Basic Sciences Timing of Intra-Arterial Neural Stem Cell Transplantation After Hypoxia–Ischemia Influences Cell Engraftment, Survival, and Differentiation Sahar Rosenblum, BS; Nancy Wang, BS; Tenille N. Smith, MS; Arjun V. Pendharkar, MS; Joshua Y. Chua, BS; Harjus Birk; Raphael Guzman, MD 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. Methods—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. Results—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 !-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. Conclusion—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. (Stroke. 2012;43:1624-1631.) Key Words: animal models ! cell transplantation ! cerebral infarct ! experimental ! stem cells
I
ntravascular 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 ab-
sence 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 Cell Culture A murine multipotent NSC line derived from the external germinal layer of the mouse cerebellum (C17.2) was used in these experi-
Received September 15, 2011; final revision received January 7, 2012; accepted February 6, 2012. From the Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA and Department of Neurosurgery, University of Basel, Spitalstrasse 21, CH-4031 Basel, Switzerland. The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111. 637884/-/DC1. Correspondence to Raphael Guzman, MD, Department of Neurosurgery, Stanford University, 300 Pasteur Drive R211, Stanford, CA 94305-5327. E-mail
[email protected] or
[email protected] © 2012 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.111.637884
Downloaded from http://stroke.ahajournals.org/ at UCLA 1624BIOMED LIBRARY SERIALS on February 22, 2015
Rosenblum et al
Timing of Intra-Arterial Stem Cell Transplantation
1625
Figure 1. Experimental timeline. This timeline represents the absolute timing of experimental HI, transplantation, BLI, and animal euthanasia for the 5 transplantation groups evaluated in this study. HI indicates hypoxia–ischemia; BLI, bioluminescent imaging.
ments.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.
Hypoxia–Ischemia Model 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 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 "L saline using a custom 10-"L 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 "L 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).
Bioluminescent Imaging BLI was conducted using the IVIS Spectrum system (Xenogen Corporation, Alameda, CA). Mice were given an intraperitoneal injection of 200 "L 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.
Immunohistochemistry 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 "m 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-!III-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.
Cell Quantification 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$/GFP$, !-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 ac-
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
1626
Stroke
June 2012
cording 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-"m 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.
Statistical Analysis 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.
Results 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 com-
pared 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 !1 and integrin #4, 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
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
Rosenblum et al
Timing of Intra-Arterial Stem Cell Transplantation
1627
Figure 2. BLI was used to quantify the average photon flux in all 5 transplantation groups 6 hours, 4 days, and 1 week after injection (A). BLI at 6 hours after NSC injection revealed a statistically significant difference between the 3-day transplantation group and the 6-hour, 7-day, and 14-day transplantation groups (A, P!0.05). This trend continued at 4 days after transplantation (P"0.0022). At 1 week after transplantation, the average photon flux in the 3-day transplantation group was higher than all other groups (A, P!0.0001). When BLI signal was normalized to the signal at 6 hours after injection (relative intensity), percent decrease in average photon flux over time was lowest in the 3-day transplantation when compared longitudinally for 1 week with all other groups (B). A representative BLI image taken at 6 hours after transplantation of in vivo luciferase activity in the 3-day transplantation group (C). BLI indicates bioluminescent imaging; NSC, neural stem cell.
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 !-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). !-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
Figure 3. Histological counts of GFP$ cells at 7 days (A) and 30 (B) days posttransplant. One week after injection, the 3-day transplantation group demonstrated significantly more GFP$ cells compared with all other groups (A, P!0.05). One month after injection, cell survival in the brain remained significantly higher in the 3-day transplantation group (B, P"0.0217). GFP indicates green fluorescent protein.
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
1628
Stroke
June 2012
Figure 4. A subset of animals were not given NSC transplants after hypoxic–ischemic insult. Their brain homogenates were analyzed to assess endogenous upregulation of VCAM-1, CCL2, and CXCL12. VCAM-1 was upregulated shortly after HI with robust expression between 24 hours and 3 days after ischemia (A). CCL2 expression was significantly higher at both 24 hours and 3 days after HI when compared with all other groups (B, P!0.05), whereas CXCL12 peaked at 3 days, reaching significance when compared with expression at both 6 and 24 hours after HI (C, P!0.05). NSC indicates neural stem cell; VCAM, vascular cell adhesion molecule; HI, hypoxia–ischemia.
but was evident 1 month after injection (online-only Data Supplement Figure II).
Discussion
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
Figure 5. Monocytic lineage marker Iba-1 was used to demonstrate whether phagocytosis contributed to the differences cell survival found among the 5 transplantation groups. At 1 week after injection, the percentage of Iba-1$/GFP$ NSCs was significantly higher in the 6-hour and 24-hour transplantation groups (A, P"0.0140). Representative images taken at 1 week after injection for the 6-hour, 24-hour, 3-day, 7-day, and 14-day transplantation groups (B) and a z-plane reconstruction showing a representation of a phagocytosed NSC (lower right corner, scale bar 5 "m). GFP indicates green fluorescent protein; NSCs, neural stem cells.
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
Rosenblum et al
Timing of Intra-Arterial Stem Cell Transplantation
1629
Figure 6. To understand the influence of brain microenvironment after HI on the phenotypic fate of NSCs, transplanted cells were stained for neural stem cell marker Nestin, immature neuronal marker !-tubulin, astrocytic marker GFAP, and oligodendrocyte marker olig2. At 1 week posttransplantation, Nestin expression remained high at 60% to 80% (A). The longer the delay in cell transplantation after HI, the higher percentage of NSCs expressing !-tubulin and lower percentage of GFAP expressing NSCs were observed. This profile was reversed when cells were transplanted at earlier time points after HI. NSCs transplanted at 3 days after HI showed a similar percentage of NSCs differentiating into both early neurons and astrocytes. No significant olig2 expression was found on transplanted NSCs (B). Confocal images representing Nestin, !-tubulin, and GFAP double-positive NSCs (C). HI indicates hypoxia–ischemia; NSCs, neural stem cells; GFAP, glial fibrillary acidic protein.
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 paren-
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
1630
Stroke
June 2012
chyma 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.
Conclusions 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.
Acknowledgments 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 American Heart Association Grant AHA-0835274N and the Bechtel Foundation (to R.G.).
Disclosures None.
References 1. Guzman R, De Los Angeles A, Cheshier S, Choi R, Hoang S, Liauw J, et al. Intracarotid injection of fluorescence activated cell-sorted CD49dpositive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model. Stroke. 2008;39:1300 –1306. 2. Guzman R, Choi R, Gera A, De Los Angeles A, Andres RH, Steinberg GK. Intravascular cell replacement therapy for stroke. Neurosurg Focus. 2008;24:E15. 3. Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke. 2009;40:510 –515. 4. Li Y, Chen J, Wang L, Lu M, Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology. 2001;56: 1666 –1672. 5. Chua JY, Pendharkar AV, Wang N, Choi R, Andres RH, Gaeta X, et al. Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes. J Cereb Blood Flow Metab. 2011; 31:1263–1271. 6. Bliss TM, Andres RH, Steinberg GK. Optimizing the success of cell transplantation therapy for stroke. Neurobiol Dis. 2010;37:275–283. 7. Andres RCR, Pendharkar AV, Gaeta X, Wang N, Nathan J, Chua JY, Lee SW, Palmer TD, Steinberg GK, Guzman R. The CCR2/CCL2 interaction mediates the transendothelial recruitment of intravascularly delivered neural stem cells to the ischemic brain. Stroke. 2011;42:2923–2931. 8. Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 2005;57:874 – 882. 9. Lee JS, Hong JM, Moon GJ, Lee PH, Ahn YH, Bang OY. A long-term follow-up study of intravenous autologous mesenchymal stem cell trans-
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
Rosenblum et al
10.
11. 12.
13.
14. 15.
16. 17.
18. 19.
Timing of Intra-Arterial Stem Cell Transplantation
plantation in patients with ischemic stroke. Stem Cells. 2010;28: 1099 –1106. Chen Y, Hallenbeck JM, Ruetzler C, Bol D, Thomas K, Berman NE, et al. Overexpression of monocyte chemoattractant protein 1 in the brain exacerbates ischemic brain injury and is associated with recruitment of inflammatory cells. J Cereb Blood Flow Metab. 2003;23:748 –755. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol. 2006;66:232–245. Hill WD, Hess DC, Martin-Studdard A, Carothers JJ, Zheng J, Hale D, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol. 2004;63:84 –96. Stumm RK, Rummel J, Junker V, Culmsee C, Pfeiffer M, Krieglstein J, et al. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci. 2002;22:5865–5878. Schroeter M, Jander S, Witte OW, Stoll G. Local immune responses in the rat cerebral cortex after middle cerebral artery occlusion. J Neuroimmunol. 1994;55:195–203. Yan YP, Sailor KA, Lang BT, Park SW, Vemuganti R, Dempsey RJ. Monocyte chemoattractant protein-1 plays a critical role in neuroblast migration after focal cerebral ischemia. J Cereb Blood Flow Metab. 2007;27:1213–1224. Guzman R, Bliss T, De Los Angeles A, Moseley M, Palmer T, Steinberg G. Neural progenitor cells transplanted into the uninjured brain undergo targeted migration after stroke onset. J Neurosci Res. 2008;86:873– 882. Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A. 2004;101:18117–18122. van Buul JD, Hordijk PL. Signaling in leukocyte transendothelial migration. Arterioscler Thromb Vasc Biol. 2004;24:824 – 833. Pendharkar AV, Chua JY, Andres RH, Wang N, Gaeta X, Wang H, et al. Biodistribution of neural stem cells after intravascular therapy for hypoxic– ischemia. Stroke. 2010;41:2064–2070.
1631
20. Ryder EF, Snyder EY, Cepko CL. Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. J Neurobiol. 1990;21:356 –375. 21. Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell. 1992;68:33–51. 22. Darsalia V, Allison SJ, Cusulin C, Monni E, Kuzdas D, Kallur T, et al. Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain. J Cereb Blood Flow Metab. 2011;31:235–242. 23. Levine S. Anoxic-ischemic encephalopathy in rats. Am J Pathol. 1960; 36:1–17. 24. Wang X, Yue TL, Barone FC, Feuerstein GZ. Monocyte chemoattractant protein-1 messenger RNA expression in rat ischemic cortex. Stroke. 1995;26:661– 665; discussion 665– 666. 25. Gourmala NG, Buttini M, Limonta S, Sauter A, Boddeke HW. Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration. J Neuroimmunol. 1997;74:35– 44. 26. Miller JT, Bartley JH, Wimborne HJ, Walker AL, Hess DC, Hill WD, et al. The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12) expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic–ischemic injury. BMC Neurosci. 2005;6:63. 27. Kokovay E, Goderie S, Wang Y, Lotz S, Lin G, Sun Y, et al. Adult SVZ lineage cells home to and leave the vascular niche via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell. 2010;7:163–173. 28. Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci U S A. 2004;101: 11839 –11844. 29. Bliss T, Guzman R, Daadi M, Steinberg GK. Cell transplantation therapy for stroke. Stroke. 2007;38(suppl):817– 826. 30. Guzman R. Cellular stroke therapy: from cell replacement to trophic support. Expert Rev Cardiovasc Ther. 2009;7:1187–1190.
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
Online Supplement Timing of Intra-Arterial Neural Stem Cell Transplantation After Hypoxia-Ischemia Influences Cell Engraftment, Survival, and Differentiation
Supplementary Methods
Magnetic Resonance Imaging MRI was performed as previously described 1 on a 7-T small animal scanner (Varian/GE Healthcare, Palo Alto, Calif). High-resolution MRI of transplanted SPIO-labeled NSCs was obtained from paraformaldehyde-fixed brains immersed in fomblin (Specialty Fluids Co, Valencia, Calif). Brains were placed in a custom-built solenoidal radiofrequency coil and imaged using a 3-dimensional FIESTA sequence (TR/TE=7.8/3.9 ms; resolution 100 µm isotropic voxels; number of excitations=1).
SPIO Labeling of Neural Stem Cells SPIO particles (Feridex IV; Berlex Laboratories, Wayne, NJ) and protamine sulfate (American Pharmaceuticals Partner, Schaumburg, IL) (5 µg/2.5 µg/ml) were incubated for 30min at 37° in culture medium before being added to the cells for 24 h as previously described 1, 2
Supplemental Figures
Supplementary Figure 1: Real-time RT-qPCR of transplanted NSCs demonstrated expression of integrin β1 and integrin α4, which dimerize to form VLA4, the receptor for VCAM-1 (A). Although CCR2 expression was low, an alternative receptor CCR43, 4 for CCL2 was expressed. CXCL12 (SDF-1) receptors CXCR4 and CXCR75 were both present (A). Boyden chamber migration assays demonstrated a dose dependent migration of NSCs to both CCL2 (MCP-1) (B) and CXCL12 (SDF-1) (C). Relative intensity was calculated by normalizing the total fluorescent readings from wells containing cells migrating to each concentration of chemoattractant to fluorescent readings from wells containing 0 ng/mL chemoattractant in PBS.
Supplementary Figure 2: High magnification z-stack images on the scanning confocal microscope were used to verify GFP+/ß-tubulin+ (A, scale bar 5µm) and GFP+/GFAP+ (B, scale bar 5µm) NSCs one week after injection. One month after transplantation, subsets of NSCs differentiated into mature NeuN+ neurons (C, scale bar 5µm).
Supplementary Figure 3: In order to correlate bioluminescent signal with the number of surviving cells in the brain, multiple dilutions of NSCs (n=3 per dilution) were stereotaxically transplanted into the striatum of normal mice. Immediately following injection, the animals were imaged, and a standard dilution curve was generated (A). A representative image showing (from left to right)
animals injected with 2.5x104, 5.0x104, or 1.0x105 NSCs (B). BLI images and the extrapolated dilution curve (C) were used to assess the number of surviving cells in each transplantation group for the first week after injection. Additionally, the correlation between histology and BLI imaging at 7 days after injection for each of the transplantation groups is pictured in (D).
Supplementary Figure 4: The endogenous microglial response after HI in animals that were not given NSC transplants was assessed using DAB staining for Iba-1. Quantification was performed by comparing the number of Iba-1+ cells in the ipsilateral HI affected areas of the cortex, striatum, and hippocampus, to equivalent ROIs in the contralateral hemisphere. A significant unilateral upregulation of microglia was found 24 hours, 3 days, 7 days, and 14 days after HI (A). Interestingly, upregulation in the ischemic hemisphere 3 days after HI was significantly different to both 24 hours and 7 days after HI. Representative images of the DAB staining 3 days after HI is shown in panel (B). Global NSC engraftment of SPIO labeled cells can be seen in panel (C).
References 1.
Pendharkar AV, Chua JY, Andres RH, Wang N, Gaeta X, Wang H, et al. Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia. Stroke 2010;41(9):2064-70.
2.
Guzman R, De Los Angeles A, Cheshier S, Choi R, Hoang S, Liauw J, et al. Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model. Stroke 2008;39(4):1300-6.
3.
Zhang T, Somasundaram R, Berencsi K, Caputo L, Gimotty P, Rani P, et al. Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4. Eur J Immunol 2006;36(2):457-67.
4.
Kim MS, Magno CL, Day CJ, Morrison NA. Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES. J Cell Biochem 2006;97(3):512-8.
5.
Schonemeier B, Schulz S, Hoellt V, Stumm R. Enhanced expression of the CXCl12/SDF1 chemokine receptor CXCR7 after cerebral ischemia in the rat brain. J Neuroimmunol 2008;198(1-2):39-45.
Timing of Intra-Arterial Neural Stem Cell Transplantation After Hypoxia−Ischemia Influences Cell Engraftment, Survival, and Differentiation Sahar Rosenblum, Nancy Wang, Tenille N. Smith, Arjun V. Pendharkar, Joshua Y. Chua, Harjus Birk and Raphael Guzman Stroke. 2012;43:1624-1631; originally published online April 24, 2012; doi: 10.1161/STROKEAHA.111.637884
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2012 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://stroke.ahajournals.org/content/43/6/1624
Data Supplement (unedited) at:
http://stroke.ahajournals.org/content/suppl/2012/04/24/STROKEAHA.111.637884.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Stroke is online at: http://stroke.ahajournals.org//subscriptions/
Downloaded from http://stroke.ahajournals.org/ at UCLA BIOMED LIBRARY SERIALS on February 22, 2015
