Transplantation of umbilical cord-derived mesenchymal stem cells into the striata of R6/2 mice: behavioral and neuropathological analysis

Abstract

Introduction: Huntington's disease (HD) is an autosomal dominant disorder caused by an expanded CAG repeat on the short arm of chromosome 4 resulting in cognitive decline, motor dysfunction, and death, typically occurring 15 to 20 years after the onset of motor symptoms. Neuropathologically, HD is characterized by a specific loss of medium spiny neurons in the caudate and the putamen, as well as subsequent neuronal loss in the cerebral cortex. The transgenic R6/2 mouse model of HD carries the N-terminal fragment of the human HD gene (145 to 155 repeats) and rapidly develops some of the behavioral characteristics that are analogous to the human form of the disease. Mesenchymal stem cells (MSCs) have shown the ability to slow the onset of behavioral and neuropathological deficits following intrastriatal transplantation in rodent models of HD. Use of MSCs derived from umbilical cord (UC) offers an attractive strategy for transplantation as these cells are isolated from a noncontroversial and inexhaustible source and can be harvested at a low cost. Because UC MSCs represent an intermediate link between adult and embryonic tissue, they may hold more pluripotent properties than adult stem cells derived from other sources.

Methods: Mesenchymal stem cells, isolated from the UC of day 15 gestation pups, were transplanted intrastriatally into 5-week-old R6/2 mice at either a low-passage (3 to 8) or high-passage (40 to 50). Mice were tested behaviorally for 6 weeks using the rotarod task, the Morris water maze, and the limb-clasping response. Following behavioral testing, tissue sections were analyzed for UC MSC survival, the immune response to the transplanted cells, and neuropathological changes.

Results: Following transplantation of UC MSCs, R6/2 mice did not display a reduction in motor deficits but there appeared to be transient sparing in a spatial memory task when compared to untreated R6/2 mice. However, R6/2 mice receiving either low- or high-passage UC MSCs displayed significantly less neuropathological deficits, relative to untreated R6/2 mice.

Conclusions: The results from this study demonstrate that UC MSCs hold promise for reducing the neuropathological deficits observed in the R6/2 rodent model of HD.

Figure 1

Immunocytochemistry (ICC) of low- and high-passaged umbilical cord mesenchymal stem cells (UC MSCs). Low- and high-passaged UC MSCs expressed the MSC marker SCA1, were negative for the hematopoietic stem cell marker CD45 and displayed typical MSC morphology. Scale bar represents 100 μm.

Figure 2

In vitro quantitative RT-PCR of BDNF mRNA expression of umbilical cord mesenchymal stem cells (UC MSCs). A significant decrease in mRNA levels of BDNF was observed in the high-passaged UC MSCs compared to low-passaged UC MSCs. (Note: † significant from high-passage UC MSCs; TTF are control cDNA isolated from mice tail-tip fibroblasts.) Bar graph represents mean value; error bars represents SEM. BDNF, brain-derived neurotrophic factor; SEM, standard error of the mean.

Figure 3

Motor coordination assessment of R6/2 mice following umbilical cord mesenchymal stem cell (UC MSC) transplantation. A significant decline in motor coordination was observed in untreated R6/2 mice when compared to WT mice. The R6/2 mice that received transplantation of high-passage UC MSCs displayed significantly longer latencies to fall, compared to untreated R6/2 mice at ten weeks of age. (Note: *significant from WT; ^#significant from R6/2; ^†significant from high-passage UC MSCs). Line graph represents mean value; error bars represents SEM. SEM, standard error of the mean; WT, wild type.

Figure 4

Spatial memory assessment of R6/2 mice following umbilical cord mesenchymal stem cell (UC MSC) transplantation. Untreated R6/2 mice displayed significant impairment in spatial memory at 9-, 10- and 11-weeks of age when compared to WT mice. Mice receiving either low- or high-passaged UC MSCs did not display sparing of this spatial memory task. (Note: *significant from WT) Bar graph represents mean value; error bars represents SEM. SEM, stanrard error of the mean; WT, wild type.

Figure 5

Limb-clasping of R6/2 mice following umbilical cord mesenchymal stem cell (UC MSC) transplantation. Untreated R6/2 mice had significantly more limb-clasping responses than did WT mice, starting at eight-weeks of age, with this impairment continuing for the duration of the study. Mice receiving either low- or high-passaged UC MSCs were similar to untreated R6/2 mice. (Note: *significant from WT; ^#significant from R6/2; ^†significant from high-passage BM MSCs). Line graph represents mean value; error bars represents SEM. BM, bone marrow; SEM, standard error of the mean; WT, wild type.

Figure 6

Measures of brain area and evidence of integrity of the metabolic tissue in the striata of mice receiving umbilical cord mesenchymal stem cell transplantations. Gross morphology of the brain near the area of transplantation can be visualized with cytochrome oxidase labeling (A). Untreated R6/2 mice and mice that received transplantation of low-passaged UCMSCs had a significant decrease in total brain area when compared to WT mice (B). Optical densitometric measures of cytochrome oxidase in the striata (outlined in dashed line) revealed significantly less metabolic activity of striatal tissue in untreated R6/2 mice, when compared to WT mice at the time of necropsy (C). R6/2 mice that received transplantation of low- or high-passaged UC MSCs had significantly higher levels of metabolic activity in striatal tissue than did untreated R6/2 mice. (Note: *significant from WT; ^#significant from R6/2). Line graph represents mean value; error bars represent SEM. SEM, standard error of the mean; UCMSCs, umbilical cord mesenchymal stem cells; WT, wild type.

Figure 7

Immunohistochemical analysis of the transplanted umbilical cord mesenchymal stem cells (UC MSCs). No neuronal or glial differentiation was observed from the transplanted UC MSCs as seen by the lack of co-localization between the UC MSCs (blue) with NeuN (red) or GFAP (green), respectively (A). A decreased significant difference in the amount of surviving transplanted cells was observed in low-passage UC MSCs, when compared to high-passage UC MSCs (B). A significant increase in GFAP optical densitometry around the transplant site was observed in the R6/2 mice that received transplantation of low-passage UC MSCs (C). GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei.