During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex.

Schwann cells (SCs) are among the most attractive cellular candidates for the development of remyelination therapies for CNS lesions. Yet, their integration in the CNS is inhibited by astrocytes and therefore the use of genetically modified SCs with improved properties is an alternative promising approach. Our strategy for ameliorating the therapeutic potential of SCs has been to alter their adhesive properties by expressing on their surface the polysialylated (PSA) form of the neural cell adhesion molecule NCAM. In the present study, SCs from transgenic GFP-mice were transduced with a retroviral vector encoding sialyl-transferase X (STX), the enzyme responsible for transferring PSA on NCAM. Engineered STX-GFP-SCs with sustained PSA expression were thus generated and were found to have improved ability to associate with astrocytes in vitro. Importantly, when these cells were transplanted in vivo in a mouse model of spinal cord injury they promoted faster and significantly greater functional recovery as compared to using SCs transduced with a control retroviral vector or no cells at all. Morphological analysis indicated that the improved locomotor recovery correlated with earlier and enhanced remyelination by grafted STX-GFP-SCs, increased remyelination by host SCs as well as enhanced differentiation/remyelination by resident oligodendrocyte precursors. Moreover, sprouting of regenerating serotonergic nerve fibres, which are known to be important for locomotion and recovery after injury, was observed into and across the lesion site. These results underline the potential therapeutic benefit of early activation of myelin-forming cells to differentiate and remyelinate severed axons thus restoring functions in CNS trauma and/or demyelinating diseases.

Schwann cells, the myelin-forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl-transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase-N stripping of PSA. PSA-positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX-transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co-cultures. Thus, migratory properties of PNS myelin-forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.

During the last decade, it has become apparent that baculoviruses not only represent a powerful expression system for production of recombinant proteins in insect cells but also can be used for transduction of dividing and nondividing mammalian cells and tissues in vitro, ex vivo, and in vivo (49). Advantages of the use of baculoviruses as gene delivery agents include their inability to replicate in mammalian cells, apparent lack of cytotoxicity, capacity to sustain large insertions of foreign DNA, ability to target many different cell types, and superior safety features relative to mammalian virus-based transduction systems (25,37,38,48). Thus, increasing interest for the development of recombinant baculoviruses as gene delivery vectors for use in human gene therapy exists.All baculovirus vectors for gene delivery in mammalian cells reported thus far have been based on the Autographa californica nuclear polyhedrosis virus (AcNPV). This is primarily due to the relatively wide host range of the virus and the multiplicity of lepidopteran cell lines in which it can be grown and propagated effectively as well as the availability of integrated methodologies allowing the rapid generation of recombinant viruses (55, 80). In contrast, there is a paucity of information regarding the suitability of other baculovirus species, particularly species with narrow host ranges to function as transduction vectors for mammalian cells.In this regard, it is also known that AcNPV has a propensity to express endogenous viral genes in nonhost insect species (12, 13). Moreover, studies involving transfection of mammalian cells with gene constructs employing baculovirus gene promoter elements have shown that certain early baculovirus promoters are marginally functional in mammalian cells and can also be activated by mammalian virus regulators (46, 66). These findings raise safety concerns related to the potential for low-level baculovirus gene expression in mammalian cells and the triggering of cellular immune responses against the transduced cells by the recipient host (61).In this work, we have explored the capacity of Bombyx mori NPV (BmNPV), the second best characterized baculovirus species after AcNPV (2,20,21,27,40,56,57,65), which has a very limited host range (58), to function as a transducing vector for mammalian cell lines and primary Schwann cells. The choice for the latter rests with the results of experimental transplantation in rodent and primate models, which has provided substantial evidence that Schwann cells are good candidates for cell therapy in human central nervous system (CNS) demyelinating diseases, such as multiple sclerosis, and trauma (5,6,9,19,26). Schwann cells provide trophic support and remyelinate demyelinated CNS axons. However, their integration in the CNS is limited. Therefore, modifying Schwann cells to express "therapeutic" factors enhancing axonal regeneration and remyelination by ex vivo gene transduction is a promising strategy to improve their capacity to repair the injured or demyelinated nervous ...