Induced Pluripotent Stem Cell-Derived Neural Stem Cell Transplantations Reduced Behavioral Deficits and Ameliorated Neuropathological Changes in YAC128 Mouse Model of Huntington's Disease

Abstract

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by neuronal loss and motor dysfunction. Although there is no effective treatment, stem cell transplantation offers a promising therapeutic strategy, but the safety and efficacy of this approach needs to be optimized. The purpose of this study was to test the potential of intra-striatal transplantation of induced pluripotent stem cell-derived neural stem cells (iPS-NSCs) for treating HD. For this purpose, we developed mouse adenovirus-generated iPSCs, differentiated them into neural stem cells in vitro, labeled them with Hoechst, and transplanted them bilaterally into striata of 10-month old wild type (WT) and HD YAC128 mice. We assessed the efficiency of these transplanted iPS-NSCs to reduce motor deficits in YAC128 mice by testing them on an accelerating rotarod task at 1 day prior to transplantation, and then weekly for 10 weeks. Our results showed an amelioration of locomotor deficits in YAC128 mice that received iPS-NSC transplantations. Following testing, the mice were sacrificed, and their brains were analyzed using immunohistochemistry and Western blot (WB). The results from our histological examinations revealed no signs of tumors and evidence that many iPS-NSCs survived and differentiated into region-specific neurons (medium spiny neurons) in both WT and HD mice, as confirmed by co-labeling of Hoechst-labeled transplanted cells with NeuN and DARPP-32. Also, counts of Hoechst-labeled cells revealed that a higher proportion were co-labeled with DARPP-32 and NeuN in HD-, compared to WT- mice, suggesting a dissimilar differentiation pattern in HD mice. Whereas significant decreases were found in counts of NeuN- and DARPP-32-labeled cells, and for neuronal density measures in striata of HD vehicle controls, such decrements were not observed in the iPS-NSCs-transplanted-HD mice. WB analysis showed increase of BDNF and TrkB levels in striata of transplanted HD mice compared to HD vehicle controls. Collectively, our data suggest that iPS-NSCs may provide an effective option for neuronal replacement therapy in HD.

Keywords: Huntington's disease; YAC128; cell transplantations; iPS-NSCs; iPSCs; neural stem cells.

Figure 1

Differentiation of iPSCs into iPS-NSCs. iPSCs were expanded to 80% confluency, and then, the iPSC media were replaced by neuronal induction media (Neurobasal-A, 1X B27-A, 1X N2, 1X NEAA, 1X Glutamax, and 5 mg/mL streptomycin and 5 UI/mL penicillin). The cells were kept in culture until they detached and formed neurospheres. The media containing detached cells were centrifuged, and the pellet was dissociated in Accutase. Cells were then re-plated in neural stem cell media (Neurobasal-A, 1X B27-A, 1X N2, 1X NEAA, 1X Glutamax, 20 ng/mL EGF, 10 ng/mL bFGF, and 5 mg/mL streptomycin and 5 UI/mL penicillin). Cells were passaged three times and then characterized using ICC.

Figure 2

Characterization of iPS-NSCs through ICC. (A) Hoechst-labeled iPS-NSCs (blue) showed positive expression of neural stem cells markers; SOX2 (green) and Nestin (red). Upper row shows a neurosphere, and lower row shows individual cells. (B) Few cells in the neurosphere showed positive expression of immature neuronal marker, β-tubulin-III (green). (C) Percentage of cells expressing Nestin (M = 61.9%, SD = 14.3), SOX2 (M = 46.1%, SD = 18.7), and β-tubulin-III (M = 11.8%, SD = 2.9).

Figure 3

Accelerating rotarod testing. Accelerod testing showed a significant decrease of the fall latency in HD vehicle control compared to WT mice (p = 0.001) but no significant decrease in the iPS-NSCs treated HD mice were found compared to WT mice (p = 0.077). At the same time, there was no overall significant difference between HD vehicle controls and iPS-NSCs treated HD mice (p = 0.336). Weekly testing on the accelerod revealed a significant difference between iPS-NSCs treated HD mice and WT mice at baseline (p = 0.017). However, iPS-NSCs treated HD mice showed no significant difference from WT mice starting from Week 4 after transplantation (p = 0.159) and up to 10 weeks after transplantation (p = 0.605). Also, there was no significant difference between iPS-NSCs treated HD mice and HD vehicle controls (p = 0.393) suggesting an intermediate treatment effect at this time point. ^*significant different from WT+HBSS, p < 0.05.

Figure 4

Striatal neuronal and medium spiny neurons counts and densities. (A) Stitched images for striatum in each group showing markers of iPS-NSCs (Hoechst labeled cells; blue), mature neurons (NeuN; green) and medium spiny neurons (DARPP-32, red). (B,D) Analysis of NeuN and DARPP-32 labeled cells (mature neurons and medium spiny neurons, respectively) in striata showed that HD+HBSS mice are significantly different from WT+HBSS mice (p = 0.002), but HD+iPS-NSCs are not significantly different from WT+HBSS mice (NeuN, p = 0.79; DARPP-32, p = 0.57). Moreover, HD+iPS-NSCs mice are significantly different from HD+HBSS (p = 0.01; p = 0.01). (C,E) Analysis of NeuN and DARPP-32 labeled cells densities (mature neurons and medium spiny neurons, respectively) in striata showed that HD+HBSS mice are significantly different from WT+HBSS mice (p = 0.01, p = 0.003, respectively), but HD+iPS-NSCs are not significantly different from WT mice (p > 0.05). Also, DARPP-32 labeled cells densities were significantly increased in iPS-NSCs-HD treated mice compared to HD vehicle controls (p = 0.047). ^*Significant different from WT+HBSS,^†significant different from HD+iPS-NSCs, p < 0.05.

Figure 5

(A) Transplanted iPS-NSCs survived and differentiated into mature neurons and medium spiny neurons in HD mice. Confocal images were captured from iPS-NSCs treated HD mouse. Hoechst labeled iPS-NSCs (blue) were found in striata 10 weeks post transplantation. Also, Hoechst labeled iPS-NSCs (blue) show co-expression of mature neurons marker (NeuN; green) and region specific neurons (DARPP-32; red). (B) Proportion of Hoechst labeled iPS-NSCs that are co-labeled with DARPP-32 is significantly higher in HD+iPS-NSCs mice compared to WT+iPS-NSCs (p = 0.029). (C) Proportion of Hoechst labeled iPS-NSCs that are co-labeled with NeuN is significantly higher in HD+iPS-NSCs mice compared to WT+iPS-NSCs (p = 0.038). ^*Significant different, p < 0.05.

Figure 6

Astrocyte response at the injection site following transplantation. Greater activation of astrocytes (GFAP; red) was observed in iPS-NSCs (Hoechst labeled cells; blue) transplanted brains in comparison to vehicle controls and in iPS-NSCs transplanted WT mice in comparison to iPS-NSCs transplanted HD mice.

Figure 7

Western blot analysis of BDNF and TrkB. (A,B) Western blot analysis showed a significant decrease of BDNF in striata of HD+HBSS compared to WT+HBSS mice (p = 0.04), but HD+iPS-NSCs were not significantly different from WT mice (p = 0.59), and HD+HBSS mice (p = 0.25) suggesting intermediate treatment effect. (A,C) Western blot analysis showed a significant decrease of total TrkB in striata of HD+HBSS compared to WT+HBSS mice (p = 0.02), but HD+iPS-NSCs were not significantly different from WT+HBSS mice (p = 0.31), and HD+HBSS (p = 0.31) suggesting intermediate treatment effect. ^*Significant different from WT+HBSS, p < 0.05.