In vitro and in situ properties of canine neural precursor cells with respect to transplantation potential

Abstract

Neurodegenerative diseases represent a broad spectrum of genetic and acquired processes that would benefit from CNS transplantation therapy. The lysosomal storage disease Mucopolysaccharidosis VII (MPS VII) is an inherited disorder caused by E-glucuronidase (GUSB) enzyme deficiency whose signs include mental retardation due to neurodegenerative disease. The existence of both small and large animal homologues of human MPS VII make this disease a valuable model for transplantation therapy of storage diseases. The studies described herein focused upon the characterization of canine neural precursor cells (CNPCs) for CNS transplantation therapy in an animal model of MPS VII. Canine NPCs were isolated from three neurogenic regions of the postnatal brain: the olfactory bulb, cerebellum and striatal subventricular zone (SVZ). The growth, expansion, and multipotency of CNPCs from unaffected and MPS VII dogs were compared. There were no differences attributable to CNPC genotype. Cerebellar-derived CNPCs grew more slowly than those from other brain regions. Cells obtained from all regions could be expanded ex vivo nearly 600-fold. Independent of genotype or region, undifferentiated CNPCs were immunopositive for nestin and glial fibrillary acidic protein (GFAP), and could be differentiated into neurons, astrocytes and oligodendrocytes. Neurogenic regions within the canine brain were evaluated for two candidate NPC cell surface markers, CD15 and CD133. We evaluated the immunophenotype of cells in the ventricular/subventricular zone and the cerebellum. CD15 immunoreactivity in the postnatal cerebellum was present in white matter tracts of folia. The embryonic ventricular zone and postnatal SVZ stained positively for CD15, but were immunonegative for CD133. The proportion of CD15-positive cells in the canine SVZ was 6.6 ± 2.2%. When cells from the SVZ were sorted into CD15-positive and negative populations, both populations could be expanded in response to basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), although CD15-positive cells more consistently so. Efficient transduction of CNPCs was achieved with lentiviral and modified, selectable retroviral vectors containing the human GUSB gene and promoter. Transplantation of the lentivirus transduced CNPCs into a xenograft model (the neonatal SCID mouse) resulted in sparse but consistent GUSB-positive cell engraftment up to six weeks post-transplantation primarily in regions immediately adjacent to the lateral ventricles, especially white matter tracts. Xenografts of cells transduced with the retroviral vector showed robust GUSB expression at 1 week post-transplantation, but significant loss of GUSB activity was observed after 4 weeks. In allograft transplants of lentivirus transduced cells, small numbers of GUSB-positive cells were detected in the canine striatum and thalamus up to the latest time-point evaluated, 4 weeks post-transplantation. At 2 weeks post-transplantation, small numbers of nestin-positive and GFAP-positive graft cells were identified, but no graft cells stained with neuronal markers. The data describe a population of postnatal cells in the dog that possess characteristics of neural precursor cells. These cells have at least limited self-renewal ability and are multipotent. It is hoped that CNPC characterization will afford a valuable model of therapeutic cellular transplantation in a large animal model of genetic neurodegenerative disease.