Article Figures & Data
Figures
- Figure 1.
Neural progenitor cells isolated from adult mouse brain express neural lineage-specific markers in vitro. A, Phase-contrast image of proliferating cultures. B, Fluorescent micrograph showing expression of neuronal (MAP2a+b; green) or astroglial (GFAP; red) markers. C, Fluorescent micrograph showing expression of an oligodendroglial (GalC) marker; cultures were counterstained for 4′,6′-diamidino-2-phenylindole (B; blue). Scale bars: A, 100 μm; (in B) B, C, 10 μm.
- Figure 2.
Survival and differentiation of NPCs in the ASMKO mouse brain 6 weeks after transplantation. Coronal sections show the distribution of BrdU-positive (red) NPCs after transplantation into the adult mouse hippocampus (A-D) and thalamus (E-H). Photomicrographs were taken at 300 μm intervals. There was clustering of cells at the implantation site (C, G), but many of the transplanted cells migrated throughout the hippocampus and thalamus, respectively. Confocal images show BrdU-immunoreactive cells (red) colocalized with NeuN immunoreactivity (green) within the granular cell layer of the hippocampus (I). Yellow indicates transplanted cells double labeled for NeuN and BrdU. Other BrdU-immunoreactive cells colocalized with GFAP-astrocyte immunoreactivity (J) or with NG2-oligodendrocyte immunoreactivity (K) in the molecular layer of the hippocampus. Scale bars: (in D) A-D, 100 μm; (in H) E-H, 100 μm; I, 5 μm; J, K, 10 μm.
- Figure 3.
Distribution and differentiation of NPCs after transplantation into the neonatal mouse hippocampus. A, B, Coronal section of the dentate gyrus of the hippocampus showing the remarkable localization of transplanted BrdU-labeled cells (A) within the granular cell layer (B). BrdU-immunoreactive cells (red) colocalized with NeuN immunoreactivity (green) mostly along the inner margins of the granular cell layer of the dentate gyrus. Inset, A confocal image showing transplanted cells within the granular cell layer double labeled for NeuN (green) and BrdU (red). Scale bars: (in B) A, B, 100 μm; inset, 5 μm.
- Figure 4.
Retroviral transduction of mouse NPCs with the human acid sphingomyelinase gene. A, B, Immunostaining of transduced NPCs for human acid sphingomyelinase (red) 1 week after selection. High magnification shows lysosomal localization of hASM (B). Nuclei are counterstained by 4′, 6′-diamidino-2-phenylindole (blue). C, Expression of hASM at the implant site. Coronal section of the thalamus of ASMKO recipients at 2 weeks after transplantation showing the presence of ASM-immunopositive cells in the area close to the injection site (arrowhead points to single ASM-labeled cells shown at higher magnification in inset). D, RT-PCR expression of hASM mRNA in tissue isolated from the transplant site of seven animals compared with two (untreated) control animals. Four of the seven transplanted animals had distinct bands of hASM mRNA (571 bp band) expression compared with control tissue samples in which no hASM band was detected. Scale bars: A, B, 10 μm; C, 100 μm; inset, 25 μm.
- Figure 5.
Reduced amount of lysosomal pathology in the hippocampus and thalamus of an ASMKO mouse brain that received ASM-transduced NPC transplants. A, B, Photomicrographs of the granular cell layer of the hippocampus from nontransplanted (A) and transplanted (B) ASMKO mice at 4 weeks after transplantation. C, D, Photomicrographs of the thalamus transplanted with nontransduced cells (C) and ASM-transduced cells (D) of ASMKO mice at 4 weeks after transplantation. Note the decrease in cytoplasmic distension in neural cells in the ASMKO mouse transplanted with ASM-expressing NPCs (B, D), whereas there remains profound vacuolation in cells on the nontransplanted side (A) or the side transplanted with nontransduced cells (C) as a result of the accumulation of distended lysosomes (arrows). Insets show higher magnifications of cells indicated by arrows. Scale bars: (in B) A-D, 20 μm; insets, 5 μm.
- Figure 6.
Regional clearance of cholesterol deposits at 2 weeks after transplantation visualized by filipin staining in the ASMKO mouse brain that received NPCs. A, Coronal view of the adult mouse hippocampus showing the broad dispersion of transplanted BrdU-immunoreactive cells. B, Adjacent sections stained with filipin showing that the regional clearance of fluorescent deposits matches closely with the distribution of the transplanted cells indicated by arrowheads. D, Higher magnification showing the substantial decrease of filip in deposits within the granular cell layer (gcl), the molecular layer, and the CA1 region of the hippocampus from the transplanted region (indicated by arrowheads in B) compared with the nontransplanted side (C). Scale bars: (in A) A, B, 200 μm; (in C) C, D, 100 μm.
- Figure 7.
Clearance of cholesterol accumulation visualized by filipin staining in different regions of ASMKO mouse brains that received ASM-expressing NPCs 4 weeks after transplantation. Coronal views of the dentate gyrus (A, B), subiculum (C, D), and thalamus (E, F) show the presence of fluorescent deposits, representing accumulated cholesterol within cells contralateral to the transplanted NPCs (A, C, E) and clearance of the fluorescent deposits ipsilateral to the transplanted ASM-expressing cells (B, D, F). Scale bar: (in A) A-F, 50 μm.
- Figure 8.
Regional clearance of cholesterol deposits visualized by filipin staining in the hippocampus 4 weeks after bilateral transplantation of ASM-transduced or ASM-nontransduced NPCs. A, B, Coronal view of the adult mouse hippocampus shows the comparable dispersion of transplanted BrdU-positive NPCs that were nontransduced (A) or ASM transduced (B). C-F, Coronal section of the pyramidal CA1 layer and dentate gyrus (C, D) and CA4 pyramidal cell layer (E, F) showing the marked reduction of fluorescent deposits visualized by filipin staining on the side that received ASM-transduced cells (D, F) versus ASM-nontransduced cells (C, E). Insets are higher-magnification views showing more clearly the cytoplasmic clearance of fluorescent deposits from the CA1 pyramidal neurons (D) and granular neurons (F) on the side of the ASM-transduced cells versus the side that received ASM-nontransduced cells (C, E). Scale bars: (in B) A, B, 200 μm; (in C) C-F, 100 μm; insets, 10 μm.
- Figure 9.
Depletion of sphingomyelin in the brains of ASMKO mice 4 weeks after bilateral transplantation of ASM-transduced or nontransduced NPCs. The sphingomyelin-specific binding protein lysenin was used to detect this lipid in sections from untreated and treated ASMKO animals. A, B, Coronal views of the striatum (A, B) and the hippocampus (C, D) showing the marked reduction in lysenin staining on the side that received ASM-transduced cells (B, D) versus ASM-nontransduced cells (A, C). Insets are higher-magnification views showing more clearly the marked depletion of sphingomyelin from the cells on the side of the ASM-transduced cells. Scale bars: (in C) A-D, 50 μm; insets, 25 μm.
Tables
- Table 1.
Cell transplant recipients by age group at time of transplantation, whether they received a unilateral or bilateral transplant, survival time, genotype, type of cell transplanted, and number of mice that were processed histologically
Age Transplant region Survival time (in weeks) Genotype Cells transplanted Number of mice Adult 8 weeks Unilateral 4 KO NPC plus ASM 2 4 KO NPC 2 9-13 weeks 1 KO NPC plus ASM 2 2 KO NPC plus ASM 4 4 KO NPC plus ASM 2 6 WT NPC 7 6 WT NPC plus ASM 6 6 KO NPC 2 6 KO NPC plus ASM 3 26-28 weeks 2 KO NPC 2 2 KO NPC plus ASM 2 8 weeks Bilateral 4 KO NPC-NPC plus ASM 4 26-28 weeks 2 KO NPC-NPC plus ASM 3 Juvenile 6 KO NPC 5 5 weeks 6 WT NPC 1 Neonate 7 KO NPC 8 4-5 d 10 WT NPC plus ASM 6 -
KO, Knock-out; WT, wild type.
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- Table 2.
Frequency of correction as shown by filipin clearance by age group at the time of transplantation and survival time after transplantation.
Age (in weeks) Total number of mice Survival time (in weeks) Number of mice with reduced filipin Number of positive transplants 8 6 4 6 6 9-10 5 6 4 4 12 8 1, 2, or 4 8 8 26-28 5 2 4 4 -
Transplants containing BrdU-immunoreactive cells were classified as positive.
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