Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression

Experimental Neurology 199 (2006) 339 – 347 www.elsevier.com/locate/yexnr

Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression André Wennersten a,⁎, Staffan Holmin a , Faiez Al Nimer a , Xia Meijer a , Lars U. Wahlberg b , Tiit Mathiesen a a

Department of Clinical Neuroscience, Section of Clinical CNS Research, Karolinska Institutet, S-171 76 Stockholm, Sweden b NS Gene A/S, Copenhagen, Denmark Received 26 June 2005; revised 22 December 2005; accepted 29 December 2005 Available online 21 February 2006

Abstract Neural stem cells have emerged as a promising therapeutic tool in CNS disease and injuries. In the clinical setting, cultured human neural stem/ progenitor cells (hNSC) are an attractive possibility for transplantation to the damaged brain. However, transplantation of hNSC requires toxic immunosuppressive treatment to avoid rejection. The aim of the current study was to evaluate if shortening the duration of immunosuppression by cyclosporin A would affect hNSC survival and differentiation after transplantation to the site of a focal brain injury in the rat. hNSC were xenografted to the hippocampus and the medial limit of an experimentally induced cortical contusion. The animals received immunosuppression for either 6 or 3 weeks or no immunosuppression. The status of the grafted human cells was analysed by immunohistochemistry. No statistically significant differences were observed between the two immunosuppressed groups regarding graft survival, migration or proliferation at 6 weeks post-transplantation. In contrast, the graft survival was extremely poor in the non-immunosuppressed group. Furthermore, the expression of the differentiation markers nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) in the transplanted cells did not differ significantly between the two immunosuppressed groups. Moreover, a fourth group of eight animals that were immunosuppressed for 3 weeks were allowed to survive for 6 months. Five of these rats demonstrated robust graft survival in the hippocampus and scattered cells in the cortex. This study demonstrates the importance of immunosuppression but also the possibility of shortening immunosuppression without impacting on the phenotype of the grafted hNSC. © 2006 Elsevier Inc. All rights reserved. Keywords: Fetal stem cell; Traumatic brain injury; Immunosuppressed; Neural progenitor; Immunesuppressed, xenotransplantation; Xenogeneic; Cyclosporine

Introduction Transplantation of neural stem cells has shown considerable therapeutic potential in several animal models of central nervous system (CNS) disorders (Aboody et al., 2000; Deacon et al., 1998; Ehtesham et al., 2002; Ogawa et al., 2002; Philips et al., 2001; Riess et al., 2002; Vescovi et al., 1999a,b). Recent findings points towards cultured human derived stem cells as an attractive choice for clinical applications due to the possibility of obtaining significant number of cells from a limited amount of material (Carpenter et al., 1999; Flax et al., 1998). Although there is an extensive body of data building up on the use of ⁎ Corresponding author. Fax: +46 8 517 717 78. E-mail address: [email protected] (A. Wennersten). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.12.035

transplanted human neural stem/progenitor cells (hNSC) in several models of CNS injury, including stroke and traumatic brain injury, studies on the effect of different amounts and duration of immunosuppression are still scarce (Jeong et al., 2003; Kelly et al., 2004; Svendsen et al., 1996; Wennersten et al., 2004). Immunosuppressive agents have potentially toxic side effects for the patient. Today, most experimental xenografting protocols use high doses of immunosuppressant throughout the lifespan of the animals (Deacon et al., 1998; Ishibashi et al., 2004; Kelly et al., 2004; Le Belle et al., 2004; Wennberg et al., 2001). A more precise knowledge on the effect of immunosuppressive treatment on the grafts and the mechanisms preceding rejection in the CNS may prevent unnecessary suppression of a patient's immune system. Furthermore, the potential benefit of a shortened period of

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immunosuppression is that long term experimental studies will be greatly facilitated. Data from behavioral testing would be easier to interpret due to the substantial reduction of stress imposed on the animals due to the immunosuppression. Finally, the susceptibility to infections would be decreased. We have previously demonstrated that cultured and cryopreserved hNSC transplanted to an experimental contusion injury in the rat survive, proliferate and differentiate 6 weeks post-grafting. In that study the animals received cyclosporin A during the entire experiment (Wennersten et al., 2004). The current study was undertaken to determine if the duration of immunosuppression by cyclosporin A could be decreased without reducing survival or affecting the phenotype of hNSC grafted to the traumatically injured brain. Material and methods Generation and in vitro culture of human neural stem/progenitor cells 7-week-old post-conception forebrain tissue was recovered from an elective first trimester routine abortion using the regular vacuum aspiration technique. The collection of residual tissue was approved by the Human Ethics Committee of the Huddinge University Hospital, Karolinska Institute, and was in accordance with the guidelines of the Swedish Society of Medicine including an informed consent from the pregnant women seeking abortions. The preparation of the tissues was done in accordance with previously published protocols (Carpenter et al., 1999). Briefly, the collected tissue was micro dissected in sterile saline and transferred to N2 medium, a defined Dulbecco's modified Eagle's medium (DMEM)/F-12 medium including 0.6% glucose, 2 mM glutamine, 5 mM HEPES buffer, insulin (25 μg/ml), transferring (100 μg/ml, Sigma), progesterone (20 nM, Sigma), Putrescine (60 μM, Sigma), Selenium chloride (30 nM, Gibco) and 2 μg/ml heparin. The tissue was triturated in DMEM/F12 medium using a glass/Teflon Potter-Elvehjelm homogenizer. The viable cells were counted with tryphan blue exclusion and the volume was adjusted by the addition of culture medium to obtain a cell density of 250,000 cells/ml. The cells were grown in the N2 medium supplemented with recombinant human epidermal growth factor (EGF; 20 ng/ml, R&D Systems), recombinant human basic-fibroblast growth factor (bFGF; 20 ng/ml, R&D Systems), and recombinant ciliary neurotrophic factor (CNTF; 10 μg/ml, R&D Systems). The cells grew as freefloating clusters (neurospheres), were passaged by mechanical dissociation every 10 days and reseeded as single cells at a density of 100,000 cells/ml. The cells for transplantation were frozen at passage 9 and recultured until passage 10 before the transplantation. Preparation of cells for transplantation The human neural stem/progenitor cells were taken for transplantation 4–5 days after the last passage as small spheres.

The spheres were collected by centrifugation at 1000 rpm for 3 min and resuspended in 1 ml DMEM/F12 medium. The sphere suspension was centrifuged again and resuspended in a smaller volume to give the equivalent of 100,000 cells/μl. Surgical procedures 36 male Sprague–Dawley rats weighing approximately 300 g were anesthetized by intramuscular injection of Hypnorm (fluanisonum, 10 mg/ml and fentanylium, 0.2 mg/ml, Janssen, Belgium) and Dormicum (midazolam, 1 mg/ml, Roche). In addition, 0.1 ml of Xylocain-Adrenalin (lidocaine hydrochloride, 5 mg/ml and adrenaline, 5 μg/ml, Astra, Sweden) was injected subcutaneously in the sagittal midline of the skull before the skin incision. The rats were put into a stereotactic frame and a craniotomy was drilled 3.0 mm posterior and 2.0 mm lateral bregma. A standardized parietal contusion was made by letting a 24-g weight fall onto a rod with a flat end diameter of 1.8 mm from a height of 6.0 cm. The rod was allowed to compress the tissue a maximum of 3.0 mm. 30 of the rats received hNSC while 6 received hNSC that had been killed by repeated freezing and thawing. Ten minutes after the injury, the cultured hNSC or killed hNSC were injected to the medial margin of the contusion (3.0 mm posterior and 1.1 mm lateral to the bregma). 1 μl was injected at a depth of 3.5 mm and 1.1 μl was injected at a depth of 2.0 mm from the cortical surface. The animals were treated with subcutaneous injections of cyclosporin A (Sandimmun, Novartis, Sweden) 4 mg/kg every Monday and Wednesday and 8 mg/kg every Friday for immunosuppression starting the day before surgery and with trimethoprim 12 mg and sulphadoxin 60 mg (Borgal Vet, Intervet, Stockholm, Sweden) to 500 ml of drinking water for infection prophylaxis. 6 animals did not receive any immunosuppression (6 weeks survival), 19 animals received cyclosporin A for 3 weeks (6 weeks or 6 months survival) and 11 animals received cyclosporin A for 6 weeks (6 weeks survival). The 6 animals that were transplanted with dead hNSC were divided between the 3 and 6 week immunosuppression group (6 weeks survival). The 6-month animals were kept in the animal care facility under standard conditions after the cessation of immunosuppression. All the animals were sacrificed by decapitation under identical anaesthesia as during the initial surgery 6 weeks or 6 months after the transplantation. The brains were immediately dissected and frozen in isopentane containing dry ice, 14 μm coronal cryosections were cut serially and stored at −20°C. Immunohistochemistry After thawing at room temperature, the sections were fixed in formaldehyde. Endogenous peroxidase was quenched for 30 min in 0.5% hydrogen peroxide diluted in PBS. The sections were incubated at room temperature for 1 h with PBS containing 1% bovine serum albumin, 0.3% Triton X-100 and 0.1% sodium azide and avidin block solution (Vector Laboratories Inc., Burlingame, CA, USA) to prevent non-specific binding. This solution, without the avidin, was used for all subsequent

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antibody dilutions. The sections were incubated with the primary antibody mouse anti-human nuclei (huN, dilution 1:200; Chemicon) or rabbit anti-human Ki-67 (dilution 1:200; DAKO) over night at 4°C. Biotin block solution (Vector Laboratories Inc., Burlingame, CA, USA) was added to the primary antibody dilution prior to application. For negative controls the same solution without primary antibody was used. In addition, the cases receiving dead hNSC were used as negative histological controls. For detection of the primary antibody an avidin conjugated secondary antibody made in horse against the appropriate species was applied. The avidin– biotin–enzyme complex (ABC) technique was used and the bound peroxidase was visualized by incubation for 3 min with a

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diaminobenzidine substrate kit (Vector Laboratories, Inc., Burlingame, CA). Single stained sections were counterstained with haematoxylin, dehydrated, and mounted with DPX. For double labeling with a second primary antibody the sections were run through the same procedure again with either mouse anti-human Nestin (dilution 1:1000; R&D Systems), rabbit antiglial fibrillary acidic protein (GFAP, dilution 1:500; DAKO) or mouse anti-neuronal nuclei (NeuN, dilution 1:500; Chemicon) antibody. This time the bound peroxidase was visualized with the Novared substrate kit (Vector Laboratories, Burlingame, CA). Great care was taken to expose the sections to the chromogenes for the same duration of time. The sections were dried on a hotplate and mounted with DPX.

Fig. 1. A collection of photomicrographs with huN-positive cells, dark brown, in one coronal section from the group receiving 3 weeks immunosuppression group (A) and one coronal section from the group receiving 6 weeks immunosuppression (B). The two sites of transplantation can be observed as areas with slightly increased cell densities proximal to the lesion and the centre of the hippocampus and the intersection between the corpus callosum, respectively. Six weeks post-transplantation the cells are typically situated along the lesion–cortex border in both groups. Migrating cells can be seen along the entire ipsilateral corpus callosum medial to the lesion and continuing across the midline and finally entering the contralateral cortex. The sections are counterstained with haematoxylin (Solid lines delineate the midline. Asterisk = lesion, i = ipsilateral cortex and ii = ipsilateral hippocampus. Scale bar = 100 μm).

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Cell counting and statistics The number of human cells was manually counted, by an investigator blinded to the groups, in four consecutive sections spaced 400 μm apart thus spanning 1200 μm in the rostrocaudal axis with the core of the transplant as centre. The amount of proliferating human cells in each case was measured by counting the total number of anti-human Ki-67 immunoreactive cells in one coronal section located central in respect to the transplant. Unpaired t test was performed using GraphPad InStat (version 3.05 for Windows NT, GraphPad Software; www.graphpad.com). Values are expressed as mean ± standard error of the mean. Results General morphology All the immunosuppressed animals that were grafted with live hNSC had surviving cells at the 6-week end point, while no animals receiving killed hNSC displayed any immunoreactivity with neither the anti-human nuclei (huN) nor the anti-human nestin antibody. In the group that were transplanted with live hNSC but denied the immunosuppressive treatment only one out of six animals displayed any surviving cells. One animal from each immunosuppressed group was excluded from further analysis because of slightly misplaced transplants. The transplanted cells were found at both sites of injection, i.e., medial limit of the lesion and the hippocampus (Fig. 1). In addition, huN-positive cells were also observed in the medial ipsilateral cortex, the ipsilateral and contralateral corpus

callosum. A few huN-positive cells were also observed in the contralateral cortex in both the group receiving 3 weeks immunosuppression and the group receiving 6 weeks immunosuppression. The grafts typically appeared as dispersed cells rostrally of the transplantation sites within the parenchyma of the cortex and hippocampus, respectively. Moving caudally, grafted cells increased in number and were clumped together in the graft core (as seen in the hippocampus of Fig. 1A). Moreover, at this coronal level human cells were found in the contralateral hemispheres. Further caudally to the transplant cores, the huNpositive cells were distributed in a similar fashion as seen rostrally to the transplant cores. The gross distribution and morphology of the grafts did not differ between the group receiving 3 weeks immunosuppression and the group receiving 6 weeks immunosuppression. Eight animals that had received immunosuppression for the first 3 weeks were allowed to survive for 6 months postoperation. Five of these rats displayed huN-positive cells in both the perilesional zone and the hippocampus (Fig. 2). Number of huN-positive cells In the 6 weeks survival groups, the total number of human cells was counted in four consecutive sections, with the transplant cores as centre, spaced 400 μm apart in each case. No huN-positive cells were detected in the animals that were grafted with dead hNSC. In the non-immunosuppressed group, only few cells (less than 15 per section) were detected in one animal. There were no differences in the numbers of huNpositive cells between the treatment groups in the ipsilateral

Fig. 2. Human cells were present in five (of eight) rats that received immunosuppression for 3 weeks and were allowed to survive for 6 months after the operation. The photomicrographs show surviving grafted cells (black) from one representative animal, at three coronal levels 400 μm apart (A–C). The wide distribution of human cells in the hippocampus was paralleled by cells in the cortical region that had migrated further than 2 mm rostral (D; −1.0 mm Bregma) and 2 mm caudal (E; −5.0 mm Bregma) from the graft core. The sections are counterstained with haematoxylin. Scale bar = 400 μm in panels A–C; scale bar = 50 μm in panels D and E.

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cortex, corpus callosum, hippocampus or the contralateral hemisphere (Fig. 3). Furthermore, the total number of huNpositive cells between the group receiving 3 weeks immunosuppression and the group receiving 6 weeks immunosuppression did not differ significantly (500 ± 99 and 438 ± 94 respectively). Ki-67—proliferation antigen To allow for an estimate of proliferating human cells the number of cells positive for the human Ki-67 antigen, present only in cells during mitosis, was counted in the perilesional cortex, the hippocampus and contralateral to the lesion. There was no significant difference in the number of human Ki-67positive cells between the group receiving 3 weeks immunosuppression and the group receiving 6 weeks immunosuppression (Fig. 4). Differentiation Immunohistochemical double staining for huN and human Nestin, GFAP or NeuN was performed to analyze the state of differentiation in the grafted cells in the ipsilateral cortex and hippocampus. huN-positive cells co-expressing either humannestin, GFAP or NeuN were found both in the ipsilateral cortex and the hippocampus in animals from both groups (Fig. 5). The proportion of huN immunoreactive cells that displayed co-expression of human nestin did not change significantly with reduced immunosuppression in neither the ipsilateral cortex nor the hippocampus (Fig. 6A). No significant difference was seen between the proportion of huN-positive cells co-expressing GFAP as a result of reduced immunosuppression in neither the ipsilateral cortex nor the hippocampus (Fig. 6B). The neuronal marker NeuN co-localized with huN in a limited number of cells. All cases displayed an average proportion lower than 1%. No significant differences were

Fig. 3. The average number of surviving human cells per section. The cells were counted in four consecutive sections 400 μm apart with the core of the transplant as centre, thus spanning a total of 1200 μm the rostro-caudal axis in each case. No significant difference was observed in the number of huN-positive cells in neither the cortex, hippocampus (h.c.), corpus callosum (c.c.) nor contralateral (contra) to the operated side between the group receiving 3 weeks immunosuppression (black bars) and the group receiving 6 weeks immunosuppression (white bars). In the group of animals that did not receive any immunosuppression, 1 of 6 had less than 15 cells per section the rest had no surviving cells.

Fig. 4. The number of cells immunoreactive for the human-Ki-67, an antigen present in proliferating cells, did not differ significantly between the two groups in the respective location. Black bars correspond to the group receiving 3 weeks immunosuppression and the white bars correspond to the group receiving 6 weeks immunosuppression (h.c. = hippocampus, contra = contralateral to the operated side).

noted between the group receiving 3 weeks immunosuppression and the group receiving 6 weeks immunosuppression. Discussion The shortening of immunosuppressive treatment from six to 3 weeks did not affect the survival, proliferation or the differentiation of fetal human neural stem/progenitor cells (hNSC) transplanted to the traumatically injured rat brain. In contrast, absence of immunosuppression was detrimental to the grafted hNSC. Although the majority of transplantation experiments using hNSC in brain transplants are xenograft models, there are still a number of unresolved issues, for instance the amount and duration of immunosuppression that is needed for graft survival. In the current report we transplanted hNSC to the experimentally injured rat brain and used two different immunosuppression protocols; the results were evaluated on the basis of the survival, differentiation, migration and proliferative activity of the transplanted cells 6 weeks post-transplantation. The cessation of immunosuppression after 3 weeks did not significantly affect the number of huN-positive cells, KI-67 expression, the fraction of huN-positive cells co-expressing NeuN, GFAP, nestin, or migration patterns. Furthermore, our observations suggest that 3 weeks of immunosuppressive treatment with cyclosporin A was compatible with graft survival at 6 months. Neural stem cells have emerged as a promising therapeutic tool in brain pathology. In the clinical setting hNSC is an attractive choice because human grafts can be expected to interact favorably with the recipient and large numbers of clonally expanded cells can be kept available for treatment when the need arrives. However, transplantation of homologous hNSC will most likely need immunosuppressive treatment to prevent rejection. Using autologous transplants could mitigate this. Such strategies may have drawbacks, for instance, in the case of disorders with a possible autoimmune or genetic component this strategy will reintroduce cells of the same background that may originally have caused the disease (Snyder et al., 2004). Moreover, for trauma patients there may be a time

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Fig. 5. Photomicrographs depicting human nuclei (huN) immunoreactive cells (dark/black stain) co-expressing human nestin (red stain; A–D), GFAP (anti-glial fibrillary acidic protein; red stain; E–H) and anti NeuN (neuronal nuclei; red stain; I–L) in the cortex (A, C, E, G, I, K) and hippocampus (B, D, F, H, J, L) from the group receiving 3 weeks immunosuppression (A, B, E, F, I, J) and the group receiving 6 weeks immunosuppression (C, D, G, H, K, L). The arrows indicate double labeled human cells that are shown in higher magnification (insets A–L). Scale bar in L = 200 μm in panels A–I and K–L and 100 μm in panel J, scale bar = 40 μm for insets in panels A–H and 10 μm for insets in panels I–L.

Fig. 6. Bar graphs showing the proportion of human cells co-expressing human nestin (A) and GFAP (B) in the cortex and hippocampus respectively. The neuronal marker NeuN co-localized with huN in a limited number of cells (average proportion below 1%; not shown in graph). The black bars represent the group receiving 3 weeks immunosuppression and the white bars represent the group receiving 6 weeks immunosuppression. There was no statistically significant difference within each location between the two groups.

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factor for efficacious treatment with transplanted cells necessitating the immediate availability of cultured hNSC and although the timing of stem cell transplantation is incompletely studied, neuroprotection may be one beneficial effect of grafting hNSC in the acute stage (Hagan et al., 2003; Ourednik et al., 2002). Mechanisms of rejection The mechanisms that lead to rejection in transplantation to the CNS are not completely understood. Several investigations suggest that the T-cell mediated immune reaction plays a significant role in graft rejection (Brevig et al., 2000; Duan et al., 2002; Mirza et al., 2004). Immunosuppressive treatment is a key determinant in graft survival in a number of xenograft paradigms (Brundin et al., 1989; Cicchetti et al., 2003; Mathiesen et al., 1989; Trojanowski et al., 1993; Wennberg et al., 2001). Moreover, in a metaanalysis from 1994, Pakzaban and Isacson concluded that the continuous treatment with cyclosporin A is the most important variable for graft survival regardless of donor species (Pakzaban and Isacson, 1994). The main attention on immunological reactions after xenografting to the CNS has been elicited by the prospect of using neural tissue from animals (mainly pigs) to treat focal CNS disorders such as Parkinson's disease and Huntington's disease (Brevig et al., 2000). Generally, CNS xenografting without immunosuppression does not allow graft survival (Brevig et al., 2000; Larsson and Widner, 2000). Cyclosporin A Cyclosporin A is a lipophilic molecule that is transported bound to plasma lipoproteins and other proteins. To exert its immunosuppressive effect it has to enter the cytosol of an activated T-cell (Akhlaghi and Trull, 2002). Inside the cell, the immunosuppressive action is mediated through inhibition of calcineurin, a calcium dependent enzyme critical for the activation of the T-cell mediated immune response (Kaminska et al., 2004). Furthermore, cyclosporin A increases the viability of rat embryonic dopamine neurons subjected to serum deprivation in vitro by inhibition of calcineurin activity (Castilho et al., 2000). It is also well established that Cyclosporin A has a neuroprotective effect after traumatic CNS injuries (Buki et al., 1999; Li et al., 2000; Scheff and Sullivan, 1999; Sullivan et al., 2000; Uchino et al., 2002). These neuroprotective properties have been linked to a direct effect on the membrane permeability transition pore (MPTP). However, calcineurin is implicated in several other aspects of the secondary injury mechanisms, including Bcl-2 family mediated apoptosis and excitotoxicity, suggesting that the neuroprotective effects of cyclosporine could be a result of inhibition of calcineurin (Shibasaki and McKeon, 1995; Wang et al., 1999). Although the mechanisms are not completely elucidated, the potentially broad protective actions provide an argument for cyclosporin A use as an immunosuppressant in cell based therapies for CNS

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injuries. However, the long term side effects of prolonged immunosuppression such as susceptibility to infections and CNS- and nephrotoxicity need consideration (Akhlaghi and Trull, 2002). The number of cells dying after CNS trauma declines extensively over the first few weeks to months (Conti et al., 1998; Wennersten et al., 2003). This would imply that the purely neuroprotective effects are most likely to be of value in the initial stages after injury. In CNS transplantation, there are reports indicating that the duration of treatment can be shortened. Even a limited period of immunosuppression compared to no immunosuppressive treatment is beneficial in transplantation of bovine or neoplastic human cells to rat CNS (Mathiesen et al., 1989; Ortega et al., 1992). Aleksandrova et al. found surviving cells 20 days posttransplantation even without any immunosuppression (Aleksandrova et al., 2002). Apart from the transplantation procedure, the animals had not been subjected to any injury to the CNS and no data of the number of surviving cells were presented, making interpretations on potential ongoing rejection difficult. However, in the current model of traumatic brain injury xenografted hNSC were rejected to a high degree if no immunosuppression was used but survived well if the immunosuppression was administered during a limited period post-surgery. Mechanisms of protection against xenograft rejection In our experiments, neither the number of surviving cells derived from the transplanted hNSC nor their distribution or expression of differentiation markers differed between the two immunosuppressed groups at 6 weeks. The equal number of cells could theoretically be explained by an increased turnover with both enhanced proliferation and cell death from rejection in the group receiving 3 weeks of immunosuppression. Such a hypothesis is, however, incompatible with comparable levels of proliferating cells in both groups. The number of Ki-67-positive human cells did not differ significantly between the two groups. Several mechanisms may explain the absence of rejection when immunosuppression is ceased: Peripheral tolerance or Tcell anergy may have been induced to the xenografted hNSC. The blood–brain barrier may have been sufficiently restored during the 3 weeks when the immune system was suppressed to prevent leukocyte infiltration of the grafts. The cultured hNSC may have a greatly reduced immunogenicity compared to fresh tissue (Al Nimer et al., 2004). There may be a prolonged rejection. Self-tolerance is naturally occurring during development when the immune system matures to loose its capacity to attack constituents of the own organism. This state can also be induced in the fully developed organism either by completely eliminating the pool of mature T-cells and then introducing graft antigens in the thymus before new thymocytes mature or by weakening the immune system at time of transplantation. The latter can be achieved either by depletion of the host T-cells or by interfering with their activation (inducing T-cell anergy) (Brevig et al., 2000; Schwartz, 2003). Larsson et al. (2003) demonstrated that a triple-blocking strategy of costimulatory signals necessary for Tcell activation induced peripheral tolerance to porcine primary

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xenografts in C57BL/6 mice. The immunosuppression in our experiments covers the period of peak inflammation caused by the trauma (Holmin et al., 1995). The activation of T-cells exposed to hNSC antigens would be suppressed during this period as a result of cyclosporin A administration. However, whether this is enough to induce peripheral tolerance or T-cell anergy remains to be demonstrated. Blood–brain barrier restoration did not stop the rejection of dissociated fetal mesencephalic mouse tissue xenografted to rats that had received 6-hydroxydopamine induced lesions (Brundin et al., 1989). This tissue was fresh and had not been kept in culture and may thus have been responsible of eliciting a more vigorous host immune response than cultured cells. In line with this hypothesis, cultured neural precursors from the embryonic pig brain have been reported to evoke a milder immune response than primary tissue (Armstrong et al., 2001). Phenotype of transplanted hNSC Modulation of the traumatic inflammation and rejection of sub-population of cells may affect the differentiation of xenografted hNSC. For this reason, we also quantified the proportion of huN-positive cells that co-expressed the markers nestin, GFAP and NeuN. The similar proportions of huN-positive cells co-expressing nestin or GFAP in both groups suggest that shortening of cyclosporin A treatment had no dramatic effect on the differentiation of hNSC xenografted to traumatically injured brain. The degree of human cells expressing neuronal markers in our experiment was considerably lower than previous studies (Kelly et al., 2004; Vescovi et al., 1999a,b). The cortical contusion injury model used in the current study, which induces a focal lesion in the cerebral cortex, may explain this. The environment may not promote neuronal differentiation alternatively it may not be able to meet the metabolic requirements of developing neurons potentially leading to their demise. Xenografted cells survived up to 6 months despite only 3 weeks of immunosuppression. The distribution of huN-positive cells with few cells in the perilesional cortex and robust survival in the hippocampus may reflect different environments in the two locations. An explanation for the apparent delayed loss of cells in the perilesional zone could be the pronounced neovascularisation seen after traumatic brain injury. The infiltration of leukocytes is a consequence of activated resident microglial cells releasing chemoattractants and acting as antigen presenting cells (Giulian et al., 1989). It is plausible that the infiltration of leukocytes is facilitated by the proximity of the grafted cells to the newly formed vessels. Further studies are, however, needed to clarify the mechanism in long term graft rejection. Conclusion Without immunosuppression transplanted hNSC perished when xenografted to a rat model of cortical contusion. However, despite earlier data indicating that continuous systemic immunosuppression is required for xenograft survival in the

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