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Human Adult Dental Pulp Stem Cells Enhance Poststroke Functional Recovery Through Non-Neural Replacement Mechanisms Wai Khay Leong, Tanya L. Henshall, [...], and Simon A. Koblar Additional article information

Abstract Human adult dental pulp stem cells (DPSCs), derived from third molar teeth, are multipotent and have the capacity to differentiate into neurons under inductive conditions both in vitro and following transplantation into the avian embryo. In this study, we demonstrate that the intracerebral transplantation of human DPSCs 24 hours following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post-treatment. At this time, 2.3 Âą 0.7% of engrafted cells had survived in the poststroke brain and demonstrated targeted migration toward the stroke lesion. In the peri-infarct striatum, transplanted DPSCs differentiated into astrocytes in preference to neurons. Our data suggest that the dominant mechanism of action underlying DPSC treatment that resulted in enhanced functional recovery is unlikely to be due to neural replacement. Functional improvement is more likely to be mediated through DPSCdependent paracrine effects. This study provides preclinical evidence for the future use of human DPSCs in cell therapy to improve outcome in stroke patients. Keywords: Dental pulp stem cells, Functional recovery, Rat model, Stem cell transplantation, Stroke

Introduction Stroke is the leading cause of long-term adult disability and the second leading single cause of death after cardiovascular disease throughout the world, affecting approximately 15 million new victims and claiming about 5.8 million lives worldwide each year [1]. Cellbased therapy has emerged as a promising approach to treat cerebral ischemia (reviewed in [2]), as it offers an extended time window for treatment as compared with thrombolysis, which requires administration within 4.5 hours of stroke onset. This study focuses on the use of human postnatal dental pulp stem cells (DPSCs) [3], which are derived from third molars (wisdom teeth) and are thought to originate from migrating cranial neural crest cells [4, 5]. Since the discovery of DPSCs a decade ago [3], an increasing number of studies have explored their intrinsic characteristics, properties, and self-renewal capacity, as well as various in vitro and in vivo differentiation and


regenerative capabilities [3, 4, 6–11]. Under inductive culture conditions, DPSCs exhibit osteogenic, dentinogenic, adipogenic, chondrogenic, and neurogenic derivative potentials [3, 6, 11]. When exposed to neuronal inductive media, human adult DPSCs differentiated into mature neuronal cells, which showed functional activity of voltage-dependent sodium and potassium channels [6, 10]. Following transplantation into the developing brain of the avian embryo, DPSCs responded to local environmental cues and acquired a neuronal morphology, and they also induced the chemoattraction of host trigeminal ganglion axons [7]. In addition, the administration of human DPSCs yielded positive therapeutic outcomes in various animal models of disease [12–17]. Intramyocardially injected human DPSCs reduced the infarct size and improved left ventricular function following acute myocardial infarction in rodents [12]. Dental pulp cells also rescued motor neurons following transplantation into hemisected spinal cord in a rat model of spinal cord injury [16], and they induced functional neovascularization in a murine model of hind limb ischemia [13]. The mechanisms of action of exogenous DPSC treatment in various in vivo experiments have been attributed to the secretion of an array of factors resulting in paracrine effects. These factors include the chemokine stromal cell-derived factor-1 (SDF-1, also known as CXCL12) [7] and the growth/trophic factors nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) [4, 16], and vascular endothelial growth factor (VEGF) [18]. These DPSC-secreted factors have been proposed to play important roles in enhancing endogenous restorative processes following injury or disease. The multilineage differentiation potential, regenerative capabilities, and paracrine effects of human DPSCs render them an attractive candidate for cell-based therapy following ischemic stroke. Furthermore, they are easily accessible, have high ex vivo proliferative capacity [3], and can thus be expanded to provide a valuable source of therapeutic material for allogeneic or autologous transplantation. In a recent study on a small number of animals, treatment with neuronal progenitor cells from wisdom teeth showed nonspecific global motor recovery poststroke, as assessed by the elevated body swing test [17]. The current study extends previously published results that demonstrated the neurogenic potential of human DPSCs [6, 7]. Human DPSCs are still a relatively untested progenitor population, and their therapeutic translation to stroke treatment is the aim of this study. In a rodent model of focal cerebral ischemia, we investigated the functional outcome of DPSC treatment using a battery of neurobehavioral tests, as well as the cellular behavior of transplanted DPSCs in the postischemic brain.

Materials and Methods Human DPSC Culture and Preparation Human DPSCs have previously been isolated from adult impacted (failed to emerge fully into its expected position) third molars by digestion of pulp tissue [3], characterized [11], and retrovirally transduced with the fluorescence marker green fluorescent protein (GFP) [7]. DPSCs at passages 3–5 were cryopreserved in 10% dimethyl sulfoxide in fetal calf


serum. Cells were thawed and passaged at least once prior to use for intracerebral transplantation. DPSCs were cultured as previously described [6], in α-modified Eagle's medium (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with 20% fetal calf serum, 100 μM l-ascorbic acid 2-phosphate (Wako, Richmond, VA, http://www.wakousa.com), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. Cells were dissociated with trypsin and resuspended in αmodified Eagle's medium without supplements at a final concentration of 1.5 × 105 cells per microliter. Cell viability was determined by the trypan blue dye exclusion method.

Rodent Middle Cerebral Artery Occlusion and Intracerebral Transplantation Fifty-four adult male Sprague-Dawley rats (Animal Resources Centre, Canning Vale, Western Australia, Australia, http://www.arc.wa.gov.au) weighing 300 ± 20 g were maintained on a 12-hour light/dark cycle with ad libitum access to food and water. Experimental procedures complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2004) and were approved by the Animal Ethics Committee at the Institute of Medical and Veterinary Science (Ethics no. 34/08). Rats were anesthetized with isoflurane and subjected to temporary, focal cerebral ischemia via intraluminal proximal middle cerebral artery occlusion (MCAo) [19, 20]. Rectal temperature was maintained at 37 ± 1°C by a heating pad, and blood oxygen saturation and pulse rate were monitored and maintained within physiological parameters with a pulse oximeter (model 8500AV; Nonin Medical, Plymouth, MN, http://www.nonin.com). In brief, the right common carotid artery, external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery were exposed through blunt dissection, followed by cauterization and ligation of the ECA. A 4-0 monofilament nylon thread (Dynek, Hendon, SA, Australia, http://www.dynek.com) with a silicone-coated tip (diameter 0.35 mm, length 5 mm) was advanced from the ECA stump, through the ICA, and up to the origin of the right middle cerebral artery (MCA). The thread was secured in place, and the incision was closed. After 2 hours of occlusion, rats were reanesthetized, and the thread was withdrawn to allow reperfusion. Rats were allowed to recover from surgery and were assessed for contralateral forelimb flexion by briefly suspending the animal by the tail and for circling behavior on the floor to verify MCAo at 2 hours. At 24 hours post-MCAo, animals were randomly selected to receive human DPSC (n = 31) or vehicle (n = 23) treatment. A total volume of 4 μl cell suspension (6 × 105 cells) was injected (1 μL/minute) into the ipsilateral parenchyma at two stereotaxic coordinates (anterior-posterior, medial-lateral relative to bregma; dorsal-ventral from dura): 2 μl into the striatum (−0.40, +4.00; −5.50 mm) and another 2 μl into the cortex (−0.40, +4.00; −1.75 mm; Fig. 1A, A,1B)1B) using a stereotaxic frame (Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). Vehicle animals were treated similarly, with the administration of α-modified Eagle's medium as the vehicle. All animals received a daily subcutaneous injection of 10 mg/kg cyclosporin A (Novartis, Basel, Switzerland, http://www.novartis.com), as shown in the timeline in Figure 1C.


Figure 1. Intracerebral transplantation sites and experimental time line. Representative coronal rat brain sections stained with 2,3,5-triphenyltetrazolium chloride to visualize infarcted (white) tissue 1 day post-MCAo (A) and anti-human mitochondrial antigen antibody ...

2,3,5-Triphenyltetrazolium Chloride Staining 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to confirm ischemia. Fresh brains were cut into 2-mm slices and incubated in 2% TTC (Sigma-Aldrich) at 37°C for 10 minutes in the dark. Brain sections were color imaged using a LiDE210 flatbed scanner (Canon, Tokyo, http://www.canon.com).

Functional Assessment Animals were trained for 3 consecutive days prior to MCAo, and the third session (day −1) was used as the preoperative baseline. Blinded assessment of behavior was performed during the light cycle at days 1 (prior to DPSC transplantation), 7, 14, 21, and 28 postMCAo (three trials in each test) (Fig. 1C). We excluded animals that failed pre-MCAo neurobehavioral training (in that the animals did not achieve baseline scores in each test) and exhibited either few to none or very severe functional deficits at least 1 week following MCAo. The experiments were undertaken with random treatment assignment and blinded outcome assessment, which conformed to Stroke Therapy Academic Industry Roundtable (STAIR) and similar guidelines [21, 22]. Neuroscore Post-MCAo gross neurological dysfunction for each animal was evaluated using a scoring system modified from previous studies [23–25] (supplemental online Table 1). Higher scores indicate greater neurological impairment. Adhesive Tape Removal Test Adhesive labels (15 mm2) were applied on the medial aspect of the rats' forepaws [26]. The order of sticky label placement onto the contralateral or ipsilateral forepaw was randomized. Upon return to the testing cage, the time spent attempting to remove the adhesive label and the total time used to remove the label (allocated maximum of 2 minutes) from each paw were recorded. The duration spent removing (or attempting to remove) the label was expressed as a percentage of the total time taken. Step Test


Each rat was held firmly using one hand to slightly raise the rat's torso and hind limbs and the other hand to restrain one of the forepaws, so as to bear weight on the other forepaw that was not constrained [27]. The rat was then moved sideways (30 cm) so that the weightbearing forepaw was stepping in the backhand direction to adjust for body movement. The order of forepaw testing was randomized. The number of steps performed with each forelimb was recorded. Rotarod Rats were placed on a rotarod device consisting of a motorized rotating ensemble of 18 × 1mm rods [28]. Animals were required to walk on the apparatus as the rotational speed was accelerated from 0 to 24 rpm within 2 minutes. The time at which the animals completed the trial, fell off the device, or gripped the rungs and spun for two consecutive rotations without attempting to walk on the rungs was recorded. Ledged/Tapered Beam Walking Animals were required to traverse an elevated beam that was tapered along its extent into a darkened box at the end [29], and their performance was videotaped. The number of foot slips made with each hind limb onto an underhanging ledge (2 cm below the upper beam surface) was calculated as a percentage of the total number of steps taken to traverse the beam. Beam walking could not be completed at day 1 post-MCAo because of the circling behavior of stroke animals.

Immunohistochemistry and Imaging At the conclusion of neurobehavioral assessment, rats were deeply anesthetized, injected with 1,000 U of heparin directly into the heart, and transcardially perfused with ice-cold saline followed by 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed and postfixed in the same fixative at 4°C overnight. For colorimetric detection of surviving human DPSCs, brains were embedded in paraffin and cut into serial 5-μm coronal sections. Sections were blocked with 3% normal horse serum in PBS and washed twice in PBS, before incubation with mouse anti-human mitochondria (1:1,500; Millipore, Billerica, MA, http://www.millipore.com), followed by biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and by streptavidin horseradish peroxidase-conjugated antibody (1:1,000; Thermo Scientific, Rockford, IL, http://www.thermoscientific.com). Antibodies were diluted in blocking solution. Antibody staining was visualized with 3,3′diaminobenzidine (Sigma-Aldrich). Bright-field images were acquired and analyzed using the NanoZoomer Digital Pathology System (Hamamatsu Photonics, Shizuoka, Japan, http://www.hamamatsu.com). The number of grafted cells remaining in the rodent brain (n = 3) at 4 weeks post-transplantation was counted in every second 5-μm coronal section, and the distribution of grafted cells was analyzed.


For double immunofluorescence studies, 100-μm free-floating sections were blocked with 10% normal horse serum in PBS containing 0.3% Triton X-100, before human DPSCs were identified using goat anti-GFP (1:250; Rockland, Gilbertsville, PA, http://www.rocklandinc.com), mature neurons using mouse anti-neuronal nuclei (NeuN; 1:500; Millipore) or astrocytes using rabbit anti-glial fibrillary acidic protein (GFAP; 1:250; DAKO, Glostrup, Denmark, http://www.dako.com). Other primary antibodies used were mouse anti-β-III tubulin (1:500; Millipore), mouse anti-nestin (1:100; Abcam, Cambridge, U.K., http://www.abcam.com), mouse anti-polysialic acid-neural cell adhesion molecule (PSANCAM; 1:200; Millipore) and mouse anti-O4 (1:500; Sigma-Aldrich). Secondary antibodies were either Cy2-conjugated anti-goat IgG, Cy3-conjugated anti-rabbit IgG, or Cy3-conjugated anti-mouse IgG (1:300; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Antibodies were diluted in blocking solution. All sections were mounted with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). To estimate the percentage of GFP-positive cells that coexpressed NeuN or GFAP, z-stack images at a magnification of ×40 were taken in five fields, which consisted of two cortical and three striatal regions in the ipsilateral hemisphere at the level of DPSC transplantation (n = 3 animals) (supplemental online Fig. 2). Confocal images were acquired sequentially using a TCS SP5 confocal microscope and were processed using its LAS AF software (Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com). Orthogonal projections were performed using ImageJ (NIH).

Measurement of Corpus Callosum Thickness Paraffin sections, at the levels of bregma and bregma ±0.1 mm, were stained with 0.1% cresyl violet (n = 6 DPSC-treated, n = 2 vehicle-treated). Measurements of corpus callosum thickness were made at the midline (midpoint between the two hemispheres) and at the lateral edge of the ventricles in both hemispheres of the brain. Measurements were expressed as a percentage of corresponding midline measurements and were done using an AxioVision imaging software (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com).

Statistical Analysis Analysis was carried out using GenStat (13th edition; VSN International, Hemel Hempstead, U.K., http://www.vsni.co.uk). The effect of treatment on behavioral test scores was analyzed using repeated-measures analysis of variance (ANOVA), with a GreenhouseGeisser correction to account for correlation between successive measurements of an individual rat, and with adjustments for prestroke data as covariates to allow for possible differences in the initial ability of the rats. A post hoc Fisher's protected least significant difference (LSD) test was carried out if the initial ANOVA was significant. Differences were considered statistically significant at p < .05. Data were presented as box plots using R (version 2.10.1; R Development Core Team, Vienna, Austria, http://www.r-project.org) or as bar graphs using Prism (version 5.02, GraphPad Software, Inc., San Diego, http://www.graphpad.com). Reported values are mean ± SEM.

Results


Transplanted Human DPSCs Enhanced Poststroke Forelimb Sensorimotor Recovery We investigated whether human DPSC treatment following focal cerebral ischemia could improve neurobehavioral function over time. We did not observe any deleterious effects following DPSC treatment in comparison with controls in the regards to morbidity or mortality rates. Mortality rates obtained following intracerebral transplantation were comparable (approximately 9%) in both treatment groups. The clinically relevant rodent model of unilateral MCAo causes substantial ischemic damage to the dorsolateral striatum and the overlying frontal/parietal cortex in the ipsilateral hemisphere of the rodent brain. As a result, animals developed neurobehavioral deficits demonstrated by an increase in neuroscore of 11.7 ± 0.4 points at day 1 poststroke (Fig. 2A). In addition, marked forelimb somatosensory and motor asymmetries were exhibited in the step test (Fig. 2B; a decrease of 82.2 ± 6.6% in the number of steps taken by the contralateral forelimb at day 1 post-MCAo) and the adhesive tape removal test (Fig. 2C; a decrease of 93.7 ± 2.8% in time attempting to remove the external stimulus from the contralateral forepaw at day 1 post-MCAo). Upon experimental stroke, loss of motor balance and coordination was also observed in the rotarod test (Fig. 2D; a decrease of 53.0 ± 2.0% in time spent on the rotarod at day 1 post-MCAo). Stroke-affected animals showed hind limb asymmetry (Fig. 2E, an increase of 21.7 ± 6.2%) in contralesional hind limb footslips during beam walking at 1 week post-MCAo. The ipsilateral limbs had normal poststroke neurobehavioral function (supplemental online Fig. 1).

Figure 2. Effects of human DPSC treatment on post-MCAo neurobehavioral outcome. Following MCAo, all animals exhibited functional deficits in all behavioral tests (day 1 or 7 vs. day −1). (A): At 4 weeks after transplantation, DPSC-treated animals showed ... All animals exhibited gradual recovery in neurobehavioral function over 4 weeks poststroke (Fig. 2). DPSC-treated animals demonstrated significantly enhanced global neurobehavioral function (Fig. 2A; p = .018 group × day interaction, repeated-measures ANOVA) and significantly lower neuroscores in comparison with the vehicle-treated group, specifically at 21 (p < .05, post hoc Fisher's protected LSD) and 28 (p < .01) days poststroke. In limb-specific neurobehavioral studies, DPSC-treated animals demonstrated significantly enhanced contralateral (stroke-affected) forelimb sensorimotor recovery in comparison with vehicle controls in the step test (Fig. 2B; p = .045 overall group effect) and the adhesive tape removal test (Fig. 2C; p = .049 overall group effect). Taken together, DPSC-treated animals showed reduced poststroke deficits in the use of the contralateral forelimb at all days measured. There was no significant difference in ipsilateral limb function between treatment groups (supplemental online Fig. 1). Performances in the


rotarod (Fig. 2D) and the tapered/ledged beam walking (Fig. 2E) tests were not statistically significantly different (p > .05) between treatment groups.

Survival of DPSCs in Ischemic Brain at 4 Weeks Post-Transplantation The findings from the neurobehavioral studies following DPSC transplantation indicated peak benefit at later time points in neurological scores and forelimb sensorimotor function. We then investigated whether the underlying cellular mechanism to explain these findings was due to neural cell replacement following DPSC transplantation into the stroke brain. We quantitated the number of donor cells that had survived at the end point of the study when peak benefit was observed. Individual DPSCs within the rodent brain were visualized using a specific antibody against human mitochondrial antigen, which does not cross-react with rat tissue. The number of labeled cells was counted manually in every second coronal brain section, that is, at 10-μm intervals. The total number of human mitochondria-positive cells detected in the brain of each animal at 4 weeks after transplantation ranged from 9,458 to 22,394 cells (Table 1). This gave an average number of 13,807 ± 4,293 remaining human DPSCs and a mean survival rate of 2.3 ± 0.7% of the originally transplanted 6 × 105 cells. Our data demonstrated that cell transplantation was successful and resulted in long-term viability, albeit at low numbers, of DPSC-derived cells in the postischemic brain.

Table 1. Survival of transplanted human DPSCs at 4 weeks poststroke

Targeted Migration of DPSCs Toward the Stroke Lesion We also analyzed the spatial distribution of human DPSCs following transplantation into the ischemic rat brain. At 24 hours post-transplantation, DPSCs were found clustered around both injection sites (data not shown). After 4 weeks, human mitochondria-labeled cells were found predominantly in the area surrounding the infarct core (Figs. 3, ,4A,4A, A,4B),4B), demonstrating that transplanted cells had migrated from the sites of transplantation preferentially toward the ischemic infarct and localized at the ischemic boundary zone (IBZ).


Figure 3. Distribution of surviving dental pulp stem cells (DPSCs) in postischemic brain. Representative coronal rat brain sections show the survival and migration pattern of transplanted DPSCs (individual cells indicated as black dots) at 4 weeks posttransplantation. ...

Figure 4. Morphology of engrafted human dental pulp stem cells (DPSCs) at 28 days poststroke. High magnification of human DPSCs (human mitochondrial antigen-positive cells stained brown) in the 4-week-post-middle cerebral artery occlusion rodent brain demonstrated ... A small number of DPSCs were observed in the hemisphere contralateral to the site of cell transplantation (Figs. 3, ,4A,4A, A,4B)4B) and in the corpus callosum (Fig. 4B). Cells had migrated at least 4 mm into the contralateral hemisphere over the 4-week period. DPSCderived cells were widely distributed in the contralateral hemisphere at low numbers as compared with the ipsilesional hemisphere, and there was no obvious migration of DPSCs into specific targeted regions of the contralateral hemisphere. On rare occasions we observed DPSC-derived cells incorporated into and around the walls of cerebral microvessels with morphological appearances consistent with endothelial cells (Fig. 4C, arrows), pericytes, or smooth muscle cells (Fig. 4C, arrowhead).

Transplanted Human DPSCs Differentiated into Neurons and Astrocytes In Vivo We then examined the morphologies of human mitochondrial antigen-positive cells in the 4-week postischemic brain. The cells appeared to have morphologies consistent with astrocytic cells that demonstrated a stellar-like appearance (Fig. 4D, arrows), whereas presumptive neuronal cells exhibited long processes (Fig. 4D, arrowhead). To verify these phenotypes, we used double immunofluorescence analysis and confocal imaging. Transplanted human DPSCs had previously been retrovirally transduced to express GFP, which allowed colabeling with either NeuN or GFAP to identify mature neurons or astrocytes, respectively. Colocalization of GFAP with GFP expression in cells within the glial scar confirmed that DPSC-derived cells had differentiated into astrocytes (Fig. 5A, A,5B).5B). Within the ipsilateral peri-infarct cortical region, NeuN and GFP doublepositive cells indicated differentiation of transplanted DPSCs into neurons (Fig. 5C, C,55D).


Figure 5. Transplanted human dental pulp stem cells (DPSCs) adopted neuronal and astrocytic phenotypes. Confocal double immunofluorescence images showed that engrafted human DPSCs (green, GFP-labeled cells) coexpressed GFAP (red) in the striatal ischemic boundary ... The distribution of astrocytes and neurons in the postischemic ipsilateral cerebral hemisphere was not uniform. We found at the IBZ in the striatal region that DPSC-derived astrocytes predominated (51.0 ± 8.6% GFAP+/GFP+ double-positive cells, expressed as a percentage of GFP single-labeled cells), whereas in comparison, there was a smaller proportion of DPSC-derived neurons (8.7 ± 6.1% NeuN+/GFP+ cells) (supplemental online Fig. 2). In the peri-infarct cortical region, the proportion of astrocytes and neurons appeared similar (38 ± 0.6% GFAP+/GFP+ cells and 32.1 ± 1.8% NeuN+/GFP+ cells respectively). Transplanted DPSCs found along the IBZ in the striatum predominantly differentiated into astrocytes, which appeared to be incorporated into the glial scar. There were more DPSCderived neurons present in the cortical peri-infarct region as compared with the striatum. Of interest, approximately one-third of GFP+ cells in the peri-infarct cortical region were also labeled with the early/intermediate neuronal marker β-III tubulin (supplemental online Fig. 3A), although it was difficult to find β-III tubulin+/GFP+ double-positive cells in the peri-infarct striatum (supplemental online Fig. 3B). Within the glial scar along the IBZ, however, many GFP+ cells also coexpressed the neural progenitor marker nestin (supplemental online Fig. 3C, 3D). There was little evidence of nestin+/GFP+ cells in the cortex. A few GFP+ cells were also positive for the migratory neuroblast marker PSANCAM in the peri-infarct cortex (supplemental online Fig. 3E). We were unable to detect PSA-NCAM+/GFP+ double-labeled cells in the rostral migratory stream (supplemental online Fig. 3F). On rare events we observed DPSC-derived oligodendrocytes (O4+/GFP+ cells) in the rodent brain at 4 weeks post-transplantation (supplemental online Fig. 4).

DPSC Treatment Reduced Poststroke Corpus Callosum Atrophy We previously demonstrated that exogenous DPSCs could influence host axonal remodeling [7]. We were therefore interested to investigate whether DPSC treatment had any effects on the loss of interhemispheric connectivity across the corpus callosum following a stroke insult. Using a similar approach [30], we found that at 4 weeks poststroke, there was a nonsignificant decrease in ipsilateral corpus callosum atrophy in DPSC-treated animals in comparison with the vehicle-treated group (DPSC-treated, 67.6 ± 12.5%, L2 measurements expressed as a percentage of corresponding midline L1 measurements; vehicle-treated, 56.6 ± 3.0%; p > .05) (Fig. 6). In DPSC-treated brains, the thickness of the corpus callosum in the ipsilateral hemisphere showed a trend toward better


preservation and attained a similar corpus callosum thickness of the contralesional (strokeunaffected) hemisphere. There was no difference in corpus callosum thickness in the contralateral hemisphere between treatment groups (DPSC-treated, 66.1 Âą 14.0%; vehicletreated, 66.5 Âą 1.6%).

Figure 6. Effects of DPSC treatment on ipsilesional CC atrophy. (A): Representative rat brain section at the level of bregma shows the two sites used for the measurement of CC thickness at the midline of the brain (L1) and at the lateral edge of the lateral ventricles ...

Discussion Our study demonstrated that intracerebrally transplanted human adult DPSCs enhanced neurobehavioral recovery in a rodent model of focal cerebral ischemia. There was a difference in the degree of benefit, with more evident contralateral forelimb functional improvement in comparison with hind limb function. The time of greatest neurobehavioral improvement was evident at 3 and 4 weeks after experimental stroke. At 4 weeks poststroke, we found that an average of 2.3% of the originally transplanted DPSCs had survived in the rodent brain. Surviving DPSCs had differentiated into astrocytes and neurons in the postischemic brain. There appeared to be an environmental influence on the lineage fate of transplanted DPSCs, with a predominance of DPSC-derived astrocytes in the striatal IBZ proximal to the stroke, whereas more DPSC-derived neurons were found in the cortical peri-infarct region (farther away from the infarct core) than in the peri-infarct striatum. Our data suggest that the cellular and molecular mechanisms underlying the DPSC-derived benefit were unlikely to be explained solely through neural cell replacement. We did not observe a similar neurobehavioral response in hind limb and trunk function in this rodent stroke model following intracerebral DPSC transplantation. One possible explanation for the difference in enhanced functional improvement between the limbs is that this rodent MCAo model produces more destruction to the sensorimotor cortical representation for forelimb function in the rodent brain because of preferential vascular supply to this region [31]. In support of this, we observed that there was a severe loss of forelimb function as measured by the step and adhesive tape removal tests, but a less dramatic impairment of hind limb function as evaluated by the tapered/ledged beam walking test immediately post-MCAo. Rotarod running and beam walking assess motor coordination and hind limb function, respectively [32], and these tests might not be sensitive enough to detect poststroke deficits. The enhanced functional improvement following DPSC treatment in comparison with vehicle-treated animals became evident over the 4 weeks of analysis of forelimb but not hind limb function.


An alternative explanation may relate to learned compensatory motor behaviors in the animal. In the rotarod running and tapered/ledged beam walking tests, through repeated testing, rodents may have learned novel ways in which stroke-unaffected limbs or tail deviation can be used to compensate for loss of function in the stroke-affected limb [33]. Forelimb-specific tasks allowed minimal use of compensatory behaviors to overcome poststroke loss of sensorimotor function, as measured by the step and adhesive tape removal tests. The adult brain has endogenous reparative and restorative processes with which to counter damage after an insult such as ischemia [34]. Cell-based treatments provide a therapeutic strategy to increase poststroke functional recovery by enhancing the brain's self-repair process [35â&#x20AC;&#x201C;37]. Our study has demonstrated that human DPSC treatment enhanced functional recovery up to 4 weeks following ischemic stroke in a rodent MCAo model. We found that, on average, 2.3% of the originally 6 Ă&#x2014; 105 DPSCs injected into the brain had survived at 4 weeks following transplantation. This would argue against the notion of DPSC-enhanced functional recovery being dependent on neural replacement alone. Our data are consistent with other studies in which minimal cell survival and neuronal differentiation were found, albeit with cell therapy benefiting functional outcome following stroke [38â&#x20AC;&#x201C;41]. A larger proportion of transplanted DPSCs was detected in one animal, with approximately double the number of cells having survived at 4 weeks posttransplantation in comparison with the other two animals. One possible explanation may relate to variation in the size of MCAo-induced cerebral infarct between animals due to differences in collateral circulation to the MCA territory, which is known when using Sprague-Dawley rats, and the impact this may have on transplanted cell survival [42, 43]. However, even with such variation, there were low numbers of DPSC-derived cells present in the 4-week postischemic brain. There was no evidence of cell preparation or technical problems that could possibly lead to the low number of surviving DPSCs. We do not exclude the possibility that transplanted cells might be immunorejected by the host brain, although all animals were given daily therapeutic doses of cyclosporin. In this study, we demonstrated the chemoattraction of transplanted DPSCs to the core region of focal ischemia from the site of initial intracerebral injection. Others have suggested that this feature of progenitor/stem cells may be crucial to postischemic functional recovery, as the capacity for brain repair is enhanced at the peri-infarct region where tissue damage is reversible [37, 44]. This targeted migration is indicative of signaling between host parenchymal cells and transplanted donor cells following ischemic stroke. It has been reported that cerebral ischemia results in the activation of endothelial cells in the IBZ, which secretes the chemokine SDF-1 [37]. Bone marrow cells [45], mesenchymal stem cells [46], and neural stem cells [47] used in models of brain injury, such as ischemia, have been shown to express the chemokine receptor CXCR4, which is the cognate receptor to SDF-1. We have demonstrated previously that when injected into the embryonic avian hindbrain, DPSCs induced axon guidance of host trigeminal ganglion axons via the SDF-1/CXCR4 chemokine signaling pathway [7]. Human DPSCs, when injected into this embryonic paradigm, also tended to aggregate, which we suspect may be due to expression of the CXCR4 receptor by the exogenous cells (supplemental online Fig. 5). We thus hypothesize that transplanted DPSCs migrated in a targeted manner toward the


stroke lesion via SDF-1/CXCR4 chemokine signaling. Additional studies will be required to substantiate this hypothesis. The previously observed effects of DPSCs on host axonal remodeling [7] led us to postulate that the cells might have a similar effect on the rescue of progressive corpus callosum atrophy following ischemic stroke. Indeed, we showed that there was a trend toward better preservation of interhemispheric axon projections in the postischemic brains of DPSC-treated animals in comparison with vehicle controls, although this did not reach statistical significance. The concept that stem/progenitor cells express an array of factors that may interact with host tissue is important and may underlie the non-neural replacement mechanisms of action of cell-based therapy that results in restoration of neurological function in a range of injury or disease scenarios [48]. Human dental pulp cells are known to secrete a range of factors, including NGF, BDNF, GDNF, and SDF-1 [4, 7]. Some of these factors have been shown to be beneficial specifically in ischemic stroke: BDNF provides trophic support and prevents parenchymal cells in the penumbra from further degradation [49], whereas GDNF secretion by reactive astrocytes along the IBZ has been associated with the rescue of dying cells in the ischemic border [50]. At 4 weeks poststroke, few DPSC-derived cells remain in the rodent brain; however, a large proportion of these cells had differentiated into GFAPexpressing cells, indicative of astrocytes. It is possible that these DPSC-derived GFAPpositive cells may also play active roles in improving neurological function after stroke, as suggested by other investigators [51]. Within the glial scar of the IBZ, expression of nestin by DPSC-derived cells suggests that these cells may undergo a recapitulation of neurodevelopmental molecular pathways. The observed engraftment of DPSC-derived cells into the wall of cerebral microvessels was a rare occurrence in this study. This observation is consistent with previous studies that showed that transplanted DPSCs [12, 52] and bone marrow-derived mesenchymal stem cells [12, 52] following myocardial infarction infrequently differentiated into endothelial, cardiac or smooth muscle cells. This suggests that transplanted stem/progenitor cells may not be involved structurally in the formation of new blood vessels following cerebral ischemia but instead may play a role in secreting factors, such as VEGF, to enhance endogenous angiogenesis and neurogenesis [53]. Recently, human VEGF was shown to be an important molecule contributing to cell-enhanced functional recovery following ischemic stroke [54]. Transplanted DPSCs may also play an immunomodulatory role through varied secreted factors to improve function after stroke. We and others have previously reported the immunomodulation properties of DPSCs to dampen immune responses, similarly to the properties described for other mesenchymal stem cell-like populations [55â&#x20AC;&#x201C;57].

Conclusion This study provides preclinical evidence of the therapeutic efficacy of human adult DPSCs to enhance the recovery of poststroke sensorimotor deficits. DPSCs isolated from third molars are a clinically accessible source of stem cells. In this study, we transplanted DPSCs


directly into the brain; however, with minimal cell survival at 4 weeks poststroke and targeted cell migration toward the stroke lesion being evident, vascular routes of administration will be worth pursuing. Additional studies will focus on the underlying mechanisms of action of candidate paracrine factors produced by human adult DPSCs.

Acknowledgments We thank Austin Milton (Basil Hetzel Institute for Medical Research, Queen Elizabeth Hospital, Woodville, South Australia, Australia) for participation in blinded assessment of neurobehavioral studies. This work was funded by grants from the Robinson Institute Centre for Stem Cell Research, the University of Adelaide, the Catholic Archdiocese of Sydney, and the Peter Couche Foundation, Stroke SA. W.K.L. is supported by graduate scholarships from the University of Adelaide and the Australian government.

Author Contributions W.K.L.: collection and assembly of data, data analysis and interpretation, manuscript writing; T.L.H.: stroke model, collection and assembly of data, data analysis and interpretation, manuscript writing; A.A.: conception and design, setup of stroke model, provision of study material, manuscript review; K.L.K.: collection and assembly of data, data analysis, manuscript writing and review; M.D.L.: involvement in study and data interpretation, manuscript review; S.C.H.: teaching of stroke model including anesthesia, support with behavioral tests, manuscript review; J.F.: statistical support and analysis; M.A.H.-B.: study discussion and manuscript review; S.W.: data collection (blinded behavioral studies), manuscript review; J.M.: data collection (immunohistochemistry), manuscript review; R.V.: technical support for study and assistance with stroke model, manuscript review; S.G.: conception and design, provision of study material, financial support, manuscript review, final approval of manuscript; S.A.K.: conception and design, financial support, data analysis and interpretation, manuscript review, final approval of manuscript.

Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest.

Article information Stem Cells Transl Med. 2012 March; 1(3): 177–187. Published online 2012 February 22. doi: 10.5966/sctm.2011-0039 PMCID: PMC3659845 Wai Khay Leong,a,b,* Tanya L. Henshall,c,* Agnes Arthur,a,c,* Karlea L. Kremer,a,b,* Martin D. Lewis,a,b Stephen C. Helps,d John Field,e Monica A. Hamilton-Bruce,f Scott Warming,b Jim Manavis,g Robert Vink,d Stan Gronthos,a,c,† and Simon A. Koblar a,e,f,† a Centre for Stem Cell Research, Robinson Institute, and Schools of bMolecular and Biomedical Science,


d

Medical Science, and Medicine, University of Adelaide, Adelaide, South Australia, Australia; c Division of Hematology and g Centre for Neurological Diseases, Hanson Institute, SA Pathology, Adelaide, South Australia, Australia; f Department of Neurology, Queen Elizabeth Hospital, Woodville, South Australia, Australia Corresponding author. *Contributed equally as first authors. †Co-senior authors. Correspondence: Simon A. Koblar, M.D., Ph.D., School of Medicine, University of Adelaide, Adelaide, SA 5005, Australia. Telephone: +61-88222-6740 (office), +61-416928658 (mobile); Fax: +61-88222-6042, e-mail: simon.koblar/at/adelaide.edu.au Received October 12, 2011; Accepted January 17, 2012. Copyright ©AlphaMed Press 1066-5099/2012/$20.00/0 Articles from Stem Cells Translational Medicine are provided here courtesy of AlphaMed Press e

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Human adult dental pulp stem cells enhance poststroke functional recovery through non  
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