Therapeutic effects of stem cells and substrate reduction in juvenile Sandhoff mice.

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the ß-subunit gene of ß-hexosaminidase A and B (Hexb gene). Substrate reduction therapy, utilizing imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ), reduces ganglioside biosynthesis and levels of stored GM2 in SD mice. Intracranial transplantation of Neural Stem Cells (NSCs) can provide enzymatic cross correction, to help reduce ganglioside storage and extend life. Here we tested the effect of NSCs and NB-DGJ, alone and together, on brain ß-hexosaminidase activity, GM2, and GA2 content in juvenile SD mice. The SD mice received either cerebral NSC transplantation at post-natal day 0 (p-0), intraperitoneal injection of NB-DGJ (500 mg/kg/day) from p-9 to p-15, or received dual treatments. The brains were analyzed at p-15. ß-galactosidase staining confirmed engraftment of lacZ-expressing NSCs in the cerebral cortex. Compared to untreated and sham-treated SD controls, NSC treatment alone provided a slight increase in Hex activity and significantly decreased GA2 content. However, NSCs had no effect on GM2 content when analyzed at p-15. NB-DGJ alone had no effect on Hex activity, but significantly reduced GM2 and GA2 content. Hex activity was slightly elevated in the NSC + drug-treated mice. GM2 and GA2 content in the dual treated mice were similar to that of the NB-DGJ treated mice. These data indicate that NB-DGJ alone was more effective in targeting storage in juvenile SD mice than were NSCs alone. No additive or synergistic effect between NSC and drug was found in these juvenile SD mice.

Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells.

In humans, beta-hexosaminidase alpha-subunit deficiency prevents the formation of a functional beta-hexosaminidase A heterodimer resulting in the severe neurodegenerative disorder, Tay-Sachs disease. To explore the feasibility of using ex vivo gene transfer in this lysosomal storage disease, we produced ecotropic retroviruses encoding the human beta-hexosaminidase alpha-subunit cDNA and transduced multipotent neural cell lines. Transduced progenitors stably expressed and secreted high levels of biologically active beta-hexosaminidase A in vitro and cross-corrected the metabolic defect in a human Tay-Sachs fibroblasts cell line in vitro. These genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and newborn mice. Engrafted brains, analyzed at various ages after transplant, produced substantial amounts of human beta-hexosaminidase alpha-subunit transcript and protein, which was enzymatically active throughout the brain at a level reported to be therapeutic in Tay-Sachs disease. These results have implications for treating neurologic diseases characterized by inherited single gene mutations.

Segregation of human neural stem cells in the developing primate forebrain.

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.

Lineage-independent determination of cell type in the embryonic mouse retina.

We previously used a retroviral vector to mark clones in the postnatal rodent retina and showed that at least two types of neurons and Müller glia can arise from a common progenitor. Here we describe the use of exo utero surgery to introduce a marker retrovirus into the proliferative zone of the retinas of embryonic day 13 and 14 mice. Analysis of marked clones in the resulting adult retinas shows that almost all progenitor cells that continued mitosis were multipotential and that a single progenitor can generate most retinal cell types. The size of marked clones indicates that retinal cells do not employ a stem cell mode of division, but instead, both daughter cells of a progenitor can continue to divide. These results suggest that cell type determination in the rodent retina is independent of lineage. We propose a model for the generation of retinal cell types in which the cessation of mitosis and cell type determination are independent events, controlled by environmental interactions.

Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.

Grafting immortalized neurons to the CNS.

The ability to transplant an unlimited supply of clonally related neural progenitors that, in the brain, have the capacity to differentiate into neurons and glia in an anatomically and, perhaps, functionally appropriate manner, may not only facilitate developmental inquiries, but may also circumvent the limitations of primary fetal tissue for neural transplantation. These types of transplants also make possible new strategies for gene therapy and repair of the CNS, including replacement of degenerated cells, engineering donor cells to be resistant to toxins, delivery of missing metabolic or other gene products, over-expression of molecules, and substitution of alternate metabolic pathways.

Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease.

Neural stem cells (NSCs) have been proposed as tools for treating neurodegeneration because of their capacity to give rise to cell types appropriate to the structure in which they are grafted. In the present work, we explore the ability of NSCs to stably express transgenes and locally deliver soluble molecules with neuroprotective activity, such as glial cell line-derived neurotrophic factor (GDNF). NSCs engineered to release GDNF engrafted well in the host striatum, integrated and gave rise to neurons, astrocytes, and oligodendrocytes, and maintained stable high levels of GDNF expression for at least 4 months. The therapeutic potential of intrastriatal GDNF-NSCs grafts was tested in a mouse 6-hydroxydopamine model of Parkinson's disease. We found that GDNF-NSCs prevented the degeneration of dopaminergic neurons in the substantia nigra and reduced behavioral impairment in these animals. Thus, our results demonstrate that NSCs efficiently express therapeutic levels of GDNF in vivo, suggesting a use for NSCs engineered to release neuroprotective molecules in the treatment of neurodegenerative disorders, including Parkinson's disease.

Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells.

Activation of the Sonic hedgehog (Shh) signal transduction pathway is essential for normal pattern formation and cellular differentiation in the developing CNS. However, it is also thought to be etiological in primitive neuroectodermal tumors. We adapted GAL4/UAS methodology to ectopically express full-length Shh in the dorsal neural tube of transgenic mouse embryos commencing at 10 d postcoitum (dpc), beyond the period of primary dorsal-ventral pattern formation and floorplate induction. Expression of Shh was maintained until birth, permitting us to investigate effects of ongoing exposure to Shh on CNS precursors in vivo. Proliferative rates of spinal cord precursors were twice that of wild-type littermates at 12.5 dpc. In contrast, at late fetal stages (18.5 dpc), cells that were Shh-responsive but postmitotic were present in persistent structures reminiscent of the ventricular zone germinal matrix. This tissue remained blocked in an undifferentiated state. These results indicate that cellular competence restricts the proliferative response to Shh in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord. Thus, Hedgehog signaling may contribute to CNS tumorigenesis by directly enhancing proliferation and preventing neural differentiation in selected precursor cells.

Brain transplantation of neural stem cells cotransduced with tyrosine hydroxylase and GTP cyclohydrolase 1 in Parkinsonian rats.

Neural stem cells (NSCs) of the central nervous system (CNS) recently have attracted a great deal of interest not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases, such as Parkinson's disease (PD). To examine if genetically modified NSCs are a suitable source for the cell and gene therapy of PD, an immortalized mouse NSC line, C17.2, was transduced with tyrosine hydroxylase (TH) gene and with GTP cyclohydrolase 1 (GTPCH1) gene, which are important enzymes in dopamine biosynthesis. The expression of TH in transduced C17.2-THGC cells was confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, and expression of GTPCH1 by RT-PCR. The level of L-DOPA released by C17.2-THGC cells, as determined by HPLC assay, was 3793 pmol/10(6) cells, which is 760-fold higher than that produced by C17.2-TH cells, indicating that GTPCH1 expression is important for L-DOPA production by transduced C17.2 cells. Following the implantation of C17.2-THGcC NSCs into the striata of parkinsonian rats, a marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats grafted with C17.2-THGC cells but not in the control rats grafted with C17.2 cells. These results indicate that genetically modified NSCs grafted into the brain of the parkinsonian rats are capable of survival, migration, and neuronal differentiation. Collectively, these results suggest that NSCs have great potential as a source of cells for cell therapy and an effective vehicle for therapeutic gene transfer in Parkinson's disease.

At the interface of the immune system and the nervous system: how neuroinflammation modulates the fate of neural progenitors in vivo.

Neural stem and progenitor cells express a variety of receptors that enable them to sense and react to signals emanating from physiological and pathophysiological conditions in the brain as well as elsewhere in the body. Many of these receptors and were first described in investigations of the immune system, particularly with respect to hematopoietic stem cells. This emerging view of neurobiology has two major implications. First, many phenomena known from the hematopoietic system may actually be generalizable to stem cells from many organ systems, reflecting the cells' progenitor-mediated regenerative potential. Second, regenerative interfaces may exist between diverse organ systems; populations of cells of neuroectodermal and hematopoietic origin may interact to play a crucial role in normal brain physiology, pathology, and repair. An understanding of the origins of signals and the neural progenitors' responses might lead to the development of effective therapeutic strategies to counterbalance acute and chronic neurodegenerative processes. Such strategies may include modifying and modulating cells with regenerative potential in subtle ways. For example, stem cells might be able to detect pathology-associated signals and be used as "interpreters" to mediate drug and other therapeutic interventions. This review has focused on the role of inflammation in brain repair. We propose that resident astroglia and blood-born cells both contribute to an inflammatory signature that is unique to each kind of neuronal degeneration or injury. These cells play a key role in coordinating the neural progenitor cell response to brain injury by exerting direct and indirect environmentally mediated influence on neural progenitor cells. We suggest that investigations of the neural progenitor-immunologic interface will provide valuable data related to the mechanisms by which endogenous and exogenous neural progenitor cells react to brain pathology, ultimately aiding in the design of more effective therapeutic applications of stem cell biology. Such improvements will include: (1) ascertaining the proper timing for implanting exogenous neural progenitor cells in relation to the administration of anti-inflammatory agents; (2) identifying what types of molecules might be administered during injury to enhance the mobilization and differentiation of endogenous and exogenous neural progenitor cells while also inhibiting the detrimental aspects of the inflammatory reaction; (3) divining clues as to which molecules may be required to change the lesioned environment in order to invite the homing of reparative neural progenitor cells.

Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.

The study of the basic physiology of the neural precursors generated during brain development is driven by two inextricably linked goals. First, such knowledge is instrumental to our understanding of how the high degree of cellular complexity of the mature central nervous system (CNS) is generated, and how to dissect the steps of proliferation, fate commitment, and differentiation that lead early pluripotent neural progenitors to give rise to mature CNS cells. Second, it is hoped that the isolation, propagation, and manipulation of brain precursors and, particularly, of multipotent neural stem cells (NSCs), will lead to therapeutic applications in neurological disorders. The debate is still open concerning the most appropriate definition of a stem cell and on how it is best identified, characterized, and manipulated. By adopting an operational definition of NSCs, we review some of the basic findings in this area and elaborate on their potential therapeutic applications. Further, we discuss recent evidence from our two groups that describe, based on that rigorous definition, the isolation and propagation of clones of NSCs from the human fetal brain and illustrate how they have begun to show promise for neural cell replacement and molecular support therapy in models of degenerative CNS diseases. The extensive propagation and engraftment potential of human CNS stem cells may, in the not-too-distant-future, be directed towards genuine clinical therapeutic ends, and may open novel and multifaceted strategies for redressing a variety of heretofore untreatable CNS dysfunctions.

Physiological relevance and functional potential of central nervous system-derived cell lines.

Central nervous system (CNS)-derived neural cell lines have proven to be extremely useful for delineating mechanisms controlling such diverse phenomena as cell lineage choice and differentiation, synaptic maturation, neurotransmitter synthesis and release, and growth factor signalling. In addition, there has been hope that such lines might play pivotal roles in CNS gene therapy and repair. The ability of some neural cell lines to integrate normally into the CNS following transplantation and to express foreign, often corrective gene products in situ might offer potential therapeutic approaches to certain neurodegenerative diseases. Five general strategies have evolved to develop neural cell lines: isolation and cloning of spontaneous or mutagenically induced malignancies, targeted oncogenesis in transgenic mice, somatic cell fusion, growth factor mediated expansion of CNS progenitor or stem cells, and retroviral transduction of neuroepithelial precursors. in this article, we detail recent progress in these areas, focusing on those cell lines that have enabled novel insight into the mechanisms controlling neuronal cell lineage choice and differentiation, both in vitro and in vivo.

Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Transplantation of neural progenitor and stem cells: developmental insights may suggest new therapies for spinal cord and other CNS dysfunction.

Multipotent neural progenitors and stem cells may integrate appropriately into the developing and degenerating central nervous system. They may also be effective in the replacement of genes, cells, and nondiffusible factors in either a widespread or a more circumscribed manner, depending on the therapeutic demands of the clinical situation. In addition, they may be uniquely responsive to some types of neurodegenerative conditions. We believe that these various appealing capabilities are the normal expression of basic biologic properties and attributes of a stem cell. The therapeutic utility of some of those properties is illustrated in this review of ongoing work in our laboratory, particularly with regard to spinal dysfunction. In these examples, we believe we have tapped into a mechanism that underlies a remarkable degree of natural plasticity programmed into the nervous system at the cellular level, and we have now exploited those properties for therapeutic ends.

Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo.

Green fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multipotent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retrovirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using helper-virus free HSV-1 amplicon vectors.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Hormonal requirements for neuronal survival in culture.

The inability to grow neurons in culture in the absence of complex and undefined biological fluids (e.g. serum) has proved a major obstacle to a rigorous formulation of the growth, nutritional and hormonal requirements of the developing nervous system. We have been successful in maintaining dissociated chick dorsal root ganglia neurons in a serum-free, defined medium composed of F12 synthetic medium and, substituting for serum, a combination of hormones (insulin, PTH, triiodothyronine, TRH, somatomedin, hydrocortisone, testosterone) and other factors (transferrin). Not only were these hormones found to be sufficient for the maintenance of neurons in vitro, but, by the selective elimination of one component from the mixture, the role of specific hormones in neutral development could be grossly assessed. The elimination of insulin proved to be most inimical to neuronal survival.

Remyelination: cellular and gene therapy.

Dysfunctional myelination or oligodendroglial abnormalities play a prominent role in a vast array of pediatric neurological diseases of genetic, inflammatory, immunological, traumatic, ischemic, developmental, metabolic, and infectious causes. Recent advances in glial cell biology have suggested that effective remyelination strategies may, indeed, be feasible. Evidence for myelin repair is accumulating in various experimental models of dysmyelinating and demyelinating disease. Attempts at remyelination have either been directed towards creating myelin de novo from exogenous sources of myelin-elaborating cells or promoting an intrinsic spontaneous remyelinating process. Ultimately, some disorders of myelin may require multiple repair strategies, not only the replacement of dysfunctional cells (oligodendroglia) but also the delivery or supplementation of gene products (i.e., growth factors, immune modulators, metabolic enzymes). Although primary oligodendrocytes or oligodendroglial precursors may be effective for glial cell replacement in certain discrete regions and circumstances and although various genetic vectors may be effective for the delivery of therapeutic molecules, multipotent neural stem cells may be most ideally suited for both gene transfer and cell replacement on transplantation into multiple regions of the central nervous system under a wide range of pathological conditions. We propose that, by virtue of their inherent biological properties, neural stem cells possess the multifaceted therapeutic capabilities that many diseases characterized by myelin dysfunction in the pediatric population may demand.

Medical management of neonatal posthemorrhagic hydrocephalus.

Posthemorrhagic hydrocephalus is a relatively common complication of premature birth. One third of patients who do not undergo spontaneous resolution require medical management aimed at normalizing intracranial pressure by correcting the imbalance between cerebrospinal fluid (CSF) production and drainage. Serial lumbar punctures intermittently remove CSF in bulk. Pharmacologic therapy decreases CSF production. Each of these therapies have attendant benefits and risks. For patients whose CSF absorption does not improve with growth and recovery, placement of an indwelling ventricular drain is ultimately required.