Preparation of a kidney-derived dECM hydrogel and collagen I hydrogel
The kidney-derived dECM hydrogel was prepared with minor modifications based on the methods of a previous study 41. Briefly, kidneys derived from minipigs (Oriental Yeast Co., Ltd, Tokyo, Japan) were cut into small pieces, which were further washed in PBS and decellularized with 0.5% sodium dodecyl sulfate (SDS; Fujifilm Wako Pure Chemical Corp.) for 10 h. The decellularized kidney pieces were rinsed with PBS for 4 days at 4 ℃. The rinsing solution was changed four times a day to remove the residual detergent. The tissues were lyophilized and milled into a powder. Moreover, the resulting powder was enzymatically digested in pepsin/HCl solution to prepare a decellularized kidney-derived collagen solution. This solution was further lyophilized and dissolved in water to prepare the hydrogel solutions at concentrations of 8 and 16 mg/ml, respectively. The solution was neutralized to pH 7.4 and subsequently used. Furthermore, collagen I hydrogels (3 mg/ml; Corning, Inc.) were prepared according to the manufacturer’s protocol.
Dorsal root ganglion isolation and ex vivo culture on the hydrogels
DRG were obtained from Sprague-Dawley (SD) rats at postnatal days 1–5 (P1-5) as previously discussed 51. The DRGs with nerve roots were placed in Hank’s Balanced Salt Solution. After removing the nerve roots under a stereoscopic microscope, the DRGs were seeded onto 8-well plates pre-coated on the previous day with collagen I hydrogel or decellularized kidney hydrogel (8 or 16 mg/ml) (n = 3). To coat the 8-well chamber, 60–80 μl of each type of hydrogel was used: collagen I hydrogel, dECM hydrogel (8 mg/ml), and dECM hydrogel (16 mg/ml). The chambers were then incubated at 37°C with 92% humidity and 5% CO2 for 15–30 min to ensure gelation. The DRGs were cultured for 5 days on each hydrogel in a Neurobasal medium (Gibco) supplemented with 100 ng/ml of nerve growth factor (NGF), 2% B27 (Gibco), 1% L-glutamine (Gibco), and 1% penicillin–streptomycin (Gibco). The medium was changed every day.
Immunocytochemistry
To perform the immunocytochemical analysis, the cells were fixed with 4% paraformaldehyde for 15 min and then rinsed three times with phosphate-buffered saline (PBS) for 5 min each. The cells were then permeabilized and blocked with 5% skim milk and incubated with a primary antibody for βIII-tubulin (mouse IgG2b, 1:500; Sigma, Inc.) at 4 °C for 24 h, followed by incubation with the corresponding secondary antibodies: Fluor 488 (1:400; Abcam, Inc.) and Hoechst 33258 (1:1000, Sigma–Aldrich) at 37 °C for 1 h. The immunofluorescence images of the stained sections were obtained using a fluorescence microscope (Leica Microsystems 173 THUNDER Imager Live Cell, LAS X Version: 3.7.5.24914).
Human neural stem/progenitor cells hNS/PC generation from iPSC
A feeder-free human iPSC line (QHJI01s04) was established at the Center for iPS Cell Research and Application from the peripheral blood cells obtained from HLA homozygous healthy donors under xeno-free and feeder-free conditions via the transduction of reprogramming factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, dominant-negative p53, and EBNA1) using episomal vectors. A clonal working cell bank of MCB003, a master cell bank of QHJI01s04, was used for hNS/PC induction. The iPSCs were supported by applying the feeder-free iPSC culture method. The hNS/PC induction was performed using the methods used in previous research 52. Experiments using hiPSCs were approved by the ethics committee of the Keio University School of Medicine (Approval Numbers: 20030092, 20130146). Informed consent was acquired from the donor from whom the hiPSCs were derived, in accordance with the guidelines outlined in the Declaration of Helsinki. All procedures were conducted following the institutional guidelines.
Animals
Adult female SD rats (pregnant rat, Sankyo Labo Service Corporation, INC., Tokyo, Japan), SD rats at postnatal days 1–5 (P–− 5), and adult (8-week-old) female athymic nude rats (F344/NJcl-rnu/rnu; weight, 110–160 g; CLEA Japan, Inc., Tokyo, Japan) were used for these experiments. The rats were assigned randomly in groups of four per cage (24 × 42 × 24 cm), irrespective of their experimental group. The animals were kept under a controlled 12/12-h light/dark cycle with regulated temperature and humidity, and they had free access to food and water. Antibiotics (orbifloxacin; Sumitomo Dainippon Pharma Animal Health, Inc., Osaka, Japan) were administered for 3 days after the surgeries. Urinary retention occurred after spinal cord transection injury, which required bladder expression for 1–2 weeks. Fluid supplementation was provided as needed for rats with poor food intake. All experimental procedures were authorized by the Experimental Animal Care Committee of Keio University, School of Medicine (assurance no. A2022-214, A2022-127) and conducted in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Additionally, the study adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to ensure transparency and reproducibility in the reporting of methods and results.
Surgical procedures
For surgical treatment, the rats were anesthetized via the subcutaneous injection of 0.4 mg/kg medetomidine hydrochloride, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol. Laminectomy was performed at T10, and the dura mater was opened longitudinally. Complete spinal cord transection at this level was then performed using microscissors. A total of 90 rats were used for the in vivo experiments. The exclusion criteria were as follows: rats displaying a BBB score of ≥ 7 within 6 weeks after SCI, rats that developed soft tissue infections, rats that died. The composition of each group was as described below: TP (+ hydrogel injection) without scar resection series (BBB score of ≥ 7 rats: n = 6, infected rats: n = 1, dead rats: n = 2), control (BBB score of ≥ 7 rats: n = 4), scar resection (BBB score of ≥ 7 rats: n = 3, infected rats: n = 1, dead rats: n = 2), TP (BBB score of ≥ 7 rats: n = 2), TP + scaffold (BBB score of ≥ 7 rats: n = 3, dead rats: n = 1).
Forty-two days after SCI, the hNS/PCs (1 × 106 cells) were transplanted into the lesion epicenter (cell transplantation: TP group) with or without an 8 μl injection of dECM hydrogel (8 mg/ml). The hNS/PCs were washed thrice with PBS and suspended in 2 μl of PBS. Subsequently, 3.3 × 105 cells/2 μl or 2 μl of PBS were injected into three locations (the epicenter and areas 1 mm rostral and caudal to it) using a 27G metal needle and a microstereotaxic injection system (KDS310; Muromachi-Kikai Co., Ltd.). Moreover, the injection depth was 0.6–0.8 mm, whereas the injection speed was 1 μl/min.
For the scar resection model, the scar tissue (1 mm wide at the lesion epicenter) was resected 42 days after SCI using microscissors. Either 8 μl of PBS (scar resection group) or dECM hydrogel (8 mg/ml) was injected into the gap created by the scar resection (scar resection + scaffold group). After the hydrogel administration, the rat was left undisturbed on a 37 °C warming incubator for about 20 min to confirm gelation. In these groups, hNS/PCs (1 × 106 cells) were injected into three locations at the lesion epicenter one week after scar resection. Conversely, the control group used a model without scar resection.
Immunohistochemistry and picrosirius staining
The rats were anesthetized, exsanguinated transcardially with heparinized saline, and euthanized using a 4% PFA solution. The spinal cord tissues were dissected and postfixed in 4% PFA followed by sequential soaking in 10% and 30% sucrose solutions. Subsequently, the tissues were then embedded in a frozen section compound and sectioned at a thickness of 16 μm for the sagittal plane. The sections were then processed for histological analysis using the following primary antibodies: anti-HNA (MAB4383, mouse IgG1, 1:100, Millipore, Inc.), anti-GFAP (16825-1-AP, rabbit IgG, 1:500, Proteintech, Inc.; mouse IgG2a, Thermo Fisher, Inc.), anti-CS56 (C8035, rabbit IgG, 1:200, Sigma–Aldrich), anti-Arg1 (ab60176, goat IgG, 1:400, Abcam, Inc.), anti-Iba1 (019-19741, rabbit IgG, 1:1000, Wako), anti-CD31 (AF3628, goat IgG, 1:100, R&D Systems, Inc.), anti-ELAVL3/4 (Hu C/D) (A21271, mouse IgG2b, 1:500, Molecular Probes, Inc.), anti-APC (ab16794, mouse IgG2b, 1:300, Abcam, Inc.), STEM121 (Y40420, mouse IgG1, 1:200, Takara Bio, Inc.), anti-NF-H (rodent-specific) (ab8135, rabbit IgG, 1:500, Abcam, Inc.), and anti-βIII-tubulin (mouse IgG2b, 1:500, Sigma, Inc.). The sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Abcam, Inc.) and Hoechst 33258 (1:1000, Sigma–Aldrich). Picrosirius staining was performed using a staining kit (ScyTek Laboratories, Inc.). The images were captured using a fluorescence microscope (Leica Microsystems 173 THUNDER Imager Live Cell (LAS X Version: 3.7.5.24914) and BZ-X710 (Keyence, Osaka, Japan)) or confocal laser-scanning microscope (LSM 780; Carl Zeiss, Jena, Germany). All the image analyses were conducted using the ImageJ software (version 2.1.0/1.53c).
Behavior analysis of the hindlimb locomotor functions
The motor function of the rats’ lower extremities was assessed weekly until 42 days after transplantation using the Basso–Beattie–Bresnahan (BBB) scale 53. Behavioral analysis was conducted by two investigators who were blinded to the experimental groups.
RNA-seq analysis
Three mm-long dissected spinal cord samples that were collected one week after scar resection (49 days after SCI) were used for the analysis, and the samples were pooled in the control (PBS, n = 3), scar resection (scar resection + PBS, n = 4), and scar resection + scaffold (scar resection + dECM hydrogel, n = 4) groups (total: n = 11). The total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, with slight modifications. The RNA from each sample was purified, and the mRNA libraries were prepared following the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, United States) protocol and sequenced on the HiSeq 2500 System (Illumina) to obtain single-end reads. Raw reads were trimmed for quality and read length using Trimmomatic 0.38. Read mapping to the reference genome (rn6) was performed, and transcript counts were obtained using StringTie version 2.1.3b. The read count of each sample was normalized to fragments per kilobase of the transcript per million mapped reads (FPKM) and transcripts per kilobase million (TPM). A differentially expressed gene (DEG) analysis was conducted on two comparison pairs as requested using DESeq2. The raw count data were normalized with DESeq2’s geometric mean-based size factors, and statistical analysis was carried out using the log2 fold change (FC) and Wald test for each pair. Significant results were identified based on a FC of ≥ 2 or ≤ 0.5 and a raw p value of < 0.05 from the Wald test.
GO enrichment analysis
In this study, GO enrichment analysis was performed using the gProfiler tool (g:GOSt: e111_eg58_p18_f463989d). The genes with an adjusted p value below 0.05 were considered significant using the Bonferroni test.
Quantification of staining
Immunohistochemical staining across all sections was quantified using ImageJ software, with threshold values kept constant throughout all analyses by observers blinded to the experimental groups and conditions. Moreover, in each DRG on the hydrogel, the five longest neurites were measured and averaged. CSPG was evaluated by measuring the CS56-positive area in the sagittal sections (2000 μm-wide regions). The average numbers of the microglia/macrophages were calculated in the sagittal sections (2000 μm-wide regions). Neovascularization was evaluated by measuring the CD31-positive area in the sagittal sections (1000 μm-wide regions). The graft volume was estimated by assessing the HNA + area. Based on the previously reported methods 54, both graft and lesion volumes were measured in entire sagittal sections. The number of regenerating host neurons positive for rodent-specific NF-H at the injury epicenter was quantified in the sagittal sections (1000 μm-wide regions). For the calculation of graft volume, we analyzed all sagittal sections to ensure accurate volume measurements. Quantification of other tissues was performed using the midsagittal section. In cases where the total number of sections was even, or when quantification on the midsagittal section was not feasible, the adjacent section was used for analysis.
Experimental designs and statistical analysis
All statistical analyses were conducted using SPSS Statistics (version 26.0.0.0; Japan IBM, Tokyo, Japan). Data were expressed as mean ± SEM. The in vitro and in vivo immunohistochemistry (IHC) staining results of three and five groups were compared using the one-way ANOVA and Tukey–Kramer test, respectively. The in vivo immunohistochemistry (IHC) staining results of two groups were compared using the Mann–Whitney U tests. Statistical significance was set at a P value of < 0.05 (as *P < 0.05).
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- Source: https://www.nature.com/articles/s41598-024-82959-7