Alexandros A. Lavdas

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro

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.

Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

ABSTRACT Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury

Functional recovery after spinal cord lesion remains an important goal. A combination of inhibitory molecules and lack of appropriate permissive factors in the lesioned spinal cord results in failure of fiber tract reconnection and function. Experimental transplantation in rodent and primate models of CNS injuries has led to the idea that Schwann cells (SCs) are promising candidates for autologous transplantation to assist myelination of lesions and to deliver therapeutic agents in the CNS. In this study, we used retroviral transduction to genetically modify SCs from transgenic GFP-mice in order to overexpress the cell adhesion molecule L1, a protein promoting neurite outgrowth and implicated in myelination. SCs transduced to express L1 or its chimeric secreted form L1-Fc were mixed and grafted rostrally to the lesion site of adult mice immediately after spinal cord compression injury. Our results indicate that 3 weeks postoperatively, but not thereafter, mice transplanted with L1/L1-Fc-expressing SCs exhibited faster locomotor recovery as compared to animals which received SCs transduced with a control vector or no cells at all. Morphological analysis indicated that the accelerated functional recovery correlated with earlier and enhanced myelination by both grafted and host SCs. Moreover, increased sprouting of serotonergic fibers into and across the lesion site was observed in the L1/L1-Fc group as compared with controls. Our results suggest that transplantation of L1-overexpressing SCs enhances early events in spinal cord repair after injury and may be considered in combinatorial strategies together with other regeneration-promoting molecules.

The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review

BACKGROUND The importance of Schwann cells in promoting nerve regeneration across a conduit has been extensively reported in the literature, and Schwann cell motility has been acknowledged as a prerequisite for myelination of the peripheral nervous system during regeneration after injury. METHODS Review of recent literature and retrospective analysis of our studies with genetically modified Schwann Cells with increased motility in order to identify the underlying mechanism of action and outline the future trends in peripheral nerve repair. FINDINGS Schwann cell transduction with the pREV-retrovirus, for expression of Sialyl-Transferase-X, resulting in conferring Polysialyl-residues (PSA) on NCAM, increases their motility in-vitro and ensures nerve regeneration through silicone tubes after end-to-side neurorraphy in the rat sciatic nerve model, thus significantly promoting fiber maturation and functional outcome. An artificial nerve graft consisting of a type I collagen tube lined with the genetically modified Schwann cells with increased motility, used to bridge a defect in end-to-end fashion in the rat sciatic nerve model, was shown to promote nerve regeneration to a level equal to that of a nerve autograft. CONCLUSIONS The use of genetically engineered Schwann cells with enhanced motility for grafting endoneural tubes promotes axonal regeneration, by virtue of the interaction of the transplanted cells with regenerating axonal growth cones as well as via the recruitment of endogenous Schwann cells. It is envisaged that mixed populations of Schwann cells, expressing PSA and one or more trophic factors, might further enhance the regenerating and remyelinating potential of the lesioned nerves.

Lentivirus-mediated expression of insulin-like growth factor-I promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons

J. Neurochem. (2010) 115, 460–474.

Cell Adhesion Molecules in Gene and Cell Therapy Approaches for Nervous System Repair

The inability of the central nervous system (CNS) to efficiently repair damages results in severe functional impairment after trauma or neurodegenerative/demyelinating diseases. Regeneration failure is attributed to inhibitory molecules creating a nonpermissive environment for axonal regrowth, and dictates the necessity for the development of novel therapeutic strategies. An emerging approach for improving regeneration is the use of gene therapy to manipulate cell adhesion molecule expression in experimental animal models of degeneration. Alternatively, cell transplantation to replace lost neurons and the grafting of myelinating cells to repair demyelinating lesions are promising approaches for treating CNS injuries and demyelination. Schwann cells (SCs), oligodendrocyte progenitors, olfactory ensheathing cells and embryonic and neural stem cells have been shown to form myelin after transplantation into the demyelinated CNS. The repair capacity of the peripheral nervous system (PNS) is much higher, but there is still a limit to the amount of nerve loss that can be bridged after injury, and longer nerve gaps call for the use of conduits populated with living cells. In both cases, the interaction of grafted cells with the host environment is of paramount importance for the incorporation and functional integration of these cells and the manipulation of cell adhesion molecules is an attractive approach towards achieving this goal. In this review we summarize data from the recent literature regarding the manipulation of cell adhesion molecule expression towards CNS and PNS repair and discuss the prospects for future therapeutic applications.

Transplantation of Embryonic Neural Stem/Precursor Cells Overexpressing BM88/Cend1 Enhances the Generation of Neuronal Cells in the Injured Mouse Cortex†‡§

The intrinsic inability of the central nervous system to efficiently repair traumatic injuries renders transplantation of neural stem/precursor cells (NPCs) a promising approach towards repair of brain lesions. In this study, NPCs derived from embryonic day 14.5 mouse cortex were genetically modified via transduction with a lentiviral vector to overexpress the neuronal lineage‐specific regulator BM88/Cend1 that coordinates cell cycle exit and differentiation of neuronal precursors. BM88/Cend1‐overexpressing NPCs exhibiting enhanced differentiation into neurons in vitro were transplanted in a mouse model of acute cortical injury and analyzed in comparison with control NPCs. Immunohistochemical analysis revealed that a smaller proportion of BM88/Cend1‐overexpressing NPCs, as compared with control NPCs, expressed the neural stem cell marker nestin 1 day after transplantation, while the percentage of nestin‐positive cells was significantly reduced thereafter in both types of cells, being almost extinct 1 week post‐grafting. Both types of cells did not proliferate up to 4 weeks in vivo, thus minimizing the risk of tumorigenesis. In comparison with control NPCs, Cend1‐overexpressing NPCs generated more neurons and less glial cells 1 month after transplantation in the lesioned cortex whereas the majority of graft‐derived neurons were identified as GABAergic interneurons. Furthermore, transplantation of Cend1‐overexpressing NPCs resulted in a marked reduction of astrogliosis around the lesioned area as compared to grafts of control NPCs. Our results suggest that transplantation of Cend1‐overexpressing NPCs exerts beneficial effects on tissue regeneration by enhancing the number of generated neurons and restricting the formation of astroglial scar, in a mouse model of cortical brain injury. STEM CELLS 2010;28:127–139

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus‐transduced mammalian cells enhance Schwann cell motility

J. Neurochem. (2010) 115, 1137–1149.