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Administration of C5a Receptor Antagonist Improves the Ecacy of Human iPSCS-derived NS/PC Transplantation in the Acute Phase Of spinal Cord Injury

Background: We previously reported the ecacy of human-induced pluripotent stem cells derived neural stem/progenitor cells (hiPSC-NS/PCs) transplantation for spinal cord injury (SCI) in the subacute phase. However, this procedure is not effective in the acute phase due to the inammatory response occurring immediately after SCI, which negatively impacts transplanted cell survival. C5a, which is one of the complement components, is a powerful chemoattractant which recruits inammatory cells through binding of the C5a receptor. We hypothesized that suppression of the inammatory response immediately after SCI using a C5a receptor antagonist (C5aRA) as an immunosuppressant would improve the ecacy of hiPSC-NS/PCs transplantation for SCI in the acute phase. Methods: Immunodecient SCID-Beige mice underwent contusion SCI at T10 and received C5aRA immediately after SCI. Inammatory cytokines and inammatory cells in the injured spinal cord tissue during the acute phase were quantied using quantitative PCR and ow-cytometry. Next, we randomly and blindly divided the SCI mice into 4 groups (PBS only, C5aRA only, PBS + transplantation (PBS+TP), C5aRA + transplantation (C5aRA+TP)). Immediately after SCI, C5aRA or PBS was injected intraperitoneally once a day for 4 consecutive days, and then, 5.0 × 10 5 hiPSC-NS/PCs were transplanted into the lesion epicenter on day 4 in the PBS+TP and C5aRA+TP groups. We evaluated cell survival rate, hindlimb motor function, and the differentiation prole of the graft hiPSC-NS/PCs. Results: C5aRA administration signicantly reduced several inammatory cytokines such as IL-1b, IL-6 and TNFα, as well as inammatory cells after SCI. Within the transplantation groups, the C5aRA+TP group had better functional improvement as compared to the PBS only group. The C5aRA+TP group also had a signicantly higher cell survival rate compared to the PBS+TP group. There were no signicant

However, critical issues remain surrounding the timing of cell transplantation and optimization of the injury site milieu. Currently, the optimal timing for transplantation is the subacute phase of SCI, which is about 7-14 days after SCI in rodents, after neuroin ammation subsides. Since the in ammatory reaction in the acute phase of injury does not allow transplanted cells to survive, researchers have been forced to wait until the subacute phase to transplant cells [6,7]. During the acute phase of injury, several neurotoxic cytokines are upregulated and in ammatory cells intrude into the lesion area [8,9]. As a consequence of this heightened in ammatory response, there is signi cant host cell death and a diminished ability for the spinal cord to recover, and thereby, functional improvement is limited even after cell transplantation.
However, if this harmful in ammatory response were to be suppressed immediately after the injury, a favorable environment for the grafted cells could be created, and moreover, it may be possible to perform cell transplantation in the acute phase.
The complement system has a very important role in initiating secondary damage during the acute phase in SCI. The complement proteins activate various neurotoxic cytokines and in ammatory cells, and this complex in ammatory cascade lead to the exacerbation of neural damage [10][11][12][13]. Thus, the in ammation process is principally triggered by the complements, and among these proteins, C5a has recently received attention as a target of anti-in ammatory treatment. C5a is a small glycoprotein (74 amino acids, about 11 kDa) and generated by the cleaving of complement C5 [12]. C5a is an anaphylatoxin, which causes in ammatory cytokine activation and leukocyte in ltration through the C5a receptor (C5aR) [14]. Consequently, C5aR antagonist (C5aRA) could be a target as a treatment for in ammatory reaction after SCI. Among C5aRA, PMX205 (hydrocinnamate-(OPdChaWR)) is often used in the eld of CNS disease experiment because PMX205 is able to penetrate blood-brain barrier and blood-spinal cord barrier [15,16], and it has been shown that PMX205 administration after SCI suppresses in ammatory cytokines and macrophage in ltration into the lesion, decreases secondary damage, and improves the recovery of locomotor function [17]. These studies indicate that administration of C5aRA can inhibit the in ammatory reaction during the acute phase of SCI and prepare the injured microenvironment for receiving transplanted NS/PCs.
The purpose of the current study is to investigate whether C5aRA improves the in ammatory environment and enables increased survival of grafted cells when transplanted during the acute phase after SCI. We also evaluated the impact of a combined therapy of hiPSC-NS/PCs transplantation and C5aRA on locomotor functional recovery.
This study is particularly important as it is the rst study to our knowledge which demonstrates immunosuppressant enables e cient hiPSC-NS/PCs transplantation during acute phase after SCI by increasing survival of grafted cells.

Animals
Adult female SCID-Beige mice (8-10 weeks, 17-22 g), which are de cient in lymphocytes and NK cells, were provided by Charles River Laboratory. All animals were housed in a temperature-and humiditycontrolled 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 In nite Horizon impactor (Precision Systems and Instrumentation, Fairfax Station, VA), as previously described [18]. The muscles were sutured and the skin was closed with wound clips. After spinal cord injury, ampicillin (12.5 mg/kg) was administrated subcutaneously.

C5a ELISA
A total of 25 mice were used in this experiment; the injured mice were sacri ced 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 sacri ced 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 [19]. 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.

mRNA-Seq
A total of 22 mice were used in this experiment; the injured mice were sacri ced 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 ltering were performed using Real-Time Analysis Software version 1.18.61 and raw reads were mapped to reference genome mm9 using Sail sh (v0.7.6) with default settings. All gene expression pro les were evaluated using Exatlas (https://lgsun.irp.nia.nih.gov/exatlas/). The extracted data were visualized with Morpheus (https://software.broadinstitute.org/morpheus).

Flow cytometry
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 uorescent 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 de ned as CD45 high CD11b + LY6G-population, and neutrophils were de ned as CD45 high CD11b-LY6G + population.
Cell culture, lentivirus transduction Cell culture of hiPSC (414C2) was performed as described previously, with subtle modi cations [20]. Brie y, 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 re y luciferase [21] under the control of an EF promoter to enable the detection of the grafted cells in living mice and in xed sections, was prepared [22], and transduced into hiPSC-NS/PCs as previously described [2].

Cell transplantation
Four days after SCI, the mice were randomly and blindly divided into four groups based on their BMS score to ensure equivalent de cits across the groups: (PBS only group, C5aRA only group, PBS + transplantation (PBS + TP) group, C5aRA + transplantation (C5aRA + TP) group), and re-anesthetized with iso urane. hiPSC-NS/PCs (5 × 10 5 cells/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 sacri ced 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 con rm the survival of the transplanted hiPSC-NS/PCs, as described previously [23] .
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 quanti ed in maximum radiance units (photons/second/centimeter squared/steradian photons/sec/cm 3

Statistical analysis
All data are presented as means ± SEM. A Mann-Whitney U test was used to identify any signi cant differences between groups with respect to the results of owcytometry and immunohistochemistry. Oneway analyses of variance (ANOVA) followed by Tukey-Kramer tests for multiple comparisons were used to detect signi cant 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 signi cance level was set at p < 0.05. Microsoft Excel 2016 and IBM SPSS Statistics (ver. 25) were used for all calculations.

C5a and C5aR expression in the spinal cord after injury
To investigate expression of C5a in the spinal cord, we evaluated the protein levels of C5a before and after injury at different time points. The results revealed that C5a protein levels were signi cantly increased 1 day after injury (8.16 ± 0.83 pg/µg protein) and maintained the high amount of protein compared with the one prior to injury(2.43 ± 0.45 pg/µg protein) at 4 (6.69 ± 0.99 pg/µg protein), 7 (9.72 ± 0.85 pg/µg protein) and 14 days (8.24 ± 0.98 pg/µg protein) (Fig. 1A). Next, we investigated the level and distribution of C5a receptor expression in the injured spinal cord using western blotting and immunohistochemistry. Western blotting showed that the level of C5a receptor expression were upregulated at 1 day after injury, then decreased at days 4 and 7 (1 day: 14.02 ± 01.54, 4 days: 4.18 ± 0.85, 7 days: 2.08 ± 0.16) (Fig. 1B-C). To examine what types of cells expressed the C5a receptor, immunohistochemical analysis was performed for the injured spinal cord. At 1 and 4 days after SCI, Iba1 + macrophages, activated microglia and some LY6G + neutrophils expressed C5a receptor ( Fig. 1D-E). These results suggest that C5a protein was produced immediately after SCI, and that C5a receptors were expressed in in ammatory cells such as macrophages, activated microglia and neutrophils during the acute phase after SCI.
Next, mRNA-Seq analysis was performed to analyze the in uence of C5aRA on the gene expression pro le in the injured spinal cord. mRNA sequencing revealed that the expression of genes associated with in ammatory cytokines was suppressed by administering C5aRA at 12 hours after SCI (Fig. 2D). With regard to the apoptotic and necroptotic markers, administration of C5aRA also downregulated several apoptotic (Caspase8 and Pidd1) and necroptotic (RIPK3 and MLKL) markers 4 days after injury (Fig. 2E).

Inhibition of C5aR increases the survival rate of grafted NS/PCs
To examine the effect of C5aRA on the survival rate of grafted cells, we assessed the luminescence of the grafted cells using BLI (Fig. 4A). The BLI analyses demonstrated that the luminescence of the grafted cells in the C5aRA + TP group was signi cantly higher than that of grafted cells in the PBS + TP group at 14 days post-SCI and thereafter (C5aRA + TP vs PBS + TP; day14: 4.86 ± 0.71 E + 07 vs 3.24 ± 0.51 E + 07, day21: 4.93 ± 0.94 E + 07 vs 2.64 ± 0.51 E + 07, day28: 6.85 ± 0.94 E + 07 vs 2.39 ± 0.46 E + 07, p < 0.05) (Fig. 4B).
We also evaluated grafted cell survival using immunohistochemistry. In the C5aRA + TP group, GFP + area had a tendency to increase compared to PBS + TP group in each axial section (Fig. 4C), and total volume of GFP + areas was signi cantly larger compared to the PBS + TP group (C5aRA; 0.29 ± 0.05mm 3 , PBS; 0.13 ± 0.03mm 3 , p < 0.05) (Fig. 4D). These results indicate that inhibition of C5aR improves the survival and proliferation of hiPSC-derived NS/PCs after the transplantation.

Discussion
As shown previously, anti-in ammatory treatment is needed to enable e cient cell transplantation therapy during acute phase because transplanted cell rarely survives in an in ammatory environment in spinal cord during acute phase after SCI, but effective immunosuppressant which increase grafted cell survival has not been found. Therefore, we hypothesized that C5aRA could be a novel immunosuppressant which strongly suppressed in ammatory reaction after SCI and enables e cient hiPSC-NS/PCs transplantation during acute phase. The present study demonstrates that C5aRA administration after SCI signi cantly reduces upregulation of IL-1β, IL-6 and TNFα, as well as the in ltration of neutrophils and macrophages. C5aRA also downregulated the expression of in ammatory cytokines and apoptosis markers, which was demonstrated by RNA sequence analysis. The combined therapy of hiPSC-NS/PCs transplantation and C5aRA decreased the grafted cell death compared to cell transplantation monotherapy. Consequently, this combined therapy signi cantly improved locomotor function after SCI. Our ndings show that C5aRA could be a promising medication to enhance the e cacy of hiPSC-NS/PCs when transplanted during the acute phase of SCI.
It is well known that in ammatory reactions play a critical role in the exacerbation of secondary damage in the SCI microenvironment [6,8,10,26]. In particular, IL-1β and TNFα contribute to upregulation of several in ammatory mediators, recruiting neutrophils and macrophages, and resulting in the apoptosis of neurons and oligodendrocytes after SCI [27][28][29][30][31]. IL-6 also promotes cytotoxic macrophage in ltration into the lesion and increases secondary damage [32,33]. Neutrophil and macrophage in ltration into the lesion area is a detrimental factor for resident cell survival and functional recovery after SCI [34][35][36][37]. A previous study showed that these in ammatory cytokines and cells were regulated by the complement C5a-C5aR axis, and that inhibition of these factors decreased resident cell death, thus improving functional restoration after SCI [17]. In the present study, we demonstrated that administration of C5aRA reduced secretion of various cytokines, in ltration of in ammatory cells, and apoptosis of resident cells in the spinal cord, suggesting that the C5aRA comprehensively suppressed main factors which contribute to secondary damage after SCI. Therefore, the intervention using C5aRA could prevent the expansion of secondary damage and create a hospitable environment for cell survival after transplantation in SCI.
Previous studies have revealed that the acute in ammatory reaction after CNS injury contributes to rejection of grafted cells in spinal cord tissue [38][39][40]. To address this challenge, several researchers have tried to improve cell engraftment by inhibiting acute in ammatory factors, but few studies have reported successful improvement in cell survival after SCI. For example, depletion of neutrophils was found to decrease the astrogliosis of the transplanted cells, but did not change the survival rate [41]. A reduction in macrophages also failed to favorably affect cell engraftment [37]. It is inferred from those results that the blockade of a single in ammatory factor cannot decrease grafted cell death. Therefore, a strong immunosuppressant capable of blocking several in ammatory reactions after SCI is indispensable for the improvement of cell survival. C5aRA was an e cacious drug against rejection of hiPSC-NS/PCs transplantation because this medication was able to suppress multiple in ammatory cells, and our results demonstrated amelioration of the cell survival rate. We can deduce from this result that C5aRA is more effective for improving the e cacy of cell transplantation into the injured spinal cord than other immunosuppressants, because the C5a-C5aR axis regulates several in ammatory reactions in the acute phase after SCI [12].
Our results demonstrated that, compared to NS/PC transplantation only, cell engraftment with C5aRA administration decreased cell death and maintained the number of cells, which contributed to functional restoration after SCI. In other words, the intensive in ammatory reaction during the acute phase of SCI did not allow transplanted cells to survive su ciently and exert their e cacy when C5aRA was not used.
Although the in vivo differentiation pro les were comparable between groups with or without the C5aRA (Fig. 5), our results indicate that it is necessary to secure a certain number of survived transplanted cell for the differentiated cells to play functional roles. By using C5aRA, it is inferred that more NS/PCs differentiated into axons to construct functional circuits, or differentiated into oligodendrocytes for remyelination, and these mechanisms led to functional recovery [2,42,43]. If cell transplantation is possible in the acute phase of injury, earlier interventions could become feasible and bring the bene ts of regenerative medicine to the SCI eld.
For the transplantation studies using human-derived cells, immunode cient animals were employed to avoid rejection of the grafted cells. For example, NOD-SCID mice which generally used in transplantation experiment lacked lymphocytes and impaired innate immune system such as neutrophils, macrophage and complement [23,[43][44][45][46]. However, NOD-SCID mice are not suitable for our current study because normal activation of the complement system is necessary to evaluate the effect of C5aRA.
Immunocompetent mice are also not appropriate for the present study owing to lymphocytes, which strongly reject xenograft [40,47]. In this study, we selected SCID-Beige mice, which lack lymphocytes but maintain a functional complement system. We demonstrated that the complement system was activated normally after SCI in SCID-Beige mice, and we could perform evaluating the in uence of C5aRA on the grafted cells and locomotor function recovery. Thus, the SCID-Beige mouse was a reasonable tool to evaluate the impact of complements on the transplanted cells in SCI. Importantly, as it is known that T cells and NK cells can express C5a receptor [48,49], this study using SCID-Beige mice might be insu cient to accurately evaluate the effect of C5aRA against cell transplantation after SCI. Therefore, further study using immunocompetent mice with lymphocyte depressants are necessary to enhance our understanding.
In this study, we showed the e cacy of the combined therapy C5aRA and hiPSC-NS/PCs transplantation for SCI in SCID-Beige mice. However, in order to apply this treatment in a clinical setting, it is necessary to perform further studies using C5aRA, which has already been clinically applied, instead of PMX205 that is not approved for marketing [16]. For example, CCX168 has completed phase trials [50] and is one of the most advanced C5aRA in clinical development. However, because CCX168 is effective only in human C5aR, further studies using human C5a knock-in mice are necessary.

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The present study demonstrates that the administration of C5aRA suppresses the in ammatory response during the acute phase of SCI. This bene cial effect led to improved transplanted hiPSC-NS/PCs survival rates as well as the enhancement of motor functional restoration. These ndings suggest that administration of C5aRA makes it possible to transplant hiPSC-NS/PCs during the acute phase of SCI.
This work opens a potentially novel strategy wherein the transplantation of neural stem cells could be combined with early decompressive surgery as a one stage treatment for severe SCI.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  In ammatory reaction after spinal cord injury with C5aR antagonist. A-C: In ammatory cytokine (IL-1 , IL-6, TNFα) activation at 3h, 6h, 12h, 1d, and 4d after SCI by RT-PCR. **; p<0.05 D: Analysis of in ammatory cytokines at 4d after SCI by RNA sequencing. Heat map of expression pro les for the 9 signi cant genes involved in in ammation as assessed by RNA-sequencing. E: Analysis of apoptotic necroptotic markers at 4d after SCI by RNA sequences. Heat map of expression pro les for the 4 signi cant genesinvolved in apoptotic and necroptotic markers as assessed by RNA-sequencing. F-G: Analysis of neutrophils and macrophages at 4d after SCI using ow cytometry. **; p<0.05