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Abstract

Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic–ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121⁺ cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121⁺ NSCs (p = 0.0125, p = 0.0098), fewer FJC⁺ neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN⁺ expression (p= 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4, VCAM-1, Thrombospondins 1 and 2, and BDNF. BDNF pretreatment of NSCs results in higher initial NSC engraftment and survival, increased neuroprotection, and greater functional recovery when compared to untreated NSCs.

Keywords

Neural stem cells (NSCs)Brain-derived neurotrophic factor (BDNF)Hypoxic–ischemic (HI) stroke Intravascular transplantation Pretreatment

Introduction

Intravascular stem cell therapies are currently being evaluated for safety and efficacy in humans (6) with the help of promising studies demonstrating cell engraftment and functional recovery following intravascular transplantation in rodent models of hypoxic–ischemic (HI) stroke (3,13,23). In the field of intravascular neural stem cell (NSC) transplantation, our lab has shown a direct correlation between the number of cells surviving in the brain after intra-arterial (IA) transplantation and positive functional outcomes (13). However, intravascular transplantation of human NSCs into rodents has often demonstrated a very low rate of cell engraftment (5). Therefore, we sought a way to improve cell engraftment following transplantation in the hopes of improving functional outcomes after stroke.

Here we chose brain-derived neurotrophic factor (BDNF) because of its well-known positive influence on NSC survival, proliferation, and apoptosis (1,7,17,19,22,39) and because NSCs of both embryonic and fetal origin have been shown to express the BDNF receptor trkB (7,19). Additionally, BDNF has been shown to influence NSC migratory pathways (17,31,39), which are of great importance to the success of intravascular therapies. In vitro pretreatment of NSCs with BDNF concentrations between 20 and 100 ng/ml has been shown to increase proliferation and migration (39). Furthermore, in vivo studies in rodent models of traumatic brain injury using NSCs overexpressing BDNF have shown both increased transplanted cell survival and better functional outcomes compared to naive cell transplants (22,25). One of these studies in particular demonstrated human NSCs over-expressing BDNF had increased survival, decreased cell death, and increased angiogenesis for as long as 8 weeks posttransplant (22). Human NSCs genetically modified to overexpress BDNF downstream target v-akt murine thymoma viral oncogene homolog 1 (Aktl; protein kinase Bα) have also demonstrated a similar increase in both cell survival and functional outcomes compared to naive cell transplants (21). These positive results lead us to hypothesize that pretreatment with BDNF may induce a similar profile in our IA transplantation model and improve cell survival, migration, and functional outcomes following experimental HI stroke.

Here we used in vivo bioluminescence imaging (BLI) and immunohistochemistry to study how BDNF pretreatment affects migration and engraftment of human NSCs in the rodent brain after HI stroke. Through immunocytochemistry, RT-qPCR, and proteomics, we studied the molecular and cellular changes of BDNF-treated NSCs in vitro in order to elucidate mechanisms through which BDNF may influence observed in vivo effects.

Materials and Methods

Cell Culture

Human embryonic-derived NSCs (SCR055; Millipore, Billerica, MA, USA) were transduced as previously described (30,34). In short, a bifusion construct containing firefly luciferase and enhanced green fluorescent protein was inserted into a lentiviral vector. The vector was obtained as a gift from the lab of Sanjiv Gambhir (Stanford University, Stanford, CA, USA). Construction and validation was performed as previously described (30,34). Viral particles were produced in 293T human embryonic kidney cells (American Type Culture Collection, Manassas, VA, USA). NSCs were suspended with high-titer (>10⁹ particles/ml) viral supernatants, centrifuged at 1,800 × g for 3 h at 32°C, and incubated overnight at 37°C in 5% CO₂. Successfully transduced cells were isolated using a three-laser fluorescence-activated cell sorter (Becton Dickinson, San Jose, CA, USA). Once sorted, NSCs were grown as a monolayer on poly-L-ornithine (P3655; Sigma-Aldrich, St. Louis, MO, USA) and laminin (L2020; Sigma-Aldrich)-coated tissue culture plates (BD Biosciences, San Jose, CA, USA) at 37°C in a 5% CO₂ humidified incubator. Cells were fed every other day with ENStem-A Neural Expansion Media (SCM004; Millipore) supplemented with basic fibroblast growth factor (bFGF; GF003; Millipore) to a final concentration of 100 ng/ml and 1% GlutaMAX (35050; Invitrogen, Waltham, MA, USA). Cells were split using Accutase (SCR005; Millipore) when plates reached 95-100% confluency.

Hypoxic–ischemic Stroke Model

All animal procedures were approved by Stanford University's Administrative Panel on Laboratory Animal Care. Brain ischemia was induced using a HI stroke model as previously described (3,13,30). Ten-week-old male nonobese diabetic/severe combined immunodeficiency mice (Taconic Farms, Oxnard, CA, USA) underwent temporary left common carotid artery occlusion with a Yasargil Titanium Aneurysm Clip (Aesculap, Center Valley, PA, USA) before being placed in hypoxic conditions (8% O₂) at 37°C for 15 min. After hypoxia, reperfusion was achieved by removal of the aneurysm clip.

Cell Transplantation

Prior to transplantation, NSCs were pretreated with BDNF (248-BD-025; R&D Systems, Minneapolis, MN, USA) at a concentration of 100 ng/ml for 1 h in a single cell suspension, and the two control groups received untreated cells or saline only. For the behavior groups, mice were randomly assigned to receive BDNF pretreated NSCs (n = 9), untreated NSCs (n = 8), or saline injection at 3 days after HI stroke (n = 10). After group assignment, the common carotid artery was reexposed, and a single cell suspension of 5 × 10⁵ NSCs or saline was injected in 5 μl using a custom 10-μl Hamilton (Reno, NV, USA) syringe with a 33-gauge needle (3,11,30). No significant differences in mortality were observed between groups. One animal was lost in both the BDNF pretreated and untreated NSC groups (no animals were lost in the control group).

Bioluminescence Imaging

BLI was conducted using the IVIS Spectrum system (Xenogen Corporation, Alameda, CA, USA). Mice were given an intraperitoneal injection of 200 μl D-luciferin [15 mg/ml in phosphate-buffered saline (PBS); Promega, Madison, WI, USA]. Whole-body images were acquired for 1 min. BLI signal was recorded as maximum photon flux (photons/second). Living Image 3.0 software (Caliper Life Sciences, Hopkinton, MA, USA) was used to quantify maximum photon flux in regions of interest (ROIs) in the head. We have previously shown that the photon flux has a strong linear correlation with the number of cells in the brain (35).

Immunohistochemistry and Immunocytochemistry

Animals were transcardially perfused with PBS (100100-49; Invitrogen) followed by 4% paraformaldehyde (O4042; Fisher Scientific, Waltham, MA, USA). After removal, brains were fixed for 48 h and placed in 30% sucrose (Sigma-Aldrich) in PBS. Brains were then cryosectioned at 30 mm using a microtome. For fluorescent immunohistochemistry, sections were blocked in PBS 0.3% triton (HFH10; Invitrogen) with 5% goat serum (S1000; Vector Labs, Burlingame, CA, USA) and 1% bovine serum albumin (BSA; A2153; Sigma-Aldrich) for 1 h at room temperature. Primary antibody was incubated overnight (12-14 h) at 4°C in a 1:10 dilution of blocking solution (5% goat serum/1% BSA) with the following antibodies: anti-SC-121 (human cytoplasmic marker; 1:1,000; Stem Cell Inc., Newark, CA, USA), anti-neuronal nuclei (NeuN; mab377; 1:250; Millipore). The secondary antibody was used at 1:500 (Alexa Fluor 488; Invitrogen) at 4°C for 6 h, followed by nuclear stain with 4,6-diamidino-2-phenylindole (DAPI; 1:5,000; AnaSpec, San Jose, CA, USA). Sections were visualized on a laser scanning confocal microscope (LSM510; Zeiss, Peabody, MA, USA).

For immunocytochemistry, BDNF-treated NSCs and untreated NSCs were grown on four-well glass chamber slides (PEZGS0416; Millipore). After 7 days, cells were fixed in ice-cold methanol (Fisher Scientific) for 25 min at 4°C. Cells were blocked for 1 h in a PBS/10% goat serum solution at room temperature. Primary antibody was incubated for 8-10 h at 4°C in PBS/1% goat serum. The appropriate secondary antibody was used at 1:500 (Alexa Fluor 488, Alexa Fluor 546; Invitrogen) at 4°C for 4 h followed by DAPI. The following antibodies were used for immunocytochemistry: anti-Nestin (PRB570C; 1:250; Covance, Princeton, NJ, USA), anti-glial fibrillary acidic protein (GFAP; Z033429; 1:500; Dako, Glostrup, Denmark), anti-oligodendrocyte lineage transcription factor 2 (Olig2; AF2418; 1:250; R&D Systems), anti-NeuN (mab377; 1:100; Millipore), anti-chemokine C-X-C motif receptor 4 (CXCR4; ab2090; 1:100; Abcam, Cambridge, UK).

Fluoro-Jade C Staining

Fluoro-Jade C (FJC) staining, a marker of neurodegeneration after stroke (8), was assessed 7 days after transplantation (10 days after HI stroke) (n = 4/group). Sections were mounted, washed in distilled water, and immersed in 1% sodium hydroxide (S5881; Sigma-Aldrich)/80% ethanol for 5 min. Following this step, slides were rinsed for 2 min in 70% ethanol, 2 min in distilled water, and then incubated in 0.06% potassium permanganate (223468; Sigma-Aldrich) solution for 10 min. Slides were then transferred into a 0.0001% solution of FJC (AG325; Millipore) dissolved in 0.1% acetic acid (695092; Sigma-Aldrich) for 10 min and rinsed. Slides were then dried on a slide warmer at 50°C for 5 min, cleared in xylene for 1 min, and coverslipped with DPX (06522; Sigma-Aldrich).

Cell Quantification

SC-121⁺ cells in the cortex were quantified in three representative sections by selecting two ROIs/section within the peri-infarct region. SC-121⁺ cells in the hippocampus were quantified in two representative sections with one ROI selected per section. Representative sections were selected within a 2-mm region, reaching from 3 mm behind the frontal cortex to 5 mm behind the frontal cortex. Representative sections were 400 μm apart (five representative sections, three from the cortex, two from the hippocampus; each representative section 400 μm apart, for a total of 2 mm of brain analyzed) and chosen by an investigator blinded to treatment groups. ROIs in peri-infarct regions were chosen based on previous data from our lab illustrating the commonly affected regions of the brain after HI stroke (30,35). A different blinded investigator then performed the unbiased counting of SC-121⁺ cells. Z-stack reconstructions were used for quantification. To evaluate the extent of neuronal degeneration, cortical and hippocampal FJC⁺ cells were quantified using five representative sections to evaluate the cortex and three representative sections to evaluate the hippocampus with five ROIs/section. Blinded investigators were used in the same fashion as was done to quantify FJC⁺ cells. All cell counts were performed using ImageJ analysis software (NIH, Bethesda, MD, USA). Lastly, NeuN was used as a marker to assess neuroprotection after cell transplantation because NeuN can be lost in areas of ischemic damage, and this loss can be attenuated by neuroprotective mechanisms (2). Specifically, NeuN⁺ staining was quantified by dividing the cortical area (mm²) in ipsilateral hemisphere demonstrating NeuN positivity by the total ipsilateral cortical area (mm²). NeuN staining was analyzed by blinded observers using ImageJ, by outlining areas of positivity defined above a fixed threshold gray value, which was set at a level just below specific staining to discard nonspecific staining. The same threshold value was used for all analyzed sections, all stained with the identical protocol.

Horizontal Ladder Behavioral Testing

Animals were trained to run across a 3-foot horizontal ladder (TAP Plastics, Mountain View, CA, USA) with evenly spaced bars as previously described (3,28) for 3 consecutive days prior to treatment, with each training session consisting of three ladder crossings per mouse. Scores were based on the function of the contralateral forepaw and were quantified on a scale from 0 to 6, where 0 represented a complete miss and 6 represented a perfect step. Following the baseline score 1 day prior to induction of HI stroke, testing was carried out 2, 6, 10, 17, 24, and 31 days after HI stroke. Animals were excluded from the study if their score did not decrease by at least 10% at 2 days after HI stroke (1 day prior to transplantation). A total of 11 animals were excluded before transplantation as a result of this criterion. Videos were scored by two blinded reviewers and averaged for the final score. If scores differed by more than two standard deviations from the average difference in scores, the mouse was reevaluated by both reviewers.

Gene Expression Analysis

Gene expression analysis was done in triplicate for both BDNF-treated NSCs and untreated NSCs using independent cultures for each sample. RNA was isolated using an RNeasy mini kit (74104; Qiagen, Valencia, CA, USA) and transcribed to cDNA using the RT² HT first-strand synthesis kit (33041; SABiosciences, Valencia, CA, USA) according to manufacturer's instructions. Gene array analysis was carried out using RT² Real-Time SYBR Green PCR Master Mix (330523; SABiosciences) on a Stratagene Mx300P (La Jolla, CA, USA). Gene expression of BDNF-treated NSCs and untreated NSCs were evaluated on chemokine (PAHS-022; SABiosciences) and adhesion molecule (PAHS-013; SABiosciences) arrays. Data were analyzed using SABiosciences data analysis software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php), and a positive result was taken as any gene with expression earlier than 34 cycles. Data were normalized using housekeeping genes and converted to a simple ratio for comparison between groups. A normalized expression ratio of 0.0 corresponds to no expression, while 1.0 indicates expression equivalent to that of the housekeeping genes. Genes with a greater than twofold change in expression were considered significant when compared between groups.

Luminex Immunological Assay

To evaluate protein expression, NSCs were plated at a density of 500,000 cells/ml of media (n = 3 for both BDNF-treated NSCs and untreated NSCs). Twenty-four hours after plating, all conditioned media were removed and replaced with new media supplemented with bFGF (100 ng/ml) and 1% GlutaMAX as described above. Supernatant was collected 48 h later, a proteinase inhibitor (P1860; Sigma-Aldrich) was added, and samples were flash frozen. In coordination with the Human Immune Monitoring Center at Stanford University, Human-51-plex kits (Affymetrix, Santa Clara, CA, USA) were used according to the manufacturer’ s recommendations to screen for secreted cytokines and inflammatory proteins. Briefly, samples were mixed with antibody-linked polystyrene beads on 96-well filter-bottom plates and incubated at room temperature for 2 h, followed by overnight incubation at 4°C. Plates were vacuum filtered and washed twice with wash buffer, then incubated with a biotinylated detection antibody for 2 h at room temperature. Samples were filtered, washed, and resuspended in streptavidin–phycoerythrin. After incubation for 40 min at room temperature and two additional vacuum washes, the samples were resuspended in Reading Buffer. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument (Austin, TX, USA) with a lower bound of 100 beads per sample per cytokine.

Boyden Chamber Migration Assay

Modified Boyden chamber migration assay (CytoSelect, CBA-106; Cell Biolabs, San Diego, CA, USA) was used to evaluate NSC migratory capacity as previously described (35). Chemoattractant stromal cell-derived factor-1 (SDF-1) (300-28A; Peprotech, Rocky Hill, NJ, USA) was placed in the chambers at concentrations of 0, 100, 500, 1,000, and 1,500 ng/ml. Monocyte chemoattractant protein 1 (MCP-1; AF-300-04; Peprotech) concentrations of 0, 5, 10, 40, and 70 ng/ml were also evaluated. A standard dilution curve (n = 3, serial dilutions from 0 to 250,000 cells) demonstrated a strong linear correlation (r = 0.9440, p < 0.0001) between number of cells and fluorescence. Total fluorescence was normalized to fluorescent readings from wells containing 0 ng/ml of SDF-1 or MCP-1 in PBS.

Statistical Analysis

All statistical analyses were completed using Prism (GraphPad, San Diego, CA, USA). Quantitative data were expressed as mean ± SEM. Means were compared using a one-way ANOVA and the Student's t-test. The Newman–Keuls method of correction was used for comparison between multiple groups. Behavioral analysis was performed using a two-way ANOVA. Correlations were computed using the Pearson correlation test. Results with p < 0.05 were considered statistically significant.

Results

Treatment of NSCs with BDNF Increases NSC Engraftment and Survival in the Brain

Survival of NSCs evaluated using in vivo BLI demonstrated a significant difference between the three treatment groups (p = 0.0011) over time (p < 0.001) (Fig. 1A, B) on two-way ANOVA analysis. On individual days of testing, significance was found between the BDNF-treated NSC group and untreated NSCs group as late as 7 days after IA injection (p < 0.049) (Fig. 1A, B). Immunohistochemistry corroborated this finding as significantly more NSCs were found in the cortex (p = 0.0126) (Fig. 1C) and hippocampus (p = 0.0098) (Fig. 1D) of animals injected with BDNF-treated NSCs. One week after transplantation, the BDNF-treated NSC group had an average photon flux of 3.85 × 10⁵ ± 8.33 × 10⁴ photons/s and histological counts of 130.5 ± 35.4 cells/mm² in the cortex and 87.7 ± 24.3 cells/mm² in the hippocampus. The untreated NSC group had an average photon flux of 3.12 × 10⁵ ± 3.39 × 10⁴ photons/s and histological counts of 67.5 ± 21.3 cells/mm² in the cortex and 46.4 ± 9.6 cells/mm² in the hippocampus. In both cell-treated groups, NSCs were found primarily in the stroked hemisphere. Although BLI signals for both cell treatment groups were significantly greater than the control group 28 days after transplant, no cells were seen histologically in any group.

Figure 1

NSC homing and survival. BLI was used to track NSC survival in vivo for 1 month after transplantation. Representative images of animals in the BDNF-treated NSC, untreated NSC, and control groups at 1, 7, and 28 days after transplantation are shown (A). BLI demonstrated a significant difference between the three treatment groups (p = 0.0011) over time (p < 0.001) (B). On individual days of testing, significantly higher photon flux was found in the BDNF-treated NSC group compared to both the untreated NSC and control groups at 2 days (p =0.045, p = 0.024, respectively), 6 days (p = 0.025, p = 0.0026), and 7 days (p = 0.049, p = 0.0021) after cell transplantation (B). Histology to assess surviving NSCs 7 days after transplantation revealed significantly more cells in the cortex (p = 0.0126) (C) and hippocampus (p = 0.0098) (D) of the BNDF-treated NSC group when compared to the untreated NSC group. Scale bar: 50 μm. Images show human cytoplasmic marker (SC-121) staining in green with nuclear staining (DAPI) in blue.

Treating NSCs with BDNF Prior to Transplant Results in Greater Functional Recovery

Using a two-way ANOVA, the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC group and control group (p < 0.001) over the 1-month period (p < 0.001) following transplantation as measured by the horizontal ladder test (Fig. 2A). On individual days of behavioral testing, a two-way ANOVA Newman–Keuls posttest revealed a significant improvement in performance in the BDNF-treated NSC group compared to the control group 14, 21, and 28 (p < 0.05) days after transplant (17, 24, and 31 days after HI stroke). Although animals injected with untreated NSCs performed better than animals in the control group, the trend did not reach significance on any individual day over the entire 1-month period. Bioluminescence signal 1 week after transplant correlated positively with functional outcome 2 weeks (r = 0.5634, p = 0.0230) and 4 weeks after transplant (r = 0.5328, p = 0.0409) (Fig. 2B). BLI also correlated with outcome at 3 weeks, but did not reach significance (r =0.4587, p = 0.0739).

Figure 2

Functional outcome. Two-way ANOVA revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC group and control group (p < 0.001) over the 1-month period (p < 0.001) following transplantation as measured by the horizontal ladder test (A). On individual days of behavioral testing, two-way ANOVA Newman–Keuls posttest revealed a significant improvement in performance in the BDNF-treated NSC group compared to the control group 14, 21, and 28 (p<0.05) days after transplant (17, 24, and 31 days after HI stroke). BLI signal 1 week after transplant correlated positively with functional outcome 2 weeks (r = 0.5634, p < 0.0230) and 1 month after transplantation (r = 0.5328, p < 0.0409) (B).

Increased Neuroprotection Is Seen Following Engraftment of BDNF-Treated NSCs

To understand the effect of transplantation on endogenous neurons, FJC⁺ neurons were quantified in the cortex and hippocampus of all groups at 7 days after transplantation (n = 4/group). The BDNF-treated NSC group had significantly fewer FJC⁺ cells in the cortex (p < 0.05) (Fig. 3A) and hippocampus (p < 0.05) (Fig. 3B) than both the untreated NSC group and control group. Although injection of untreated NSCs resulted in some neuroprotection, the decrease in FJC⁺ cells did not reach significance when compared to the control group. Per engrafted NSCs, significantly fewer FJC⁺ cells were found in the cortex (p=0.0133) and hippocampus (p = 0.0052) of the BDNF-treated NSC group than in the untreated NSC group (Fig. 3C). There was a significant negative correlation between the number of SC121⁺ NSCs and FJC⁺ cells in the cortex (r = −0.7715, p = 0.0149) (Fig. 3D) and hippocampus (r = −0.6724, p = 0.0472) (Fig. 3D) for all cell-treated animals. The ratio of stroked hemisphere volume to contralateral hemisphere volume for each animal was assessed ex vivo 28 days after transplantation. The largest ratio (stroked hemisphere with the volume closest to the contralateral hemisphere) was found in the BDNF-treated NSC group, but did not reach significance over the untreated NSC or control groups (data not shown). Lastly, the area demonstrating NeuN⁺ staining in the cortex relative to the total ipsilateral cortical area was significantly greater in the BDNF-treated NSC group compared to the untreated NSC group (p = 0.0354) (Fig. 4) and control group (p = 0.0246) (Fig. 4).

Figure 3

NSC-mediated neuroprotection. FJC⁺ degenerating neurons were quantified in the hippocampus and cortex at 7 days after transplantation (n = 4/group). The BDNF-treated NSC group had significantly fewer FJC⁺ cells in the cortex (p < 0.05) (A) and hippocampus (p < 0.05) (B) than both the untreated NSC and control groups. Significantly 5 fewer FJC cells per engrafted NSC were found in the cortex (p = 0.0133) and hippocampus (p = 0.0052) of the BDNF-treated NSC group than in the untreated NSC group (C). There was a significant negative correlation between the number of SC121⁺ NSCs and FJC⁺ cells in the cortex (r = −0.7715, p = 0.0149) (D) and hippocampus (r = −0.6724, H p = 0.0472) (D) for all cell-treated animals. Scale bar: 100 μm.

Figure 4

NSC-mediated neuroprotection. The area of NeuN positivity relative to the total cortical area in the hemisphere ipsilateral to HI stroke was found to be significantly greater in the BDNF-treated NSC group compared to the untreated NSC group (p = 0.0354) and saline control (p = 0.0246). Representative images are also given for each group. Scale bar: 200 μm.

NSCs Remain in an Undifferentiated State After Treatment with BDNF

Seven days after BDNF treatment, cultured NSCs were analyzed by immunocytochemistry for stem cell marker nestin, neuronal marker NeuN, oligodendrocyte marker Olig2, and astrocytic marker GFAP. No difference in number of NeuN⁺, Olig2⁺, and GFAP⁺ cells was found between BDNF-treated cells and untreated cells (data not shown). Over 98% of cells in both groups continued to express nestin 1 week after treatment.

NSCs Treated with BDNF Demonstrate Improved Migration to SDF-1

Immunocytochemistry and gene expression analysis confirmed constitutive expression of the SDF-1 receptor CXCR4 on NSCs (Fig. 5A), and gene expression analysis indicated that treatment with BDNF resulted in a 1.75-fold increase in CXCR4 expression. Normalization of CXCR4 gene expression revealed BDNF-treated NSCs with CXCR4 expression greater than 90% of housekeeping gene expression levels, while in the untreated NSCs, CXCR4 expression was only 60% of housekeeping gene expression levels (Fig. 5A). Evaluation of the migratory potential of NSCs using a modified Boyden chamber assay revealed that treatment with BDNF stimulated robust dose-dependent migration toward SDF-1 (Fig. 5B). The total number of cells migrating was significantly greater for BDNF-treated NSCs compared to untreated NSCs at SDF-1 concentrations of 100 ng/ml (p = 0.0245), 500 ng/ml (p =0.0179), and 1,500 ng/ml (p = 0.0369). Additionally NSCs demonstrated a nonsignificant increase in migratory capacity with increasing doses of MCP-1 (data not shown).

Figure 5

NSC migration. Immunohistochemical staining of NSCs for SDF-1 receptor chemokine C-X-C motif receptor 4 (CXCR4) reveals constitutive expression (A). Normalization of CXCR4 gene expression revealed BDNF-treated NSCs with CXCR4 expression higher than 90% of housekeeping gene expression levels, while in the untreated NSCs, CXCR4 expression was only 60% of housekeeping gene expression (n = 3) (A). A modified Boyden chamber assay revealed that treatment with BDNF led to a dose-dependent migration toward SDF-1 (n = 3) (B). The total number of cells migrating was significantly greater for BDNF-treated NSCs compared to untreated NSCs at SDF-1 concentrations of 100 ng/ml (p = 0.0245), 500 ng/ml (p = 0.0179), and 1,500 ng/ml (p = 0.0369). Scale bar: 20 μm.

BDNF Induces Secretion of Chemokines, Adhesion Molecules, and Growth Factors From NSCs

In vitro evaluation of the NSC protein secretion profile found significant increases in, for example, intercellular adhesion molecule-1 (ICAM-1) (p = 0.0002), macrophage colony-stimulating factor (p < 0.0038), vascular endothelial growth factor (VEGF) (p < 0.0001), and vascular cell adhesion molecule-1 (VCAM-1) (p = 0.0029) and decreases in interferon-α (p = 0.0096) and chemokine (C-C motif) ligand-7 (p = 0.0039) following treatment with BDNF (Fig. 6A).

Figure 6

Proteomics and gene expression. In vitro evaluation of the NSC protein secretion profile (A) and gene expression (B) following BDNF pretreatment (black bars) and controls (white bars). CCL7, chemokine C-C ligand 7; IFN, interferon; VCAM-1, vascular cell adhesion molecule-1; GM-CSF, granulocyte macrophage colony-stimulating factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; M-CSF, macrophage colony-stimulating factor; ICAM-1, intercellular adhesion molecule-1; NCAM-1, neural cell adhesion molecule-1; THBS1, thrombospondin 1.

Changes in NSC Gene Expression Following BDNF Treatment

After BDNF treatment, adhesion molecules VCAM-1 and neural cell adhesion molecule-1 (NCAM-1) and thrombospondins 1 (THBS1) and 2 were upregulated on NSCs (Fig. 6B). Treatment with BDNF also resulted in a higher expression of BDNF itself.

Discussion

In this study, BDNF pretreatment of NSCs elicited many desired effects including increased NSC survival as measured by BLI and immunohistochemistry, decreased endogenous cell death as measured by FJC⁺ staining (Fig. 3A, B), increased NeuN⁺ cortical area (Fig. 4), greater NSC migratory capacity (Fig. 5B), and improved functional outcomes compared to animals injected with untreated NSCs or saline (Fig. 2A). The effects of BDNF pretreatment on cell survival was seen at 7 days posttransplantation, but at 28 days (31 days after HI stroke) no surviving cells were found. Despite this fact, greater functional outcomes were observed in the BNDF-treated NSC group compared to untreated NSCs and control groups at 28 days after transplantation.

We hypothesize that the first 1-2 weeks following a HI stroke in this particular model are the most important factors in the determination of functional gains later. This notion is supported by the fact that proinflammatory factors and a milieu of cytotoxic immune cells and factors are recruited to the brain in the acute time period after stroke (18). We postulate that in this model, greater survival and improved migration of NSCs to the peri-infarct areas results in an increased capacity to mediate the acute cytotoxic poststroke environment. It is in part by this mechanism that functional improvements at later time points are observed. This is supported by our data showing the greatest number of SC-121⁺ cells (Fig. 1C, D) and the lowest number of FJC⁺ neurons (Fig. 3A) at 7 days after transplantation in the BDNF-treated NSC group. Moreover, there was a significant negative correlation between number of FJC⁺ neurons and transplanted NSCs (Fig. 3D). These data correlate with increased functional outcomes in the BDNF-treated NSC group at 28 days after transplant.

Overall, the intensity of the immediate endogenous response following stroke greatly influences peri-infarct cell death or survival, which is strongly linked to stroke outcomes (27). Cells with an increased ability to migrate and survive have a greater potential to secrete immunomodulatory and neuroprotective factors and influence penumbral tissue survival. This further supports the idea that NSC survival in the brain during the acute time period after stroke is an essential contributor to long-term functional gains.

In this study, a BDNF pretreatment concentration of 100 ng/ml was chosen for 1 h. Cells were treated just prior to injection while in a single cell suspension, a nonideal environment for this particular monolayer-grown cell line. Thus, while it cannot be definitively concluded that a longer treatment time might have a similar or even greater outcome, it was chosen as the shortest time in which we were likely to still see effects as a result of pretreatment. Moreover, when deciding whether cells should be pretreated or genetically modified, we concluded that pretreatment was a more rapidly translatable therapy since genetic modification is not acceptable for NSC therapy in humans. Of course BDNF pretreatment does not elicit constitutive expression, and thus the desired effect of treatment may have been retarded after transplantation.

Pretreatment of NSCs with BDNF increased the average number of transplanted cells found in the cortex (Fig. 1C) and hippocampus (Fig. 1D). In order to better understand the mechanisms behind the higher rate of engraftment after BDNF treatment, we evaluated changes in adhesion molecule and chemokine receptor expression of NSCs in vitro. Here we found that treatment with BDNF results in a dramatic increase in CXCR4 expression and significantly improved migration in response to SDF-1 in vitro (Fig. 5). Previous data from our lab and others have found that SDF-1 is highly upregulated 3 days after stroke (15,35), and the interaction of SDF-1 with CXCR4 plays an important role in facilitating migration of cells in the ischemic brain (36). Our data corroborates recent evidence that overexpression of CXCR4 in intravascularly transplanted mesenchymal stem cells improves engraftment (37), and we postulate that the significant change in migratory ability of NSCs seen after BDNF pretreatment could contribute to greater cell engraftment. In addition expression of many other chemokine receptors (CCR1, CCR4, CCR6, CCR7, CXCR3, CXCR6; data not shown), chemokine ligands (CXCL1, CXCL6, CXCL9) (Fig. 6A, B), and adhesion molecules [NCAM-1 (12,26,33), VCAM-1 (13), and ICAM] (Fig. 6A, B) known to be important for NSC chemotaxis and migration were also increased after BDNF treatment.

We also attempted to elucidate the mechanism by which BDNF-treated NSCs elicit functional benefits after transplantation. While the mechanisms driving neuroprotection after stem cell treatment are not known (20), recent studies have found that VEGF secretion has a neuroprotective effect in vitro (38) and in vivo (20,32). We found that VEGF secretion was strongly enhanced by BDNF treatment (Fig. 6A), and our data match previous studies reporting that increased VEGF expression results in reduced apoptosis and greater survival of transplanted NSCs (20). In addition to enhancing angiogenesis and promoting functional recovery following stroke (9,16), VEGF is also known to influence microglial activation (14,29). The bottom line here is that neuroprotective effects seen following NSC engraftment may be driven by secreted factors such as VEGF, interacting either directly with neurons or indirectly through other endogenous cells such as microglia. Additionally, thrombospondin 1 and 2 are important for the recovery of synapses and axonal plasticity in the penumbra (4,10,24) after stroke, and we found that expression of both is increased following BDNF pretreatment.

In summation, with many changes seen after BDNF pretreatment, it is likely that multiple mechanisms of interaction are involved in the neuroprotective and functional benefits seen following transplantation.

Conclusions

We have demonstrated that pretreatment of NSCs with BDNF prior to IA injection improves functional outcomes after HI stroke. We have also shown that increased expression of chemokine receptors and adhesion molecules following BDNF pretreatment contributes to improved migration, and helps promote cerebral engraftment. Furthermore, BDNF pretreatment of NSCs induces expression and secretion of proteins that mediated neuroprotection. Therefore, BDNF treatment of NSCs is a simple manipulation that can be used to improve the success of cell therapies for stroke and potentially other central nervous system diseases. Further studies are needed to elucidate whether neural stem cell-secreted factors interact either directly with neurons or indirectly through other endogenous cells such as microglia or astrocytes.

Footnotes

Acknowledgments

We wish to thank Dr. Sanjiv Sam Gambhir, Stanford University, for the viral transduction of the cells, and Elizabeth Hoyte for the preparation of the illustrations. This work was supported by the American Heart Association Grant AHA-0835274N, the Swiss National Science Foundation grant 310030_146632, and the Bechtel Foundation (to R. Guzman). The authors declare no conflict of interest.

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BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery after Transplantation in Hypoxic–Ischemic Stroke

Abstract

Keywords

Introduction

Materials and Methods

Cell Culture

Hypoxic–ischemic Stroke Model

Cell Transplantation

Bioluminescence Imaging

Immunohistochemistry and Immunocytochemistry

Fluoro-Jade C Staining

Cell Quantification

Horizontal Ladder Behavioral Testing

Gene Expression Analysis

Luminex Immunological Assay

Boyden Chamber Migration Assay

Statistical Analysis

Results

Treatment of NSCs with BDNF Increases NSC Engraftment and Survival in the Brain

Treating NSCs with BDNF Prior to Transplant Results in Greater Functional Recovery

Increased Neuroprotection Is Seen Following Engraftment of BDNF-Treated NSCs

NSCs Remain in an Undifferentiated State After Treatment with BDNF

NSCs Treated with BDNF Demonstrate Improved Migration to SDF-1

BDNF Induces Secretion of Chemokines, Adhesion Molecules, and Growth Factors From NSCs

Changes in NSC Gene Expression Following BDNF Treatment

Discussion

Conclusions

Footnotes

Acknowledgments

References