Validation and characterisation of a new method for in vivo assessment of human donor cells

This thesis encompasses a range of experiments designed to characterise and validate a method of desensitising rodent hosts in the neonatal period to human tissue in order to promote the survival of human striatal grafts in the adult host. Thus, the successful application of this method is important to allow the preclinical testing of potential human donor cells for therapeutic transplantation, specifically in neurological disease. Demonstrating safety and functionality of transplanted human cells in rodent hosts requires long term assessment of surviving grafts, for which current immune suppression methods are insufficient. The experiments presented in this thesis were therefore designed to determine the optimum parameters of a previously described method of desensitising rats to human tissue and to validate this method in mice. The findings provide further support for the neonatal desensitisation method in rat hosts, and suggest the potential for use of non-neural tissue types for desensitisation of neonates. The data presented in this thesis also has implications for the mechanisms underlying the success of the method in the rat. However interpretation was difficult as graft survival was generally poor and even mouse to mouse allografts did not survive to the level expected. Thus this highlights the need to reassess standard immunosuppression protocols in mice, and determine what differs between the rat and mouse rejection response to xenografts

Xeno-free expansion of late-adherent human olfactory mucosa cells: Towards an allogeneic therapy for neural regeneration

Human olfactory mucosa cells (hOMCs) are anchorage dependent cells that have potential for treatment of spinal cord injury. However, current hOMC therapies relied on autologous transplantation and it is not feasible to prepare and characterize sufficient quantities of cells (in the order of 107 - 108 cells) within a timeframe to treat acute injury. Thus an allogeneic (universal) off-the-shelf approach would offer an alternative for this case. We incorporated the regulator-approved c-MycERTAM gene (ReNeuron) into primary late-adherent hOMCs to extend their ex vivo proliferation in the presence of the synthetic drug 4-hydroxytamoxifen (4-OHT). Polyclonal populations of hOMCs were generated and characterized, with an ultimate goal of developing a potential cell therapy product for application in spinal cord injury. Due to the lack of scalability, the availability of labour intensive manual processes and fetal bovine serum (FBS) supplementation, we aimed to develop a xeno-free process for the expansion of a these cells. An initial issue for the manufacture of hOMCs is that key bioprocess parameters have not been established. In this work, we performed cell growth characterization to provide information about their growth i.e. effect of initial cell seeding density, long-term culture, and metabolite profiles to ultimately define the expansion process window. Although widely used, FBS is a finite resource that raises concerns about the presence of adventitious agents. Alternative human-derived (xeno-free) or chemically-defined (serum-free) supplements were assessed for their ability to sustain cell growth. From these studies, human platelet lysate supplementation at 2-5% (% v/v) was found to be a viable xeno-free option to sustain growth of hOMCs with no adverse effects on their phenotype. Finally, we sought to replace the current manually intensive monolayer expansion process with a more flexible and scalable platform such as suspension culture on animal-free microcarriers. Successful expansion of c- MycERTAM-derived late-adherent hOMCs on plastic microcarriers at 80-mL scale was achieved to establish a suspension culture expansion platform for the translation of a potential candidate cell therapy for neural regeneration. In summary, we show a systematic approach to address main hOMC bioprocessing challenges for an allogeneic therapy to treat patients suffering from spinal cord injury

The effect of tissue preparation and donor age on striatal graft morphology in the mouse

Huntington's disease (HD) is a progressive neurodegenerative disease in which striatal medium spiny neurons (MSNs) are lost. Neuronal replacement therapies aim to replace MSNs through striatal transplantation of donor MSN progenitors, which successfully improve HD-like deficits in rat HD models and have provided functional improvement in patients. Transplants in mouse models of HD are more variable and have lower cell survival than equivalent rat grafts, yet mice constitute the majority of transgenic HD models. Improving the quality and consistency of mouse transplants would open up access to this wider range of rodent models and facilitate research to increase understanding of graft mechanisms, which is essential to progress transplantation as a therapy for HD. Here we determined how donor age, cell preparation, and donor/host strain choice influenced the quality of primary embryonic grafts in quinolinic acid lesion mouse models of HD. Both a within-strain (W-S) and a between-strain (B-S) donor/host paradigm were used to compare transplants of donor tissues derived from mice at embryonic day E12 and E14 prepared either as dissociated suspensions or as minimally manipulated tissue pieces (TP). Good graft survival was observed, although graft volume and cellular composition were highly variable. The effect of cell preparation on grafts differed significantly depending on donor age, with E14 cell suspensions yielding larger grafts compared to TP. Conversely, TP were more effective when derived from E12 donor tissue. A W-S model produced larger grafts with greater MSN content, and while high levels of activated microglia were observed across all groups, a greater number was found in B-S transplants. In summary, we show that the effect of tissue preparation on graft morphology is contingent on the age of donor tissue used. The presence of microglial activation in all groups highlights the host immune response as an important consideration in mouse transplantation

Male and female mice lacking Neuroligin-3 modify the behavior of their wild-type littermates

In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior criticallydepends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying adeletion of Nlgn3, a gene associated with autism spectrum disorders, and their wild-type littermates induceschanges in each other’s behavior. We have found that, when raised together, male Nlgn3 knockout mice and theirwild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockoutmice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells intransgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, furtherindicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-typeanimals’ behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to theirpeers’ behavior but modified the social behavior of their littermates. Altogether, our findings show that theenvironment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. Inaddition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of micelacking Nlgn3, being more influential in males than females

An alginate-based encapsulation system for delivery of therapeutic cells to the CNS

Treatment options for neurodegenerative conditions such as Parkinson's disease have included the delivery of cells which release dopamine or neurotrophic factors to the brain. Here, we report the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner. An optimal alginate formulation was selected with a view to providing a sustained physical barrier between engrafted cells and host tissue, enabling exchange of small molecules while blocking components of the host immune response. In addition, a potent immunosuppressant, FK506, was incorporated into the outer layer of alginate beads using electrosprayed poly-ε-caprolactone core–shell nanoparticles with prolonged release profiles. The stiffness, porosity, stability and ability of the alginate beads to support and protect encapsulated SH-SY5Y cells was demonstrated, and the release profile of FK506 and its effect on T-cell proliferation in vitro was characterized. Collectively, our results indicate this multi-layer encapsulation technology has the potential to be suitable for use in CNS cell delivery, to protect implanted cells from host immune responses whilst providing permeability to nutrients and released therapeutic molecules

Local delivery of tacrolimus using electrospun poly-ε-caprolactone nanofibres suppresses the T-cell response to peripheral nerve allografts

OBJECTIVE: Repair of nerve gap injuries can be achieved through nerve autografting, but this approach is restricted by limited tissue supply and donor site morbidity. The use of living nerve allografts would provide an abundant tissue source, improving outcomes following peripheral nerve injury. Currently this approach is not used due to the requirement for systemic immunosuppression, to prevent donor-derived cells within the transplanted nerve causing an immune response, which is associated with severe adverse effects. The aim of this study was to develop a method for delivering immunosuppression locally, then to test its effectiveness in reducing the immune response to transplanted tissue in a rat model of nerve allograft repair. APPROACH: A coaxial electrospinning approach was used to produce poly-ε-caprolactone fibre sheets loaded with the immunosuppressant tacrolimus. The material was characterised in terms of structure and tacrolimus release, then tested in vivo through implantation in a rat sciatic nerve allograft model with immunologically mismatched host and donor tissue. MAIN RESULTS: Following successful drug encapsulation, the fibre sheets showed nanofibrous structure and controlled release of tacrolimus over several weeks. Materials containing tacrolimus (and blank material controls) were implanted around the nerve graft at the time of allograft or autograft repair. The fibre sheets were well tolerated by the animals and tacrolimus release resulted in a significant reduction in lymphocyte infiltration at three weeks post-transplantation. SIGNIFICANCE: These findings demonstrate proof of concept for a novel nanofibrous biomaterial-based targeted drug delivery strategy for immunosuppression in peripheral nerve allografting

Neonatal desensitisation for the study of regenerative medicine

Cell replacement is a therapeutic option for numerous diseases of the CNS. Current research has identified a number of potential human donor cell types, for which preclinical testing through xenotransplantation in animal models is imperative. Immune modulation is necessary to promote donor cell survival for sufficient time to assess safety and efficacy. Neonatal desensitization can promote survival of human donor cells in adult rat hosts with little impact on the health of the host and for substantially longer than conventional methods, and has subsequently been applied in a range of studies with variable outcomes. Reviewing these findings may provide insight into the method and its potential for use in preclinical studies in regenerative medicine

Direct comparison of rat- and human-derived ganglionic eminence tissue grafts on motor function

Huntington’s disease (HD) is a debilitating, genetically-inherited neurodegenerative disorder that results in early loss of medium spiny neurons from the striatum and subsequent degeneration of cortical and other subcortical brain regions. Behavioural changes manifest as a range of motor, cognitive and neuropsychiatric impairments. It has been established that replacement of the degenerated medium spiny neurons with rat-derived fetal whole ganglionic eminence (rWGE) tissue can alleviate motor and cognitive deficits in preclinical rodent models of HD. However, clinical application of this cell replacement therapy requires the use of human-derived (hWGE), not rWGE, tissue. Despite this, little is currently known about the functional efficacy of hWGE. The aim of this study was to directly compare the ability of the gold-standard rWGE grafts, against the clinically-relevant hWGE grafts, on a range of behavioural tests of motor function. Lister-hooded rats either remained as unoperated controls or received unilateral excitotoxic lesions of the lateral neostriatum. Subsets of lesioned rats then received transplants of either rWGE or hWGE primary fetal tissue into the lateral striatum. All rats were tested post-lesion and post-graft on the following tests of motor function: staircase test, apomorphine-induced rotation, cylinder test, adjusting steps test and vibrissae-evoked touch test. At 21 weeks post-graft, brain tissue was taken for histological analysis. The results revealed comparable improvements in apomorphine-induced rotational bias and the vibrissae test, despite larger graft volumes in the hWGE cohort. hWGE grafts, but not rWGE grafts, stabilised behavioural performance on the adjusting steps test. These results have implications for clinical application of cell replacement therapies, as well as providing a foundation for the development of stem cell-derived cell therapy products

Is the adult mouse striatum a hostile host for neural transplant survival?

Human donor cells, including neurally directed embryonic stem cells and induced pluripotent stem cells with the potential to be used for neural transplantation in a range of neurodegenerative disorders, must first be tested preclinically in rodent models of disease to demonstrate safety and efficacy. One strategy for circumventing the rejection of xenotransplanted human cells is to desensitize the host animal to human cells in the early neonatal period so that a subsequent transplant in adulthood is not immunorejected. This method has been robustly validated in the rat, but currently not in the mouse in which most transgenic models of neurodegeneration have been generated. Thus, we set out to determine whether this could be achieved through modification of the existing rat protocol. Mice were inoculated in the neonatal period with a suspension of human embryonic cortical tissue of varying cell numbers, and received a subsequent human embryonic cortical tissue cell transplant in adulthood. Graft survival was compared with those in mice immunosuppressed with cyclosporine A and those receiving allografts of mouse whole ganglionic eminence tissue. Poor survival was found across all groups, suggesting a general problem with the use of mouse hosts for testing human donor cells