Transplanted human neural precursor cells migrate widely but show no lesion-specific tropism in the 6-hydroxydopamine rat model of Parkinson's disease.

Parkinson's disease (PD), while primarily associated with degeneration of nigrostriatal dopamine neurons, is now increasingly recognized to have more widespread cell loss and so the most effective cell replacement therapy should target all these neuronal losses. Neural precursor cells might be ideal in this regard as in certain circumstances they have been shown to migrate widely following transplantation into the CNS. The aim of this study was to investigate whether transplanted human expanded neural precursor cells (hENPs) could migrate to sites of established or evolving pathology in the adult brain using the 6-hydroxydopamine (6-OHDA) rat model of PD. hENPs were grafted into the striatum prior to, at the same time as, or after the animals received a 6-OHDA lesion to the medial forebrain bundle. The presence of donor cells was then assessed in a distant site of cell loss (substantia nigra) or sites where cell death would not be expected (frontal cortex and globus pallidus). Donor cells were found distant from the site of implantation but the migration of these hENPs was not significantly greater in the 6-OHDA-lesioned brain and the cells did not specifically target the site of cell loss in the substantia nigra. The temporal relationship of grafting relative to the lesion, and therefore dopaminergic cell death, did not affect the migration of hENPs nor their differentiation. We conclude that while transplanted hENPs are capable of migration away from the site of implantation, they show no specific tropism for sites of ongoing or established nigral dopaminergic cell loss in this lesion model. Therefore, the use of such cells to replace the range of neurons lost in PD is likely to require a deeper understanding of the migratory cues in the damaged adult brain and some manipulation of these cells prior to transplantation.

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.

Neural stem cells: from cell biology to cell replacement.

A large number of crippling neurological conditions result from the loss of certain cell populations from the nervous system through disease or injury, and these cells are not intrinsically replaced. Mounting evidence now suggests that replacement of depleted cell populations by transplantation may be of functional benefit in many such diseases. A diverse range of cell populations is vulnerable, and the loss of specific populations results in circumscribed deficits in different conditions. This diversity presents a considerable challenge if cell replacement therapy is to become widely applicable in the clinical domain, because each condition has specific requirements for the phenotype, developmental stage, and number of cells required. An ideal cell for universal application in cell replacement therapy would possess several key properties: it would be highly proliferative, allowing the ex vivo production of large numbers of cells from minimal donor material; it would also remain immature and phenotypically plastic such that it could differentiate into appropriate neural and glial cell types on, or prior to, transplantation. Critically, both proliferation and differentiation would be controllable. This review considers some of the evidence that stem cells exist in the central nervous system and that they may possess characteristics that make them ideal for broad application in cell replacement therapy.

Hibernated human fetal striatal tissue: successful transplantation in a rat model of Huntington's disease.

The use of fresh human fetal tissue in neural transplantation presents considerable logistical difficulties and limits the clinical applicability of this promising therapy. This study compared the survival of human fetal striatal tissue that had been stored for 24 h in a defined hibernation medium with that of fresh human fetal striatal tissue following xenotransplantation in a rat model of Huntington's disease (HD). Six to 7 weeks postgrafting, there was no significant difference between fresh and hibernated grafts in volume or in various striatal phenotypic markers, although there was a trend towards decreased graft volume. We conclude that short-term hibernation of this tissue is without significant adverse effects on the survival of grafted human fetal striatal tissue. This has important implications for the practical implementation of clinical neural transplant programs in HD.

Survival, neuronal differentiation, and fiber outgrowth of propagated human neural precursor grafts in an animal model of Huntington's disease.

Expanded neural precursor cells provide an attractive alternative to primary fetal tissue for cell replacement therapies in neurodegenerative diseases. In this study we transplanted epigenetically propagated human neural precursor cells into a rat model of Huntington's disease. Neural precursors survived transplantation and large numbers differentiated to express neuronal antigens, including some that expressed DARPP-32, indicating a mature striatal phenotype had been adopted. Neuronal fibers from the grafts projected diffusely throughout the host brain, although there was no evidence that outgrowth was specifically target directed. This study supports the contention that propagated human neural precursors may ultimately be of use in therapeutic neural transplantation paradigms for diseases such as Huntington's disease.

Staging and preparation of human fetal striatal tissue for neural transplantation in Huntington's disease.

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.

A new method for the rapid and long term growth of human neural precursor cells.

A reliable source of human neural tissue would be of immense practical value to both neuroscientists and clinical neural transplantation trials. In this study, human precursor cells were isolated from the developing human cortex and, in the presence of both epidermal and fibroblast growth factor-2, grew in culture as sphere shaped clusters. Using traditional passaging techniques and culture mediums the rate of growth was extremely slow, and only a 12-fold expansion in total cell number could be achieved. However, when intact spheres were sectioned into quarters, rather than mechanically dissociated, cell cell contacts were maintained and cellular trauma minimised which permitted the rapid and continual growth of each individual quarter. Using this method we have achieved a 1.5 million-fold increase in precursor cell number over a period of less than 200 days. Upon differentiation by exposure to a substrate, cells migrated out from the spheres and formed a monolayer of astrocytes and neurons. No oligodendrocytes were found to develop from these human neural precursor cells at late passages when whole spheres were differentiated. This simple and novel culture method allows the rapid expansion of large numbers of non-transformed human neural precursor cells which may be of use in drug discovery, ex vivo gene therapy and clinical neural transplantation.

Cellular and molecular aspects of striatal development.

The striatum is a key component of the basal ganglia and there is considerable evidence that it has an important role in motor, cognitive and limbic functions. However, very little is known about how this forebrain structure develops. This review considers the role of cellular and molecular mechanisms involved in the development of the striatum, and the potential application of this knowledge to the understanding of the pathology and treatment of primary disease of this structure.

Neural stem cell technology as a novel treatment for Parkinson's disease.

The transplantation of human fetal ventral mesencephalic (VM) tissue for patients with advanced Parkinson's disease (PD) has now proved to be of benefit in early clinical trials (1-3). This has been clearly seen in terms of improved motor function, which has been correlated with increased fluorodopa signal on positron emission tomographic scanning at the site of the implant and the presence of abundant tyrosine hydroxylase (TH)-positive neurons in those patients who have come to postmortem analysis (4,5). However, although the concept of restoration of function through neural transplantation is promising, there are major practical as well as ethical problems with the use of aborted human fetal tissue. In particular, aborted fetal tissue is not available in many countries, and even where it can be obtained, isolation of the VM from the large numbers of fetuses the procedure requires presents major logistical difficulties. For example, in PD the best results have been obtained using an average of six to eight fetuses per patient. Therefore, the search for alternative sources of tissue for transplantation is imperative if the procedure is to be widely adopted in the clinical domain. A number of possibilities are currently being explored experimentally (see Table 1), although all of them present difficulties that must be overcome before they can be adopted clinically (reviewed in ref. 6). Table 1 Alternatives to Primary Human Neuronal Cells for Transplantation in PD Dopamine-containing polymers that release dopamine slowly over months/years. Catecholamine-producing cells found naturally within the adult, which may thus be suitable for autotransplantation, e.g., adrenal medulla, carotid body, superior cervical ganglion. Catecholamine-producing cell lines that may be encapsulated to prevent rejection and spread of the tumour cells out into the host brain, e.g., PC12 cells. Cells transfected with tyrosine hydroxylase, which potentially allows for the possibility of autotransplantation, e.g., skin fibroblasts. Xenografts of dopamine-rich tissue, e.g., embryonic porcine ventral mesencephalic tissue. Neural stem cells. Embryonic stem (ES) cells.

Neurodegeneration: a failure of neuroregeneration?

Evidence suggests that the brain, like many other tissues, is in a state of dynamic equilibrium. It has an endogenous population of stem cells that proliferate in response to environmental and pharmacological manipulations and that can replace cells lost in some experimental lesions. However, the fact remains that neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases are characterised by continuous loss of neurons that are not replaced. In this hypothesis, we postulate that a primary deficit in neural stem-cell proliferation; migration, or differentiation, or both, might contribute to net cell loss and neuronal circuit disruption in these disorders. Experimental validation of this hypothesis would not only substantially advance understanding of the pathogenesis of these diseases, but could also have profound implications for future treatment of these incurable disorders.

Anaesthetic waste gas scavenging systems.

The Department of Health and Social Security has recently recommended that waste anaesthetic scavenging systems should be installed to reduce pollution in operating theatres. Three passive and two active systems were compared to see how effectively they reduced concentrations of halothane in the atmosphere. All five systems reduced halothane levels significantly, the combination of an active system and semiclosed circuitry being the most effective. All obvious leaks from equipment were controlled in this study, but normally such leaks contribute significantly to atmospheric pollution. Some of the benefits of a scavenging system may be lost if gases can still escape through leaks.

Molecular cloning, chromosomal localization, and expression of murine dipeptidyl peptidase I.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.

Death of dopaminergic neurons in vitro and in nigral grafts: reevaluating the role of caspase activation.

Caspases are cysteine proteases involved in apoptotic cell death, and pharmacological caspase inhibition has been demonstrated to prevent neuronal cell death in certain experimental paradigms. In this study, the role of caspase-1 and -3 in the death of dopaminergic neurons derived from the E14 rat ventral mesencephalon (VM) has been examined in two model systems using peptide caspase inhibitors. First, cell death was induced in vitro by withdrawing serum after 2 days. Different doses of caspase-1 (IL-1beta converting enzyme) and caspase-3 inhibitors (Ac-DEVD-cmk) were added to the medium at the time of serum withdrawal, and the ability of the inhibitors to promote dopaminergic neuronal survival and prevent activation of caspase-3 was assessed at 7 days. Immunostaining using tyrosine hydroxylase (TH) and cleaved caspase-3 antibodies demonstrated that caspase-1 and -3 inhibitors reduce caspase-3 activation as well as overall cell death. This did not, however, improve the survival of TH-positive neurons, although it did appear to promote their maturation. The second paradigm investigated the effects of these inhibitors in the 6-hydroxydopamine rat model of PD, and similarly, addition of caspase-1 or -3 inhibitor during tissue preparation or immediately prior to grafting of VM tissue did not promote dopaminergic neuronal survival. These results demonstrate that the reduction of apoptotic cell death by pharmacological inhibition of caspase-1 and -3 does not increase dopaminergic neuronal survival in these paradigms and suggest either that caspase-3 activation is not the major determinant of dopaminergic neuronal death in vitro and in grafts or that the ability of caspase inhibitors to rescue cells depends upon the degree of apoptotic stress. This implies that strategies to improve dopaminergic cell survival in clinical programmes of transplantation for PD will need to target other pathways of cell death.

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.