Assessing Human Embryonic Stem Cell-Derived Dopaminergic Neuron Progenitor Transplants Using Non-invasive Imaging Techniques.

PURPOSE: Human pluripotent stem cell (hPSC)-derived dopaminergic neuron progenitor cells (DAPCs) are a potential therapy for Parkinson's disease (PD). However, their intracranial administration raises safety concerns including uncontrolled proliferation, migration and inflammation. Here, we apply a bimodal imaging approach to investigate the fate of DAPC transplants in the rat striatum. PROCEDURES: DAPCs co-expressing luciferase and ZsGreen or labelled with micron-sized particles of iron oxide (MPIOs) were transplanted in the striatum of RNU rats (n = 6 per group). DAPCs were tracked in vivo using bioluminescence and magnetic resonance (MR) imaging modalities. RESULTS: Transgene silencing in differentiating DAPCs accompanied with signal attenuation due to animal growth rendered the bioluminescence undetectable by week 2 post intrastriatal transplantation. However, MR imaging of MPIO-labelled DAPCs showed that transplanted cells remained at the site of injection for over 120 days. Post-mortem histological analysis of DAPC transplants demonstrated that labelling with either luciferase/ZsGreen or MPIOs did not affect the ability of cells to differentiate into mature dopaminergic neurons. Importantly, labelled cells did not elicit increased glial reactivity compared to non-labelled cells. CONCLUSIONS: In summary, our findings support the transplantation of hPSC-derived DAPCs as a safe treatment for PD.

Dopaminergic manipulations and its effects on neurogenesis and motor function in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder that is classically defined by a triad of movement and cognitive and psychiatric abnormalities with a well-established pathology that affects the dopaminergic systems of the brain. This has classically been described in terms of an early loss of dopamine D2 receptors (D2R), although interestingly the treatments most effectively used to treat patients with HD block these same receptors. We therefore sought to examine the dopaminergic system in HD not only in terms of striatal function but also at extrastriatal sites especially the hippocampus, given that transgenic (Tg) mice also exhibit deficits in hippocampal-dependent cognitive tests and a reduction in adult hippocampal neurogenesis. We showed that there was an early reduction of D2R in both the striatum and dentate gyrus (DG) of the hippocampus in the R6/1 transgenic HD mouse ahead of any overt motor signs and before striatal neuronal loss. Despite downregulation of D2Rs in these sites, further reduction of the dopaminergic input to these sites by either medial forebrain bundle lesions or receptor blockade using sulpiride was able to improve both deficits in motor performance and adult hippocampal neurogenesis. In contrast, a reduction in dopaminergic innervation of the neurogenic niches resulted in impaired neurogenesis in healthy WT mice. This study therefore provides evidence that D2R blockade improves hippocampal and striatal deficits in HD mice although the underlying mechanism for this is unclear, and suggests that agents working within this network may have greater effects than previously thought.

Clusterin secreted by astrocytes enhances neuronal differentiation from human neural precursor cells.

Neuronal differentiation from expanded human ventral mesencephalic neural precursor cells (NPCs) is very limited. Astrocytes are known to secrete neurotrophic factors, and so in order to enhance neuronal survival from NPCs, we tested the effect of regional astrocyte-conditioned medium (ACM) from the rat cortex, hippocampus and midbrain on this process. Human NPC's were expanded in FGF-2 before differentiation for 1 or 4 weeks in ACM. The results show that ACM from the hippocampus and midbrain increase the number of neurons from expanded human NPCs, an effect that was not observed with cortical ACM. In addition, both hippocampal and midbrain ACM increased the number and length of phosphorylated neurofilaments. MALDI-TOF analysis used to determine differences in media revealed that although all three regional ACMs had cystatin C, α-2 macroglobulin, extracellular matrix glycoprotein and vimentin, only hippocampal and midbrain ACM also contained clusterin, which when immunodepleted from midbrain ACM eliminated the observed effects on neuronal differentiation. Furthermore, clusterin is a highly glycosylated protein that has no effect on cell proliferation but decreases apoptotic nuclei and causes a sustained increase in phosphorylated extracellular signal-regulated kinase, implicating its role in cell survival and differentiation. These findings further reveal differential effects of regional astrocytes on NPC behavior and identify clusterin as an important mediator of NPC-derived neuronal survival and differentiation.

Neuropeptide Y modifies the disease course in the R6/2 transgenic model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by progressive neuronal dysfunction and cell loss, especially striatal GABAergic neurons, generating motor, cognitive and affective problems. Although the disease-causing gene is known, the exact mechanism by which it induces its pathological effect remains unknown, and no cure is currently available for this disease. Interestingly, striatal neurons that express neuropeptide Y (NPY) are preferentially spared in HD and the number of such cells is increased in the striatum of HD patients. Furthermore, neurogenesis in the subventricular zone (SVZ) also appears to be up-regulated in HD patients, and previously we also demonstrated in wild-type mice that intracerebroventricular (ICV) NPY promotes SVZ neurogenesis with migration of the newborn cells towards the striatum where they differentiate into GABAergic neurons. Therefore, we sought to determine whether NPY could be of therapeutic benefit in a transgenic mouse model of HD (R6/2) through an action on SVZ neurogenesis. We found that a single ICV injection of NPY in R6/2 mice increased survival time through reduced weight loss as well as having a beneficial effect on motor function as evidenced by improving rotarod performance and reducing paw-clasping. We also demonstrated that the degree of cerebral and striatal atrophy was reduced following such a single NPY injection and that whilst the peptide also increased the number of BrdU-positive cells in the SVZ (but not in the dentate gyrus) of R6/2 mice, this was not sufficient to account for the changes in anatomy and function that we found.. These results suggest that NPY may be of some therapeutic interest in patients with HD, although further work is needed to ascertain exactly how it mediates its beneficial effects.

A functional role for adult hippocampal neurogenesis in spatial pattern separation.

The dentate gyrus (DG) of the mammalian hippocampus is hypothesized to mediate pattern separation-the formation of distinct and orthogonal representations of mnemonic information-and also undergoes neurogenesis throughout life. How neurogenesis contributes to hippocampal function is largely unknown. Using adult mice in which hippocampal neurogenesis was ablated, we found specific impairments in spatial discrimination with two behavioral assays: (i) a spatial navigation radial arm maze task and (ii) a spatial, but non-navigable, task in the mouse touch screen. Mice with ablated neurogenesis were impaired when stimuli were presented with little spatial separation, but not when stimuli were more widely separated in space. Thus, newborn neurons may be necessary for normal pattern separation function in the DG of adult mice.

The search for a curative cell therapy in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder, characterised by the progressive loss of dopaminergic neurons in the substantia nigra, and typically treated by dopamine replacement. This treatment, although very effective in the early stages of the disease, is not curative and has side-effects. As such there has been a search for a more definitive treatment for this condition, which has mainly concentrated on replacing the lost neurons with neural grafts. Possible cell sources for replacement range from autologous grafts of dopamine secreting cells to allografts of fetal ventral mesencephalon and neural precursor cells derived from fetal tissue or embryonic stem cells. Some of these cells have been the subject of clinical trials, which to date have produced disparate outcomes. Therefore, whilst cell therapies remain a promising treatment for PD, there is need for further refinement of the techniques involved in this experimental procedure, before any new trials in patients are undertaken.

The effect of truncated human alpha-synuclein (1-120) on dopaminergic cells in a transgenic mouse model of Parkinson's disease.

Alpha-Synuclein is thought to play an important role in the pathology of Parkinson's disease (PD). Truncated forms of this protein can be found in PD brain extracts, and these species aggregate faster and are more susceptible to oxidative stress than the full-length protein. We investigated the effect of truncated alpha-synuclein on dopaminergic cells using a transgenic mouse expressing alpha-synuclein (1-120) driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background. We found a selective reduction in the yield of dopaminergic cells from transgenic embryonic ventral mesencephalic cell cultures. However, in vivo the substantia nigra/ventral tegmentum dopaminergic cell counts were not reduced in transgenics, although these mice are known to have reduced striatal dopamine. When transplanted to the striatum in the unilateral 6-hydroxydopamine-lesioned mouse model of PD, dopaminergic cells derived from transgenic embryonic ventral mesencephala were significantly smaller at 6 weeks, and showed a trend towards being less effective at ameliorating rotational asymmetry than those from control alpha-synuclein null mice. These results suggest that alpha-synuclein (1-120) renders dopaminergic cells more susceptible to stress, which may have important implications as to how this truncated protein might contribute to dopaminergic cell death in sporadic PD.

Long-term survival and integration of porcine expanded neural precursor cell grafts in a rat model of Parkinson's disease.

Porcine fetal neural tissue has been considered as an alternative source to human allografts for transplantation in neurodegenerative disorders by virtue of the fact that it can overcome the ethical and practical difficulties using human fetal neural tissue. However, primary porcine neural xenografts are rejected while porcine expanded neural precursor neural cells (PNPCs) seem to be less immunogenic and thus survive better [Armstrong, R.J., Harrower, T.P., Hurelbrink, C.B., McLaughin, M., Ratcliffe, E.L., Tyers, P., Richards, A., Dunnett, S.B., Rosser, A.E., Barker, R.A., 2001a. Porcine neural xenografts in the immunocompetent rat: immune response following grafting of expanded neural precursor cells. Neuroscience 106, 201-216]. In this study, we extended these observations to investigate the long-term survival of such transplants in immunosuppressed rats. Unilateral 6 OHDA lesioned rats received grafts into the dopamine denervated striatum of either primary porcine fetal neural tissue dissected from the E26 cortex or cortically derived neural stem cells which had been derived from the same source but expanded in vitro for 21 days. All cortically derived neural stem cell grafts survived up to 5 months in contrast to the poor survival of primary porcine xenografts. Histological analysis demonstrated good graft integration with fibers extending into the surrounding host tissue including white matter with synapse formation, and in addition there was evidence of host vascularization and myelinated fibers within the graft area. This study has therefore shown for the first time the reliable long-term survival of grafts derived from porcine expanded neural precursors in a rat model of PD, with maturation and integration into the host brain. This demonstrates that such xenografted cells may be able to recreate the damaged circuitry in PD although strategies for dopaminergic differentiation of the porcine neural precursor cell remain to be refined.

Porcine neural xenografts in the immunocompetent rat: immune response following grafting of expanded neural precursor cells.

Intracerebral neural xenografts elicit a host immune response that results in their rapid rejection. This forms a key barrier to the therapeutic use of xenogeneic tissue transplantation for conditions such as Parkinson's disease. The current study sought to provide insight into the cellular components of donor cell suspensions that are important in stimulating the host rejection response and thereby to suggest rational manipulations of xenogeneic donor tissue that might ultimately enhance its clinical utility. The neural stem cell mitogens, epidermal growth factor and fibroblast growth factor-2, have been used to isolate and expand populations of primordial neural precursor cells from the embryonic pig brain. The immune response elicited by these cells on transplantation into the non-immunosuppressed rat has been fully characterised. In the first experiments, expanded neural precursors were grafted into the hemi-parkinsonian, non-immunosuppressed Sprague-Dawley rat and graft status and host response examined 10, 21, 35 and 60 days post-transplantation. While equivalent primary tissue grafts were completely eliminated at 35 days, grafts of expanded neural precursors with healthy neurofilament-positive projections were present at all time-points, and two large grafts remained even at 60 days. Some grafts appeared to elicit minimal host immune responses at the time-points they were examined, although most did appear to be undergoing a rejection process since a co-ordinated response involving host cytotoxic T-lymphocytes, microglia/macrophages, immunoglobulin M and complement could be demonstrated to varying degrees. Subsequent experiments went on to demonstrate further that expanded precursor populations and primary tissue suspensions differed in their immunogenic profile. Firstly, when primary tissue was injected intraperitoneally into immunocompetent rats a vigorous primary humoral response was generated. No such response was detected following injection of expanded neural precursors. Secondly, flow cytometric analysis revealed small but significant levels of class II porcine major histocompatibility complex expression in primary cell suspensions but no such expression in expanded precursor populations.The results of this study therefore demonstrate that the immunogenicity of porcine neural cell suspensions used for intracerebral grafting is reduced when neural stem cell mitogens are used to expand precursor cells. The implications of these findings in the development of novel xenogeneic cellular therapies for neurodegenerative conditions such as Parkinson's disease are discussed.

The morphological development of neurons derived from EGF- and FGF-2-driven human CNS precursors depends on their site of integration in the neonatal rat brain.

Neural precursor cells derived from the developing human brain were expanded in vitro under the influence of fibroblast growth factor-2 (FGF-2) and epidermal growth hormone (EGF), and were then transplanted into different regions of the neonatal rat brain. Four weeks later neurons were seen to have developed from human embryonic precursors, using a human-specific antibody to tau (htau). There were morphological differences between implanted neurons developing in the hippocampus, striatum and neocortex, which were confirmed by cell volume measurements, although no specific neurochemical phenotypes were identified. Htau-positive fibres were seen to project extensively along fibre pathways appropriate for the site of neuronal integration. This study demonstrates that, following cell division in vitro, neurons differentiating from human precursor cell populations retain the ability to respond appropriately to regional determinants present in the neonatal rat brain. This is important for the application of such cells in CNS repair strategies, in particular neural transplantation.

Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson's disease.

Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal growth factor and fibroblast growth factor-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by 20 weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.

Characterisation of the immune response in a neural xenograft rejection paradigm.

We have looked at both donor and host MHC expression in a neural xenograft rejection paradigm. Grafts of either mouse corpus callosum or an SV40 large T transformed astrocytic cell line were placed in the mid-brain of neonatal rats. Three weeks later graft rejection was induced by the application of a skin graft of the same donor origin. MHC expression in the neural graft and the host brain was examined histologically four and ten days after the animals had received a skin graft. Donor MHC expression was detected in the corpus callosal grafts at both time points and preceded host MHC expression and the lymphocytic infiltrate. The grafts of the transformed cell line could not be induced to express MHC antigens under the experimental protocol used nor were they rejected. The migratory patterns of the transformed cells were compared to the well characterised migration patterns of astrocytes from the corpus callosal grafts.

Co-expression of MAP-2 and GFAP in cells developing from rat EGF responsive precursor cells.

In this study we have performed a detailed analysis of EGF-responsive precursors as they develop into neurons and astrocytes using antibodies to nestin, microtubule-associated protein 2 (MAP-2c and MAP-2ab) and glial fibriallary acidic protein (GFAP). Surprisingly, at early time points, most GFAP-positive cells also stained for MAP-2c, and we postulate that this may be a normal stage of astroglial development. At 7 days most of the cells had developed into astrocytes and MAP-2ab-positive cells only represented 5% of the total neuronal population. This study shows that (i) MAP-2c is a marker for early precursors, (ii) the majority of cells developing from. EGF-responsive precursors develop into glia and (iii) only a small population of cells arising from expanded populations of EGF-responsive precursors develop into neurons expressing MAP-2ab. Thus, certain critical signals important for full neuronal differentiation may be missing from this system.

Structure and properties of a herpesvirus of turkeys recombinant in which US1, US10 and SORF3 genes have been replaced by a lacZ expression cassette.

In the process of generating an insertional mutant of herpesvirus of turkeys (HVT) expressing lacZ at the protein kinase (PK) locus, we isolated a recombinant which contained an intact PK gene but the short unique regions US1, US10 and SORF3 had been deleted and replaced by the lacZ cassette. Moreover, the virus contained duplicate copies of gD, gI and gE in an opposite orientation flanking lacZ, US2 and PK which were contiguous. These results are of interest in relation to the flexibility of the short unique segment (Us) and of the inverted repeats flanking Us of the alpha-herpesviruses. The recombinant expressed beta-galactosidase and was genetically stable in vitro and in vivo. Chickens inoculated with the virus developed antibodies to HVT antigens and to beta-galactosidase but the replication of the recombinant in vivo was impaired in comparison to parental HVT as shown by a reduction in the proportion of infected lymphocytes.

Transfection with hsp70i protects rat dorsal root ganglia neurones and glia from heat stress.

Studies have shown that the induction of heat shock proteins (hsp's) in the CNS is protective against excitotoxicity and correlative evidence also suggest that hsp's may be protective against ischemic stress and free radical damage. In fibroblasts and Chinese hamster ovary cells expression of hsp70i has been shown to be essential if the cells are to survive an heat stress. To investigate the effect of constitutive overexpression of hsp72 inducible (hsp70i) in neurones and glia we transfected rat dorsal root ganglia (DRG) with a plasmid construct containing the human EF-1 alpha-promoter and human hsp70i. The results showed that prior transfection with hsp70i protected both neurones and glia from heat stress. These data support the hypothesis that overexpression of hsp70i plays an important role in enhancing the survival of neuronal cells following stress and suggests that the induction of a stress response in the CNS may provide an alternative form of treatment for neurodegenerative diseases.

Transfection-mediated expression of human Hsp70i protects rat dorsal root ganglian neurones and glia from severe heat stress.

Considerable evidence suggests that the expression of heat shock proteins prior to a toxic insult (e.g. ischaemia, excitoxins, heat) can confer protection to neurones and glia. It is not certain which hsp(s) are involved in conveying these neuroprotective effects. Here we show that calcium phosphate-mediated transfection of dorsal root ganglia with an EF-1 alpha promoter-hsp70i expression vector significantly increased the survival of neurones and glia exposed to a severe heat stress. These data suggest that overexpression of hsp70i plays an important role in protecting neurones and glia from the denaturing effects of severe thermal stress. Inducing the expression of specific hsps may lead to the development of novel treatment strategies for CNS diseases.

Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus.

A herpesvirus of turkeys (HVT) recombinant containing a 3.9 kbp fragment of Marek's disease virus (MDV) DNA encoding MDV glycoprotein B (gB), stably integrated into the thymidine kinase (TK) gene of HVT, has been constructed. The replication of the recombinant in chick embryo fibroblasts (CEF) was comparable to that of wild-type HVT. The recombinant expressed authentic MDV gB and its processed forms (110K, 65K and 48K) in CEF as shown by immunoblotting using an MDV-specific anti-peptide serum. Northern blot analysis showed that MDV gB mRNA was transcribed from MDV promoter sequences flanking the MDV gB open reading frame and also from the HVT TK promoter. However, the level of replication of the recombinant in vivo appeared to be lower than wild-type HVT as shown by the titres of HVT antibodies, determined by ELISA. Pathogenicity tests showed that the recombinant was safe and did not cause microscopic or gross Marek's disease lesions or other abnormalities. The results suggest that HVT has potential as a vector for recombinant vaccines.