Adult female SCID-Beige mice (8–10 weeks, 17–22 g), which are deficient in lymphocytes and NK cells, were provided by Charles River Laboratory. All animals were housed in a temperature- and humidity- controlled environment. All experimental procedures were approved by the ethics committee of Keio University (Assurance No. 13020) and were in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). The number of mice used in this study is shown in Supplemental File 1.
Spinal cord injury
The mice were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Laminectomy was performed at the 10th thoracic spinal vertebra (T10), and the dorsal surface of the dura matter was exposed. Moderate (70 kdyn) contusion injury was induced at the level of T10 using an Infinite Horizon impactor (Precision Systems and Instrumentation, Fairfax Station, VA), as previously described . The muscles were sutured and the skin was closed with wound clips. After spinal cord injury, ampicillin (12.5 mg/kg) was administrated subcutaneously.
A total of 25 mice were used in this experiment; the injured mice were sacrificed at 4 times (n = 5 /each survival time) with 5 naive controls. The times included 1, 4, 7, and 14 days post-injury. The mice were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg) intraperitoneally and transcardially perfused with PBS. Six millimeters of spinal cord, centered at the lesion epicenter, were dissected and immediately frozen in liquid nitrogen. As a control, samples of naïve spinal cords were harvested using the same protocol (N = 5). Dissected spinal cords were homogenized in lysis buffer with phosphatase and protease inhibitors and centrifuged at 10000 × g for 5 min. The supernatants were collected and protein concentration was evaluated using a Bradford assay. C5a concentration was determined using the ELISA kit (R&D system) according to the manufacturer’s instructions.
C5a receptor western blotting
A total of 16 mice were used in this experiment; the injured mice were sacrificed at 3 times (n = 4 /each survival time) with 4 naive controls. The times included 1, 4, and 7 days post-injury. Spinal cords centered at the lesion were dissected, as mentioned previously. Dissected spinal cords were homogenized in lysis buffer with phosphatase and protease inhibitors and centrifuged. The supernatant was dissolved in 4 × Laemmli sample buffer and heat-denatured at 95 °C for 5 min. As a control, samples of naïve spinal cords were harvested using the same protocol (N = 4). Samples were electrophoretically dissociated on 10% SDS-PAGE and transferred to membrane as described . The membranes were blocked for 1 h at room temperature with block solution (Blocking one, nacalai tesque, Japan) and incubated with diluted C5aR antibody (1:1000, Rat, Bio-Rad, USA) at 4 °C overnight. After, the membranes were subjected to a reaction with HRP-conjugated anti-rat IgG at room temperature for 1 h. After the membranes were washed, the HRP activity was detected using an ECL kit. The image was scanned with the ImageQuant LAS4000 mini (GE Healthcare Life Sciences, USA), and the data were analyzed using ImageJ. β-actin (1:2000, Rat) was used as an internal control.
C5a receptor antagonist administration
We used PMX205 (Tocris Bioscience, England) as a C5a receptor antagonist, as previously reported . Immediately after SCI, the mice randomly received PMX205(1 mg/kg body weight)  diluted in 1% ethanol or PBS containing 1% ethanol intraperitoneally once a day for 4 consecutive days, or until sacrificed.
A total of 50 mice were used in this experiment; the injured mice were sacrificed at 4 times (C5aRA group; N = 4 each time point, PBS group; N = 5 each time point) with 5 naive controls. The times included 6 h, 12 h, 1 day, and 4 days after SCI. Six mm spinal cord sections centered at the lesion were harvested and immediately frozen in liquid nitrogen (C5aRA group; N = 4 each time point, PBS group; N = 5 each time point). Total RNA was isolated from the frozen spinal cords using the RNeasy Mini Kit (Qiagen Inc., Hilgen, Germany), in accordance with the manufacturer’s instructions. As a control, total RNA was isolated from naïve spinal cords by the same protocol (N = 5). A reverse transcription reaction was performed using Reverse transcriptase kit (ReverTra Ace; Toyobo, Japan). RT-PCR was performed using primers specific to the genes of interest and TaqMan Fast Advanced Master Mix (Thermo Fischer Scientific, USA). The data were normalized to the expression level of β−actin (ACTB).
The following manufactured primers (Applied Biosystems; http://www.appliedbiosystems.com) against mouse DNA sequences were used: interleukin-1β (Il1β)-Mm00434228_m1, interleukin-6 (Il6)-Mm00446190_m1, TNF-Mm00443258_m1, ACTB-Mm02619580_g1.
A total of 22 mice were used in this experiment; the injured mice were sacrificed at 4 times (C5aRA group; N = 2 each time point, PBS group; N = 2 each time point) with 2 naive controls. The times included 3 h, 6 h, 12 h, 1 day, and 4 days after SCI. Six mm spinal cord sections centered at the lesion were harvested and samples were processed for RNA preparation and library preparation for mRNA-SEq. Samples for mRNA-Seq were prepared using the TruSeq RNA Sample Prep Kit (Illumina) in accordance with the manufacture’s instructions. As a control, samples were prepared from naïve spinal cords using the same procedure. The sequencing library was sequenced with the HiSeq 2500 (Illumina). Base-calling and chastity filtering were performed using Real-Time Analysis Software version 1.18.61 and raw reads were mapped to reference genome mm9 using Sailfish (v0.7.6) with default settings. All gene expression profiles were evaluated using Exatlas (https://lgsun.irp.nia.nih.gov/exatlas/). The extracted data were visualized with Morpheus (https://software.broadinstitute.org/morpheus).
After the mice were transcardially perfused with PBS, 6 mm spinal cord sections centered at the lesion were harvested (Neutrophils; N = 6 each group, Macrophage; N = 6 each group), digested with collagenase (Accumax; Innovative cell technologies, USA), and passed through a wire mesh screen (Sigma-Aldrich Canada Ltd., Canada) to acquire a single-cell suspension. The cells were incubated on ice for 30 min with Fc blocker followed by an additional 30 min on ice with fluorescent antibodies. To exclude the dead cells, 7-AAD was added. Flow cytometric analysis was carried out using a FACS Verse (Becton Dickinson, USA), and the data were analyzed using Cell Quest software. The samples were immunolabeled with rat anti-CD11b-BV421 (1:200; BD Horizon, USA), rat anti-LY6G-PE (1:200; BD Horizon, USA) and rat anti-CD45-APC (1:200; BD Horizon, USA). Macrophages were defined as CD45high CD11b+ LY6G- population, and neutrophils were defined as CD45high CD11b- LY6G+ population.
Cell culture, lentivirus transduction
Cell culture of hiPSC (414C2) was performed as described previously, with subtle modifications . Briefly, hiPSC grown on gelatin-coated (0.1%) tissue culture dishes were used for EB formation. EBs were then enzymatically dissociated into single cells and cultured in serum-free media hormone mix (MHM) for 10–12 days to allow the formation of neurospheres. Neurospheres were dissociated into single cells and cultured in the same method for passage. A lentivirus that expressed ffLuc, which is a fusion protein consisting of yellow variant of Aequorea GFP and a firefly luciferase  under the control of an EF promoter to enable the detection of the grafted cells in living mice and in fixed sections, was prepared , and transduced into hiPSC-NS/PCs as previously described .
Neural differentiation and immunohistochemistryDissociated third-passage ffLuc induced NS/PCs (1.0 × 105/well) were plated in poly-L-ornithine /fibronectin coated 8 well chamber slides (Thermo Fischer Scientific, USA), and PMX205 (20µ g/well) or PBS was added to each chamber. These cells were cultured in medium without growth factors at 37 °C under 5% CO2 for 14 days in total. Differentiated cells were fixed at 0.1M PBS containing 4% PFA and stained with the following primary antibodies overnight at 4 °C; anti-βIII Tubulin (mouse IgG2b, 1:300; SIGMA, T8660), anti-O4 (mouse IgM, 1:200; Thermo Fischer, MAB1326) and anti-GFAP (Rabbit, 1:2000; Proteintech, AG10423). These sections were incubated with Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Thermo Fisher Scientific, USA), and Hoechst 33258 (10 µg/ml, Sigma-Aldrich) as a nuclear counterstaining. All images were obtained using a fluorescence microscope (BZ 9000; Keyence Co., Japan) or a confocal laser scanning microscope (LSM 700; Carl Zeiss, Germany). For quantification of βIII Tubulin-, O4-, and GFAP-positive cells, five regions within the chamber were randomly selected and counted under × 40 magnification.
Four days after SCI, the mice were randomly and blindly divided into four groups based on their BMS score to ensure equivalent deficits across the groups: (PBS only group, C5aRA only group, PBS + transplantation (PBS + TP) group, C5aRA + transplantation (C5aRA + TP) group), and re-anesthetized with isoflurane. hiPSC- NS/PCs (5 × 105cells/2µ l) or PBS were transplanted into the lesion epicenter using a metal needle at a rate of 1 µl/minute (PBS only;N = 20, C5aRA only;N = 19, PBS + TP;N = 15, C5aRA + TP;N = 16). After transplantation, the skin was closed with wound clips and ampicillin (12.5 mg/kg) was injected subcutaneously. These mice were sacrificed 42 days after the SCI.
A Xenogen-IVIS spectrum CCD optical macroscopic imaging system (PerkinElmer, USA) was used in vitro and in vivo for bioluminescence imaging (BLI) to confirm the survival of the transplanted hiPSC-NS/PCs, as described previously  .
In vitro, the hiPSC-NS/PCs were plated to 8-chamber wells and D-luciferin (1 mg/well) was then added to each well. The luminescent signal was detected immediately using a Xenogen-IVIS spectrum cooled charged-coupled device (CCD) optical macroscopic imaging system (Caliper Life Sciences, USA) (N = 3 each group).
In vivo, the mice were injected D-Luciferin (300 mg/kg body weight) intraperitoneally, and placed in a light-tight chamber 15 minutes after injection. The peak of the signal intensity was between 15 and 30 minutes after injection. The integration time was 5 seconds to 2 minutes, depends on the intensity of signals emitted from luciferase-expressing grafted cells. BLI signals were quantified in maximum radiance units (photons/second/centimeter squared/steradian photons/sec/cm3/sr) and presented as log10 values (PBS + TP;N = 15, C5aRA + TP;N = 16).
The mice were anesthetized and transcardially perfused with 0.1 M PBS containing 4% PFA. Their spinal cords were harvested and immersed in 0.1 M PBS containing 4% PFA at 4 °C for 24 h. The tissues were transferred into 10% sucrose for 24 h followed by 30% sucrose for 24 h. The sections were embedded in Optimal Cutting Temperature compound (Sakura Finetek Japan, Japan) and immediately frozen by liquid nitrogen and stored at -30 °C. The tissues were sectioned at 20 µm in axial or 16 µm sagittal planes using a cryostat (Leica CM3050 S; Leica Microsystems, Germany). The sections were stained with the following primary antibodies overnight at 4 °C; anti-CD88(Rat, 1:400; Biorad, MCA2456), anti-C5R1 (Rabbit, 1:1000; Abcam, ab59390), anti-Iba1 (Rabbit, 1:400; WAKO, CDP0133), anti-LY6G (Rat, 1:1000; Novusbio, NBP200441), anti-GFP (Rabbit, 1:2000; MBL), anti-human nuclear antigen (mouse IgG, 1:100; Millipore, MAB4383), anti-human Nestin (Rabbit, 1:200; IBL: 18741), anti-Ki67 (rabbit IgG, 1:200; Leica, NCL-Ki67p), anti-pan-ELAVL (human IgG, 1:1000, a gift from Dr. Robert Darnell; The Rockefeller University, New York, USA), anti-APC (mouse IgG2b, 1:200; Millipore, OP80), and anti-GFAP (Rabbit, 1:2000; Proteintech, AG10423). anti-CD88 and anti-C5R1 antibodies were used as a C5a receptor detection. These sections were incubated with Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Thermo Fisher Scientific, USA), and Hoechst 33258 (10 µg/ml, Sigma-Aldrich, USA) as a nuclear counterstaining. All images were obtained using a fluorescence microscope (BZ 9000; Keyence Co., Japan) or a confocal laser scanning microscope (LSM 700; Carl Zeiss, Germany). For Quantification of survival rate of grafted cells, anti-GFP (Rabbit, 1:2000; MBL) and Hoechst 33258 (10 µg/ml, Sigma-Aldrich, USA) as a primary antibody and the appropriate secondary antibodies were used. The images were captured at the lesion epicenter and 0.2, 0.4, 0.6, 0.8, and 1.0 mm rostral and caudal to the epicenter in axial sections using a fluorescence microscope (BZ 9000; Keyence Co., Japan), and the area of survived grafted cells was assessed by measuring the GFP positive areas on each cross section using ImageJ (https://imagej.nih-.gov/ij/). For quantification of APC-, GFAP-, pan-ELAVL-, Ki67-, Nestin- and OCT4-positive cells, five regions within the area 2 mm rostral and caudal to the lesion epicenter were randomly selected and counted under × 63 magnification.
Locomotor function analysis
The hindlimb locomotor function analyses were evaluated using the Basso Mouse Scale (BMS) score at 0, 1, and 4 days as well as weekly after SCI, up to 35 days  (PBS only;N = 20, C5aRA only;N = 19, PBS + TP;N = 15, C5aRA + TP;N = 16). Motor coordination was evaluated using a rotating rod apparatus (Rotarod, Muromachikikai Co., Japan), which is composed of a 3 cm diameter plastic rod rotated at 20 revolutions per min. The mice were tested by monitoring the time spent on the rod (PBS only;N = 20, C5aRA only;N = 19, PBS + TP;N = 15, C5aRA + TP;N = 16). The gait performance of the mice was assessed through treadmill gait analysis (DigiGait system; Mouse Specifics, USA) (PBS only;N = 11, C5aRA only;N = 11, PBS + TP;N = 8, C5aRA + TP;N = 8).
All data are presented as means ± SEM. A Mann-Whitney U test was used to identify any significant differences between groups with respect to the results of flowcytometry and immunohistochemistry. One-way analyses of variance (ANOVA) followed by Tukey–Kramer tests for multiple comparisons were used to detect significant differences in stride length, stance angle, and rotarod score between the four groups. Two-way repeated-measures ANOVA followed by Tukey–Kramer tests were used for the others. For all statistical analyses, the significance level was set at p < 0.05. Microsoft Excel 2016 and IBM SPSS Statistics (ver. 25) were used for all calculations.