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Address correspondence to Josef Priller, Department of Neurology, Charité, Humboldt-University, Schumannstrasse 20/21, D-10117 Berlin, Germany. Tel.: (49) 30-450-560140. Fax: (49) 30-450-560932. E-mail: josef.priller{at}charite.de
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Abstract |
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Key Words: bone marrow transplantation; gene transfer; green fluorescent protein; nervous system; Purkinje cells
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Introduction |
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Results and discussion |
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In contrast to previous findings, our data suggest that neuronal differentiation of BM cells is a rare biological event and occurs late, after BMT. The observation period in the present study almost covered the entire life span of a laboratory mouse. The discrepancy may relate to the fact that we scored only those GFP-expressing cells as neurons that showed the characteristic morphology with axon/dendrites and expression of neuronal antigens. In the regions of adult neurogenesis, such as the olfactory bulb, engrafted cells underwent apoptosis before full neuronal differentiation (unpublished data). Interestingly, a recent BMT study in mice using GFP-marked BM cells revealed that no donor-derived neurons were detectable in the brain at 6 mo posttransplantation, which is in perfect agreement with our findings (Nakano et al., 2001). In terms of macrophage/microglial engraftment and lack of astroglial differentiation of BM-derived cells, our data are also in line with recently published results (Brazelton et al., 2000, Nakano et al., 2001). With regard to the cerebellum, it remains enigmatic why engraftment of Purkinje cells was seen at 12 mo, but not at 4 mo post-BMT. Moreover, it is unclear why BM cells did not contribute to the population of granule cells, which are the most numerous neurons in the brain. It is tempting to speculate that late neogenesis of Purkinje cells represents a physiological response to ageing when almost 30% of the Purkinje cell population, but none of the cerebellar granule cells, are lost (Sturrock, 1990). The decrease in Purkinje cell number is noticeable in mice at 15 mo, but not at 6 mo of age. Importantly, recent experimental evidence suggests that BM stem cells have regenerative capacity (Ferrari et al., 1998; Lagasse et al., 2000; Orlic et al., 2001).
Purkinje cells are large, GABAergic neurons that serve as sole output of the cerebellar cortex (Voogd and Glickstein, 1998). Chickquail chimera work revealed that they arise from clonally related embryonic founder cells whose progeny populate each side of the cerebellum, respecting the cerebellar midline (Alvarez Otero et al., 1993). In this context, it is interesting to note that we found GFP-expressing Purkinje cells in both cerebellar hemispheres, suggesting that the factors governing neogenesis of Purkinje cells in the adult may be different from those during cerebellar development. It remains to be determined which types of BM stem cells give rise to Purkinje neurons in the adult brain. In the absence of a consensus regarding which markers are consistently expressed on BM stem cell populations, an analysis of the genomic insertion sites of the retrovirus may in the future help to define the clonal precursors of BM-derived Purkinje cells. Mesenchymal stem cells have been shown to generate neurons in vitro and after intraventricular injection into neonatal mouse brains (Kopen et al., 1999; Sanchez-Ramos et al., 2000; Woodbury et al., 2000), but failed to engraft in the brain parenchyma following in utero transplantation (Liechty et al., 2000).
Several technical points need to be addressed. The DNA content of certain large neurons, notably Purkinje cells, has been suggested to exceed diploid levels, but a subsequent critical appraisal of techniques contradicted the findings (Mann et al., 1978). Hyperdiploid DNA values may represent changes in chromatin compaction and the presence of single-stranded DNA related to the metabolic state of Purkinje cells (Bernocchi et al., 1986). Thus, fusion of host Purkinje cells with GFP-marked blood-borne cells is an unlikely scenario. No temporal or spatial correlation between the appearance of donor-derived Purkinje cells and the engraftment of macrophages in the cerebellum was observed, and GFP-marked Purkinje cells did not express Iba1 (unpublished data). Although GFP may have promoted the differentiation of BM cells into neurons, an overwhelming body of evidence suggests that the fluorophore is biologically inert (Goldman and Roy, 2000). Finally, using transgenic mice that ubiquitously express GFP, we could exclude the possibility that GFP labeling of Purkinje cells was a mere result of recombinant retrovirus taken up by host neurons. The bicistronic vector used in this study provided high and stable gene expression in hematopoietic cells in the absence of replication-competent retrovirus.
The data suggest that BM cells can, upon adoptive transplantation, generate Purkinje cells in the adult brain. These findings may have important implications for the therapy of CNS disorders that are associated with Purkinje cell loss, as engraftment of BM cells might be enhanced during pathology. Interestingly, reduced ataxia and increased life span was observed in mouse models of Niemann-Pick disease after transplantation of normal BM, and Purkinje cells were present in the cerebellum despite the lack of enzyme activity in the brain (Miranda et al., 1998).
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Materials and methods |
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BM was harvested from 812-wk-old male C57BL/6 mice 2 d after treatment with 150 mg/kg 5-fluoruracil (Sigma-Aldrich). After prestimulation for 48 h in DME supplemented with 15% heat-inactivated fetal calf serum (Biochrom), 20 ng/ml murine interleukin-3, 50 ng/ml human interleukin-6 (PromoCell), and 50 ng/ml rat stem cell factor (provided by Amgen). BM cells were cocultured with irradiated (1,300 cGy) MGirL22Y virusproducer cells in the presence of 6 µg/ml polybrene (Sigma-Aldrich). After 48 h, nonadherent BM cells were rinsed off the producer cell monolayer and transplanted into lethally irradiated (two doses of 550 cGy separated by 3 h) adult male C57BL/6 mice. Alternatively, BM was harvested form "green mice" (Okabe et al., 1997) and transplanted directly into lethally irradiated adult male C57BL/6 mice. Recipient mice received 5 x 106 cells by tail vein injection. For in vitro clonogenic progenitor assays, transduced BM cells were suspended in 2 ml of methylcellulose supplemented with interleukin-3, interleukin-6, erythropoietin, and stem cell factor (StemCell Technologies), supporting the growth of myeloid colonies.
Flow cytometric analysis of GFP expression in peripheral blood cells
1, 4, 8, and 12 mo after BMT, peripheral blood obtained by tail vein puncture was examined by fluorescent-activated cell sorting (Becton Dickinson). After erylysis, cells labeled with Mac-1-Pe, B220-Pe, Gr-1-Pe (PharMingen), and CD4-Pe (Becton Dickinson) were analyzed for GFP expression. Dead cells (propidium iodidepositive) were excluded. Data were evaluated using the CellquestTM software.
Immunohistochemical analysis of donor cell engraftment in the cerebellum
BM chimeric mice (n = 24) were killed between 2 wk and 15 mo posttransplantion, and perfused transcardially with 4% paraformaldehyde in PBS. 30 µm vibratome or 20 µm cryosections were obtained from the brains. For immunohistochemistry, IgG block was performed and sections were incubated in PBS supplemented with 510% normal goat serum and 0.5% Triton X-100. Primary antibodies were added overnight at dilutions of 1:200 for anti-calbindin-D28K (mouse monoclonal; provided by Dr. Veh, Charité, Berlin, Germany), 1:250 for antiglutamic acid decarboxylase (GAD) (rabbit polyclonal; Chemicon), 1:100 for anti-NeuN (mouse monoclonal; Chemicon), 1:100 for anti-Iba1 (rabbit polyclonal; provided by Dr. Imai, National Institute of Neuroscience, Tokyo, Japan), 1:50 for anti-F4/80 (rat monoclonal; Serotec), 1:2,500 for anti-GFAP (rabbit polyclonal; Dako), and 1:100 for anti-S100ß (rabbit polyclonal; provided by Dr. Veh, Charité, Berlin, Germany). Texas redconjugated secondary antibodies (goat antimouse and goat antirabbit; Molecular Probes) were added at 1:200 for 1 h. Omission of primary antibodies served as negative control and resulted in no detectable staining. Digital micrographs of GFP and Texas red fluorescence were obtained on a conventional fluorescence microscope equipped with a color scanner (Leica) or on a confocal laser scanning microscope (ZEISS).
Quantification of engraftment
Semiquantitative analysis of Purkinje cell engraftment was performed in three representative animals. Every seventh cerebellar section was analyzed, and the number of GFP-expressing Purkinje cells was determined in relation to the total number of calbindin- or GAD-stained Purkinje cells in these sections. Moreover, the remaining GFP-expressing microglia and macrophages, demonstrating Iba1 immunoreactivity, were counted. In two representative chimeras (retroviral and transgenic GFP expression), every single section from the olfactory bulb to the cerebellum was analyzed for GFP-marked neurons.
Ultrastructural analysis of engrafted cells
Preembedding immunohistochemistry was performed on 80-µm vibratome sections as described above. A preabsorbed polyclonal anti-GFP antibody (CLONTECH Laboratories, Inc.) was used at a dilution of 1:250. Omission of primary antibody served as negative control. Addition of the secondary biotinylated goat antirabbit antibody (Vector Laboratories) was followed by incubation with the avidinbiotin peroxidase complex (ABC; Vector Laboratories) and 3,3'-diaminobenzidine/H2O2 (Sigma-Aldrich) according to the manufacturer's instructions. Sections were then fixed in 2% glutaraldehyde/PBS and immersed in 2% Dalton's osmium. After dehydration, the tissue was embedded in araldite. For high resolution light microscopy, 1-µm semithin araldite sections were scanned with a Leica color scanner. Each cell immunoreactive for GFP was sought in adjacent ultrathin sections cut on a LKB-ultramicrotome. The sections were mounted on mesh copper grids and processed for electron microscopy.
Intravital microscopy
In vivo confocal laser imaging of GFP-expressing cells was performed according to a modified protocol of Villringer et al. (1991). Anesthesia was induced in BM chimeric mice by inhalation of 2% halothane in a mixture of 30% O2/70% N2O. Anesthesia was maintained throughout the experiment by intraperitoneal administration of 750 mg/kg urethane and 150 mg/kg -chloralose. The femoral artery and vein were cannulated and mean arterial blood pressure was monitored. Systemic arterial pressure was 82 ± 8 mm Hg (mean ± SD; n = 3). Body temperature was maintained between 36.5°C and 37.5°C using a heating pad. Mice were intubated and ventilated with a mixture of O2 and air. End tidal CO2 partial pressure was monitored and kept between 2.53.5% by adjusting the respiratory rate, tidal volume, and inspiration time. After fixation to a stereotaxic frame, a craniotomy was performed over the parietal cortex. The dura mater was removed and a closed cranial window was implanted. Mice received 250 mg/kg rhodamine B dextran (Sigma-Aldrich) by intravenous injection. Subsequently, the brains were examined for GFP-expressing cells through the coverglass of the cranial window for 3 h using a confocal laser scanning microscope (Leica).
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Footnotes |
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Acknowledgments |
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This work was partly supported by Deutsche Forschungsgemeinschaft and the Herrmann and Lilly Schilling Foundation.
Submitted: 22 May 2001
Revised: 13 September 2001
Accepted: 18 September 2001
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References |
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