Ex vivo infection of human embryonic spinal cord neurons prior to transplantation into adult mouse cord

Abstract

Background: Genetically modified pseudorabies virus (Prv) proved suitable for the delivery of foreign genes to rodent embryonic neurons ex vivo and maintaining foreign gene expression after transplantation into spinal cord in our earlier study. The question arose of whether human embryonic neurons, which are known to be more resistant to Prv, could also be infected with a mutant Prv. Specifically, we investigated whether a mutant Prv with deleted ribonucleotide reductase and early protein 0 genes has the potential to deliver marker genes (gfp and beta-gal) into human embryonic spinal cord neurons and whether the infected neurons maintain expression after transplantation into adult mouse cord.

Results: The results revealed that the mutant Prv effectively infected human embryonic spinal cord neurons ex vivo and the grafted cells exhibited reporter gene expression for several weeks. Grafting of infected human embryonic cells into the spinal cord of immunodeficient (rnu-/rnu-) mice resulted in the infection of some of the host neurons.

Discussion: These results suggest that Prv is suitable for the delivery of foreign genes into transplantable human cells. This delivery method may offer a new approach to use genetically modified cells for grafting in animal models where spinal cord neuronal loss or axon degeneration occurs.

Figure 1

Untreated and virus treated human embryonic spinal cord cells. A and B shows 8.5 weeks-old spinal cord cells incubated for 12 h with the Prv. In A, the human cell marker hu-NCAM is shown in red and GFP is green. In B, the GFAP+ cells are red and GFP is green. B shows embryonic spinal cord cells at higher magnification. The cord is poor in cells and the two types of immunolabelling can be clearly distinguished: there is no overlap between the GFP and the GFAP labellings. Note the relatively well-developed glial cells with extensive processes. In C, result of the mock-infection can be seen: Embryonic cells are positive for NCAM, but contain no GFP. Scale bar in A-C: 50 μm. D, E and F show 12-weeks-old untreated human embryonic spinal cord cells processed for GFAP and hu-NCAM double labelling immunohistochemistry. In D, the glial cells are green, and in E, the human cell marker hu-NCAM is red. In F, the merged image of D and E is shown. It should be noted, that the two types of immunolabelling do not overlap. The glial cells shown in E carry a radial glia-like morphology. Scale bar: 250 μm

Figure 2

Electron microscopic photographs of human embryonic spinal cord tissue treated with the Prv for 12 h. In A, the green fluorescent protein immunohistochemistry end-product is labelled by silver granules. The boxed area at high magnification reveals the reaction product in the cytoplasm. In B, the virion antigens are visualized in the infected human embryonic spinal cord cells. The boxed area shows at high magnification the presence of the virion antigen on the cell membrane.

Figure 3

Human spinal cord graft cells infected with Prv-rrep0lacgfp virus before transplantation. The cells containing hu-NCAM (human cells) are red, GFP⁺ cells are green and the cells expressing both antigens are yellow. Confocal microscopic photographs (A, B and C) illustrate the same grafted spinal cord tissue after a survival time of 1 week in the dorsal horn of the host cord. In D, E and F human neurons can be seen 3 weeks after transplantation: the human neurons express GFP and possess well-defined processes. In G, host cells (arrows) are shown expressing GFP near the human graft 3 weeks after transplantation. Scale bar: 40 μm