Implantation of undifferentiated and pre-differentiated human neural stem cells in the R6/2 transgenic mouse model of Huntington's disease

Abstract

Background: Cell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology.

Results: In this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3.

Conclusions: Although the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line's therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.

Figure 1

Viability and neuronal phenotype of re-seeded cultures. Harvesting and re-seeding of long-term differentiated cultures did not significantly affect their viability (A, Live/Dead stain), neuronal differentiation (B,β-III-tubulin, Tuj), DARPP-32 (C), or astrocytic differentiation (D).

Figure 2

Body weight. Weight gain between groups was equivalent up to 7 weeks of age when animals were grafted. Post-implantation WT mice with vehicle injection continued to gain weight. All R6/2 mice started to lose weight 3 weeks post-grafting (11 weeks of age). There was no effect of the implantation of undifferentiated or differentiated cells on body weight.

Figure 3

Behaviour.A. Rotarod: no effect of genotype on rotarod performance was detected one week pre- or post-grafting. However, a significant impairment was evident in R6/2 mice at 3 and 5 weeks post-grafting which was not improved through the implantation of undifferentiated or differentiated cells. B. Open field: the total exploratory activity of R6/2 was reduced compared to WT controls at all time points tested. Neither undifferentiated, nor differentiated, cells attenuated the deterioration of R6/2 exploratory behaviour. C. Grip strength: all R6/2 mice showed significantly impaired performance compared to WT mice at all time points. No beneficial effect of treatment was evident. (* p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 4

MRI. Volumetric analysis revealed a significant atrophy in striatal (A), cortical (B), and hippocampal tissue (C). Lateral ventricles (D) were minimally enlarged, but no significant statistical effect was evident. There was no treatment effect of the implantation of either undifferentiated or differentiated cells. (* p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 5

Survival of the transplanted cells. A small population of implanted cells survived (A). Human cells (human nuclear antigen + cells in pink) were mostly found within the injection tract in the striatum. A select few individual cells were observed migrating along the corpus callosum. Stereological cell counts revealed no significant difference between undifferentiated and differentiated cell implantation (B). However, cell survival was very variable with some animals having no surviving cells. Neuronal differentiation, as determined by FOX3 staining, of implanted cells was very poor (C&D). Most implanted cells differentiated into astrocytes (E), whereas others did neither express markers of neurons nor astrocytes. (Scale bar 200 μM).