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 (PϽ0.05). Early transplantation at 6 and 24 hours after HI resulted in significantly higher expression of glial fibrillary acidic protein (Pϭ0.0140), whereas late transplantation at 7 and 14 days after HI resulted in higher expression of ␤-tubulin (PϽ0.0001). 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 ofNSCs 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 Ⅲ cell transplantation Ⅲ cerebral infarct Ⅲ experimental Ⅲ stem cells I ntravascular transplantation of neural stem cells (NSCs) represents a promising therapeutic opportunity for stroke. [1][2][3][4][5][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 therap...

Purpose The purpose of this study is to evaluate the 18 kDa translocator protein (TSPO) radioligand [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]PBR06) as a positron emission tomography (PET) imaging biomarker of stroke-induced neuroinflammation in a rodent model. Procedures Stroke was induced by transient middle cerebral artery occlusion in Balb/c mice. Dynamic PET/CT imaging with displacement and preblocking using PK111195 was performed 3 days later. PET data were correlated with immunohistochemistry (IHC) for the activated microglial markers TSPO and CD68 and with autoradiography. Results [18F]PBR06 accumulation peaked within the first 5 min postinjection, then decreased gradually, remaining significantly higher in infarct compared to noninfarct regions. Displacement or preblocking with PK11195 eliminated the difference in [18F]PBR06 uptake between infarct and noninfarct regions. Autoradiography and IHC correlated well spatially with uptake on PET. Conclusions [18F]PBR06 PET specifically images TSPO in microglial neuroinflammation in a mouse model of stroke and shows promise for imaging and monitoring microglial activation/neuroinflammation in other disease models.

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 embryonicderived 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 BDNFtreated 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.

Neonatal hypoxic-ischemic insults are a significant cause of pediatric encephalopathy, developmental delays, and spastic cerebral palsy. Although the developing brain's plasticity allows for remarkable self-repair, severe disruption of normal myelination and cortical development upon neonatal brain injury are likely to generate life-persisting sensory-motor and cognitive deficits in the growing child. Currently, no treatments are available that can address the long-term consequences. Thus, regenerative medicine appears as a promising avenue to help restore normal developmental processes in affected infants. Stem cell therapy has proven effective in promoting functional recovery in animal models of neonatal hypoxic-ischemic injury and therefore represents a hopeful therapy for this unmet medical condition. Neural stem cells derived from pluripotent stem cells or fetal tissues as well as umbilical cord blood and mesenchymal stem cells have all shown initial success in improving functional outcomes. However, much still remains to be understood about how those stem cells can safely be administered to infants and what their repair mechanisms in the brain are. In this review, we discuss updated research into pathophysiological mechanisms of neonatal brain injury, the types of stem cell therapies currently being tested in this context, and the potential mechanisms through which exogenous stem cells might interact with and influence the developing brain.

Stroke is the third leading cause of death and the leading cause of adult disability in North America. Emphasis has been placed on developing treatments that reduce the devastating long-term impacts of this disease, and preclinical research on stem cell therapy has demonstrated promising results. However, questions about the optimal cell delivery method and timing of cell transplantation are not fully answered. Recent findings suggest that intravascular stem cell delivery is a safe and efficacious alternative to stereotactic cell injections. It also offers advantages should repeat treatments prove beneficial. Recent reports further suggest that intra-arterial injection results in a wider distribution of cells throughout the stroked hemisphere with a significantly greater cell engraftment compared to intravenous injection. In this review, we describe the benefits and potential risks associated with intravascular stem cell delivery and compare intra-arterial to intravenous cell transplantation methods. We discuss the importance of cell biodistribution and timing of transplantation in driving cell survival. We examine current proposed mechanisms involved in cell migration and functional recovery and discuss future directions for intravascular stem cell therapy research.

Introduction: Cell based therapies have shown a considerable ability to improve functional outcome when administered after experimental stroke. Intra-arterial (IA) cell transplantation has been shown to be a valuable alternative transplantation method for cell treatment. The success of IA therapy depends on the targeted homing and subsequent migration of cells to the injured brain area. This process is reliant on neural stem cell (NSC) expression of chemokines and their receptors. Understanding the differences in cell expression of these molecules and the resulting differences in chemotaxis will help identify ideal cell types for transplantation. Previously, we compared mRNA from human and mouse NSCs and found that only a 10% overlap in chemokine expression. We wanted to determine if there are also variations between different cell types from the same species. We compared NSCs obtained from induced pluripotent stem cells (iPS NSCs), to fetal NSCs (F NSC). We used RT-qPCR, in vitro cell migration, and quantification of cell secretion to compare chemokine and ligand expression in F NSCs and iPS NSCs. Methods: Secondary neurospheres originating from FVB mice were generated from either fetal primary NSCs from E17.5 mice (F NSC), or derived by transducing neurospheres originating from E3 mice with Oct4 (iPS NSC). Cells were grown as neurospheres in growth media treated with EGF and FGF. RT-qPCR was performed on extracted RNA using microarray technology for chemokine receptors and ligands. Luminex immunological assay was completed to assess secreted cytokines and inflammatory proteins. The Boyden chamber migration assay was used to determine migration to SDF-1 or MCP-1 at varying concentrations. Results: RNA expression was detected for 58% of the factors assessed. F NSCs alone expressed 6% (e.g. mmp2, Cxcl10), iPS NSCs alone expressed 16% (e.g. CCL2, CCL7, LIF), and both iPS NSCs and F NSCs expressed 20% (e.g. BDNF, HIF1a, CXCR4) of the detected factors. We assessed 26 secreted proteins, 73% occurred at detectable levels, of these 74% were expressed more highly in F NSCs than in iPS NSCs, including, il-1α, CCL3, CCL5, TNF-α, and VEGF; iPS NSCs secreted higher levels of MCP3, Il-3, and substantially more MCP-1 (113.7 ± 1.44 vs 52.78 ±4.221 pg/ml). iPS NSCs also had a higher migratory response to MCP-1 than F NSCs did (p < 0.001). Cell migration to SDF-1 did not differ between cell types (p = 0.905). Conclusions: Several chemokines and their receptors were differentially expressed in mouse iPS and F NSCs. These differences may result in variations in the NSC response to the ischemic brain. The profile of chemokine and chemokine receptor expression on NSCs should be strongly considered when selecting cell types for post stroke treatment.

Intro: Increasing cell migration through upregulation of chemokine and adhesion molecule receptors could improve intravascular cell treatment for stroke and BDNF has been shown to induce these pathways. Therefore we tested whether BDNF cell-pretreatment would improve cell migration and functional recovery in an experimental stroke model. Methods: hES-derived NPCs (5x10 5 in 5µl saline) pre-treated with BDNF for 5 hours and harboring a reporter gene construct containing renilla luciferase and eGFP in serum free media, non-treated hES-derived NPCs (5×10 5 in 5[l saline) in media, and media control with BDNF were delivered to the brain via the ipsilateral carotid artery at 3 days after hypoxic-ischemic stroke in NODSCID mice (n=11/group). Cell engraftment was monitored by in-vivo bioluminescence imaging (BLI). The ladder test was used to assess behavioral recovery throughout a 4 week time course. Brain homogenates from animals at 28 days were analyzed using RT-qPCR for common chemokines, adhesion molecules, and neurotrophins. Mechanisms of cell migration were evaluated by assessing cell receptor expression of chemokines and adhesion molecules on hES-derived NPC and by analyzing the change in expression profile in the mouse brain at 3 days after stroke. Boyden-chamber migration assays were used to evaluate cell migratory potential in vitro . RESULTS: One day after cell transplantation the subset of animals transplanted with BDNF-pretreated cells showed significantly higher BLI signal at 1 (p=0.021) and 7 days after transplantation (p=0.002). Histological analysis also revealed engraftment of hES-derived NPC at 1 week after transplantation. Behavioral assessment revealed significant functional recovery in the BDNF pre-treated group throughout the 28 day time course (ANOVA, p<0.05). BDNF-pretreatment of hES-derived NPCs upregulated CXCR4 expression 12.5 times and in vitro led to significantly greater migration in response to CXCL12 (CXCR4 ligand) compared to untreated cells. At 28 days after transplantation, neurotrophic factors IL6, IL10, Ntrk1 were upregulated 3.3, 3.4, and 3.3 times. Common T-cell and neutrophil cytokine receptors IL8rb, IL8ra and IL1a were all downregulated, while several chemokines that increase migration of inflammatory cells were downregulated including CCL2, CCL5, CCL8, and CCL12. Anti-Angiogenic factor Adamts8 was also downrgulated in the brains of animals transplanted with BDNF pre-treated cells. Lastly, MMP3, MMP8, and MMP9 were downregulated at 28 days after stroke indicating increased blood brain barrier integrity. Conclusion: Intravascular transplantation of BDNF pre-treated hES-derived NPCs elicits functional gains via increased migration of cells, immunomodulation, increased BBB integrity, and by influencing the upregulation of neur0protective factors.

Introduction: Cerebral palsy (CP) is the most common cause of motor disability in children, and chronic deficits are associated with white matter injury. Neonatal hypoxic-ischemic insults, an important cause of CP, induce oligodendrocyte apoptosis and impair normal myelin development. No CP treatments target myelination making regenerative medicine a promising research frontier. We investigated the effect of human embryonic-derived neural stem cell (NSC) treatment on oligodendrocytes and myelination following hypoxia-ischemia (HI). Methods: Neonatal Wistar rat pups underwent left CCA ligation followed by placement in 8% O2 at 37°C on post-natal day 7 (P7). Following T2w MRI on P9, immunosuppressed pups received intra-arterial transplant of 500k fLuc/eGFP transduced NSCs or saline on P10. BrdU was administered intraperitoneally from P11 - P18. In vivo bioluminescence images (BLI) were obtained 1 - 10 days (d) after injection. Myelination was evaluated using luxol fast blue (LFB) and myelin basic protein (MBP) staining 10 and 30 d after treatment. Oligodendrogenesis was quantified using BrdU staining in conjunction with Olig2, NG2, and CC1. RT-qPCR was performed on neonatal brain isolates and NSC mRNA. Luminex immunological assay was used to quantify NSC protein secretion. Functional recovery was assessed using the novel object recognition (NOR) task at P30. Results: Stroke size between groups was not significantly different 3, 10, and 30 d after treatment. BLI demonstrated significant NSC homing to the ischemic hemisphere days 1 - 7 (p=0.001) after transplant. Histology confirmed initial NSC localization to corpus callosum and cortex with migration into external capsule and corona radiata 30 d after transplant. NSC-treated pups had significantly more BrdU+ cells near the lateral ventricle (p=0.036) and in the corpus callosum (p=0.020) than controls. In addition to more Olig2+ and NG2+ cells in the striatum, NSC-treated pups had significantly more BrdU+/Olig2+ cells in the corpus callosum (p<0.05) than controls 30 d after treatment. LFB and MBP staining demonstrated greater myelination 10 and 30 d after treatment in corpus callosum (p=0.022, p<0.05) and striatum (p=0.017, p=0.001) of NSC-treated pups. Stat3 (2.82), IL-6 (1.48), and IL-6Rβ (1.73) mRNA was upregulated in the brains of NSC-treated pups. Proteomic and mRNA data confirm NSC expression of VEGF (17.9pg/mL, 4.35) and CXCL1 (3.6pg/mL, 10.27). NSC-treated pups performed better on NOR (p=0.016). Conclusions: Intra-arterial NSC transplant after hypoxia-ischemia results in NSC engraftment into white matter tracts, increased oligodendrocyte proliferation, and improved myelination. NSC-derived proteins may drive the distinct changes in gene expression occurring in the brain after NSC treatment and may mediate functional recovery via activation of endogenous self-repair mechanisms, including oligodendrogenesis.