Research reportTransplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response
Introduction
Intracerebral neural grafting and cell replacement therapy have been employed clinically to ameliorate the symptoms of neurological movement disorders such as Parkinson’s disease and Huntington’s disease [4]. These methods are now proposed for therapeutic intervention in stroke [6], [24], [38]. However, it is important to evaluate the immunological mechanisms that may determine integration and acceptance, or the rejection of such grafts [8], [9].
Incompatibility between graft donor and recipient host major histocompatibility complex (MHC) molecules is considered as a main source of the sensitisation of the host immune system to antigens from the graft, with subsequent immune-mediated rejection [28], [43], [47], [53]. The smaller the disparity between the MHC molecules of the host and the transplanted cells, the better grafts survive. It has been argued that murine cells transplanted into rat hosts should be considered concordant xenotransplants, because rats lack preformed antibodies against mouse antigens and are therefore not susceptible to the same type of immune response as observed from a host with preformed antibodies (a discordant xenograft, for example grafting of rabbit or guinea pigs with mouse cells) [25]. Selection of an MHC compatible donor tissue therefore influences the type of immune response to the graft.
Different types of cells also express different levels of MHC antigens and can therefore influence the sensitisation of the immune system. For instance, fetal CNS tissue grafts contain elements of blood vessels and microglia which constitutively express or can be induced to express MHC antigens, and are capable of provoking a cellular immune response [54]. Despite this, fetal neural allografts can survive for long periods within the host CNS, although further immunosuppression (for example with concomitant cyclosporin A treatment) is necessary to ensure longer-term survival of such grafts [47]. As alternatives, graft suspensions or grafts lacking endothelial cells show better survival and are less likely to express MHC antigens that sensitise the host immune system [2], [42]. Consequently, a desirable criterion for transplantation would be to select a transplant source which constitutively expresses no or very little MHC antigens.
In particular, neural progenitors and stem cells are promising, as these have the potential to be maintained in vitro, and are less likely to present MHC antigens at the time of transplantation [28], yet little information is available on the immunological response to transplanted stem cells at present. Although it is unclear how the immune system will respond to grafted stem cells, immunosuppressive therapy, aimed to increase the survival of transplanted cells, is administered routinely. However, it is possible that a similar survival can be obtained without CSA treatment. An evaluation of the necessity for CSA treatment in animal models is important, because long-term administration in the clinic may affect renal function and the quality of life of the patient.
The survival of transplanted cells not only depends on the type of graft and immunosuppressive treatment, but also on the site of implantation itself [40], [46]. Mason and colleagues [33] showed that grafts implanted into the ventricles were more quickly rejected than grafts placed in the parenchyma. Modo et al. [34] also noted that stem cells from the MHP36 cell line transplanted into the intact contralateral hemisphere in rats with stroke damage showed a better survival than cells implanted into the lesioned hemisphere or ventricles. Possibly improved survival of contralateral grafts may depend on the less compromised state of the intact hemisphere compared to the lesioned hemisphere which has leukocytes and macrophages infiltrating to clear the ischaemic debris. However, cells implanted into the ischaemic lesion showed graft survival which corroborated other studies using different types of lesions [18], [21], [44], [45], [50], [51]. In all studies, a short regime of immunosuppression for 2 weeks with CSA on alternative days was found to be sufficient for long-term survival of MHP36 for longer than 10 months following grafting [50] and in non-human primates [51].
We propose that a rejection of transplanted cells at a period of 4 weeks post-ischaemia, and 2 weeks post-implantation should induce a further upregulation of MHC antigens in response to the implantation and a recruitment of immune effector cells. For instance, Larsson et al. [25], [26] have indicated that 2 weeks is a crucial time point marking the initial immune response to transplanted cells characterized by the infiltration of leukocytes and macrophages with a second episode of immune response setting in much later (at about 12 weeks post-grafting) which would reflect a cellular immune rejection of extended fetal grafts. However, in our previous experiments, graft survival was excellent at later time points and we therefore focused on the peak of the early immune response which is thought to be reduced by the initial administration of CSA during the 2 weeks post-grafting. Thus, a major influx of leukocytes and upregulation of MHC class I and II antigens, in addition to a failure of grafts to survive would be indicative of an immune rejection of grafted stem cells.
In the present study, we investigated whether the survival of MHP36 cells within the brain is influenced by: (i) immunosuppression (CSA vs. no CSA), (ii) the local (intact vs. lesioned hemisphere), or (iii) global (lesioned vs. sham) brain environment. A proliferation assay of lymphocytes from the cervical lymph nodes was used to determine if any differential sensitisation of lymphocytes occurs under various transplant conditions. The survival of MHP36 cells within the brains was graded semi-quantitatively to indicate the optimal condition for cell survival. Brain sections were additionally examined for immunological indications of graft rejection [(expression of MHC class I and II, CD45 (leukocyte common antigen), and CD11b (C3bi receptor, expressed on macrophages)]. These three lines of investigation (proliferation assay, grading of graft survival, and immunohistochemical identification of an immune response) were used to determine whether grafted cells elicited an immune rejection response within this critical period of transplantation in the rodent MCAo model of stroke.
Section snippets
Animals
Sprague–Dawley rats (Charles River, UK) were acclimatised for at least a week with five rats per cage. Animals weighed between 250 and 270 g upon arrival. A 10-h light, 14-h dark schedule was maintained throughout the experiment. Food and water were available ad libitum. All procedures were in accordance with the UK Animals (Scientific) Procedures Act 1986 and the ethical review process of the Institute of Psychiatry, University of London.
Surgery
Animals between 280 and 330 g were either allocated to
Evaluation of grafted cells
Transplanted cells were clearly detectable by PKH26 fluorescence which delineated the cell membrane (Fig. 1). The macroscopic appearance of the graft, i.e. the distribution of cells labelled with PKH26, indicated that grafted cells resided in the striatum and overlying cortex (around the injection site) with cells migrating out from the implantation site along the corpus callosum to colonise the opposite hemisphere (for both ipsi- and contralateral transplants). Injection tracts of the
Discussion
Intracerebral transplants are rarely rejected within hours (the product of preformed antibodies attacking the endothelium of whole organ transplants), but an infiltration of lymphocytes sensitised to drained MHC antigens is observed within a few days post-grafting. The acute response to transplanted cells is prompt and evident within a week post-transplantation [1], [28], [40], whereas chronic rejection develops slowly and continuously [9], [43]. Several factors are known to be involved in host
Acknowledgements
The authors would like to thank Chantal Heuschling and Andrew Chadwick for their assistance. MM is supported by pre-doctoral scholarships from NATO and the Ministère de l’Education du Luxembourg. This study was supported by ReNeuron Ltd (http://www.reneuron.com).
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