Accelerated atherosclerosis in beta-thalassemia.

Children with beta-thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. To evaluate this, wild-type (WT, littermates) and BT (Hbbth3/+) mice, both male and female, were placed on a 3-mo high-fat diet with low-density lipoprotein receptor suppression via overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). Mechanistically, we hypothesize that heme-mediated oxidative stress creates a proatherogenic environment in BT because BT is a hemolytic anemia that has increased free heme and exhausted hemopexin, heme's endogenous scavenger, in the vasculature. We evaluated the effect of hemopexin (HPX) therapy, mediated via an adeno-associated virus, to the progression of atherosclerosis in BT and a phenylhydrazine-induced model of intravascular hemolysis. In addition, we evaluated the effect of deferiprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice. Aortic en face and aortic root lesion area analysis revealed elevated plaque accumulation in both male and female BT mice compared with WT mice. Hemopexin therapy was able to decrease plaque accumulation in both BT mice and mice on our phenylhydrazine (PHZ)-induced model of hemolysis. DFP decreased atherosclerosis in BT mice but did not provide an additive benefit to HPX therapy. Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.NEW & NOTEWORTHY This work definitively shows for the first time that beta-thalassemia leads to accelerated atherosclerosis. We demonstrated that intravascular hemolysis is a prominent feature in beta-thalassemia and the resulting increases in free heme are mechanistically relevant. Adeno-associated virus (AAV)-hemopexin therapy led to decreased free heme and atherosclerotic plaque area in both beta-thalassemia and phenylhydrazine-treated mice. Deferiprone-mediated iron chelation led to deceased plaque accumulation in beta-thalassemia mice but provided no additive benefit to hemopexin therapy.

Renal protection by atorvastatin in a murine model of sickle cell nephropathy.

Recent studies have demonstrated pleiotropic effects of statins in various mouse models of kidney disease. In this study, Townes humanized sickle cell mice were treated for 8 weeks with atorvastatin at a dose of 10 mg/kg/day starting at 10 weeks of age. Treatment with atorvastatin significantly reduced albuminuria, and improved both urine concentrating ability and glomerular filtration rate. Atorvastatin also decreased markers of kidney injury and endothelial activation, and ameliorated oxidant stress in renal tissues and peripheral macrophages. Atorvastatin downregulated the expression of mRNA levels of the NADPH oxidases, Cybb (also termed Nox2) and Nox4, which are major sources of oxidant stress in the kidney. These findings highlight the pleiotropic effects of atorvastatin and suggest that it may provide beneficial effects in sickle cell nephropathy.

Impaired Collateral Vessel Formation in Sickle Cell Disease.

OBJECTIVE: The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. APPROACH AND RESULTS: We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=10 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice. CONCLUSIONS: Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease.

Using microfluidics to investigate hematopoietic stem cell and microniche interactions at the single cell level.

In recent years, microfluidic devices have become widely used in biology, and with the advantage of requiring low sample volumes, enables previously technologically infeasible experiments in hematopoietic stem cell (HSC) research. Here, we introduce a microfluidic device to investigate dynamic interactions between HSC and model niches in vitro. The device comprises a pneumatic valve which enables the culturing of different types of niche cells in different parts of the same device. Single HSCs can then be injected into the microfluidic device, manipulated, and placed onto different niches within the same device as controlled by the user. Here, we describe the device fabrication method, the HSC collection methodology, and the operational procedure for the device.

TNF-α, IFN-γ, IL-10, and IL-4 levels were elevated in a murine model of human sickle cell anemia maintained on a high protein/calorie diet.

Increased frequency and risk of infection is one of the well described complications of sickle cell anemia (SCA). Dietary supplementation in children with SCA and growth retardation improved growth and decreased incidence of infection. We investigated the impact of a high protein diet on weight gain, hematological profile, and immune cytokine levels in the Berkeley model of SCA, 16 of which were randomized to either regular mouse diet with 20% of calories from protein (n = 8) or a test feed with 35% of calories from protein (n = 8). Control mice (C57BL/6, n = 16) were correspondingly randomized, and were all feed ad libitum for three months with actual intake estimated by subtracting the weight of gnaw waste from that of the feed given. Blood was collected at sacrifice by cardiac puncture and plasma levels of T helper cell 1 (TH1) and TH2 associated cytokines were measured using a multiplex antibody immobilized bead assay. SCA mice receiving the 35% protein diet had modest improvements in weight, red blood cell count, and hemoglobin level, with a slight decrease in reticulocyte count compared with SCA mice on the regular mouse diet. Furthermore, they also had significantly higher plasma levels of cytokines tumor necrosis factor (TNF)-α (P = 0.02), interferon (IFN)-γ (P = 0.01), interleukin 10 (IL-10; P = 0.02), and IL-4 (P = 0.02) compared with those that received the 20% protein diet. We conclude that providing additional protein calories to transgenic SCA mice increased the plasma levels of acute inflammatory cytokines associated with immune response to infection, which might partly explain decreased episodes of infection observed among supplemented children with SCA.

Platelet biogenesis and functions require correct protein O-glycosylation.

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc(-/y)). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc(-/y) platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbß3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc(-/y) platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

Hematopoietic stem cell function in a murine model of sickle cell disease.

Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model. In vitro colony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit(+), Sca-(1+), Lineage(-)) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G(0) phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells. In vivo analysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.

Stem cell survival and functional outcome after traumatic brain injury is dependent on transplant timing and location.

PURPOSE: Recent work indicates that transplanted neural stem cells (NSCs) can survive, migrate to the injury site, and facilitate recovery from traumatic brain injury (TBI). The present study manipulated timing and location of NSC transplants following controlled cortical impact injury (CCI) in mice to determine optimal transplant conditions. METHODS: In Experiment 1 (timing), NSCs (E14.5 mouse) were injected into the host striatum, ipsilateral to the injury, at 2, 7, or 14 days. In Experiment 2 (location), NSCs or vehicle were injected into the mouse striatum (7 days post-CCI) either ipsilateral or contralateral to the injury and cognitive and motor abilities were assessed from weeks 1-8 post-transplant. Histological measures of NSC survival, migration, and differentiation were taken at 6 and 8 weeks post-transplant. RESULTS: The results demonstrate that: (1) 2-7 days post-injury is the optimal time-range for delivering NSCs; (2) time of transplantation does not affect short-term phenotypic differentiation; (3) transplant location affects survival, migration, phenotype, and functional efficacy; and (4) NSC-mediated functional recovery is not contingent upon NSC migration or phenotypic differentiation. CONCLUSIONS: These findings provide further support for the idea that mechanisms other than the replacement of damaged neurons or glia, such as NSC-induced increases in protective neurotrophic factors, may be responsible for the functional recovery observed in this model of TBI.

Laminin and fibronectin scaffolds enhance neural stem cell transplantation into the injured brain.

Cell transplantation offers the potential to treat central nervous system injuries, largely because multiple mechanisms can be targeted in a sustained fashion. It is crucial that cells are transplanted into an environment that is favourable for extended survival and integration within the host tissue. Given the success of using fetal tissue grafts for traumatic brain injury, it may be beneficial to mimic key aspects of these grafts (e.g. three-dimensionality, cell-cell and cell-matrix support) to deliver cells. Extracellular matrix proteins such as fibronectin and laminin are involved in neural development and may provide adhesive support for donor cells and mediate subsequent cell signalling events. In this study, neural stem cells were transplanted into the traumatically injured mouse brain within a tissue-engineered construct containing either a laminin- or fibronectin-based scaffold. Cells delivered within the scaffolds were more widely distributed in the injured brain compared to cells delivered in media alone. There were no differences in donor cell survival at 1 week post-transplant; however, by 8 weeks post-transplant, cells delivered within the scaffolds showed improved survival compared to those transplanted in media alone. Survival was more enhanced with the laminin-based scaffold compared to the fibronectin-based scaffold. Furthermore, behavioural analyses indicated that mice receiving neural stem cells within the laminin-based scaffold performed significantly better than untreated mice on a spatial learning task, supporting the notion that functional recovery correlates positively with donor cell survival. Together these results suggest that the use of appropriate extracellular matrix-based scaffolds can be exploited to improve cell transplantation therapy.

C-reactive protein and interleukin-6 are decreased in transgenic sickle cell mice fed a high protein diet.

Sickle cell disease is associated with hypermetabolism and a consequent shortage of substrates for normal growth and healthy immune response. The protein:energy ratio is a major determinant of dietary adequacy; the requirement for optimal growth of control mice is 20% of energy from dietary protein. This study investigated the efficacy of increased dietary protein for improving weight gain and reducing inflammation in the Berkeley sickle cell mouse model (S). The study examined the effect of diet on weight gain and circulating levels of 2 inflammatory proteins, C-reactive protein (CRP), and cytokine interleukin-6 (IL-6). Male C57BL/6 (C) control (n = 8) and S mice (n = 8) were randomized at weaning to 40 d of isoenergetic diets containing 20% (normal) and 35% (high) of energy from protein (C20, C35, S20, S35), replacing dextrin. Rate of weight gain was calculated and plasma CRP and IL-6 concentrations determined by ELISA. Liver mRNA expression of these proteins was measured by real-time PCR and L-arginase by colorimetric assay. S35 mice tended to gain weight more rapidly than S20 mice (P = 0.06) and more rapidly than C35 mice (P < 0.01). Circulating CRP and IL-6 levels were also lower in S35 mice than in S20 mice (P < 0.05), as was liver CRP mRNA expression (P < 0.01). These results demonstrate that introducing a high protein diet at weaning attenuates the steady-state inflammation in this S mouse model. Dietary L-arginine availability was investigated as a possible mechanism for increased nitric oxide production and consequent reduced inflammation.

Complete regression of large solid tumors using engineered drug-resistant hematopoietic cells and anti-CD137 immunotherapy.

Combining chemotherapy and immunotherapy is problematic because chemotherapy can ablate the immune responses initiated by modulators of the immune system. We hypothesized that protection of immunocompetent cells from the toxic effects of chemotherapy, using drug resistance gene therapy strategies, would allow the combined use of chemotherapy and immunotherapy. In wild-type mice, the antitumor effectiveness of an immunotherapy regimen employing an agonistic anti-CD137 antibody is diminished with escalating doses of the antifolate trimetrexate (TMTX). Using retroviral gene transfer of a mutant form of dihydrofolate reductase (L22Y-DHFR), hematopoietic stem cells were genetically engineered to withstand the toxic effects of TMTX. Mice transplanted with L22Y-DHFR-modified bone marrow were then challenged with AG104 sarcoma cells and treated with TMTX only, anti-CD137 only, or a combination of chemotherapy and immunotherapy. Although tumor burden was transiently decreased during TMTX administration, no mice treated with TMTX alone survived the tumor challenge, whereas approximately 40% of transplanted mice treated with anti-CD137 alone survived. However, 100% of mice survived with complete tumor regression after transplantation with L22Y-DHFR-transduced bone marrow followed by combined treatment with TMTX and anti-CD137. In addition, adoptive transfer of splenocytes from cured mice extended the survival of tumor- bearing animals by approximately 3 weeks compared with controls. Therefore, protection of the hematopoietic system can allow for the combined administration of chemotherapy and immunotherapy, which results in complete tumor clearance.

Enforced expression of cytosolic 5'-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection.

PURPOSE: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. METHOD: In this study, we investigate retroviral transfer of cytosolic 5'-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2'-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. RESULTS: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. CONCLUSION: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker.

Donor marker infidelity in transgenic hematopoietic stem cells.

Transgenic marking approaches are increasingly used to evaluate the developmental potential of stem cells. However, cell fate mapping studies using different transgenic marking systems have produced conflicting results. These disparate findings may be due in part to the infidelity of donor marker gene expression. Analysis of hematopoietic stem cells (c-Kit+, Sca-1+, lineage marker- [KSL]) from a transgenic mouse (1Osb) engineered to ubiquitously express the enhanced green fluorescent protein (EGFP) reveals two distinct populations. Forty percent of KSL cells demonstrate intermediate levels of EGFP fluorescence and differentiate into subpopulations of B cells, T cells, and myeloid cells that do not express EGFP. By contrast, progeny of the remaining 60% of KSL cells are almost exclusively EGFP bright. Long-term multilineage hematopoietic reconstitution and serial transplantation experiments show that these differences in EGFP are a property of self-renewing stem cells. Furthermore, both the transgene integration site and the activation status of a cell are important determinants of EGFP expression. These results indicate that a combination of donor cell markers is required to reliably track the full differentiation potential of transgenic stem cells.

Highly efficient transduction of repopulating bone marrow cells using rapidly concentrated polymer-complexed retrovirus.

Using the cationic polymer, Polybrene, and the anionic polymer, chondroitin sulfate C, we concentrated recombinant retrovirus pseudotyped with an ecotropic envelope, which is susceptible to inactivation by high-speed concentration methods. To evaluate gene marking, murine bone marrow was harvested from C3H mice, transduced with polymer-concentrated GFP virus, and transplanted into lethally irradiated recipients. Total gene marking in mice averaged 30-35% at 8 weeks post-transplant and transgene expression remained stable for over 16 weeks. Using the polymer concentration method, a second retroviral vector encoding the drug resistant variant of dihydrofolate reductase (L22Y-DHFR) was concentrated and tested. Approximately 40% of transduced murine bone marrow progenitor cells were protected against trimetrexate concentrations that completely eliminated the growth of non-modified cells. These results show that anionic and cationic polymers can be combined to rapidly concentrate viruses that are normally difficult to concentrate, and the concentrated virus efficiently transduces hematopoietic stem cells.

Neural progenitor cell transplants promote long-term functional recovery after traumatic brain injury.

Studies demonstrating the versatility of neural progenitor cells (NPCs) have recently rekindled interest in neurotransplantation methods aimed at treating traumatic brain injury (TBI). However, few studies have evaluated the safety and functional efficacy of transplanted NPCs beyond a few months. The purpose of this study was to assess the long-term survival, migration, differentiation and functional significance of NPCs transplanted into a mouse model of TBI out to 1 year post-transplant. NPCs were derived from E14.5 mouse brains containing a transgene-expressing green fluorescent protein (GFP) and cultured as neurospheres in FGF2-containing medium. Neurospheres were injected into the ipsilateral striatum of adult C57BL/6 mice 1 week following unilateral cortical impact injury. Behavioral testing revealed significant improvements in motor abilities in NPC-treated mice as early as 1 week, and the recovery was sustained out to 1 year post-transplant. In addition, mice receiving NPC transplants showed significant improvement in spatial learning abilities at 3 months and 1 year, whereas an intermediate treatment effect on this behavioral parameter was detected at 1 month. At 14 months post-transplant, GFP(+) NPCs were observed throughout the injured hippocampus and adjacent cortical regions of transplanted brains. Immunohistochemical analysis revealed that the majority of transplanted cells co-labeled for NG2, an oligodendrocyte progenitor cell marker, but not for neuronal, astrocytic or microglial markers. In conclusion, transplanted NPCs survive in the host brain up to 14 months, migrate to the site of injury, enhance motor and cognitive recovery, and may play a role in trophic support following TBI.

Noscapine crosses the blood-brain barrier and inhibits glioblastoma growth.

The opium alkaloid noscapine is a commonly used antitussive agent available in Europe, Asia, and South America. Although the mechanism by which it suppresses coughing is currently unknown, it is presumed to involve the central nervous system. In addition to its antitussive action, noscapine also binds to tubulin and alters microtubule dynamics in vitro and in vivo. In this study, we show that noscapine inhibits the proliferation of rat C6 glioma cells in vitro (IC(50) = 100 microm) and effectively crosses the blood-brain barrier at rates similar to the ones found for agents such as morphine and [Met]enkephalin that have potent central nervous system activity (P < or = 0.05). Daily oral noscapine treatment (300 mg/kg) administered to immunodeficient mice having stereotactically implanted rat C6 glioblasoma into the striatum revealed a significant reduction of tumor volume (P < or = 0.05). This was achieved with no identifiable toxicity to the duodenum, spleen, liver, or hematopoietic cells as determined by pathological microscopic examination of these tissues and flow cytometry. Furthermore, noscapine treatment resulted in little evidence of toxicity to dorsal root ganglia cultures as measured by inhibition of neurite outgrowth and yielded no evidence of peripheral neuropathy in animals. However, evidence of vasodilation was observed in noscapine-treated brain tissue. These unique properties of noscapine, including its ability to cross the blood-brain barrier, interfere with microtubule dynamics, arrest tumor cell division, reduce tumor growth, and minimally affect other dividing tissues and peripheral nerves, warrant additional investigation of its therapeutic potential.

Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease.

Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT.

Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P

Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain.

Multipotential stem cells are an attractive choice for cell therapy after traumatic brain injury (TBI), as replacement of multiple cell types may be required for functional recovery. In the present study, neural stem cells (NSCs) derived from the germinal zone of E14.5 GFP-expressing mouse brains were cultured as neurospheres in FGF2-enhanced medium. When FGF2 was removed in vitro, NSCs expressed phenotypic markers for neurons. astrocytes, and oligodendrocytes and exhibited migratory behavior in the presence of adsorbed fibronectin (FN). NSCs (10(5) cells) were transplanted into mouse brains 1 week after a unilateral, controlled, cortical contusion (depth = 1 mm, velocity = 6 m/s, duration = 150 ms) (n = 19). NSCs were injected either directly into the injury cavity with or without an injectable FN-based scaffold [collagen I (CnI)/FN gel; n = 14] or into the striatum below the injury cavity (n = 5). At all time points examined (1 week to 3 months posttransplant), GFP+ cells were confined to the ipsilateral host brain tissue. At 1 week, cells injected into the injury cavity lined the injury penumbra while cells inserted directly into the striatum remained in or around the needle track. Striatal transplants had a lower number of surviving GFP+ cells relative to cavity injections at the 1 week time point (p < 0.01). At the longer survival times (3 weeks-3 months), 63-76% of transplanted cells migrated into the fimbria hippocampus regardless of injection site, perhaps due to cues from the degenerating hippocampus. Furthermore, cells injected into the cavity within a FN-containing matrix showed increased survival and migration at 3 weeks (p < 0.05 for both) relative to injections of cells alone. These results suggest that FGF2-responsive NSCs present a promising approach for cellular therapy following trauma and that the transplant location and environment may play an important role in graft survival and integration.