Brain Advance Access originally published online on October 6, 2004
Brain 2004 127(11):2518-2532; doi:10.1093/brain/awh273
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Brain Vol. 127 No. 11 © Guarantors of Brain 2004; all rights reserved
Wild-type bone marrow cells ameliorate the phenotype of SOD1-G93A ALS mice and contribute to CNS, heart and skeletal muscle tissues
1 Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, IRCCS Ospedale Maggiore Policlinico, Milano, 2 Centro di Eccellenza per lo Studio delle Malattie Neurodegenerative, Università degli Studi di Milano, Milano and 3 IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy
Correspondence to: Professor Giacomo P. Comi, Dipartimento di Scienze Neurologiche, Università di Milano, Padiglione Ponti, Ospedale Maggiore Policlinico, Via Francesco Sforza 35, 20122 Milan, Italy E-mail: giacomo.comi{at}unimi.it
Received February 11, 2004. Revised May 27, 2004. Second revision on June 24, 2004. Accepted June 27, 2004.
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Summary |
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Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease without any effective therapy. To evaluate the potential of wild-type bone marrow (BM)-derived stem cells to modify the ALS phenotype, we generated BM chimeric Cu/Zn superoxide dismutase (SOD1) mice by transplantation of BM cells derived from mice expressing green fluorescent protein (GFP) in all tissues and from Thy1-YFP mice that express a spectral variant of GFP (yellow fluorescent protein) in neurons only. In the recipient cerebral cortex, we observed rare GFP+ and YFP+ neurons, which were probably generated by cell fusion, as demonstrated by fluorescence in situ hybridization (FISH) analysis, suggesting that this phenomenon is not limited to Purkinje cells. GFP-positive microglial cells were extensively present in both the brain and spinal cord of the affected animals. Completely differentiated and immature GFP+ myofibres were also present in the heart and skeletal muscles of SOD1 mice, confirming that BM cells can participate in striated muscle tissue regeneration. Moreover, wild-type BM chimeric SOD1 mice showed a significantly delayed disease onset and an increased life span, probably due to a positive ‘non-neuronal environmental’ effect rather than to neuronogenesis. This improvement in SOD1-G93A mouse survival is comparable with that previously obtained using some safer pharmacological agents. BM transplantation-related complications in humans preclude its clinical application for ALS treatment. However, our data suggest that further studies aimed at improving the degree of tissue chimerism by BM-derived cells may provide valuable insights into strategies to slow ALS progression.
Key Words: amyotrophic lateral sclerosis; motor neuron; stem cells; transplantation; SOD
Abbreviations: ALS = amyotrophic lateral sclerosis; BM = bone marrow; FACS = fluorescence-activated cell sorting; FISH = fluorescence in situ hybridization; GFP = green fluorescent protein; hUCB = human umbilical cord blood; NeuN = nuclear neural-specific antigen; NF = neurofilament; SOD1 = Cu/Zn superoxide dismutase; TuJ1 = class III ß-tubulin; YFP = yellow fluorescent protein
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Introduction |
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TopSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective loss of motor neurons in the spinal cord, brainstem and cerebral cortex. About 10% of cases are dominantly inherited and
20% of these arise from mutations in the gene for Cu/Zn superoxide dismutase (SOD1) (Rosen, 1993
). Although a dose-dependent toxic gain of function plays a role in this process, the mechanisms whereby mutant SOD1 causes motor neuron death are not known.
Several transgenic mice expressing mutated human SOD1 have been created. One of these lines is the G93A mouse that expresses a mutant SOD1 carrying the Gly93Ala missense mutation and that develops an ALS-like motor neuron disease (Gurney et al., 1994). These animals exhibit a predictable disease onset at 90 days, with leg tremor, decreased stride and muscle strength, and death after almost 30 days (Gurney et al., 1994). These characteristics make the SOD1 Gly93Ala mice strain a useful tool for testing new therapeutic strategies.
Stem cell transplantation is a potential strategy for the treatment of ALS; transplanted cells might be expected to have beneficial effects either by replacing damaged CNS cells or by providing support to motor neurons through neurotrophic factor production or even by scavenging of agents toxic to motor neurons.
Chimeric mice, generated by injection of wild-type embryonic stem cells into SOD1 blastocysts, demonstrated that ‘environmental’ non-neuronal cells, in some cases representing a small minority of total cells, can ameliorate degeneration and survival of SOD1 mice (Clement et al., 2003).
Recent evidence suggested that bone marrow (BM)-derived stem cells can contribute to the regeneration of several tissues such as muscle (skeletal and cardiac tissues), liver and brain. Two mechanisms have been suggested to explain this phenomenon: de novo cell generation through ‘transdifferentiation’, and cell fusion.
It has been reported that after BM transplantation, BM donor-derived cells, expressing neural markers, were found in the brain and spinal cord of the recipients, in both mouse (Eglitis et al., 1997; Brazelton et al., 2000; Mezey et al., 2000; Priller et al., 2001; Corti et al., 2002a,b; Wagers et al., 2002) and human brains (Mezey et al., 2003; Weimann et al., 2003a; Cogle et al., 2004). At present, there is evidence that this neuronal conversion might be due to cell fusion, particularly in the case of Purkinje neurons (Alvarez-Dolado et al., 2003; Weimann et al., 2003b).
As an alternative to BM cells, human umbilical cord (hUCB) cells have been proposed as a stem cell source that could acquire a neuroectodermal phenotype. It has been described previously that intravenous administration of a high dose of hUCB cells into SOD1 irradiated mice increased their life span (Ende et al., 2000; Garbuzova-Davis et al., 2003).
To investigate the contribution of BM stem cells to the ALS phenotype, we generated BM chimeric SOD1 mice by transplanting BM cells derived from mice expressing green fluorescent protein (GFP) into all tissues (Okabe et al., 1997) and Thy-1-yellow fluorescent protein (YFP) neuron-specific transgenic mice (Feng et al., 2000). With this model, we also wanted to evaluate if the contribution of BM cells to CNS and mesodermal tissues is associated with de novo cell generation or with cell fusion.
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Materials and methods |
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Animals
Transgenic TgN(SOD1-G93A)1GUR mice overexpress human SOD1 carrying the Gly93 to alanine mutation. Transgenic mice were genotyped using a described polymerase chain reaction (PCR) method (Gurney et al., 1994
).
Transgenic TgN(ACTbEGFP)1Osb mice expressing an ‘enhanced’ GFP cDNA under the control of a chicken ß-actin promoter and cytomegalovirus enhancer were used as BM donors. Their transgene is widely expressed in all tissues, with the exception of erythrocytes and hair (Okabe et al., 1997).
B6.Cg-TgN(Thy1-YFP)16Jrs mice express spectral variants of GFP (such as YFP) at high levels in motor and sensory neurons, as well as in subsets of central neurons (Feng et al., 2000). Genotyping was performed by PCR as described (Feng et al., 2000).
The transgenic construct contains a YFP gene under the direction of regulatory elements derived from the mouse Thy1 gene. These elements are composed of a 6.5 kb fragment obtained from the 5' portion of the Thy1 gene, extending from the promoter to the intron following exon 4. Exon 3 and its flanking introns are absent. The deleted sequences are required for expression in non-neural cells but not in neurons. The remaining sequence is required for neuronal expression (Feng et al., 2000).
All transgenic animals were purchased from the Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed according to institutional guidelines that are in compliance with national (D.I. no. 116, G.U. suppl. 40, Feb. 18, 1992, Circolare No.8, G.U., 14 Luglio 1994) and international law and policies (EEC Council Directive 86/609, OJ L358, 1 Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996).
Whole BM transplantation
BM cells (30 x 106 cells) from GFP, YFP and SOD1-G93A mice were transplanted intraperitoneally after irradiation (800 rad) in SOD1-G93A mice aged 4 weeks. Twelve SOD1 mice were transplanted with GFP-derived BM, 12 with Thy1-YFP BM and 10 with SOD1-G93A BM, and all were evaluated up to end stage.
The study was designed so that littermates were distributed equally into the wild-type transplanted mice, mutant SOD1 mice and untreated mice. The study was also equally divided between males and females. Analytical information on littermates and gender group composition is provided in the Supplementary data available at Brain Online.
For motor neuron and axon count, another subgroup of animals was sacrificed at 100 days. This subgroup included SOD1 mice transplanted with GFP BM (n = 4); Thy1-YFP BM (n = 4); mutant SOD1 BM (n = 3) mice; and SOD1-untransplanted mice (n = 5).
To evaluate tissue chimerism, we also transplanted non-transgenic littermates with GFP and YFP BM cells. Sex-mismatched transplantation was performed by transplanting male BM cells into female recipients, and vice versa.
Behaviour and survival
Transplanted mice were observed daily for survival. A corresponding number of transgenic littermates was used as controls. Motor function was tested by using an accelerating rotarod device (4–40 r.p.m. Rota-Rod 7650; Ugo Basile, Comerio, Italy) weekly, starting from the day before transplantation (28 days of age). The time during which mice remained on the rotarod was registered.
Mortality was scored as the age at death, when the mouse was unable to right itself within 30 s when placed on its back in a supine position (Li et al., 2000).
Statistical analysis
Survival evaluation was performed by Kaplan–Meier analysis. The rotarod test was analysed by ANOVA (analysis of variance) followed by a Tukey post hoc analysis for multiple comparison.
Evaluation of BM repopulation
The degree of GFP BM chimerism was evaluated by fluorescence-activated cell sorting (FACS; Vantage SE, BD Biosciences, San Diego, CA) and by direct observation by fluorescence microscopy in transplanted mice. YFP chimerism was assessed by PCR for the YFP gene on BM cells as described (Feng et al., 2000).
Flow cytometry and FACS analysis of YFP BM cells
To determine whether BM cells expressed YFP, whole unfractionated BM from YFP trangenic mice, GFP trangenic mice and wild-type mice was analysed using FACS (Vantage, BD Biosciences, San Diego, CA).
Data were displayed as a contour plot, as a function of side-scattered versus GFP fluorescence. All three cell types were processed simultaneously.
Tissue analysis
The animals were sacrificed, perfused and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4).
The brain, spinal cord, muscles and heart were isolated, immersed in paraformaldehyde solution for 1 h, then in 20% sucrose solution in PBS (pH 7.4) overnight and frozen in Tissue Tek OCT compound with liquid nitrogen. The tissues were cryosectioned (10 µm) and mounted on gelatinized glass slides.
Cerebral tissue was cut in the coronal plane from the frontal lobes. Spinal cord, cervical, thoracic and lumbar levels were each evaluated in the axial plane. Regarding brain and spinal cord, every first section (10 µm) from one in five series was sampled using a random starting point (30 sections per animal). The cerebellum was cryosectioned in the sagittal plane at 20 µm, to detect Purkinje cells.
All CNS sections were blocked with 1% fetal calf serum in PBS and permeabilized with 0.25% Triton X-100. Triton permeabilization was not performed on muscle and heart sections. Sections were processed for multiple markers to determine the cellular phenotype of GFP/YFP-labelled cells. Anti-GFP antibody rabbit serum Alexa 488 (1 : 400 dilution; Molecular Probes, Eugene, OR) was used to reveal GFP/YFP positivity in double immunostaining (Wu et al., 2000). Slides were observed under a conventional fluorescence microscope (Zeiss Axiophot, Germany).
Laser scanning confocal (Bio-Rad, UK) microscopic analyses were performed to evaluate co-expression of GFP/YFP and non-haematopoietic antigens.
Motor neuron and axon count
For motor neuron counting, the lumbar spinal cord region (L1–L5) was processed for cryostat sectioning. Serial cross-sections at 10 µm thickness were processed by Nissl staining. We examined every fifth section at 20x magnification for neuron counting. At least 10 sections from the lumbar region were analysed. For axon count, the tissue was dissected, immersed in 2.5% glutaraldehyde overnight and then post-fixed in 2% osmium tetroxide. Samples were then dehydrated in ethanol and embedded in Epon. Semi-thin transverse sections (1 µm) were stained with toluidine blue. L5 roots were examined for axon counting on the optic microscope.
CNS immunohistochemistry
Primary antibodies were added overnight at 4°C at dilutions of 1 : 200 for nuclear neural-specific antigen (NeuN, mouse monoclonal antibody; Chemicon, Temecula, CA), 1 : 200 for neurofilament (NF, mouse monoclonal antibody; Chemicon), 1 : 200 for class III ß-tubulin (TuJ1, mouse monoclonal antibody; Chemicon), 1 : 200 for microtubule-associated protein 2 (mouse monoclonal antibody, Sigma-Aldrich, Saint Louis, MO); 1 : 200 for oligodendrocyte marker O4 (mouse monoclonal antibody; Chemicon) and 1 : 200 for glial fibrillary acidic protein (mouse monoclonal antibody, R-phycoerythrin-conjugated; Sigma-Aldrich). R-phycoerythrin-conjugated goat anti-mouse antibody(1 : 100; Dako, Carpenteria, CA) was used for 1 h at room temperature as secondary antibody, when unconjugated primary antibody was used.
To evaluate the BM contribution to the microglial cell compartment, brain and spinal cord sections (10 µm) were stained with rabbit anti-GFP and F4/80 R-phycoerythrin-conjugated antibody (1 : 100; Serotec, Raleigh, NC) (n = 12 SOD1 and n = 12 non-transgenic mice, that received GFP BM). For each mouse, 16 sections for brain and spinal cord level were selected from anatomically matched levels. Cell counts were performed at 40x magnification on eight fields from each section. We considered only those F4/80+ cells with the morphological features of ramified microglial cells (multiple branched cellular processes and a small cell soma).
Co-expression of GFP/YFP and tissue-specific markers was evaluated by laser confocal scanning (Bio-Rad, UK) microscopic analysis. The criteria for scoring were a clear double staining, with homogeneous GFP/YFP expression, along with an intact cellular morphology.
For brain and spinal cord, every first section (10 µm) from one in five series was sampled using a random starting point (30 sections for animal) to estimate the number of GFP/YFP cells, using a confocal microscope at 60x oil objective. In the animal group transplanted with SOD1 BM cells, we estimated the number of donor-derived cells by counting Y chromosome-positive cells. To evaluate the BM donor-derived microglial cells, we counted the number of Y-positive cells that express F4/80.
To obtain an unbiased stereological estimate of positive cells, optical dissectors and random sampling were used in the CNS. Optical dissectors sized 100 x 70 x 10 µm were randomly sampled and the number of positive cells in each dissector was quantified.
Data were analysed statistically by Student's t test.
Fluorescence in situ hybridization (FISH) analysis
Thin sections (10 µm) of brain from YFP-transplanted mice were incubated for 1 h with anti-GFP antibody rabbit serum Alexa 488 (1 : 400 dilution; Molecular Probes, Eugene, OR) followed by incubation with a TSA kit horseradish peroxidase goat anti-rabbit IgG and Alexa Fluor 488 Tyramide (Molecular Probes).
After immunoreaction, sections were rinsed with 2x standard saline citrate stock solution (SSC) for 10 min, and denaturated in 70% formamide in 2x SSC at 60°C for 10 min. The Cy-3 Y chromosome probe (Cambio Ltd, Cambridge, UK) was denaturated and incubated with slides at 60°C for 10 min.
The sealed slides were placed horizontally in a humid chamber and hybridized at 37°C for 18 h. Then the probe was washed off in 50% formamide 2x SSC and in 2x SSC, respectively, for 5 min at 37°C. The nuclei were then counterstained with DAPI (4',6-diamidino-2-phenylindole).
Muscle and heart immunohistochemistry
Tibialis anterior, quadriceps and paravertebral muscles were taken from G93A and littermate mice. Double expression of GFP with the myogenic proteins desmin (1 : 40; Novocastra, Newcastle upon Tyne, UK) and 
-SR-actin (1 : 40; Novocastra) was evaluated by confocal microscopy (Bio-Rad, UK).
For heart tissue analysis, the markers desmin (1 : 40; Novocastra), -SR-actin (1 : 40; Novocastra) and -actinin (1 : 100; Novocastra) were analysed by confocal microscopy (Bio-Rad, UK).
Expression of Ki67 antigen, a nuclear marker expressed in all cycling cells (G1, S, G2 and early mitosis), was evaluated in transplanted and untreated SOD1 mice and corresponding non-transgenic littermates using a rabbit polyclonal anti-Ki67 antibody (Novocastra) (Scholzen et al., 2000). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated swine anti-rabbit (1 : 40; Dako) was used for 1 h at room temperature as secondary antibody.
Morphological evaluation was done with haematoxilin and eosin staining of cardiac tissue. The number of double-positive GFP fibres was expressed as a percentage of total fibres.
Data were analysed statistically by Student's t test.
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Results |
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TopSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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To investigate the contribution of BM stem cells to ALS phenotype, we generated BM chimeric SOD1-G93A mice by transplanting BM cells derived from mice expressing GFP into all tissues (Okabe et al., 1997
) and Thy1-YFP neuron-specific transgenic mice (Feng et al., 2000). Three different groups of mice were used as controls: (i) untreated transgenic SOD1-G93A littermates of transplanted SOD1 mice; (ii) SOD1-G93A mice that underwent the same irradiation protocol as those transplanted with GFP or YFP BM, but that were intraperitoneally transplanted with BM cells (30 x 106 cells) from a SOD1-G93A donor; and (iii) non-transgenic littermates, i.e. mice from the same progeny who tested negative for SOD1-G93A mutation; this last group was used to compare the degree of tissue chimerism with that of their mutated littermates.
The transplantation procedure was well tolerated: all recipient animals survived the transplantation protocol. We used a sublethal irradiation of 800 rad as conditional treatment for BM transplantation. After this irradiation dose without BM transplantation, 50% of untransplanted SOD1 mice and 40% of non-transgenic littermates died within 30 days. This survival rate is consistent with previously described data for this dose of radiation (Soderberg et al., 1987).
GFP BM-transplanted mice (n = 12) and Thy1-YFP mice (n = 12) showed a prolonged mean survival time of 12–13 days compared with untreated SOD1 littermates and 10–11 days compared with a third group of SOD1-G93A mice (n = 10) receiving SOD1-G93A BM cells (GFP BM, mean 139 ± 8 days; Thy-1-YFP BM, 140 ± 6; SOD1-untransplanted mice 127 ± 2 days; SOD1-G93A BM, 129 ± 5; combined GFP + YFP versus untreated, 
2 = 24.11, P < 0.0001; combined GFP + YFP versus SOD1 BM 2 = 8.55, P = 0.0034) (Fig. 1A). A gender effect on survival was not detected in the present study, although mean survival time was mildly delayed in female mice belonging to all SOD1 groups (GFP + YFP-treated male versus GFP + YFP-treated female 2 = 1.143992, P = 0.2848).
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Analytical data on group composition (number of litters and gender) and comparisons between subgroups are available as supplementary data.
The longest life span in each condition was 138 days for untreated SOD1-G93A mice, 140 days for transgenic animals transplanted with SOD1-derived BM, 160 days for GFP-transplanted mice and 163 days for YFP-transplanted mice.
Neuromuscular function was tested on GFP-transplanted (n = 12) and untreated control (n = 12) animals by rotarod performance. Between 91 and 105 days of age, untreated animals showed a marked decrease in performance, whereas the treated animals displayed the greatest deficits 14 days later (Fig. 1B).
At 100 days, we also observed that average motor neuron counts in the lumbar spinal cord in wild-type BM-transplanted animals [GFP-BM mice (n = 4), 18.7 ± 1.3; Thy1-YFP BM mice (n = 4), 19.7 ± 1.7] were significantly different from those of mice transplanted with mutant SOD1 BM (n = 3; 12.3 ± 0.5) and SOD1-untransplanted mice (n = 5; 11.4 ± 1.1) which showed a substantial loss of motor neurons (Figs 1C and D, and 7J–L).
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At this time, the axon number in the L5 ventral root in wild-type BM-transplanted mice [GFP BM mice (n = 4), 680.75 ± 53; Thy1-BM YFP mice (n = 4): 687.5 ± 60] was significantly different from that of mice transplanted with mutant SOD1-BM (n = 3; 480.33 ± 46) and SOD1-untransplanted mice (n = 3; 456 ± 68) which showed a substantial loss of axons (Figs 1E and 7M–O). There were no significant differences in axon counts between BM-transplanted and untreated mice at end stage (Fig. 1F).
GFP-transplanted animals achieved a high degree of BM chimerism as evaluated by FACS (mean 66.8%, 14.2 SD) (Fig. 2A) and by direct observation. YFP BM reconstitution was assessed by PCR for the YFP transgene in BM cells (data not shown).
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No expression of YFP was detectable either in non-neural cells or in BM, as demonstrated by FACS analysis (Fig. 2B) and as previously described (Feng et al., 2000
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BM-derived neurons in SOD1 mice
Examination of SOD1 brain tissue revealed the presence of rare GFP- and YFP-positive fully differentiated neurons in the cerebral cortex (Fig. 3; Table 1). GFP+ and YFP+ neurons showed the presence of neuritic extensions. The activation of the Thy1 neuron-specific transgene provides direct evidence of the acquisition of neuronal phenotype by BM-derived cells.
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GFP-positive cells, localized in the Purkinje cells layer and displaying a morphology indistinguishable from host Purkinje cells, were detected in the cerebellum (Fig. 4). Since B6.Cg-TgN(Thy1-YFP)16Jrs mice do not express the YFP reporter gene in Purkinje neurons (Feng et al., 2000
), not surprisingly no YFP-Purkinje-positive cells were detected in YFP-transplanted animals.
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To test the contribution of cell fusion to the generation of BM-derived neurons, we analysed the nuclear content of these cells and we found that some GFP+ and YFP+ neurons presented a double nuclear content (Fig. 3B). In the brain of male SOD1 mice that received female YFP BM, the neuron-specific reporter gene was present together with a Y chromosome-positive nucleus in some cells, suggesting the occurrence of cell fusion (Fig. 5). De novo YFP expression confirms that fused BM nuclei can reprogramme their gene expression towards a neuronal gene pattern. Other YFP-positive cells showed only one Y chromosome-negative nucleus. On the other hand, the opposite sex-mismatched experimental condition led to the observation that YFP cells were also Y chromosome positive.
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Furthermore, to characterize the neuronal phenotype of GFP+ and YFP+ cells which displayed a neuronal morphology, we evaluated the co-expression of neuronal-specific markers in these cells. When analysed by immunohistochemistry, they were positive for TuJ1, NeuN and NF (Fig. 6). GFP Purkinje cells with fully developed morphology expressed calbindin-D28-K (Fig. 4).
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In the spinal cord, very few GFP+ and YFP+ cells expressed both markers for immature neurons, such as TuJ1, and for differentiated neurons, such as NF and NeuN (Fig. 7A–F). These cells were present in both anterior and posterior spinal cord horns. However, we did not observe GFP+ neuronal cells with a large body, such as motor neurons, or cells with extension outside the spinal cord.
GFP donor-derived cells with small dimension were present in all analysed cerebral areas (cortical and subcortical forebrain areas, cerebellum) and spinal cord of SOD1 mice. Most of them were found in vessels and in perivascular and leptomeningeal sites. Brain and spinal cord samples derived from SOD1-G93A mice contained more GFP+ cells than those derived from their non-transgenic littermates (P < 0.01). A relevant proportion (mean 1.5 ± 0.45%) of all cell types in SOD1 were GFP+. These cells were, for the majority, macrophage and microglial cells (Fig. 7G and H). Furthermore, in GFP BM-transplanted SOD1 mice, a large proportion of the microglial compartment (26.6 ± 7%) was of BM origin.
In animals transplanted with SOD1 mutant BM cells, we also used sex-mismatched transplantation, thus we could follow the level of donor cell infiltration in recipient mice by FISH for the Y chromosome (Fig. 7I). The amount of donor-derived Y cells was 1.7 ± 0.62%. The proportion of donor-derived Y microglia was 28.5 ± 8%.
Contribution of BM cells to skeletal and cardiac muscles
In all BM-transplanted mice, GFP-positive muscle fibres were detected in quadriceps, tibialis anterior and paravertebral muscles. SOD1 mice showed a significantly higher percentage of GFP+ myofibres than control non-transgenic littermates (in quadriceps, SOD1, 0.03 ± 0.005%; controls, 0.006 ± 0.004%; P < 0.01). GFP+ muscle fibres were either dispersed or in clusters, thus suggesting the contribution of single cell clones.
GFP+ myofibres co-expressed skeletal muscle antigens such as desmin and -sarcomeric actin (Fig. 8).
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BM-derived GFP cells contribute significantly to myocardial regeneration in SOD1 mice, while only sporadic immature myofibres were observed in their littermates. Indeed, SOD1 mouse hearts showed both mature myocytes (0.01 ± 0.005% of overall myofibres) and small developing myocytes (1 ± 0.03%) (Fig. 9). Non-transgenic littermates displayed no mature GFP myocytes <0.001%, and they showed a lower level of developing myocytes 0.02 ± 0.01% (P < 0.01).
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The GFP+ cardiomyocytes co-expressed cardiac antigens desmin,
-actinin and -sarcomeric actin.
Generation of cardiomyocytes from BM has been demonstrated almost exclusively in pathological hearts (ischaemic and dystrophic models) and their frequency seemed to be proportional to heart injury (Bittner et al., 1999; Orlic et al., 2001; Jackson et al., 2001).
To investigate whether the heart of SOD1 mice undergoes any degenerative–regenerative process, we performed morphological analysis and immunohistochemistry using anti-Ki67 antibodies.
Nuclear antigen Ki67 has been shown to be expressed in cycling cells (Scholzen et al., 2000) and has been detected previously in ischaemic heart tissues (Beltrami et al., 2001). Haematoxylin and eosin staining failed to reveal any macroscopic alteration in the cardiomyocyte organization. Nonetheless, a high level of Ki67 immunostaining was found in 4-month-old SOD1 mice. No Ki67 positivity or <0.001% of Ki67-positive nuclei per heart section was found in non-transgenic littermates of the same age and in transgenic SOD1 animals at 1 month of age (Fig. 9). Ki67 positivity in 4-month-old SOD1 mice was accounted for by a mean of 0.8 ± 0.2% of positive cardiac nuclei. Forty percent of these nuclei were located in cardiac fibres. In SOD1 GFP-transplanted animals, 30% of all Ki67-positive cells also expressed GFP.
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Discussion |
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TopSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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In this work, we have found that wild-type BM transplantation can ameliorate the disease phenotype of SOD1 mice, an animal model of ALS. We hypothesized that this beneficial effect is due to a ‘non-neuronal environmental change’ achieved by BM transplantation. In the CNS tissues, BM-derived stem cells contribute to the generation of microglia and rarely to neuron formation, probably by cell fusion. Furthermore, BM cells also participate in the regeneration of mesodermal tissues: skeletal muscle and heart.
Recently, it has been demonstrated that chimeric mice generated by injection of wild-type embryonic stem cells into SOD1 blastocysts showed an extended life span (Clement et al., 2003). Wild-type non-neuronal cells, in some cases representing a small minority of total cells, can decrease the degeneration process in motor neurons and ameliorate survival of mice expressing mutant SOD1 compared with those in parental SOD1 mutant mice. On the other hand, the SOD1 negative effect on adjacent non-motor neuronal cells (interneurons, astrocytes and microglia) seems to be one of the major contributors to the pathogenesis of SOD1 mutations (Clement et al., 2003).
In our study, a significant proportion of the microglial compartment appears to be generated from BM, and this environmental cell replacement can account for the observed phenotypic improvement.
Our study also provides new data on the issue of BM contribution to neuronogenesis; indeed, we observed rare GFP+ and YFP+ neurons co-expressing specific neuronal markers, both in the brain and in the spinal cord.
Examination of nuclear content and FISH analysis confirmed that fully differentiated neurons are probably generated from fusion. However, a fraction of neurons derived from female BM is negative at FISH analysis for the Y chromosome: these data could be ascribed to incomplete detection, though occasional differentiative events cannot be completely ruled out.
We observed the presence of rare fully differentiated neurons other than Purkinje cells which up to now represent the only neurons with a specific neuron morphology, observed by other groups (Priller et al., 2001; Wagers et al., 2002; Alvarez-Dolado et al., 2003; Weimann et al., 2003a). However, as previously described in the case of Purkinje cells (Weimann et al., 2003b), the fusion of BM nuclei with neurons leads to BM nuclear reprogramming towards a neuronal phenotype as demonstrated by de novo Thy1 neuronal-specific transgene expression.
Moreover, we found very few GFP+ and YFP+ cells in the spinal cord which were positive for markers of immature neurons (TuJ1), and for differentiated neurons (NF and NeuN). However, we did not observe GFP+ neuronal cells with a classical neuronal morphology such as motor neurons, or cells with extension outside the spinal cord.
The observed degree of BM contribution to skeletal and cardiac muscle regeneration suggests that the SOD1-G93A genetic background also has widespread degenerative effects on extra-nervous tissues.
It has been demonstrated that the overexpression of normal human SOD1 in mice causes a muscular dystrophy phenotype (Rando et al., 1998). These transgenic mice presented atrophy of muscles, particularly quadriceps. Morphological analysis revealed muscle necrosis and regeneration, fibre splitting and increased connective tissue. Muscle degeneration in this model was attributed to an increased susceptibility to oxidative stress (Rando et al., 1998). Furthermore, a severe muscle dystrophic phenotype was observed in transgenic mice carrying the muscle-specific deletion of survival motor neuron gene exon 7, therefore supporting a primary muscle involvement also in lower motor neuron disorders (Cifuentes-Diaz et al., 2001).
The generation of cardiac fibres from BM has been observed only in infarcted or dystrophic heart and not in normal tissue (Bittner et al., 1999; Jackson et al., 2001; Orlic et al., 2001). The detection of BM generated fibres in SOD1 heart prompted us to investigate whether a pathological process was present in that tissue. The morphological evaluation did not reveal abnormalities; however, we detected the expression of the nuclear Ki67 protein in a fraction of cardiomyocytes. Ki67 is an antigen associated with cell division that has been detected previously in infarcted heart (Scholzen et al., 2000; Beltrami et al., 2001).
The observation that 30% of Ki67-positive cells expressed GFP supports the hypothesis whereby a significant proportion of cycling cells in the heart are derived from BM cells. The observed degenerative–regenerative processes in both cardiac and skeletal muscle tissues are likely to be due to the effect of mutated SOD1 expression. Indeed, it has been demonstrated that altered SOD1 expression may affect heart tissue post-ischaemic repair: overexpression of normal human SOD1 decreases the cellular injury that occurs following re-perfusion of ischaemic tissues; in contrast, knockout mice for SOD1 and manganese superoxide dismutase (SOD2) displayed impaired post-ischaemic recovery of contractile heart function (Wang et al., 1998; Asimakis et al., 2002). The application of BM transplantation to animal models of neurodegenerative disorders may unravel hitherto unidentified tissue involvement in these disorders.
Several studies have documented the contribution of BM stem cells to the regeneration of CNS and mesodermal tissues, in either normal or injured animal models. It has been described previously that an intravenous administration of a high dose of hUCB cells into SOD1 irradiated mice increased their life span.
However, in their report, Ende et al. (2000) did not investigate the fate of transplanted cells in the CNS extensively. Another study (Garbuzova-Davis et al., 2003) confirmed that administration of hUCB cells in SOD1 mice improved survival, also providing data about the integration of exogenous cells into the brain and spinal cord of recipient animals. The CNS hUCB-derived cells express neuroectodermal markers, but the human origin of these cells was demonstrated only through an antibody against human nuclei. Furthermore, the issue of cell fusion was not addressed.
These observations suggested that haematopoietic stem cells can ameliorate the phenotype of SOD1 mice. However, immunocompetent mice may not be particularly receptive to xenogenic intravenous transplantation of hUCB cells. In this regard, a combined model (such as double transgenic SOD1 mutant mice/NOD-SCID or NOD-RAG mice) may clarify this issue.
From a clinical point of view, that goes beyond the aim of the present experiments, there are a number of logistic and biological differences between adult allogenic BM transplantation and hUBC, which may make one cell source more advantageous than the other in haematopoietic transplantation (for a review see Grewal et al., 2003). In particular, the stem cell dose represents a limit in the case of hUBC as it requires a pool of hUBC donor cells to achieve an amount of stem cell sufficient for an adult. Provided that this limitation can be overcome, hUCB cells are likely to be more widely used in the future.
However, the goal of our study was not to propose whole BM transplantation tout-court as a therapy for ALS but to provide some basic biological information about the possible effect of wild-type stem cells on disease progression. This may promote further studies on the behaviour and properties of somatic stem cells as a cell source for the treatment of neurodegenerative diseases.
Allogenic BM transplantation actually suffers high morbidity and mortality. BM transplantation for malignancy and for non-malignant diseases presents similar morbidity and mortality rates. The reported overall mortality rate from BM transplantation-related complications is 15%: this percentage is estimated at 10% in HLA (human leukocyte antigen)-identical transplants, and at up to 25% for related non-HLA-identical transplants (for more complete and accurate data see Kaufman et al., 1999; Grewal et al., 2003). Any human study aiming at applying similar protocols is likely to report comparable mortality rates.
In our study, we irradiated the mice before transplantation, but in a human setting conditional therapy may alternatively involve chemotherapeutic agents. Furthermore, non-myeloablative stem cell transplantation or mini-transplant has been developed recently as a less toxic treatment regimen for haematological diseases (Barlogie et al., 2004).
The life span increase observed after wild-type BM transplantation is comparable with that obtained with other safer pharmacological strategies such as riluzole and minocycline. Studies by Gurney et al. (1996) reported a 10% effect of riluzole on the survival rate (10–15 days) in a SOD1-G93A mouse model. Administration of minocycline, beginning in the late pre-symptomatic stage (7 or 9 months of age), delayed the onset of motor neuron degeneration and muscle strength decline, and increased the longevity of SOD1-G37R mice, by 5 weeks for 70% of tested mice (Kriz et al., 2002). It has been described recently that VEGF (vascular endothelial growth factor) delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model by 30% (Azzouz et al., 2004).
We would like to point out that the benefit observed from the BM transplantation protocol in G93A mice is relatively low compared with the risk of allogenic BM transplantation. However, our data suggest that further studies aimed at improving the degree of tissue chimerism by using BM-derived cells may provide valuable insights into strategies to slow ALS progression.
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The financial support of the following research grants to G.P.C. is gratefully acknowledged: Italian Ministry of Health, Ricerca Finalizzata 2001 ‘Isolamento, espansione e caratterizzazione di cellule staminali a scopo di trapianto e riparazione cellulare’; MIUR (Ministero Istruzione Università e Ricerca Scientifica) Italian Ministery FIRB 2002 ‘Animali geneticamente modificati per lo studio di patologie neurodegenerative’; Progetto a Concorso Ospedale Maggiore Policlinico 2003. The technical assistance of Dr Gigliola Fagiolari is gratefully acknowledged. We wish to thank especially the ‘Associazione Amici del Centro Dino Ferrari’ for their support.
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