Infection of human embryonic neurons

We first investigated the toxicity of the Prv-rrep0lacgfp virus to the human embryonic spinal cord neurons after incubation times of 6 h and 12 h with the virus at a titer of 10⁸ p.f.u. using the Trypan Blue exclusion test. Untreated human embryonic spinal cord pieces served as control. After the 6 h incubation with the Prv, 79% ± 6 (SEM) of the cells remained viable, this number decreased to 58% ± 8 (SEM) after incubation for 12 h. The proportion of viable cells in the Prv-incubated tissue pieces did not differ significantly (paired t-test) from that in the control tissues at any time point (p_6h = 0.65; p_12h = 0.4).

Next, the ratio of cells expressing foreign genes was determined in cryostat sections of the human embryonic tissues incubated with Prv-rrep0lacgfp virus at a titer of 10⁸ p.f.u. for 6 h or 12 h, using double labelling immunohistochemistry against GFP and hu-NCAM. In previous experiments the Prv-rrep0lacgfp virus was able to infect rat embryonic neurons efficiently after incubation for 3-6 h in a solution containing 10⁸ p.f.u./ml of Prv and the incubation resulted in an infection rate of 100% [10]. However, in our present experiments incubation for 6 h time was found not to be long enough to achieve the infection of the majority of the human embryonic tissue (not shown). After incubation for 12 h, however, the majority of the cells (85% ± 2.8 (SEM) exhibited strong β-gal or GFP labelling throughout the whole piece of the cord (Fig. 1A).

We also examined the cell specificity of the Prv infection in cryostat sections of the human tissues incubated with the Prv for 12 h by using GFAP and GFP double-labelling immunohistochemistry. The virus induced GFP expression in many cells of the embryonic tissues, but none of them was GFAP-positive (Fig. 1B). Mock-infection without virus resulted in intense hu-NCAM labelling of the embryonic spinal cord cells without GFP immunolabelling (Fig. 1C). The specificity of hu-NCAM immunolabelling was tested on human embryonic spinal cord cells: the hu-NCAM antibody did not label GFAP-positive cells in the spinal cord (Fig. 1D,E,F), and it therefore appeared evident that the Prv infected neurons only. The GFAP immunolabelled glial cells had a radial glia-like appearance in the peripheral parts of the embryonic cords (Fig. 1D and 1F), while more centrally located glial cells had an appearance of cells with more developed processes (Fig. 1B). The presence of radial glia in embryonic human cords during this early phase of neural development has already been confirmed [14].

The presence of the virion antigens and the expressed GFP were monitored ultrastructurally, too. The Prv antigens were confined to the cell membranes of the infected neurons and no viral antigens or the whole virion were found in the cytoplasm of the cells. The expressed GFP was present in high amounts in the cytoplasm of the cells and was not concentrated in a given part of the cytoplasm, but was dispersed equally in the soma and in the processes of the infected human neurons. The infected human cells displayed a similar morphology to that of the untreated ones (Fig. 2.).

Marker gene expression by grafted human cells

The foreign gene expression and the survival of the infected human cells grafted into the spinal cord of immunodeficient nude mice were studied next. The product of the gfp gene and the presence of the hu-NCAM were detected by double labelling immunohistochemistry 1 week and 3 weeks following grafting. Although the volume of the transplanted tissue was constant in each experiment, the number of the surviving human cells varied in a broad range: after 1 week, the numbers of surviving cells ranged between 410 and 2357 cells (1007.83 ± 824.8 SEM); after survival for 3 weeks 586-1358 cells remained (736.29 ± 309.63 SEM). To study the time course of foreign gene expression, we determined the number of the GFP-positive human cells in consecutive sections of the grafted cords. After survival for 7 days 79% ± 1.25 (SEM) of the human cells expressed GFP and the number decreased to 50% ± 3.12 (SEM) after survival for 3 weeks. On survival for 1 week the human cells appeared virtually undifferentiated as they had no visible processes. After survival for 3 weeks many grafted cells had well-developed, long and branching processes (Fig. 3D, E, F).

In 2 animals after survival for 7 days and in 3 animals after survival for 3 weeks GFP-expressing host neurons were found no farther than 500 μm away from the graft-host interface. The numbers of GFP-positive host cells observed in the spinal cords of the nude mice after the 1 and 3-week survival periods did not differ significantly: 26 ± 8 (SEM) cells.

Cells and viruses

The generation and characterization of the neuro-attenuated pseudorabies virus (Prv-rrep0lacgfp) used in our experiments has already been described [10]. Briefly, the virus strain contains two mutations: a frame shift mutation in the small subunit of the ribonucleotide reductase gene and a large deletion in the early protein 0 gene. The gfp and lacZ reporter genes were inserted in place of the early protein 0 gene. Prv was propagated in porcine kidney (PK-15) cells. The GFP gene was placed under the control of a chimeric promoter composed of latency-associated transcript promoter (LAP) and human cytomegalovirus major IE promoter [10].

Infection of the donor embryos

The human embryonic spinal cords derived from 8-12 weeks old embryos were obtained from abortions and used with the permission of the Human Ethical Committee of University of Szeged. The human tissues were used according to the guidelines of Helsinki Declaration of Human Rights. The lumbar enlargement of the spinal cord was dissected and the ventral portion was chopped into 1-2 mm pieces. The pieces of tissue were incubated with the virus for 6 and 12 hours at 37°C in a Sanyo CO₂ thermostat. In each experiment 1 ml virus suspension containing 10⁸ plaque-forming units (p.f.u./ml) of virus particles was used for the infection. Following incubation the samples were washed three times in Hanks' solution to remove excess virions. We separated 8 infected spinal cord pieces and allowed in Hanks' solution for 13 hours then processed for either Trypan Blue staining or immunohistochemistry. However, infected tissues marked for transplantation were used up immediately. Mock-infection was performed by keeping embryonic spinal cords tissues in Hanks' solution for 24 hours.

Surgery

Adult homozygous BALB/c rnu-/rnu- nude mice (Charles River Laboratories, Inc., Gödöllő, Budapest, n = 15) were used as hosts for transplantation. The mice were athymic and therefore had no T-cell-based immune response. All surgical interventions were performed under deep anaesthesia achieved with a combination of Hypnorm (0.0045 ml/10 g body weight, Hypnorm, Janssen) and Diazepam (0.0075 ml/10 g body weight, from 5 mg/ml stock solution). On postnatal day 3, the left sciatic nerve of the mice was crushed to bring about motoneuron depletion in the spinal segments L4-L5. The animals were allowed to recover and used for grafting when they reached full maturity (18-20 g). After sciatic nerve crush, the animals exhibited permanent partial paralysis in the affected hindlimb. At the age of 8 weeks, the mice were anaesthetized again as described above and, after skin incision and muscle dissection, L1 laminectomy was performed corresponding to segment L4 of the spinal cord. The dura was opened to expose the cord and 0.5 μl of solid embryonic cord pieces were pressure-injected into the segment L4 via a Hamilton syringe equipped with a glass micropipette. Finally, the muscles were closed over the laminectomy site and the skin was sutured. After survival time of 7 days or 3 weeks, the animals were perfused transcardially with 4% phosphate-buffered paraformaldehyde (pH = 7.4).

Histochemistry

The Trypan Blue exclusion test was performed on the Prv-treated human embryonic spinal cord cells after Prv incubation for 6 h or 12 h. The tissues were first trypsinated with a mixture of trypsin and EDTA, and the dissociated cells were then incubated with Trypan Blue solution (Sigma Aldrich, Budapest, Hungary) for 20 min, followed by a brief wash in physiological saline. The numbers of labelled and unlabelled cells were determined by using a Bürker chamber. The above procedure was carried out on untreated human embryonic spinal cord tissues, too. The proportions of viable cells were determined in the Prv-treated and and in the control samples.

The embryonic tissues incubated with the Prv and the grafted spinal cords were postfixed in 4% paraformaldehyde and then cryoprotected in 30% sucrose in phosphate-buffered saline overnight. Serial cryostat sections (25 μm thick) were cut in a cryostat (Leica CM1850, Bensheim, Germany) and mounted onto gelatine-coated microscopy slides.

The following antibodies were used to detect antigens: polyclonal rabbit antibody against glial fibrillary acidic protein (GFAP, 1:1000, Dakopatts, Denmark) labelling astrocytes and radial glial cells which are typical glial cells in the foetal CNS, polyclonal rabbit antibody against the whole virion (1:10.000, generous gift of Lynn Enquist, Princeton University, Princeton, NJ, USA), monoclonal mouse antibody against β-galactosidase (β-gal; 1:200, Boehringer Mannheim, Germany) and polyclonal chicken antibody against GFP (AB160901, 1:4000; Chemicon International, Temecula, CA, USA). For clarity, only the results of GFP immunohistochemistry are shown. The human neurons were detected by monoclonal mouse antibody against the human neural cell adhesion molecule (hu-NCAM, clone CD56, 1:400; IQ Products, The Netherlands). The antibody recognizes hu-NCAMs expressed on human postmitotic neurons [18]. The primary antibodies were visualized with appropriate biotinylated secondary antibodies and various fluorescent chromophores. Some immunohistochemical reactions were tyramide-amplified by using TSA-Cy3 and TSA-FITC (Perkin Elmer Life Sciences, Boston, MA, USA).

The sections were investigated in an Olympus BX50 fluorescent microscope and photographs were taken with a DP70 digital camera (Olympus Ltd., Tokyo, Japan). Some sections were investigated in a Zeiss LSM510 Meta confocal microscope, too. The labelled cells in the embryonic cords and in the grafts were identified and counted: hu-NCAM or GFP-positive cells were counted only if the cell profile was not present in the consecutive section.

The subcellular localization of virions and GFP was investigated in some Prv-infected embryonic tissues by using electron microscopic immunohistochemistry. Vibratome sections (40 μm thick) were processed for immunohistochemistry. In the immunohistochemical procedure after the ABC step, 3,3'-diaminobenzidine tetrahydrochloride (Sigma Aldrich Co. Ltd., Budapest, Hungary) was added and the oxidized coloured end-product was silver-intensified [19]. Sections were postfixed with 1% aqueous OsO₄ and then embedded in Durcupan (Fluka, Buchs, Switzerland). Ultrathin sections (50-60 nm) were cut on an ultramicrotome (Leica Ultracut R, Bensheim, Germany) and the sections were treated with uranyl acetate and investigated in an electron microscope (JEOL JEM 1010, JEOL Ltd, Tokyo, Japan).

Statistical analysis

Whenever needed, the paired t-test was used for the statistical analysis of data.