in 0.1 M PB, nanogold signals were enhanced with silver enhancement solution for 2.5 min at 25 °C. Gold-labeled sections were postfixed with 1.0% OsO4 for 90 min at 4 °C, en bloc stained with uranyl acetate for 20 min at 25 °C, dehydrated through a graded ethanol series and embedded into pure Epon. Ultrathin Sects. (80 nm) were prepared with an ultramicrotome (UC7, Leica Microsystems) and stained with uranyl acetate and lead citrate. The sections were imaged by transmission electron microscopy (JEM1400 plus, JEOL, Tokyo, Japan).


Electrophysiological experiments (MEP: motor-evoked potential) were performed using a Neuropack S1 MEB9402 signal processor (Nihon Kohden, Tokyo, Japan) 84 days after SCI (n = 4 each). The surface of the T2 spinal cord was stimulated, and needle electrodes were used to record the signal from the hindlimb. An active electrode was placed in the quadriceps muscle, a reference electrode was placed near the distal quadriceps muscle tendon, and a ground electrode was placed in the back muscle. The stimulus parameters were an intensity of 2.0 mA, a duration of 0.2 ms, and an interstimulus interval of 1 Hz. The maximal amplitude was measured in a peak-to-peak manner. Peak latency was measured as the length of time from the stimulation to the highest point of the MEP wave.

Neural differentiation analysis in vitro

The hiPSC-NS/PCs cells were cultured for 5 days in a floating culture, and the neurospheres were administered rhHGF (20 ng/per) or PBS. After fifteen minutes, it embedded in iPGell (PG20-1; Genostaff, Tokyo, Japan) following the manufacturer’s instructions, and frozen and sectioned at a 16 μm thickness on a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany). Tissue sections were stained with the following primary antibodies: anti-SOX2 (mouse IgG2a, 1: 40, RD systems, Inc., MAB2018), anti-Nestin (mouse IgG1, 1: 200, Millipore, Inc., MA5326), anti-c-Met (mouseIgG1, 1: 200, Cell signaling, Inc., 25H2), anti-phosphorylated c-Met (rabbit IgG, 1:100, Invitrogen, Inc., 44-882G). All samples were incubated overnight at 4 ℃ and then incubated with Alexa Fluor-conjugated secondary antibodies (1: 500, Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Nuclei were stained with Hoechst 33,258 (10 μg/ml, Sigma-Aldrich), then examined under a confocal laser-scanning microscope (LSM 700, Carl Zeiss, Jena, Germany).

Quantification and statistical analyses

Statistical analysis was performed with SPSS Statistics (version; Japan IBM, Tokyo, Japan). All data are presented as the mean ± SEM. The Mann‒Whitney U test was used for comparison between the 2 groups. One-way ANOVA followed by the Tukey–Kramer test was applied for group analysis. Two-way ANOVA followed by the Tukey–Kramer test was used for group and behavioral analyses. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001.

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