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. 2002 May;109(9):1183-91.
doi: 10.1172/JCI14862.

Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span

Hee Kyung Jin  1 , Janet E Carter, George W Huntley, Edward H Schuchman
Affiliations

Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span

Hee Kyung Jin et al. J Clin Invest. 2002 May.

Abstract

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow-derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice, but none of the untreated controls, survived for > or = 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD, although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low, but surviving Purkinje cells contained the retrovirally expressed human enzyme, and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow-derived MSCs.

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Figures

Figure 1
Figure 1
Survival and migration of MSCs in the ASMKO mouse brain 1 month after injection. Above each micrograph is a schematic depiction of the brain indicating the position of the injection site (dotted arrows) and the area shown in the photographs below (highlighted in red). Arrowheads indicate blue-green nuclei of transplanted GFP-expressing MSCs counterstained with DAPI. (a) Clustering of MSCs at the dentate gyrus. (b) Lateral migration, at a position 2.06 mm posterior to bregma. (c and d) Lateral migration of MSCs 2.70 mm posterior to bregma. GCL, granule cell layer; CA3, CA3 region of the hippocampus. Original magnification ×20.
Figure 2
Figure 2
Survival of ASMKO mice receiving intracerebral MSC transplants. By 28 weeks after transplant (31 weeks of age), all of the untreated ASMKO mice (squares) had died, whereas seven of eight treated ASMKO animals (triangles) and all of the normal animals (circles) survived. The longest-surviving treated ASMKO mouse lived to be about 47 weeks old (44 weeks after transplant). n = 8 for each group.
Figure 3
Figure 3
Body weights and rotarod scores for treated and untreated male ASMKO mice. (a) The mean body weights of treated (filled circles) and untreated (open circles) ASMKO male mice (n = 4) are shown beginning at 17 weeks after transplant (20 weeks of age). The mean body weight (38 g) of normal male animals (+/+) is also indicated. (b) Rotarod scores of the same treated (filled circles) and untreated (open circles) male mice were averaged and plotted beginning 17 weeks after transplant (20 weeks of age). The mean rotarod score (300 s) of normal male animals (+/+) is indicated. All of the untreated control animals died by 28 weeks after transplant (see Figure 2). *P < 0.05, treated versus untreated, per one-tailed Student t test.
Figure 4
Figure 4
Body weights and rotarod scores for treated and untreated female ASMKO mice. (a) The mean body weights of treated (filled circles) and untreated (open circles) ASMKO female mice (n = 3) are shown beginning at 17 weeks after transplant (20 weeks of age). The mean body weight (29.5 g) of normal female animals (+/+) is also indicated. (b) Rotarod scores of individual female treated (filled circles) and untreated (open circles) mice were averaged and plotted beginning 17 weeks after transplant (20 weeks of age). The mean rotarod score (300 s) of normal female animals (+/+) is indicated. All of the untreated animals died by 28 weeks after transplant (see Figure 2). *P < 0.05, treated versus untreated, per one-tailed Student t test.
Figure 5
Figure 5
Purkinje cell immunohistochemistry at 4, 12, and 24 weeks after transplantation. Visualization of Purkinje cells was performed using anti-calbindin antibodies as described in Methods. Arrows indicate Purkinje cells. Note the extensive vacuolization and absence of Purkinje cells in the untreated ASMKO mice at 12 and 24 weeks after transplant, compared with the treated animals. The sections shown are from position A in Figure 7a, 75 μm from the cerebellar injection site.
Figure 6
Figure 6
Histograms showing Purkinje cell numbers in treated ASMKO mice at 4, 12, and 24 weeks after transplantation. Quantitation was performed as described in Methods. The data shown in the histograms correspond to the images shown in Figure 5 (position A in Figure 7a). Data are expressed as mean ± SEM (n = 4). Black bars indicate normal control animals, white bars indicate treated ASMKO animals, and gray bars indicate untreated ASMKO control animals. *P < 0.05, treated versus untreated ASMKO mice, per one-tailed Student t test.
Figure 7
Figure 7
Purkinje cell survival in transplanted ASMKO mice is a function of distance from the cerebellar injection site. (a) Schematic depiction of a mouse brain showing the location of the hippocampal (filled circle) and cerebellar (filled square) injection sites. A–D indicate the location of sections used for the quantification shown in b (75–300 μm from the cerebellar injection site). The data shown in Figures 5 and 6 are from position A, indicated by the arrow in b. Quantification was performed as described in Methods. Open symbols, normal animals; filled symbols, treated ASMKO animals; circles, 4 weeks after transplant; squares, 12 weeks after transplant; triangles, 24 weeks after transplant.
Figure 8
Figure 8
Expression of human ASM and depletion of sphingomyelin in the brains of transplanted ASMKO mice. The sections shown were taken from 4-month-old animals, 75 μm from the cerebellar injection site. (a) Anti–human ASM antibodies were used to detect the retrovirally expressed enzyme in cerebellar sections of normal, untreated ASMKO, and treated ASMKO animals. Note that a strong positive (brown) signal was seen in Purkinje cells from the treated animals. No signal was seen in Purkinje cells from normal animals, demonstrating the specificity of the reaction. (b) The sphingomyelin-specific binding protein, lysenin, was used to detect this lipid in cerebellar sections from normal, untreated ASMKO, and treated ASMKO animals. Note that the sections from untreated ASMKO animals had a very strong lysenin signal compared with normal or treated ASMKO animals.
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References

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