Neural stem cells, neural progenitors, and neurotrophic factors

Neural stem cells (NSCs) have been proposed as a promising cellular source for the treatment of diseases in nervous systems. NSCs can self-renew and generate major cell types of the mammalian central nervous system throughout adulthood. NSCs exist not only in the embryo, but also in the adult brain neurogenic region: the subventricular zone (SVZ) of the lateral ventricle. Embryonic stem (ES) cells acquire NSC identity with a default mechanism. Under the regulations of leukemia inhibitory factor (LIF) and fibroblast growth factors, the NSCs then become neural progenitors. Neurotrophic and differentiation factors that regulate gene expression for controlling neural cell fate and function determine the differentiation of neural progenitors in the developing mammalian brain. For clinical application of NSCs in neurodegenerative disorders and damaged neurons, there are several critical problems that remain to be resolved: 1) how to obtain enough NSCs from reliable sources for autologous transplantation; 2) how to regulate neural plasticity of different adult stem cells; 3) how to control differentiation of NSCs in the adult nervous system. In order to understand the mechanisms that control NSC differentiation and behavior, we review the ontogeny of NSCs and other stem cell plasticity of neuronal differentiation. The role of NSCs and their regulation by neurotrophic factors in CNS development are also reviewed.     

神经前体细胞在中枢神经系统基因治疗中的应用

基因治疗有望为中枢神经系统疾病提供一种有效的治疗手段,神经前体细胞由于其良好的生物学特性,因此目前被认为是中枢神经系统转基因治疗较为理想的载体细胞.现仅针对神经前体细胞在中枢神经系统基因治疗中的研究进展作一综述.     

Neural development by transplanted human embryonal carcinoma stem cells expressing green fluorescent protein

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.     

Profile of gene expression in the subventricular zone after traumatic brain injury

Neural stem cells, which reside in the subventricular zone (SVZ) and dentate gyrus (DG) of adult mammals, give rise to new neurons throughout life. However, these neural stem cells do not appear to contribute to regeneration in the damaged central nervous system. Following traumatic brain injury (TBI) in adult rats, the number of proliferating cells labeled with bromodeoxyuridine (BrdU) is significantly increased in the bilateral SVZ and DG; however, these proliferating cells do not contribute to effective regeneration in the damaged area. To gain insight into the molecular mechanisms of these biological actions, changes in gene expression in the SVZ after brain trauma were examined by cDNA microarray. Of 9,596 genes screened, 97 were upregulated and 204 were downregulated. Classifying these genes according to their function suggests that TBI affects a broad range of cellular functions. The validity of the data was confirmed by RT-PCR. The expression of some genes localized in the SVZ was confirmed by in situ hybridization. This combined strategy is effective for comprehensive analysis of the pathophysiological changes in the SVZ after brain injury and should contribute to the understanding of the molecular events that occur after injury. In the future, this may enable regeneration of the damaged central nervous system.     

Inhibition of chromatin condensation prevents transgene silencing in a neural progenitor cell line transplanted to the rat brain

The use of ex vivo gene therapy in the central nervous system has so far suffered from transgene downregulation. Condensation of the transgenic sequences has been proposed to be a mechanism involved in this silencing. In this study we inhibited either histone deacetylation or DNA methylation in neural progenitor cell lines, transduced with a lentiviral vector carrying green fluorescent protein (GFP), prior to grafting them into the rat striatum. The expression of GFP was significantly higher in grafts pretreated with either of the inhibitors. After 1 week in vivo we detected an 11-fold increase in the number of GFP-expressing cells due to the inhibition of DNA methylation in vitro with azadeoxycytidine and a ninefold increase when inhibiting histone deacetylation with trichostatin A. This suggests that a pretreatment paradigm could be used to increase efficacy of ex vivo delivery of a therapeutic protein locally in the brain.     

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.     

Efficacy of nonviral gene transfer in the canine brain

OBJECT: The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain. METHODS: Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression. RESULTS: Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS). CONCLUSIONS: Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.     

Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression.

BACKGROUND: The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo. METHODS: An adenovirus vector containing the luciferase (Ad-Luc) or beta-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy. RESULTS: In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells. CONCLUSIONS: This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.     

Use of human neural tissue for the generation of progenitors

Accumulating evidence suggests that a better understanding of normal human brain stem cells and tumor stem cells (TSCs) will have profound implications for treating central nervous system disease during the next decade. Neurosurgeons routinely resect excess surgical tissue containing either normal brain stem cells or TSCs. These cells are immediately available for expansion and use in basic biological assays, animal implantation, and comparative analysis studies. Although normal stem cells have much slower kinetics of expansion than TSCs, they are easily expandable and can be frozen for future use in stem cell banks. This nearly limitless resource holds promise for understanding the basic biology of normal brain stem cells and TSCs, which will likely direct the next major shift in therapeutics for brain tumors, brain and spinal cord injury, and neurodegenerative disease. This report reviews the progress that has been made in harvesting and expanding both normal and tumor-derived stem cells and emphasizes the integral role neurosurgeons will play in moving the neural stem cell field forward.     

The adult neural stem cell niche: lessons for future neural cell replacement strategies

Transplantation of neural stem cells (NSCs) and the mobilization of endogenous neural precursors in the adult brain have been proposed as therapies for a wide range of central nervous system disorders, including neurodegenerative disease (eg, Parkinson's disease), demyelinating disorders (eg, multiple sclerosis), stroke, and trauma. Although there is great hope for the success of such therapies, the clinical development of NSC-based therapies is still in its infancy. A greater understanding of how to control the proliferation, migration, differentiation, and survival of NSCs and their progeny is critical for the development of cell replacement therapies. NSCs are partially regulated by the specialized microenvironment--or "niche"--in which these cells reside. The adult rodent brain retains NSCs in two separate niches that continually generate new neurons: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus subgranular zone (SGZ) of the hippocampus. Similar niches may be found in the human brain. In tis article, the authors briefly review their current understanding of the SVZ and SGZ niches. Lessons learned from these niches may allow one to manipulate NSCs better in culture for therapeutic transplantation and possibly even to mobilize endogenous precursors to repair diseased or injured brain.     

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.     

Restorative surgery of the central nervous system by means of tissue engineering using NeuroGel implants

A novel approach aimed at restoring tissue structure and function and enhancing axonal recovery in damaged parts of the central nervous system is described. In contrast to contemporary neurotransplantation technologies which focus on tissue reconstruction of neural parenchyma by cell replacement, this approach is based on repair by tissue engineering. The technique involves the implantation of a 3-dimensional polymer hydrogel into the site of injury. The physical properties of the hydrogel induce the organisation of migrating wound-healing cells and regenerating axons within its 3-dimensional structure. Two complementary approaches are described and illustrated using results obtained in vivo and in vitro: (1) implantation into the brain and spinal cord of the polymer hydrogel NeuroGel, which has a defined macromolecular structure that enhances tissue-building capabilities, and the implantation of advanced hydrogel derivatives carrying biologically active molecules to promote selective cell interactions, and (2) biohybrid hydrogels that contain entrapped developing neural tissue cells, embryonic carcinoma-derived neurons, or genetically modified cells which secrete neurotrophic factors. These techniques create bioartificial tissues with neural tissue specificity. The potential of this biomaterial-based approach to neural tissue engineering for restorative neurosurgery is discussed.     

Anesthesia for endovascular neurosurgery and interventional neuroradiology

This review outlines the roles of anesthesiologists in the management of patients undergoing invasive endovascular procedures to treat vascular diseases, primarily of the central nervous system. This practice usually is termed interventional neuroradiology or endovascular neurosurgery. The discussion emphasizes perioperative and anesthetic management strategies to prevent complications and minimize their effects if they occur. Planning anesthetic and perioperative management is predicated on understanding the goals of the therapeutic intervention and anticipating potential problems.     

Anesthesia for endovascular neurosurgery and interventional neuroradiology

This article outlines the roles of the anesthesiologist in the management of patients undergoing invasive endovascular procedures to treat vascular diseases, primarily of the central nervous system. This practice is usually termed interventional neuroradiology or endovascular neurosurgery. The article emphasizes perioperative and anesthetic management strategies to prevent complications and minimize their effects if they occur. Planning the anesthetic and perioperative management is predicated on understanding the goals of the therapeutic intervention and anticipating potential problems.     

Neurotransplantation: lux et veritas, fiction or reality?

Neurotransplantation remains a much-debated frontier in contemporary neurosurgery and neuroscience, with roots dating to the late 19th century. Contemporary applications are far-reaching, and ongoing laboratory research and clinical trials seek to define the mechanisms at play in neurotransplant engraftment and growth, while advancing the field forward into the 21st century. Neural transplantation therapy remains an attractive idea for treating central nervous system (CNS) and peripheral nervous system (PNS) pathologies. Phase I and phase II clinical trials assessing safety and efficacy are currently underway for various disorders. The remainder of this review will focus on ongoing clinical trials and more recent research advances involving neural transplantation therapy for neuronal death, axonal injury, peripheral nerve lesions, and cancer. The field of neural transplantation, while promising, is not without ethical and scientific dilemmas; this review will conclude with a discussion of the challenges researchers and clinicians face as the field of neural transplantation moves forward.     

Neural transplantation

Neural transplantation has recently emerged as an exciting extension of neural regeneration and plasticity studies. In this review, the roots of current attempts at autologous and heterologous grafting of neural tissue are traced. Grafts of peripheral and central nervous tissue have been shown to be viable after implantation in a variety of locations in adult animals' brain and spinal cord, and survival data are impressive. Donor tissue is optimal when harvested from fetuses, and successful growth and differentiation of neural grafts have been demonstrated in host animals in a broad age range. A variety of morphologic, physiologic, and behavioral parameters suggest a certain degree of integration of graft tissue into the host central nervous system, although technical limitations do not yet allow definitive statements regarding the extent of functional reinnervation. Perhaps the most promising and innovative of current studies are those that utilize a combination of peripheral and central neural tissue as transplant material. There are a number of possible applications of neural transplantation to clinical neurology and neurosurgery, some of which are discussed.     

In vivo gene transfer into testis and sperm: developments and future application

Despite significant advances in the treatment of infertility via assisted reproductive technology (ART), the underlying causes of idiopathic male infertility still remain unclear. Accumulating evidence suggests that disorders associated with testicular gene expression may play an important role in male infertility. To be able to fully study the molecular mechanisms underlying spermatogenesis and fertilization, it is necessary to manipulate gene expression in male germ cells. Since there is still no reliable method of recapitulating spermatogenesis culture, the development of alternative transgenic approaches is paramount in the study of gene function in testis and sperm. Established methods of creating transgenic animals rely heavily upon injection of DNA into the pronucleus or the injection of transfected embryonic stem cells into blastocysts to form chimeras. Despite the success of these two approaches for making transgenic and knockout animals, concerns remain over costs and the efficiency of transgene integration. Consequently, efforts are in hand to evaluate alternative methodologies. At present, there is much interest in developing approaches that utilize spermatozoa as vectors for gene transfer. These approaches, including testis mediated gene transfer (TMGT) and sperm mediated gene transfer (SMGT), have great potential as tools for infertility research and in the creation of transgenic animals. The aim of this short review is to briefly describe developments in this field and discuss how these gene transfer methods might be used effectively in future research and clinical arenas.