Exp Neurobiol 2018; 27(6): 489-507
Published online December 28, 2018
© The Korean Society for Brain and Neural Sciences
Dong Hoon Hwang1*, Hee Hwan Park1,3, Hae Young Shin1,4, Yuexian Cui1,5, and Byung Gon Kim1,2,3*
1Department of Brain Science, Ajou University School of Medicine, Suwon 16499, Korea.
2Department of Neurology, Ajou University School of Medicine, Suwon 16499, Korea.
3Neuroscience Graduate Program, Department of Biomedical Sciences, Ajou University School of Medicine, Suwon 16499, Korea.
4Logos Biosystems, Anyang 14055, Korea.
5Department of Neurology, Yanbian University Hospital, Yanji 133000, Jilin, China.
Correspondence to: *To whom correspondence should be addressed.
Dong Hoon Hwang, TEL: 82-31-219-4561, FAX: 82-31-219-4444
Byung Gon Kim, TEL: 82-31-219-4495, FAX: 82-31-219-4444
Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/−) for
Transplantation of neural stem cells (NSCs) or progenitors, either primary or derived from pluripotent stem cells, is one of the most promising strategies that may lead to meaningful functional recovery in disabled patients with spinal cord injury (SCI). Multiple mechanisms, including provision of growth factors, immune modulation, and replacement of myelin-forming oligodendrocytes, have been proposed to explain the preclinical benefits of NSC transplantation [1,2,3,4,5]. Recently, a series of studies proposed the exciting possibility that NSCs can participate in the formation of alternative neural circuits, conveying supraspinal input to the spinal motor center [6,7,8,9]. For grafted NSCs to integrate into the novel networks, a sufficient number would have to survive and migrate to appropriate positions to establish synaptic connections. However, achieving an adequate level of survival and migration of NSCs in the injured CNS is a daunting task. Previous studies have reported that a large fraction of transplanted NSCs die and that extent of migration is frequently limited [1,10,11,12,13,14,15].
Activity-based neurorehabilitation has proven its potential in improving functional recovery after SCI [16,17,18]. Furthermore, it has been demonstrated that activation of the spinal motor circuit using various neuromodulation tools is effective in restoring lost motor functions [19,20,21,22]. We have previously noticed that combined treadmill training (TMT) substantially enhanced survival of NSC grafts in the lesioned spinal cord . Given the increasing relevance of activity-based therapies in SCI, elucidating the mechanisms underlying the interactions between biological behaviors of NSCs and neurorehabilitation interventions would have profound implications in designing future therapeutic strategies for functional repair of the injured spinal cord.
We previously reported that TMT increased the level of IGF-1 in cerebrospinal fluid and that intrathecal infusion of neutralizing antibodies against IGF-1 markedly attenuated TMT-induced enhancement of NSC graft survival in the lesioned spinal cord, suggesting the involvement of IGF-1 signaling in the enhancement of NSC survival by TMT . The current study was designed to obtain genetic evidence of cell-autonomous roles of IGF-1 signaling in transplanted NSCs to determine their biology such as survival and migration in the lesioned spinal cord. We hypothesized that IGF-1 signaling in transplanted NSCs would have more profound consequences when recipient animals are subjected to TMT. To this end, we utilized haploinsufficiency of
All experiments were conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Ajou University School of Medicine. The manuscript has been prepared following the ARRIVE guidelines. Mice of the
Neurosphere assay was performed following a previously published protocol . Tertiary neurospheres, which are formed after two passages, were used for measurement of the number and size of neurospheres. Secondary neurospheres from each group were collected and centrifuged, and the supernatant was removed. Then, Accumax dissociation medium was added, and the neurospheres were incubated for 5 min. Dissociated cells were plated at a density of 105 cells per 100 mm petri dish. Culture medium was changed every day, with fresh bFGF and EGF added during the entire culture period. Neurospheres were allowed to grow for 7 days, after which the number and size of tertiary spheres were determined. Four independent cultures were used for the neurosphere assay. Neurospheres were counted under a confocal microscope (FV300, Olympus) at a 10× magnification, with a minimum cutoff diameter of 20 µm. Three images were randomly acquired from each plate, and the diameter of the neurospheres in these images was measured using the Image J software. For in vitro NSC survival assay, dissociated NSCs cultured in a 96-well plate were exposed to either 400 µM 3-morpholinosydnonimine (SIN-1, Enzo Life Sciences, Inc.) or 1 mM hydrogen peroxide (H2O2, Sigma), which produce reactive nitrogen species (RNS) or reactive oxygen species (ROS), respectively. Nitric oxide (NO)-associated radical species can kill transplanted NSCs in the injured spinal cord, and SIN-1 was used to produce RNS to damage cultured NSCs . H2O2 has been frequently used to induce oxidative stress in stem cells [27,28]. Cultured NSCs were incubated in medium containing either SIN-1 or H2O2 for 48 h with or without IGF-1 at a concentration of 20 ng/ml. Three independent cultures were performed per experimental condition (N=3), with each culture replicated thrice. The percentage of surviving NSCs was determined using Cell Counting Kit-8 (Dojindo Laboratories) according to the manufacturer's instructions.
Nine-week-old C57BL/6 females (20~25 g) (Orient Bio), which had completed 1 week of treadmill pretraining (see below), were subjected to a contusion injury. Animals were anesthetized by intraperitoneal injection of ketamine (90 µg/g) and xylazine (10 µg/g) mixture. After laminectomy was performed, the dorsal surface of the spinal cord at the 9th vertebral level was injured using the Infinite Horizon Impactor (Precision Systems and Instrumentation), with a force of 70 kdyn. Subsequently, animals were randomly divided into 4 groups depending on whether they received NSC grafts that were wild-type (+/+) or heterozygous (+/−) for
We performed TMT using a 10-channel Flat Treadmill System for mice (model IW-FT, IWOO Scientific). Each channel consists of a 50×450 mm runway. The TMT protocol adopted in our previous study  was slightly modified for mice. All animals received pretraining for 7 days before injury; TMT was initiated 3 days after injury for animals that were assigned to the groups with TMT. Most mice were able to exhibit spontaneous stepping by 7 days at a slow treadmill speed (5 m/min). Temporarily suspended after transplantation, TMT was restarted 3 days later and continued until the animals were sacrificed for histological analysis. TMT was performed at a slow speed (5 m/min) until animals regained weight-supported plantar stepping, and thereafter, the speed was gradually increased up to 10 m/min. The training was performed 6 days per week, with 3 sessions per day. Each session consisted of 20 min of training and 5 min of break. Mice assigned to the groups without TMT, they were handled and brought to the training room together with those in the groups with TMT, but excluded from the actual training.
A total of 36 mice were included for the behavioral analysis with 9 animals per group. Basso Mouse Scale for locomotion (BMS), ladder walk test, and digitized gait analysis using the Catwalk system (Noldus Information Technology) were used to assess recovery of locomotor function. We assessed BMS 24 h after injury and once a week thereafter. For the ladder walk test, the animals were pretrained to walk on ladder rungs for 7 days before surgery, and tested 4 and 8 weeks after injury. The number of hind-paw placement errors per run was counted and the average percentage of errors was obtained from four runs for each animal. The Catwalk analysis was performed 8 weeks after injury. Animals were pretrained for walking on the Catwalk runway before surgery and retrained for 5 days before final testing. On the test day, 4 uninterrupted crossings were recorded. Before computerized analysis, individual footprints from 4 different paws were manually confirmed. The following parameters were automatically calculated: stride length, base of support, and rotation angle. The angle of hind-paw rotation was defined as the angle (in degrees) of the hind-paw axis relative to the horizontal plane. As described previously , the relative position of the fore-paws and hind-paws was obtained by measuring the distance between the center pads of the fore-paw and hind-paw prints.
After cardiac perfusion with 4% paraformaldehyde, the spinal cord was dissected and post-fixed, followed by cryoprotection in a graded series of sucrose solutions. Longitudinal sections (20 µm-thick) of the spinal cord were cut using a cryostat (CM 1900, Leica) and thaw-mounted onto Super Frost Plus slides (Thermo Fisher Scientific). For immunohistochemistry, sections were incubated overnight at 4℃ with the following primary antibodies: anti-RFP (1:500, mouse polyclonal; #R10367, Invitrogen), anti-adenomatous polyposis coli (APC) clone CC1 (APC-CC1) (1:200, mouse monoclonal; #OP80, Calbiochem), anti-glial fibrillary acidic protein (GFAP) (1:500, rabbit polyclonal; #Z0334, DAKO) and anti-doublecortin (DCX) (1:400, rabbit polyclonal; #4604S, Cell Signaling Technology). After washing, slides were incubated with appropriate secondary antibodies conjugated to the Alexa Fluor fluorescent dyes. Slides were examined using a confocal laser scanning microscope (LSM 800, Carl Zeiss).
Stereological cell counting was performed as previously described . Longitudinal sections in a horizontal plane were systematically selected at an intersection interval of 300 µm (section sampling fraction=1/15). The sampling grid dimension was 500×500 µm2 with a 200×200 µm2 counting grid (area sampling fraction=0.16). Using a dissector height of 10 µm in sections with an average post-processing thickness of 18 µm, the height sampling fraction was 0.56 (10/18). The migratory distance of RFP-positive grafts for each animal was determined as the longest longitudinal distance in either rostral or caudal direction from the epicenter out of 5 consecutive longitudinal sections with a 300-µm intersection distance. It is conceivable that the longitudinal migratory distance can be influenced by the extent of graft survival; the more NSCs survive, the longer they distribute longitudinally. To control for the different extents of graft survival, a migration index was generated by dividing the longest longitudinal migratory distance by the longest transverse diameter of the RFP-positive area.
The IGF-1-induced NSCs migration was assessed using Boyden chamber assay kit (CytoSelect™ 24-well cell migration assay kit, #CBA-107; CELL BIOLABS Inc.), where an upper chamber was separated from a well by a polycarbonate membrane insert (12-µm pore size). Dissociated NSCs were plated on a culture insert nested inside of a 24-well culture plate at a density of 5×104 cells/well. StemPro® NSC serum-free medium containing bFGF and EGF was added to an upper chamber within an insert, while IGF-1 was added as a chemoattractant to the same medium in a 24-well with the same insert. The Boyden chamber was incubated at 37℃ with 5% CO2 for 48 hours. After non-migratory cells on the upper surface of the insert were removed by a cotton swab provided in the kit, migratory cells attached to the bottom side of the insert were stained using the cell-staining solution provided in the kit for 10 min at room temperature. The rate of NSCs migration was calculated by counting cells in 3 random fields of each well using a 10× objective lens (BX51, Olympus). For in vitro scratch assay, dissociated NSCs were plated on a 24-well culture plate coated with Poly-D-lysine at a density of 5×104 cells. StemPro® NSC serum-free medium containing bFGF and EGF was added and maintained throughout the imaging session without changing. An artificial scratch was created with a white 10-µl pipette tip at the center of the mono-layered cultured cells. NSCs migrating toward the scratch were monitored real-time for up to 4 consecutive days using JuLI™Stage (NanoEntek). Areas occupied by migrating cells within the scratch were automatically calculated by JuLI™STAT software.
Neurospheres derived from
Error bars in all graphs represent mean±standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism (version 5.0) and SPSS software (version 23). Unpaired Student's
To examine influence of IGF-1/IGF-1R signaling on transplanted NSCs in the lesioned spinal cord, we attempted to generate NSCs with deleted
We have previously observed that transplanted NSCs at the epicenter region in the spinal cord are under RNS- and ROS-induced cellular stresses . Treatment of either +/+ or +/− NSCs with SIN-1 or H2O2, generating RNS or ROS, respectively, significantly reduced the number of viable NSCs (Fig. 1C, D). Addition of IGF-1 to the culture medium significantly prevented RNS- or ROS-induced NSC death, while IGF-1 treatment did not increase the number of NSCs without cellular stresses. The IGF-1-induced robust pro-survival effects were almost completely abrogated in +/− NSCs (Fig. 1C, D). These data suggest that deleting only one allele of
We assessed whether IGF-1R signaling in transplanted NSCs could affect TMT-supported locomotor recovery of mice following contusion injury. Half the animals received +/+ NSC grafts and the other received +/− grafts, and half the animals in each graft group underwent TMT up to 8 weeks after transplantation. The mice receiving +/+ NSC grafts began to exhibit improved locomotor quality, as measured using BMS, compared with those receiving +/− grafts at 3 weeks after injury and thereafter (Fig. 2A). TMT effect on BMS was noted at 4 weeks and thereafter in mice with +/+ NSC grafts, showing the best recovery of BMS in mice that received +/+ grafts and underwent TMT. Mice that received +/− grafts also exhibited gradual improvement in locomotor function, but the extent of recovery was inferior to that of the +/+ graft group. Although TMT effect was also observed in mice with +/− NSC grafts, TMT-induced recovery was delayed and less pronounced in this group than that in the +/+ graft group. Repeated-measures two-way ANOVA revealed a significant interaction between treatment conditions and the time points after SCI (p<0.001).
In the ladder walk test, mice that received +/+ NSC grafts tended to exhibit hind-paw placement errors less frequently than those with +/− grafts, regardless of TMT, at both 4- and 8-week time points (Fig. 2B, C). TMT reduced the number of errors in both genotype groups, but more evidently in +/+ graft group, resulting in the smallest number of errors in mice with +/+ grafts as well as TMT. At both time points, the effects of both genotype and TMT factors were statistically significant by two-way ANOVA (4-week genotype,
We compared the survival of NSC grafts heterozygous for
We found that different experimental conditions resulted in marked differences in the extent of NSC migration (Fig. 3). When we focused on the distance of the migrating NSCs from the epicenter, +/+ NSC grafts showed a tendency to migrate further than +/− grafts, either rostrally or caudally, at 8 weeks after transplantation (Fig. 5A~D). At this time point, mice that were subjected to TMT exhibited longer migration of +/+ NSCs (Fig. 5A, B). NSCs positioned at the leading edge of the grafts frequently showed elongation of cytoplasmic processes in a rostro-caudal direction. However, this influence of TMT was not obvious in mice that received +/− NSC grafts (Fig. 5C, D). Furthermore, elongated morphology was rarely observed in +/− NSCs. We obtained the migration index (the longest longitudinal migratory distance divided by the longest transverse diameter) for each animal from the longitudinal spinal cord section showing the longest migration. The effect of genotype on the migration index was already significant at 4 weeks by two-way ANOVA (
Given the crucial role of the
Previous studies have reported highly migratory behavior of NSCs in vivo [30,31], but it remains unclear whether NSCs are inherently motile, and if so, how their motility is regulated. Since basal migratory capacity of NSCs was observed in vitro (Fig. 6), we continuously monitored the behavior of cultured neurospheres using a live cell imaging apparatus. Surprisingly, wild-type neurospheres were highly motile (Supplementary video 1). The majority of neurospheres frequently changed their positions by a distance of up to several hundreds of micrometers (Fig. 7A), although not all neurospheres were motile. Individual NSCs occasionally migrated away from the original neurosphere; on the contrary, separate neurospheres occasionally merged together to form larger neurospheres (Supplementary video 1). The highly motile activity of neurospheres was accompanied by dynamic formation and/or retraction of cytoplasmic protrusions resembling filopodia or lamellipodia (Fig. 7B). The motile paths and the changes in cytoplasmic protrusions were quantified by tracking the marks at the center of neurospheres and tip of cytoplasmic protrusions, respectively (Fig. 7A~C). The spontaneous motility of neurospheres with dynamic cytoplasmic protrusions were quite stable during the 4-day culture duration (Fig. 7D, E), indicating that the neurosphere motility was not a transient phenomenon due to a stabilization process following the initial plating of cells. To examine potential signaling pathways involved in the spontaneous motility of neurospheres, cultured neurospheres were treated with various pharmacological inhibitors (Fig. 8). The extent of both neurosphere movement and cytoplasmic protrusions were potently attenuated only by LY294002, a PI3K inhibitor.
Next, we tested whether IGF-1 signaling could regulate neurosphere motility. When IGF-1 was added to the culture medium, not only was the movement of individual neurospheres enhanced but also the proportion of motile neurospheres was increased (Fig. 9A, B) (Supplementary video 2). At the same time, cytoplasmic protrusions were much more dynamic in the IGF-1-treated condition. On the contrary, pharmacological inhibition of IGF-1R using PPP attenuated the extent of neurosphere movement and cytoplasmic protrusions (Fig. 9C) (Supplementary video 3). Finally, neurospheres obtained from NSCs (+/−) for
We have previously found that the IGF-1 concentration in cerebrospinal fluid was increased by almost three folds in animals with SCI following TMT with a protocol similar to the one used in this study . We further demonstrated that the increased IGF-1 exerted protective effects on transplanted NSCs because intrathecal infusion of neutralizing antibodies against IGF-1 markedly attenuated TMT-induced enhancement of NSC graft survival . The current study showed that the protective effects of IGF-1 were cell-autonomous for transplanted NSCs through IGF-1R. Our experiments revealed that deficiency of one allele of
In the behavioral assessment of locomotor recovery, improvement of BMS score was attenuated in mice that received +/− NSC grafts compared with those with +/+ grafts, demonstrating that the recovery of locomotor quality is dependent on the
Although TMT tended to improve the survival of +/+ NSC grafts, the pro-survival effect of TMT was much less obvious in this study than that found in our previous study, where TMT enhanced the survival rate by more than 5-fold in rat SCI model . In our previous study using a rat model, the basal (without TMT) survival rate of transplanted rat NSCs at 8 weeks was only 4.6%, which was less than half of the survival rate in the mice that received +/+ NSCs without TMT (11.0%). The higher basal survival rate in the current study could result in the reduction of TMT effects enhancing NSC survival. It is well known that a contusive injury leads to the formation of cystic cavities in rats but not in mice . It is conceivable that the lack of tissue matrix would provide unfavorable influence on the survival of transplanted NSCs in a rat SCI model . We also speculate that the requirement of IGF-1 signaling to support survival might be different between mouse and rat NSCs. IGF-1 signaling in mouse NSCs might be sufficiently active to support survival without the TMT-induced increase in the availability of IGF-1, whereas the increase in IGF-1 levels might be essential to step up the activation status of IGF-1 signaling in rat NSCs, leading to additional benefits for survival. In contrast to its influence on survival, TMT robustly influenced the migration of transplanted NSCs. The TMT effect was evident only in +/+ NSC grafts, suggesting that the TMT-induced enhancement of NSC migration may be highly dependent on IGF-1 signaling in transplanted NSCs. Therefore, it is possible that IGF-1 signaling in mouse NSCs relevant to survival and migration may have a different threshold for full enactment; a low threshold for survival might make the further activation of IGF-1 signaling by TMT ineffective, while a high threshold for migration might allow the extra IGF-1 activation to contribute to enhanced migration.
It is well known that IGF-1 signaling is implicated in the migration of cancer cells [37,38,39]. However, only a few studies have shown potential involvement of IGF-1 in neural cell migration during development [40,41]. Interestingly, NSCs possess migratory ability toward injury or tumor foci. Several cytokines and growth factors and their receptors have been discovered to mediate the migration in these pathologic settings [30,31,42,43], but a potential role of IGF-1 signaling in this process has not been reported. Our in vitro experiments directly demonstrate that IGF-1 can induce chemotactic migration of NSCs, and that IGF-1R signaling in NSCs is required for the migration behavior. The signaling pathways that mediate robust migratory activity of NSCs toward glioma converge on PI3K . Since PI3K is also an essential downstream target of IGF-1 signaling, it would be no surprise that IGF-1 plays a role in regulating the migration of NSCs.
Intriguingly, we observed dynamic motility of NSCs. Cultured neurospheres spontaneously moved from one place to another, and dynamic cytoplasmic protrusions and/or retractions were noticed along with the neurosphere movement. Actin-based cellular motility has been intensively studied in migratory cell types including leukocytes and tumor cells. Dynamic actin assemblies in the lamellipodia and filopodia, cytoplasmic protrusions at the moving edge of cells, provide contractile forces for cell migration [45,46]. Therefore, the amoeboid movement of NSCs observed in the present study may underlie the migratory behavior of NSCs in vitro and in the lesioned spinal cord in vivo. Indeed, it has been consistently demonstrated that NSCs are able to migrate toward or home in the site of brain injury [30,47,48], and the strong migratory ability of NSCs has been exploited to target brain tumor cells [49,50,51,52]. To the best of our knowledge, however, there is no report on the actin-based amoeboid motility of NSCs characterized by lamellipodia- or filopodia-like cytoplasmic protrusions. Only one study examined contractility and actin assembly of NSCs in the context of migration, but it did not directly demonstrate formation of actin-based cytoplasmic protrusions .
In the present study, the dynamic motility of NSCs was found to be dependent on IGF-1 signaling. Addition of IGF-1 and inhibition of IGF-1R increased and decreased the motility, respectively. Importantly, deletion of one allele of
In conclusion, the current study demonstrates that IGF-1 signaling in transplanted NSCs plays an important role in the regulation of survival and migration of NSC grafts in the lesioned spinal cord. Our data also provide evidence that the benefits of TMT in locomotor recovery are in part dependent on active IGF-1 signaling in the transplanted NSCs. In addition, TMT robustly enhanced migration of NSC grafts only with a full complement of
Time-lapse movie of wild-type neurospheres.en-27-489-s001.mp4
Time-lapse movie of wild-type neurospheres treated with IGF-1.en-27-489-s002.mp4
Time-lapse movie of wild-type neurospheres treated with an IGF-1R inhibitor PPP.en-27-489-s003.mp4
Time-lapse movie of neurospheres heterozygous for