Timing of Intra-Arterial Neural Stem Cell Transplantation After Hypoxia–Ischemia Influences Cell Engraftment, Survival, and Differentiation
Background and Purpose—
Intra-arterial neural stem cell (NSC) transplantation shows promise as a minimally invasive therapeutic option for stroke. We assessed the effect of timing of transplantation on cell engraftment, survival, and differentiation.
Mouse NSCs transduced with a green fluorescent protein and renilla luciferase reporter gene were transplanted into animals 6 and 24 hours and 3, 7, and 14 days after hypoxia–ischemia (HI). Bioluminescent imaging was used to assess cell survival at 6 hours and 4 and 7 days after transplantation. Immunohistochemistry was used to assess NSC survival and phenotypic differentiation 1 month after transplantation. NSC receptor expression and brain gene expression were evaluated using real-time reverse transcription–quantitative polymerase chain reaction to elucidate mechanisms of cell migration. Boyden chamber assays were used to assess cell migratory potential in vitro.
NSC transplantation 3 days after HI resulted in significantly higher cell engraftment and survival at 7 and 30 days compared with all other groups (P<0.05). Early transplantation at 6 and 24 hours after HI resulted in significantly higher expression of glial fibrillary acidic protein (P=0.0140), whereas late transplantation at 7 and 14 days after HI resulted in higher expression of β-tubulin (P<0.0001). Corroborating the high cell engraftment 3 days after HI was robust expression of vascular cell adhesion molecule-1, CCL2, and CXCL12 in brain homogenates 3 days after HI.
Intra-arterial transplantation 3 days after HI results in the highest cell engraftment. Early transplantation of NSCs leads to greater differentiation into astrocytes, whereas transplantation at later time points leads to greater differentiation into neurons.
Intravascular transplantation of neural stem cells (NSCs) represents a promising therapeutic opportunity for stroke.1–6 Several studies have demonstrated cell engraftment and functional recovery in rodent models of hypoxia-ischemia,1,4,7 and these treatments are being evaluated for safety and efficacy in humans.8,9 After cerebral ischemia, circulating peripheral immune cells are recruited by a chemoattractive gradient.10–14 Using a similar mechanism, NSCs are able to use endogenous adhesion and chemoattractant molecules to extravasate from the vascular compartment and migrate to the ischemic lesion.1,10–12,15–18
We have previously studied the mechanism of NSC homing to the ischemic penumbra, implicating the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1)1 and the chemokines CCL2 and CXCL12 in this process.7 We also demonstrated increased NSC engraftment using intra-arterial delivery compared with intravenous delivery,19 and the absence of microstrokes after NSC injection.5 However, the optimal timing of intra-arterial NSC transplantation remains largely uncharacterized. If intravascular NSC therapies are to come to fruition, it is important to establish the ideal time of transplantation that will lead to optimal cell engraftment and survival.
We used in vivo bioluminescent imaging (BLI) and immunohistochemistry to study the ideal time for NSC transplantation. We attempted to elucidate the molecular and cellular mechanisms behind the optimal transplantation time window. Finally, we determined the effect of time of NSC transplantation on phenotypic fate.
Materials and Methods
A murine multipotent NSC line derived from the external germinal layer of the mouse cerebellum (C17.2) was used in these experiments.20 These NSCs have been shown to readily differentiate into noncerebellar neurons, astrocytes, and oligodendrocytes both in vitro and in vivo.2,21 Cells were transduced with a viral construct containing green fluorescent protein (GFP) and renilla luciferase as previously described.19 Cells were thawed at passage 5 and cultured as previously described1 in Dulbecco modified Eagle medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 5% horse serum (Gibco), and 1% l-glutamine (2 mmol/L; Gibco) at 37°C in 5% CO2.
All animal procedures were approved by Stanford University's Administrative Panel on Laboratory Animal Care. Brain ischemia was induced using a hypoxia–ischemia stroke model as previously described.1,19 Nine-week-old male C57BL6 mice (Charles River, Wilmington, MA) underwent temporary left common carotid artery occlusion with an aneurysm clip before being placed in hypoxic conditions (8% O2) at 37°C for 20 minutes. After hypoxia, reperfusion was achieved by removal of the aneurysm clip. Sham animals underwent identical surgeries without carotid artery ligation or exposure to hypoxic conditions.
Cell transplantation was performed on 5 distinct groups at different time points after stroke induction (Figure 1). Mice were randomly assigned to a poststroke transplantation time point of 6 hours, 24 hours, 3 days, 7 days, or 14 days. Each of these 5 groups is referred to as the 6-hour, 24-hour, 3-day, 7-day, and 14-day transplantation groups (n=8 per group). After group assignment, the common carotid artery was re-exposed, and a single cell suspension of 5×105 NSCs was injected in 5 μL saline using a custom 10-μL Hamilton syringe with a 33-G needle.5,19 No differences in mortality were observed between groups.
A standard dilution curve for BLI signal was created by stereotaxically injecting 2.5×104, 5.0×104, or 1.0×105 NSCs in 2 μL saline into the striatum (anteroposterior +0.8; lateral +2.2; vertical −3.0) of naive mice (n=3 per dilution). Animals were imaged after transplantation and the BLI signal (photons/s) plotted against the known transplanted cell number (online-only Data Supplement Figure IA).
BLI was conducted using the IVIS Spectrum system (Xenogen Corporation, Alameda, CA). Mice were given an intraperitoneal injection of 200 μL d-luciferin (15 mg/mL in phosphate-buffered saline [PBS]; Promega, Madison, WI). Whole-body images were acquired for 1 minute. The BLI signal was recorded as maximum photon flux (photons/s). Living Image 3.0 software (Caliper Life Sciences, Hopkinton, MA) was used to quantify maximum photon flux in regions of interest in the head.
Animals were transcardially perfused with PBS followed by 4% paraformaldehyde. After removal, brains were fixed for 48 hours and placed in 30% sucrose in PBS. Brains were then cryo-sectioned at 30 μm using a microtome. For fluorescent immunohistochemistry, sections were blocked in PBS 0.3% triton with 5% goat serum and 1% bovine serum albumin for 1 hour at room temperature. Primary antibody was incubated overnight (12–14 hours) at 4°C in a 1:10 dilution of blocking solution (5% goat serum/1% bovine serum albumin) with the following antibodies: anti-PECAM (CD31; R&D Systems 550274; 1:500), anti-GFP (Invitrogen A11120/A11122; 1:200), antiglial fibrillary acidic protein (Pharmingen 556330; 1:200), antiolig2 (R&D Systems AF2418; 1:250), anti-βIII-tubulin (Covance mms-435P; 1:500), antinestin (Abcam ab5968; 1:200), anti-Iba-1 (Wako 019-19741; 1:1000), antineuronal nuclei (Millipore mab377; 1:100). The appropriate secondary antibody was used at 1:500 (Invitrogen Alexa Fluor 488; 546) at 4°C for 6 hours followed by nuclear stain with 4,6-diamidino-2-phenylindole (AnaSpec, San Jose, CA; 1:5000). Sections were visualized on a laser scanning confocal microscope (LSM510; Zeiss).
For 3,3′-diaminobenzidine immunohistochemistry, sections were washed in a solution of 3% hydrogen peroxide (H2O2) and 30% methanol in PBS to quench endogenous peroxidase activity. Next sections were blocked and incubated in rabbit anti-Iba-1 primary antibody as described previously. After washing, sections were placed in biotinylated secondary antibody (Vector Laboratories, BA-1000; 1:250) for 4 hours at room temperature. Sections were incubated in avidin and biotinylated horseradish peroxidase for 30 minutes according to manufacturer instructions (Vector Laboratories; PK-6100) and developed in 3,3′-diaminobenzidine (Vector Laboratories, SK-4100) for 40 seconds.
GFP+ cells were quantified in the penumbral regions of the ipsilateral cortex and striatum by taking 2 regions of interest per section in 4 consecutive sections from each brain. Quantification of cell survival and differentiation were performed by an individual blinded to all conditions. Phenotypic characterization of Iba-1+/GFP+, Nestin+/GFP+, β-tubulin+/GFP+, glial fibrillary acidic protein+/GFP+, and olig2+/GFP+ were performed in both cortical and striatal regions. Z-stack reconstructions were performed on all double fluorescent immunohistochemical stains to verify colocalization of antibodies (online-only Data Supplement Figure II). Cell counts were performed using ImageJ image analysis software (National Institutes of Health, Bethesda, MD).
Gene Expression Analysis
A subset of animals were subjected to hypoxia–ischemia (n=3 per group) but were not given cell transplantation. A standardized section of the ischemic hemisphere was extracted in RNAlater (Qiagen 76106) 6 hours, 24 hours, 3 days, 7 days, and 14 days after hypoxia–ischemic stroke and immediately stored in RNAlater according to manufacturer instructions. RNA was isolated using an RNeasy mini kit (Qiagen 74104) and transcribed to cDNA using the RT2 HT first-strand synthesis kit (SABiosciences) according to manufacturer instructions. Individual assays using cDNA and primers for CCL2, CXCL12, and VCAM-1 (SABiosciences PPM03151F, PPM02965E, PPM03208B) were normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (SABiosciences PPM02946E) for each of the 5 experimental groups using RT2 real-time SYBR Green PCR Master Mix (SABiosciences 330523) on a Stratagene Mx300P. For each of 5 groups, the fold change in RNA expression of the stroked hemisphere over the contralateral hemisphere was calculated. Gene array analysis was performed on NSCs using SABiosciences RT2 profiler polymerase chain reaction arrays for adhesion molecules (Catalog No. PAMM-013A) and chemokines (Catalog No. PAMM-022A). Data were normalized using multiple housekeeping genes, and a positive result was taken as any genes with expression earlier than 33 cycles per manufacturer instruction. These were converted to simple ratios of housekeeping gene/gene of interest for easy visualization. The ratio of 0.60 (housekeeping/gene in question) corresponds to expression at 33 cycles.
Boyden Chamber Migration Assay
Modified Boyden chamber migration assay (Cytoselect; Cell Biolabs, San Diego, CA; CBA-106) was used to evaluate NSC migratory capacity toward CCL2 (monocyte chemoattractant protein-1) and CXCL12 (stromal-derived factor-1). Cells were seeded into a membrane insert with a synthetic polycarbonate 8-μm pore membrane and incubated at 37°C in a humidified atmosphere with 5% CO2. The chamber below the membrane contained chemoattractant (CCL2 at 0 ng/mL, 5 ng/mL, 10 ng/mL, 40 ng/mL, 70 ng/mL or CXCL12 at 0 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL, 1500 ng/mL; Peprotech). Cells were allowed to migrate across the membrane toward the chemoattractant for 1 hour. Cells that had migrated through the membrane were detached, lysed, and incubated at room temperature for 20 minutes with CyQuant GR dye, a fluorescent DNA binding dye. Fluorescence at 480 nm/520 nm was subsequently quantified with scanning fluorometer (Flexstation II 384; Molecular Devices, Sunnyvale, CA) and SoftMax Pro V5 software (Molecular Devices). A standard dilution curve (n=3 for 6 serial dilutions from 5.0×104 to 0) was created with each assay and demonstrated a strong linear correlation (r=0.865) between number of cells and the fluorescence output measured in this experiment. Specific migration of NSCs was assessed by normalizing total fluorescence to fluorescent readings from wells containing 0 ng/mL of chemoattractant in PBS.
Quantitative data were expressed as mean±SEM. Means were compared using a 1-way analysis of variance and the Student t test. The Newman-Keuls method of correction was used for comparison between multiple groups, correlation was described using the Pearson correlation coefficient, and P<0.05 was considered statistically significant.
Transplantation of NSCs 3 Days After Hypoxia–Ischemia Resulted in Optimal Cell Survival
NSCs were injected 6 hours, 24 hours, 3 days, 7 days, and 14 days after hypoxia–ischemia (HI; Figure 1). BLI signal was measured at 6 hours, 4 days, and 7 days after injection (Figure 2) for each transplantation group. When BLI signal was measured 6 hours after injection, the 3-day transplantation group yielded significantly higher photon flux than the 6-hour, 7-day, and 14-day transplantation groups (P<0.05). This difference was also present 4 days after injection (P=0.0022). One week after cell injection, the 3-day transplantation group had significantly higher photon flux compared with all other groups including the 24-hour transplantation group (P<0.0001). Relative intensity was calculated by normalizing the photon flux at each imaging time point to the photon flux at the 6-hour imaging time point. Over the first week after injection, the 3-day transplantation group demonstrated an 18% drop in signal. In comparison, the 6-hour, 24-hour, 7-day, and 14-day transplantation groups exhibited 51%, 26%, 72%, and 72% drop in signal, respectively, over the first 7 days after transplantation (Figure 2B). Based on the BLI standard dilution curve, the extrapolated number of cells surviving in the brain 1 week after injection was 1.0×105, 1.9×105, 3.0×105, 6.6×104, and 6.4×104 in the 6-hour, 24-hour, 3-day, 7-day, and 14-day transplantation groups, respectively (online-only Data Supplement Figure IIIC).
To corroborate the BLI findings, GFP+ NSCs were quantified in the ischemic penumbra 1 week after injection for each transplantation group using immunohistochemistry (Figure 3A). We found a mean of 850±146 cells/mm2 in the stroked hemisphere of the 3-day transplantation group, representing a significantly higher number of NSCs compared with all other transplantation groups (P<0.0001; Figure 3A). We also confirmed preferential migration to the ischemic hemisphere over the contralateral hemisphere for all transplantation groups (data not shown).
Cell engraftment was also quantified 30 days after injection (Figure 3B). One month after injection, we found a mean of 533±78 cells/mm2 in the 3-day transplantation group, representing significantly higher cell counts than all other transplantation groups (P=0.0217).
Adhesion Molecule VCAM-1 and Chemokines CCL2 and CXCL12 Are Upregulated After HI
To analyze the possible molecular mechanisms driving differences in NSC engraftment among the transplantation time points, brain homogenates from animals only subjected to HI were assayed for the expression of adhesion molecule VCAM-1 and chemokines CCL2 and CXCL12 at 6 hours, 24 hours, 3 days, 7 days, and 14 days after HI. Ischemic hemisphere RNA levels were expressed as changes in fold regulation compared with the nonstroked hemisphere. Robust expression of VCAM-1 and CCL2 was observed in brain homogenates 24 hours and 3 days after HI and at 3 days after HI for CXCL12 (Figure 4A–C). These data were confirmed by Western blot analysis (data not shown). Conjugate receptors to the aforementioned chemokines and adhesion molecules were also confirmed to be present on the NSCs used for transplantation (online-only Data Supplement Figure I). To confirm that NSCs respond to these chemoattractive signals, an in vitro migration assay using CCL2 and CXCL12 was performed using the Boyden Chamber Migration Assay. NSCs were found to migrate in a dose-dependent manner to both CCL2 and CXCL12 (online-only Data Supplement Figure IB–C). In addition, NSCs expressed integrin β1 and integrin α4, which dimerize to form VLA4, the receptor for VCAM-1 (online-only Data Supplement Figure IA).
Phagocytosis of Transplanted NSCs Is Increased at 6 and 24 Hours After HI
To assess whether phagocytosis of transplanted cells contributed to the observed differences in cell counts, the number of Iba-1+ cells that colocalized with GFP were quantified by immunohistochemistry 1 week after injection (Figure 5). When expressed as a percentage of the total number of GFP+ cells, the 6-hour and 24-hour transplantation groups demonstrated significantly elevated levels of phagocytosed transplanted NSCs compared with the 3-day, 7-day, and 14-day transplantation groups (P=0.0140; Figure 5A).
Phenotypic Fate of Transplanted Cells
To determine whether timing of transplantation had an effect on phenotypic fate, transplanted NSCs were analyzed for the neural stem cell marker Nestin, the immature neuronal marker β-tubulin, the astrocytic marker glial fibrillary acidic protein, and oligodendrocytic marker olig2 (Figure 6).
One week after injection, 60% to 80% of transplanted NSCs expressed Nestin in all transplantation groups (Figure 6A). β-tubulin expression was significantly higher in the 7-day and 14-day transplantation groups (P<0.0001; Figure 6B). Glial fibrillary acidic protein expression was significantly higher at the earlier transplantation time points of 6 and 24 hours after HI (P=0.0140; Figure 6B). Olig2 expression was not observed in any NSCs within any of the transplantation groups. The mature neuronal marker NeuN was not observed in transplanted NSCs 1 week after injection but was evident 1 month after injection (online-only Data Supplement Figure II).
Determining the optimal timing of NSC transplantation is critical to the development of intravascular cell-based therapies for stroke.2,3,6,22 In this study we used a model of diffuse ischemia that has been previously described as a model of adult ischemia23 and is used commonly in studies of neonatal stroke. In this model, the ischemic injury leads to unilateral changes in gene expression and microglial activation (Figure 4; online-only Data Supplement Figure IV). In addition, we have previously shown that this model is well suited to demonstrate the strength of intravascular cell transplantation.1,7 We used an in vivo BLI system to evaluate the degree of engraftment at varying time points after HI. The greatest bioluminescent signal at all time points imaged was observed when NSCs were transplanted 3 days after HI, indicating greater survival of NSCs in this group. In previous studies, we transplanted NSCs between 24 and 48 hours after HI,1,5,19 but these findings suggest that the 3-day transplantation time point improves NSC engraftment both 1 week and 1 month after injection. The 3-day transplantation group had only an 18% drop (8.1×105 photons/s or 3.0×105 surviving NSCs; online-only Data Supplement Figure IIIC) in BLI signal 1 week after injection compared with a 26% drop (5.1×105 photons/s or 1.9×105 surviving NSCs; online-only Data Supplement Figure IIIC) when NSCs were injected 24 hours after HI. Additionally, injection at 7 days or 14 days after HI resulted in a 70% drop in signal (Figure 2B), representing 6.6×104 and 6.4×104 surviving NSCs respectively (online-only Data Supplement Figure IIIC). These findings were corroborated using immunohistochemistry. In the 3-day transplantation group, we found a mean of 850±146 cells/mm2 in the ischemic penumbra 1 week after injection. In this same group 30 days after injection, we found a mean of 533±78 cells/mm2 in the ischemic penumbra. However, despite finding these differences in survival and engraftment of NSCs, we did not observe differences in the location of stem cell migration and homing as a function of the timing of transplantation. The global distribution of NSCs can be seen in online-only Data Supplement Figure IVC.
To explain the observed differences in engraftment, we examined the molecular mechanisms behind cellular adhesion and chemoattraction. We have previously demonstrated that the transendothelial migration of NSCs into the parenchyma is highly dependent on the interaction of the adhesion molecule VCAM-1 and the cell surface integrin CD49d.1 Furthermore, an increase in the number of cells homing to the brain has been correlated with positive behavioral outcome.1 We observed the robust expression of the adhesion molecule VCAM-1 between 24 hours and 3 days after HI, corresponding with increased homing of NSCs transplanted during that time. CD49d was also expressed on transplanted NSCs suggesting that this is a critical system for stem cell recruitment and adhesion. Additionally, the chemokine CCL2 (monocyte chemoattractant protein-1) was significantly upregulated 24 hours after HI and persisted for another 2 days. Our findings corroborate other studies on the time course of CCL2 expression in rodent models of stroke.11,15,24,25 Moreover, we demonstrate an in vitro dose-dependent response in migration of NSCs to CCL2, providing additional evidence that this system promotes higher NSC engraftment between 24 hours and 3 days after HI. CXCL12 was also highly upregulated 3 days after HI, reaching significance when compared with CXCL12 expression within the first 24 hours after HI. The CXCL12/CXCR4 interaction has been well characterized12,13,26,27 and has demonstrated a dose-dependent positive correlation with number of NSCs present in the postischemic brain.17 Taken together, these data suggest that between 24 hours and 3 days after HI, the constellation of adhesion and chemoattractive molecules is optimal for NSC recruitment to an ischemic lesion. Further studies with selective blocking or receptor knock out models will be necessary to prove the relation of chemokine and adhesion molecule expression and the time dependence of cell homing.
We also found that NSCs transplanted within the first 24 hours were more likely to be phagocytozed in the postischemic brain. Transplantation 3 days after HI resulted in significantly less cells colocalized with the pan-monocytic (all monocytic lineage cells including microglia) marker Iba-1. This finding is consistent with previously reported data describing a negative correlation between Iba-4-positive cells and survival of transplanted NSCs in the ischemic brain.28 Quantification of Iba-1 expression over a 14-day period after HI in animals that were not given transplants showed a peak 3 days after HI in the cortex, striatum, and hippocampus (online-only Data Supplement Figure IVA). We can hypothesize that when injected at 3 days after HI, NSCs are subjected to a waning cytotoxic and inflammatory milieu, whereas NSCs transplanted 6 hours or 24 hours after HI are exposed to a detrimental environment with a robust influx and activation of monocytic cells.
Varying the time of transplantation also influenced NSC differentiation. NSCs injected within the first 24 hours after HI gave rise to more astrocytic lineage cells, whereas transplanting cells 1 to 2 weeks after HI yielded more immature neuronal cells. These data suggest that the postischemic environment is constantly evolving and influencing the phenotypic fate of transplanted NSCs. Interestingly, transplanting NSCs 3 days after HI resulted in a more even distribution between neuronal and astroglial cell fates. As reported previously,1 we found no differentiation into oligodendrocytes at any time point.
There is still debate on whether the positive functional outcomes reported in many transplantation studies are mediated by cell replacement and/or trophic support,29,30 and our data suggest that timing of cell transplantation is a critical determinant of cell engraftment, survival, and phenotypic fate. As we begin to better understand the mechanisms driving functional recovery after stem cell therapy, it is important to be cognizant of how the timing of transplantation impacts a cell's ability to interact with the host.
In this mouse model of HI, we demonstrate how transplantation of NSCs 3 days after HI results in the greatest cell engraftment. Moreover, we describe how the temporal expression of adhesion and chemoattractant molecules impact the mechanisms important for NSC extravasation, targeted migration, and protection from the endogenous inflammatory response. Finally, we show that time of transplantation influences the phenotypic fate of transplanted NSCs by promoting either astrocytic or neuronal differentiation. Further studies characterizing the optimal timing of transplantation will be critical for the translation of intravascular cell-based therapies for stroke.
We thank Evan Snyder, The Burnham Institute, for the donation of the C17.2 neural stem cell line; Dr Sanjiv Sam Gambhir, Stanford University, for the viral transduction of the cells; and Elizabeth Hoyte for preparation of the illustrations.
Sources of Funding
This work was supported by the
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