Background
The injured spinal cord exhibits little spontaneous recovery and, as a result, many spinal cord injury (SCI) patients suffer from permanent functional impairments, such as motor and sensory dysfunction, and bladder and rectal disturbance. However, some previous reports have shown that neural stem/progenitor cells (NS/PCs) transplanted into the injured spinal cord of rodents[
1‐
6] and non-human primates[
7], 7–10 days post-injury (DPI), promote functional recovery after SCI. These reports indicate that NS/PC transplantation has therapeutic potential for SCI when performed during the sub-acute phase. However, patients continue to seek new therapies for SCI many years after their original injury, and most are therefore in the chronic phase. Although many researchers have sought to achieve functional recovery at the chronic phase of SCI by NS/PC transplantation, with one exception[
8], no significant recovery of motor function has been obtained in animal models of chronic-phase SCI[
9‐
12]. Despite differences in the survival rate, the cell types derived from the grafted NS/PCs and the distribution of grafted cells transplanted at the sub-acute versus the chronic phase remain unknown. Thus, it remains unanswered as to why grafted NS/PCs do not exert therapeutic benefits in the injured spinal cord at the chronic phase. To address this question, this study analyzed fetus-derived NS/PCs transplanted into the injured spinal cord of mice at 9 DPI and 42 DPI.
To assess the survival rate of grafted cells, we performed quantitative analysis using bioluminescence imaging (BLI) on a weekly basis until 42 days after transplantation. BLI is a powerful tool for the detection of exclusively living grafted cells that stably express luciferase in living animals after administration of luciferin, the luciferase substrate, because the luciferin-luciferase reaction depends on oxygen and ATP[
13]. In this study, no significant difference in the survival rate of grafted cells between the sub-acute and chronic transplanted (TP) groups was observed at each experimental time point. Immunohistology also revealed no significant difference in the differentiation pattern of grafted NS/PCs between the two groups. In addition, inflammatory cytokines and growth factors, which influence the survival rate and differentiation characteristics of grafted cells, were expressed at similar levels at both phases. By contrast, the grafted cells were distributed broadly from the epicenter to rostral and caudal sites in the sub-acute TP group, whereas they remained near the lesion epicenter, due to extensive glial scarring, in the chronic TP group. Moreover, prominent macrophages distributed at and around the lesion epicenter in the sub-acute phase by immunohistochemistry, and microarray analysis demonstrated that the expression of Arginase-1, which is associated with M2 macrophages, was up-regulated significantly at the sub-acute phase than the chronic phase. These findings indicated that the characteristics of post-SCI inflammation are different between the sub-acute and chronic phases.
Consequently, the grafted NS/PCs did not promote any motor functional or histological recovery in the chronic TP group, while the sub-acute TP group demonstrated significant recovery compared with the vehicle control group. Taken together, these data suggest that a combination therapy of NS/PC transplantation with control of glial scar formation or inflammatory reaction may be critical to achieving functional recovery for chronic SCI.
Discussion
Previous studies have indicated that NS/PC transplantation at the sub-acute phase has therapeutic potential for SCI[
1‐
7,
18]. At the chronic phase, however, NS/PC transplantation does not result in functional recovery. Many researchers have sought to improve motor function in chronic SCI by NS/PC transplantation. However, most studies obtained no significant functional recovery, and no consensus exists concerning the survival and/or fate of grafted cells in chronic-phase transplantation[
9‐
12]. For example, some reports demonstrated that cells grafted at the chronic phase of SCI have a poor survival rate[
9,
12], while Cusimano et al. reported no significant difference in survival rate between NS/PC transplantation at the sub-acute versus the chronic phase[
11]. However, these findings were based on histological examinations only. To clarify this issue, we performed sub-acute- and chronic-phase transplantations of NS/PCs derived from transgenic mice that ubiquitously express ffLuc-cp156 on a C57BL/6J background[
14] to treat SCI in adult mice. These NS/PCs showed strong bioluminescent and fluorescent signals originating from ffLuc-cp156[
14], enabling the easy detection of surviving grafted cells in living animals noninvasively by BLI[
4,
13] and confirmation of integrated cells within the injured spinal cord by immunohistology for GFP without using a lentiviral vector.
Using cells from these mice, we demonstrated that the NS/PCs grafted during the chronic phase of SCI had a similar survival rate to those transplanted at the sub-acute phase. We also observed no significant difference between the two groups in the differentiation phenotypes of the grafted cells. The survival and/or differentiation potential of grafted cells can change remarkably due to microenvironmental changes in the injured spinal cord[
19]. A previous study showed that grafted NS/PCs mainly differentiate into astrocytes after transplantation at the acute phase[
4]. Inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, also increase dramatically in the injured spinal cord during the acute phase[
20,
21], and these cytokines induce NS/PCs to differentiate into astrocytes. Moreover, growth factors affect the fate of grafted NS/PCs. For example, EGF, FGF, NT3, and PDGF have demonstrated beneficial effects on the survival of NS/PCs[
10,
12,
22]. HGF and PDGF promote neuronal differentiation[
23,
24], whereas CNTF enhances glial differentiation
in vitro[
25]. To assess microenvironmental changes in the injured spinal cord, we performed a microarray analysis to exhaustively compare the gene expression profiles between the sub-acute and chronic phases of SCI. The expression levels of all cytokines and growth factors did not significantly differ, consistent with our BLI and histological results on the survival and/or fate of the grafted cells. These data suggested that the microenvironments of the injured spinal cord were not significantly different at the sub-acute and chronic SCI phases with respect to the expression of factors known to influence the survival rate and differentiation of grafted NS/PCs.
Many reports concern possible mechanisms underlying the therapeutic effects of NS/PC transplantation for SCI; for example, grafted cells contribute to the reconstruction of neural circuits and remyelination[
2,
6,
17,
26]. NS/PCs also secrete neurotrophic factors, which support the survival of host neural cells and enhance axonal growth and angiogenesis[
27‐
30]. In this study, motor function recovery was observed in the sub-acute TP group, but not in the chronic TP group. Consistent with this result, more NF-H
+ neuronal fibers and 5HT
+ serotonergic fibers, which are involved in motor function recovery[
30‐
32], were observed in the sub-acute TP group than in the sub-acute PBS group. Moreover, Luxol Fast Blue (LFB) staining revealed significantly larger myelinated areas in the sub-acute TP group than in the sub-acute PBS group. By contrast, no significant differences in NF-H
+ neuronal fibers, 5HT
+ serotonergic fibers, or LFB
+ myelinated areas were observed between the chronic TP and chronic PBS groups. Furthermore, no MEP waves were detected in most mice in the chronic TP group.
As a possible reason for these findings, we refer to the poor migration of grafted cells in the chronic TP group. Since glial scar formation prevented grafted NS/PCs from migrating away from the graft site and integrating with the host spinal cord, the regeneration of neural circuits, remyelination, and/or preservation of host neural cells did not occur in areas rostral and caudal to the injured spinal cord of the chronic TP group. Consistent with this scenario, many previous reports indicate that insufficient migration of grafted cells results in poor functional recovery after SCI[
33,
34].
Our microarray and histological analyses suggested that more macrophages or microglial cells, including a substantial number of M2 macrophages, were distributed around the lesion epicenter at the sub-acute phase than at the chronic phase. In agreement with this finding, previous studies have reported that infiltration of macrophages and microglial cells peaks at approximately 7 DPI[
35,
36]. Moreover, M2 macrophages have an anti-inflammatory role and promote axonal growth after SCI[
37‐
39], and their phagocytic activity was shown to contribute to tissue repair in the injured CNS by reducing myelin debris[
40‐
42]. In addition, Busch et al. demonstrated that activated macrophages induced axonal dieback in the injured CNS[
43], which was prevented by switching the infiltrating macrophages from M1 to M2 phenotype[
39]. Furthermore, grafted NS/PCs also exert immunomodulatory effects through interactions with infiltrating immune cells[
44,
45]. Especially in the sub-acute phase, grafted NS/PCs can affect the properties of infiltrating macrophages, inducing their shift from M1 to M2 in the injured spinal cord[
11,
39]. Therefore, NS/PCs transplanted at the sub-acute stage may alter the microenvironment to favor M2, resulting in a synergistic effect that supports the increase in neuronal fibers and functional recovery. These findings indicate that a novel immunomodulatory strategy, such as the administration of a mediator of phenotypic switching from M1 to M2 or the combined transplantation of NS/PCs and M2 macrophages, may have therapeutic potential in chronic SCI. In contrast, previous studies reported that M1 macrophages impairs recovery of SCI through the production of oxidative metabolites or pro-inflammatory cytokines including TNF-α, which induced the apoptotic cell death of neural precursor cells[
42,
46,
47]. However, the expressions of genes associated with M1 macrophages showed no significant difference among the sub-acute and chronic phases. These findings are consistent with the similar survival rate of the grafted NS/PCs in both TP groups.
Taken together, these results suggest that during the chronic phase of SCI, the injured spinal cord microenvironment appears to be unfavorable for the therapeutic mechanisms of NS/PC transplantation, owing to extensive glial scarring[
48] and/or the phenotype of infiltrating macrophages. Accordingly, to improve the therapeutic potential of NS/PC transplantation performed at the chronic phase, altering the microenvironment in the injured spinal cord is likely to be important. For example, suppression of axonal growth inhibitors may improve the microenvironment. CSPGs, which are the most prevalent axonal growth inhibitors, are produced by reactive astrocytes and involved in glial scarring[
48]. Chondroitinase ABC (ChABC), which digests CSPG, promotes axonal growth and the migration of grafted NS/PCs[
49,
50]. Karimi-Abdolrezaee et al. described that CSPGs hinder the survival of grafted cells and the combined therapy of ChABC administration and NS/PCs transplantation promotes the migration and integration of grafted cells[
51]. While our data demonstrated that CSPGs prevent the migration of grafted cells, but show no influence on their survival, we have great hopes that a combination of NS/PC transplantation and the administration of a suppressor of axonal growth inhibitor may be effective for inducing functional recovery in chronic SCI.
Methods
NS/PC culture and analysis
NS/PCs were cultured and expanded, as previously reported[
52]. Briefly, the striata of transgenic mice ubiquitously expressing ffLuc-cp156, a fusion protein of firefly luciferase and a circularly permuted Venus protein[
14], were dissociated using a fire-polished glass pipette on embryonic day 14. Venus is a fluorescent protein with fast and efficient maturation that was originally engineered from GFP[
53], and therefore grafted cells can be detected as fluorescent Venus signals using anti-GFP antibody[
17,
26]. Dissociated cells were collected by centrifugation and re-suspended in culture medium, which consisted of Dulbecco’s modified Eagle medium/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a previously described hormone mixture[
52]. Human recombinant FGF-2 (Peprotech, Rocky Hill, NJ, USA) and EGF (Peprotech) (20 ng/ml each) were added every 2 days. The cells formed floating cell clusters (neurospheres) within 2–3 days. After propagation for 3 passages, the neurospheres were used for
in vitro BLI, differentiation, and proliferation assays, and for cell transplantation.
For differentiation analysis, the neurospheres were plated onto poly-L-ornithine/fibronectin-coated 8-well chamber slides (Iwaki; Asahi Glass Co Ltd., Tokyo, Japan) at a density of 1 × 105 cells/ml and cultured in culture medium without serum or growth factors at 37°C in 5% CO2 and 95% air for 7 days. The differentiated cells were then fixed with 4% paraformaldehyde in 0.1 M PBS and stained with the following primary antibodies for immunocytochemistry: anti-Tuj-1 (mouse IgG, 1:1000, Sigma-Aldrich), anti-GFAP (rat IgG, 1:1000, Invitrogen, Carlsbad, CA, USA), and anti-CNPase (mouse IgG1, 1:1000, Sigma-Aldrich). Nuclei were stained with Hoechst 33258 (10 μg/ml, Sigma-Aldrich). In vitro images were obtained using a fluorescence microscope (BZ-9000; Keyence Co., Osaka, Japan).
The proliferation assay was performed by measuring ATP, which indirectly reflects the number of viable cells[
15,
17,
54]. In brief, NS/PCs were first incubated in culture medium without serum or growth factors in 48-well cell-culture plates (Corning Inc., Corning, NY, USA) at 37°C in 5% CO
2 and 95% air for 24 or 72 h. D-luciferin was then added to each well, and the luminescent signal was detected immediately using a Xenogen-IVIS spectrum cooled charged-coupled device (CCD) optical macroscopic imaging system (Caliper Life Sciences, Hopkinton, MA, USA). To determine the population doubling time, the ATP assay was modified, as described elsewhere[
15,
17,
54].
SCI model
Adult female C57BL/6J mice (8–10 weeks old, 18–22 g, n = 52; Clea, Tokyo, Japan) were anesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After laminectomy at the 10th thoracic spinal vertebra (Th10), the dorsal surface of the dura mater was exposed. Contusive SCI was induced using a commercially available SCI device (IH Impactor, Precision Systems and Instrumentation, Lexington, KY, USA), as previously described[
4]. This device creates a reliable contusion injury by rapidly applying a force-defined impact (60 kdyn) with a stainless steel-tipped impactor. All experiments were approved by the ethics committee of Keio University and fully in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).
Microarray analysis
The injured mice were anesthetized and transcardially perfused with heparinized saline (5 U/ml) at 9 DPI or 42 DPI (n = 3 each). Dissected segments of spinal cord at the Th10 level were rapidly frozen and placed in TRIzol (Invitrogen). Total RNA was isolated using an RNeasy Mini Kit (Qiagen Inc., Hilgen, Germany), in accordance with the manufacturer’s instructions. As a control, samples of naïve spinal cord were harvested by the same protocol. For microarray analysis, RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and 100 ng of total RNA was reverse transcribed, biotin labeled, and hybridized to a GeneChip® Mouse Genome 430 2.0 Array (Affymetrix Inc., Santa Clara, CA, USA). The array was then washed and stained in a Fluidics Station 450, according to the manufacturer’s instructions[
55,
56]. The microarrays were scanned using a GeneChip Scanner 3000 7G, and the raw image files were converted to normalized signal intensity values using the MAS 5.0 algorithm. PCA was carried out with Gene Spring GX software (Agilent Technologies Inc.), using the full set of normalized data. For the clustering analysis, the normalized data were narrowed down by the cut-off values of expression levels (>50) and by fold change (>1.5, versus the signal of intact spinal cord), and statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test (
P < 0.05). The heat map was visualized with Gene Spring GX.
NS/PC transplantation
Various numbers of NS/PCs (approximate range 2.5 × 10
4 to 5 × 10
5 cells/2 μl) were transplanted into uninjured naïve mice (n = 3, each), and NS/PCs (5 × 10
5 cells/2 μl) were transplanted at 9 DPI (sub-acute TP group, n = 10) or 42 DPI (chronic TP group, n = 10) as previously reported[
4,
16,
17,
26,
30,
57]. The NS/PCs were transplanted into the lesion epicenter with a glass micropipette at a rate of 1 μl/min using a Hamilton syringe (25 μl) and a stereotaxic microinjector (KDS 310, Muromachikikai Co. Ltd., Tokyo, Japan). PBS (2 μl) was similarly injected into the lesion epicenter of the control mice at each time point (sub-acute and chronic PBS groups, n = 10 each).
Bioluminescence imaging
A Xenogen-IVIS spectrum CCD optical macroscopic imaging system was used for
in vitro and
in vivo BLI as previously reported[
4,
16,
17]. In brief, the signal intensity of NS/PCs
in vitro was assessed using plated cells at various cell numbers (approximate range 1 × 10
2 to 1 × 10
4 cells/well), and BLI was performed immediately after adding D-luciferin (150 μg/ml) (n = 3). The integration time was fixed at 1 min for each image.
In vivo imaging was performed 15 min after the i.p. injection of D-luciferin (0.3 mg/g body weight) with the field-of-view set at 13.2 cm, because the photon count was most stable during this period. The intensity peaked between 10 and 30 min after the i.p. injection of D-luciferin. The integration time was fixed at 5 min for each image. All images were analyzed with Living Image software (Caliper Life Sciences), and the optical signal intensity was expressed as photon flux (photon count) in units of photons/s/cm2/steradian. Each result was displayed as a pseudo-colored photon count image superimposed on a gray-scale anatomic image. To quantify the measured light, a region of interest was defined in the cell-implanted area, and all values at the same region of interest were examined.
Behavioral analyses
The motor function of each mouse was evaluated weekly using the BMS up to 49 DPI in the sub-acute TP and PBS groups and up to 84 DPI in the chronic TP and PBS groups (n = 10 per group)[
58]. This assessment was performed by two investigators blinded to the identity of the experimental mice.
Motor coordination was evaluated using a rotating rod apparatus (Rotarod, Muromachikikai Co., Ltd.), which consisted of a plastic rod (3 cm diameter, 8 cm length) with a gritted surface flanked by two large discs (40 cm diameter) (n = 10 per group). At 42 days after cell transplantation or PBS injection, each mouse was placed on the rod while it rotated at 20 rpm for 2 min sessions[
59]. Three trials were conducted, and the maximum number of seconds the mouse stayed on the rod was recorded.
Gait analysis was performed using the DigiGait system (Mouse Specifics, Quincy, MA, USA) (n = 10 per group)[
17,
26,
60]. Each mouse demonstrated weight-supported hindlimb stepping at 42 days after cell transplantation or PBS injection. The stride length was determined on a treadmill set to a speed of 7 cm/s.
Electrophysiology
Electrophysiological experiments were performed using a Neuropack S1 MEB-9402 (Nihon Kohden, Tokyo, Japan) at 42 days after cell transplantation or PBS injection (n = 7 per group)[
26]. The animals were anesthetized with an i.p. injection of ketamine (60 mg/kg) and xylazine (6 mg/kg), and the stimulation was applied through the occipitocervical area of the spinal cord and the hindlimb by needle electrodes. The active electrode was placed in the quadriceps muscle belly, and the reference electrode was placed near the distal quadriceps tendon. The ground electrode was placed on the tail. A stimulus of 0.4 mA intensity, 0.2 ms duration, and 1 Hz interstimulus interval were used. The latency was measured as the length of time from the stimulus to the onset of first response wave. The amplitude was measured from the initiation point of the first response wave to its highest point.
Histological analyses
Injured animals were deeply anesthetized and transcardially perfused with 4% PFA in 0.1 M PBS at 9 DPI or 42 DPI (n = 3 each). The treated animals were similarly prepared 42 days after cell transplantation or PBS injection. The spinal cords were removed, postfixed overnight in 4% PFA, soaked overnight in 10% sucrose, followed by 30% sucrose, embedded in Optimal Cutting Temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), frozen, and sectioned in the sagittal or axial plane at 12 μm thickness on a cryostat (CM3050 S; Leica Microsystems, Wetzlar, Germany). The injured spinal cord sections were histologically evaluated by HE staining and immunohistochemistry, followed by quantitative analysis. The sections of transplanted spinal cord were subjected to HE staining, LFB staining, and immunohistochemistry followed by quantitative analyses.
For immunohistochemistry, tissue sections were stained with the following primary antibodies: anti-GFP (rabbit IgG, 1:200; Frontier Institute, Hokkaido, Japan), anti-Hu (human IgG, 1:1000, a gift from Dr. Robert Darnell, The Rockefeller University, New York, NY, USA), anti-GFAP (rat IgG, 1:200, Invitrogen), anti-APC CC-1 (mouse IgG, 1:200; Calbiochem, San Diego, CA, USA), anti-nestin (mouse IgG1, 1:500; BD Bioscience Pharmingen, San Jose, California, USA), anti-CS56 (a marker for CSPG, mouse IgM, 1:200; Sigma-Aldrich), anti-Iba1 (a marker for microglia/macrophages, rabbit IgG, 1:200; Wako Pure Chemical Industries, Osaka, Japan), anti-arginase-1 (a marker for M2 macrophages, goat IgG, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LAMP2 (a marker for endosomes or lysosomes, rat IgG, 1:200; Abcam, Cambridge, UK), anti-NF-H (mouse IgG1, 1:200; Chemicon, Millipore, Billerica, MA, USA), and anti-5HT (goat IgG, 1:200; Immunostar, Hudson, WI, USA). For immunohistochemistry with anti-GFP, -NF-H, and -5HT, a biotinylated secondary antibody (Jackson Immunoresearch Laboratory Inc., West Grove, PA, USA) was used after exposing the sections to 0.3% H2O2 for 30 min at room temperature to inactivate endogenous peroxidases. The signals were enhanced with the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). Nuclei were stained with Hoechst 33258 (10 μg/ml, Sigma-Aldrich). All images were obtained using a fluorescence microscope (BZ 9000; Keyence Co.) or a confocal laser scanning microscope (LSM 700; Carl Zeiss, Munich, Germany).
Quantitative analyses
Quantitative analyses of the histological findings (HE, LFB staining, and immunostaining for CS56, Iba1, NF-H, 5HT, GFP/each phenotypic marker, and arginase-1/Iba1) were performed using a BZ 9000 microscope and Dynamic Cell Count BZ-HIC software (Keyence Co.). The threshold values were maintained at a constant level for all analyses. The GFP+ area was quantified using images of axial sections of the lesion epicenter and 0.5 mm, 1.0 mm, 2.0 mm, 3.0 mm, and 4.0 mm rostral and caudal to the epicenter at 100× magnification (n = 4 each). To determine the spinal cord area, HE-stained images of axial sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 100× magnification were used (n = 3 each). Quantitative analysis of the LFB+ area was similarly performed using axial sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 100× magnification (n = 3 each). CS56+ areas as well as Iba1+ areas were quantified in the midsagittal sections of injured spinal cords at 100× magnification (n = 3 each).
To quantify NF-H+ fibers, four regions were automatically captured within the midsagittal sections of the lesion epicenter and 4.0 mm rostral and caudal to the epicenter at 400× magnification (n = 3 each). To assess the 5HT+ fibers, five automatically captured regions within the axial sections were analyzed at the lumbar intumescence, which was 6–8 mm caudal to the lesion epicenter (n = 3 each).
To quantify the proportion of each cell phenotype among the grafted cells in vivo, five regions were captured within sagittal sections at 200× magnification using an LSM 700 confocal laser scanning microscope. GFP and phenotypic marker double-positive cells were counted in each section (n = 3 each). To quantify the infiltrated M2 macrophages in the injured spinal cord, the number of Iba1 and arginase-1 double-positive cells was counted within five regions of sagittal sections of the lesion epicenter at 200× magnification with an LSM 700 confocal laser scanning microscope (n = 3 each).
Statistical analysis
All data are reported as the mean ± SEM. An unpaired two-tailed Student’s t test was used to evaluate the differences between groups with respect to microarray gene expression profile, in vivo BLI analysis, in vivo differentiation assays, and analyses of the CS56+, Iba1+ and GFP+ areas, and arginase-1+/Iba1+ cells. One-way ANOVA followed by the Tukey-Kramer test for multiple comparisons was used in the analyses of the HE, LFB, NF-H+, and 5HT+ areas and the Rotarod and DigiGait results. Repeated-measures two-way ANOVA followed by the Tukey-Kramer test was used for the BMS analysis. P values < 0.05 were considered statistically significant.
Acknowledgments
We appreciate the help of Dr. A. Iwanami, Dr. Y. Takahashi, Dr. M. Shinozaki, Dr. T. Konomi, Dr. R. Zhang, Dr. G. Itakura, Dr. S. Tashiro, Dr. S. Kawabata, Dr. Y. Nishiyama, and Dr. K. Hori, members of the spinal cord research team at the Department of Orthopaedic Surgery, Rehabilitation Medicine and Physiology, Keio University School of Medicine. We also thank Ms. T. Harada, Ms. S. Miyao, Ms. M. Mizutani, and Ms. H. Shimada for their assistance with the experiments and animal care.
This work was supported by grants from the Japan Science and Technology–California Institute for Regenerative Medicine collaborative program; Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (SPS) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); the Project for Realization of Regenerative Medicine; Support for Core Institutes for iPS Cell Research from the MEXT; Keio Gijuku Academic Development Funds; by a Grant-in-Aid for the Global COE program from MEXT to Keio University; and by a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the MEXT.
Competing interests
The authors have declared that no competing interests exist.
Authors’ contributions
SN, AY, OT, HO, and MN designed the research; SN, AY, HI, MT, and HE performed research; SN, AY, and HI analyzed the data; SN, HO, and MN wrote the paper; and MN and HO supervised all the experiments. All authors read and approved the final manucript.