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Shin-Ichi Inoue, Kaori Ishikawa, Kazuto Nakada, Akitsugu Sato, Hiroyuki Miyoshi, Jun-Ichi Hayashi, Suppression of disease phenotypes of adult mito-mice carrying pathogenic mtDNA by bone marrow transplantation, Human Molecular Genetics, Volume 15, Issue 11, 1 June 2006, Pages 1801–1807, https://doi.org/10.1093/hmg/ddl102
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Abstract
For directly addressing the issue of gene therapy of adult patients with mitochondrial diseases, we carried out bone marrow transplantation to adult mito-mice with mutated mtDNA and expressing respiration defects for improvement of disease phenotypes. We supposed that bone marrow cells transdifferentiated into various tissues, so that their transplantation would suppress disease phenotypes. The results showed improvement of survival and delayed expression of renal failure. As most mito-mice without a transplant died due to renal failure, we examined whether transplanted bone marrow cells transdifferentiated into renal tissues carrying improved renal function. Histochemical analyses showed that the suppression of disease phenotypes was not due to transdifferentiation, but due to suppression of apoptosis of renal cells. Thus, bone marrow cells possess a novel function of supporting tissues by suppressing apoptosis induced by respiration defects.
INTRODUCTION
Accumulation of mtDNAs with pathogenic mutations has been shown to induce respiration defects and expression of various mitochondrial diseases in human subjects (1,2). Furthermore, these mutated mtDNAs were shown to be closely associated with aging and age-related disorders including diabetes and neurodegenerative diseases (1,2). In our previous study (3), we generated the first mouse models of mitochondrial diseases, mito-mice, and provided direct evidence that pathogenic mutations in mtDNAs were responsible for their disease phenotypes. In mito-mice, disease phenotypes appeared only when mtDNA with a large-scale (4696 bp) deletion mutation (ΔmtDNA) accumulated sufficiently for expression of respiration defects, which were caused by the reduction of mitochondrial translation due to the loss of six tRNA genes in mtDNA (3).
Mito-mice were subsequently used for precise investigation of pathogenesis of mitochondrial diseases (4,5) and should be effective for developing procedures to suppress their disease phenotypes. For preventing mitochondrial defects, the amount of pathogenic mtDNA has to be diluted to below the threshold value required to induce respiration defects. For example, replacing ΔmtDNA of mito-mice zygotes by mtDNA from normal zygotes using nuclear transplantation completely avoided the expression of disease phenotypes throughout their lives (6). Thus, nuclear transplantation can be applied to the progeny of patients with mitochondrial diseases, while it is impossible to replace mtDNA of every somatic cell of patients by normal mtDNA. So, development of new procedures is required for effective therapy of adult patients.
Although it is difficult to dilute pathogenic mtDNA in all somatic cells of adult patients, bone marrow transplantation from normal subjects may be effective for suppressing disease phenotypes, assuming that bone marrow cells include stem cells with proliferating and multipotent capacities. Therefore, we supposed that their transplantation would supply functional somatic cells continuously for regenerating functional tissues by their replication and subsequent transdifferentiation. Recently, many studies have provided evidence that mouse bone marrow-derived cells transdifferentiated into various cell types (7–17), whereas other studies have shown that the apparent multipotency of bone marrow-derived cells was due to their fusion with differentiated cells (18–22).
Thus, bone marrow transplantation may restore the function of tissues in patients of mitochondrial diseases, irrespective of whether the cells differentiate independently or by their fusion with differentiated recipient cells. This study addressed the issue of developing a suitable therapy for adult mito-mice by applying bone marrow transplantation and showed that this procedure caused reduction of disease phenotypes.
RESULTS
Transplantation of bone marrow cells from normal mice to mito-mice
For application of the therapy to adult mito-mice expressing respiration defects and resultant disease phenotypes, we transplanted bone marrow cells from normal mice to mito-mice expecting that their transdifferentiation to renal cells might restore renal function.
We selected 29 mito-mice (4-week-old) carrying 60–80% ΔmtDNA (68% on average) in their tails and divided them into two groups, each carrying 68% ΔmtDNA on average. One group of 15 mito-mice was given bone marrow transplants, and the other group of 14 mito-mice was not given transplants. The mito-mice in the transplantation group were used as recipients of bone marrow cells after lethal X-ray irradiation for disruption of their own bone marrow cells. As donors of normal bone marrow cells, we used normal B6-GFP mice, which expressed endogenous GFP gene ubiquitously by a chicken β-actin promoter (23), so that transplanted bone marrow-derived cells could be identified in the mito-mice.
Bone marrow cells were transplanted to 8-week-old mito-mice. About 3 months after their transplantation, we analyzed the peripheral blood leukocytes in the recipient mito-mice by flow cytometry (Fig. 1). The results showed that circulating blood cells were reconstituted by bone marrow-derived GFP-positive cells, which included myeloid cells (23.5±5.9%), T-lymphoid cells (9.2±1.2%) and B-lymphoid cells (67.0±6.8%). These observations indicated that the bone marrow cells were successfully transplanted and that they differentiated into GFP expressing blood cells.
Effects of bone marrow transplantation on disease phenotypes of mito-mice
First, we examined the effect of bone marrow transplantation to mito-mice on their survival (Fig. 2A). Whereas 14 non-transplanted mito-mice began to die at 25 weeks old, and all of them died within 32 weeks after birth, the group of 15 mito-mice with transplants began to die 5 weeks later, and all of them had died by 42 weeks after birth. Although the mito-mice with transplants did not survive >1 year, their average lifespan was enhanced ∼14% (4.4 weeks) compared with that of mito-mice without transplants (Fig. 2A).
As most mito-mice have a high concentration of blood urea nitrogen (BUN), reflecting renal dysfunction and die due to renal failure (3), we monitored BUN in both groups of mito-mice (Fig. 2B). The concentration of BUN progressively increased from 24 weeks after birth in mito-mice without transplants, but not until 29 weeks in those with transplants (Fig. 2B). However, it eventually showed a significant increase in mito-mice with transplants by 31–36 weeks after birth.
To exclude the possibility that suppression of renal dysfunction in mito-mice with transplants was due to irradiation, we transplanted bone marrow cells carrying 82 and 85% ΔmtDNA from mito-mice to six lethally irradiated 8-week-old mito-mice carrying 60–77% ΔmtDNA in their tails at 4 weeks after birth. Average proportions of ΔmtDNA in peripheral blood cells of transplanted mito-mice (mito-mice/mito-BMT) was 75.4±2.9% and was very close to those of non-transplanted mito-mice (78.1±3.2%) at 24 weeks after birth. Our previous study showed that respiratory function was reduced to <50% in cultivated cells carrying >75% ΔmtDNA (6). Thus, respiration defects would be expressed in peripheral blood cells of both mito-mice/mito-BMT and non-transplanted mito-mice. Mito-mice/mito-BMT as well as non-transplanted mito-mice showed progressive increase in the concentration of BUN and died within 31 weeks after birth (Fig. 2), indicating that the therapeutic effect of bone marrow transplantation was not due to the side effect of irradiation.
Next, morphological and histochemical analyses of renal tissues were carried out for precise investigation of the pathogenesis using one mito-mouse with a transplant and one without a transplant possessing 89 and 90% ΔmtDNA, respectively, in their renal tissues. The mito-mouse without a transplant had enlarged kidneys showing ischemia and a granulated surface (Fig. 3A). The renal tissues showed dilation of the cortical proximal and distal tubules (Fig. 3B) and significant reduction of cytochrome c oxidase (COX) activity (Fig. 3C). On the other hand, most of these abnormalities were not found in the mito-mouse with a transplant (Figs 2 and 3). However, COX histochemistry provided evidence for the absence of recovery of reduced COX activity in renal tissues (Fig. 3C and D), even though they appeared normal morphologically (Fig. 3B). Thus, bone marrow cells were effective for stabilizing the structure of renal tubules without activating the respiratory function in renal tissues. Similar improvements were observed in four other mito-mice with transplants possessing >80% ΔmtDNA in their renal tissues (data not shown).
Examination of transdifferentiation and apoptosis in renal tissues
How do transplanted bone marrow cells suppress renal dysfunction in mito-mice? One possibility is that they supplied functional renal cells by their own transdifferentiation or their fusion with recipient renal cells. Using fluorescence microscopy, we tested whether GFP-positive renal cells, which should be derived from transplanted bone marrow cells, were present in transplanted mito-mice.
Co-localization of GFP signals and whole leukocyte marker CD45 signals was observed in interstitial and glomerula sites (Fig. 4A), suggesting differentiation of bone marrow cells into blood cells, but not renal tissues. Moreover, GFP-positive cells were not localized in renal tubules (Fig. 4) and did not express CD31, a marker of endothelial cells in renal tissues (Fig. 4B). GFP signals were not detected in the other channels (data not shown). These observations indicated the absence of extensive transdifferentiation of bone marrow cells into renal tissues or their fusion with renal cells. This concept was supported by the observation that no increase in COX-positive cells was observed in renal tissues by bone marrow transplantation (Fig. 3). The frequency of transdifferentiation of bone marrow cells was also very low in other tissues (Table 1) and was not higher in mito-mice than in normal mice.
Another possible explanation for the positive effect of bone marrow transplantation is the suppression of apoptosis in renal cells expressing respiration defects. To test this idea, we carried out terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay for the identification of apoptotic cells (24). TUNEL-positive renal cells were observed in both mito-mice with and without a transplant (Fig. 5A, arrowhead), but their number was substantially lower in transplanted mito-mouse (Fig. 5B). These observations suggest that accumulation of ΔmtDNA and resultant expression of respiration defects induced apoptosis and renal dysfunction and that bone marrow transplantation suppressed renal dysfunction not only by their transdifferentiation, but also by suppressing apoptosis.
DISCUSSION
It has been proposed that bone marrow cells participate in the regeneration of mouse kidneys by their transdifferentiation (25), particularly when kidneys are damaged by ischemia (16,26). Thus, we expected that transdifferentiation of bone marrow cells would also be greater in tissues of mito-mice than in those of normal mice, as the kidneys of mito-mice showed chronic ischemia and died because of renal failure (3). However, this study indicated that ischemia induced by respiration defects in mito-mice did not enhance transdifferentiation of transplanted bone marrow cells (Fig. 4).
Although our observations appear to contradict previous reports (16,25,26), another recent study also showed the absence of transdifferentiation of bone marrow cells (27). This apparent discrepancy with other reports could be explained by differences in the procedures used for distinguishing transplanted bone marrow cells from recipient cells in the studies that showed transdifferentiation (16,25,26). For example, one problem was X-gal staining for identification of β-galactosidase (β-gal)-positive renal cells after bone marrow transplantation from β-gal-transgenic mice into wild-type mice (16), as suppression of the endogenous activity of β-gal simultaneously excluded the signals of β-gal-positive renal cells in transplanted mice (27), suggesting the absence of transdifferentiation of bone marrow cells into renal cells. Another problem was that Y-chromosome fluorescence in situ hybridization (Y-FISH) was used for detection of Y-chromosome-positive renal cells after transplantation of bone marrow cells from male mice into female mice (25,26). In this case, precise investigation of co-localization showed that the apparent co-localization of broad Y-FISH-positive signals of bone marrow cells and renal differentiation markers within the same renal cells was simply due to overlapping of bone marrow-derived cells and recipient renal cells (27).
This study showed enhanced survival and suppression of renal failure in mito-mice by bone marrow transplantation, whereas most bone marrow-derived cells differentiated into blood cells, and their transdifferentiation into other tissues as well as into renal tissues was only rarely observed (Table 1). This concept was supported by the finding that respiratory function was not recovered in renal cells of transplanted mito-mice (Fig. 3).
Then, the question arises of how transplantation of bone marrow cells improved disease phenotypes without transdifferentiation of these cells into renal cells and without recovery of respiratory function in renal cells (Figs 3 and 4). One possible explanation is that transplanted bone marrow cells and their differentiated blood cells express normal respiratory function, and secret sufficient amounts of growth factors, such as vascular endothelial growth factor, resulting in enhanced replication and regeneration of host renal cells. Recently, introduction of side population cells, which were prepared from the kidney and supposed to maintain stem cell-like potentials (28), improved cisplatin-induced damage of the kidneys by inducing regeneration of renal tissues, but not by their own differentiation into renal cells (29), suggesting possible involvement of growth factors in this process. However, in mito-mice with transplants, enhanced secretion of growth factors and resultant activation of regeneration would not be involved in suppression of renal failure, as decrease rather than increase in the number of proliferating cells in renal tissues was observed by bone marrow transplantation (Fig. 6).
Another possibility to explain the question is that the normal respiratory function of differentiated blood cells from transplanted bone marrow cells secreted sufficient amounts of some factors required to inhibit apoptosis of renal cells expressing respiration defects in mito-mice. This idea was supported by the findings of suppressed apoptosis and detachment of cells from renal tissues in mito-mice with transplants (Fig. 5). Probably, inhibition of apoptosis by bone marrow transplantation resulted in maintenance of the normal structure and suppression of renal failure, although we could not exclude the possibility that the inhibition of apoptosis is the consequence of maintenance of the normal structure and the suppression of renal failure by bone marrow transplantation.
It is also possible that bone marrow-derived GFP/CD45 positive cells in the kidneys of transplanted mito-mice partially differentiated into capsular or tubular epithelial cells, even though the bone marrow-derived cells express a leukocyte marker. GFP/CD45 cells appeared to be present in the wall of Bowman's capsule. Thus, partially differentiated GFP/CD45 cells could support renal functions of transplanted mito-mice.
For complete recovery of renal failure, introduction of normal cells with the potentials to be renal cells expressing normal respiratory function is required. There is the question of what kind of normal cells are effective for supplying differentiated normal renal cells. Many studies have shown that transplantation of proliferated mesenchymal stem cells helps in repair of injured renal cells (30,31), although, in these studies, it was not clear whether this repair was attained by transdifferentiation of mesenchymal stem cells into renal cells. Recent reports also showed the presence of renal stem cells (32). Moreover, differentiation of ES cells into renal tubular cells has been reported (33). Therefore, we are investigating whether transplantation of mesenchymal stem cells, renal stem cells or ES-derived renal cells into mito-mice is more effective than that of bone marrow cells for substantial recovery of renal function by their differentiation and supplying sufficient amounts of renal cells expressing normal respiratory function.
MATERIALS AND METHODS
Mice and bone marrow transplantation
Mito-mice were generated by introduction of ΔmtDNA from cultivated cells into fertilized eggs of B6 strain mice using cell fusion techniques as described previously (3,5). The proportion of ΔmtDNA in mito-mice was deduced from tail DNA samples, because it was very similar in all the tissues of the same individual mouse (3–6). Before bone marrow transplantation, recipient 8-week-old male mito-mice was irradiated (9 Gy). Bone marrow cells were isolated from the femur and tibia of 8-week-old GFP transgenic B6 mice (23) by flushing with PBS. We injected totals of 1×106 bone marrow cells in 0.2 ml PBS into tail veins of the irradiated mito-mice. Three months after bone marrow transplantation, the peripheral blood of the mito-mice was labeled with biotinylated anti-Ly5.2, streptavidin-conjugated APC, PE-conjugated anti-Mac1, Gr1, CD4, CD8 and B220, respectively, for flow cytometry.
Quantitative estimation of ΔmtDNA
Southern blot analysis was carried out as described previously (3,4). Briefly, total DNA (2.0–3.0 µg) extracted from tail samples was digested with the restriction enzyme XhoI. Restriction fragments were separated in 1.0% agarose gel, transferred to a nylon membrane and hybridized with [α-32P] dATP-labeled mouse mtDNA probes. The membrane was washed and exposed to an imaging plate for 3 h and the radioactivities of fragments were measured with a bioimaging analyzer, Fujix BAS 2000 (Fuji Photo Film).
BUN measurement
Blood was obtained from a retro-orbital vessel, and BUN concentrations were measured using a Urea NB test kit (Wako Pure Chemical).
Histological analyses
Histochemical analysis of COX activity was carried out by examining the rate of cyanide-sensitive oxidation of reduced cytochrome (34). For estimation of the number of COX-positive renal cells, the sections were counterstained with hematoxylin. For histopathological analysis, 10 µm sections of kidney were stained with hematoxylin and eosin.
Immunohistochemical staining
We used primary antibodies to albumin (1:500, Biogenesis), CD45 (1:500, Becton Dickinson), CD31 (1:500, Becton Dickinson), cytokeratin AE1/AE3 (DAKO), F4/80 (Dainippon Pharm), laminin-α2 (Sigma), NeuN (Chemicon), α-actinin (1:500, Sigma Aldrich) and PCNA (1:200, Santa Cruz). The kidneys and other tissues obtained from mito-mice were fixed with 4% paraformaldehyde and dehydrated with 30% sucrose in PBS. After 1 day with several change of dehydration liquid, tissues were embedded in OTC compound and frozen with dry ice. Tissues were embedded in paraffin, when antibodies to PCNA were used for immunohistochemistry.
Sections (5 or 10 µm) were stained with primary antibodies and were visualized with secondary antibodies conjugated with Alexa Fluor 594 (Molecular Probes). For removing OTC compound, sections were washed three times with TBS, and xylene and ethanol were used for removing paraffin. Sections were rinsed in TBS and were incubated at room temperature for 20 min using blocking solution (TBS-0.3% Triton X solution supplemented with 10% goat serum; Funakoshi), and incubated overnight with optically diluted primary antibodies in blocking solution. Then, sections were washed with TBS-0.3% Triton X solution and incubated overnight with secondary antibodies. After washing three times with TBS-0.3% Triton X solution, sections were mounted with mounting medium including DAPI.
TUNEL staining
Paraffin-embedded kidneys were cut into 8 µm sections. Paraffin was removed from the sections with xylene and with ethanol, and sections were rinsed in PBS. TUNEL staining assay for detection of apoptotic nuclei (24) was carried out with an In Situ Apoptosis Detection Kit (Takara Bio Inc.). The sections were counterstained with hematoxylin. We counted the number of positively stained apoptotic nuclei in 7–11 fields (one field was 722×539 µm) in each section under an OLYMPUS BX51 microscope.
ACKNOWLEDGEMENTS
This work was supported in part by Grants-in-Aid for Creative Scientific Research from Japan Society for the Promotion of Science (JSPS) to J.-I.H., and by Grants-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to J.-I.H.
Conflict of Interest statement. None declared.
Tissues . | Average number of cells per section . | Number of sections analyzed . | Number of transdifferentiated cells . |
---|---|---|---|
Kidney | 77 000 | 588 | 0 |
Muscle | 2 200 | 100 | 0 |
Muscle (regenerated)a | 2 200 | 80 | 1 |
Liver | 44 000 | 180 | 1 |
Heart | 19 000 | 280 | 8 |
Pancreas | 16 000 | 170 | 6 |
Brain | 52 000 | 90 | 0 |
Eye | 34 000 | 40 | 0 |
Intestine | 17 000 | 106 | 0 |
Lung | 28 000 | 55 | 0 |
Stomach | 42 000 | 61 | 0 |
Tissues . | Average number of cells per section . | Number of sections analyzed . | Number of transdifferentiated cells . |
---|---|---|---|
Kidney | 77 000 | 588 | 0 |
Muscle | 2 200 | 100 | 0 |
Muscle (regenerated)a | 2 200 | 80 | 1 |
Liver | 44 000 | 180 | 1 |
Heart | 19 000 | 280 | 8 |
Pancreas | 16 000 | 170 | 6 |
Brain | 52 000 | 90 | 0 |
Eye | 34 000 | 40 | 0 |
Intestine | 17 000 | 106 | 0 |
Lung | 28 000 | 55 | 0 |
Stomach | 42 000 | 61 | 0 |
Frozen sections of transplanted mito-mice (n=8) were analyzed for the number of transdifferentiated cells. We used primary antibody to Laminin-α2 for detection of skeletal muscle myofibers, albumin for detection of hepatocytes, α-actinin for detection of cardiac myocytes and cytokeratin AE1/AE3 for detection of acinous cells in the pancreas.
aLeft tibialis anterior muscles were regenerated by injection of cardiotoxin (10 mg/kg) at 3 months after bone marrow transplantation.
Tissues . | Average number of cells per section . | Number of sections analyzed . | Number of transdifferentiated cells . |
---|---|---|---|
Kidney | 77 000 | 588 | 0 |
Muscle | 2 200 | 100 | 0 |
Muscle (regenerated)a | 2 200 | 80 | 1 |
Liver | 44 000 | 180 | 1 |
Heart | 19 000 | 280 | 8 |
Pancreas | 16 000 | 170 | 6 |
Brain | 52 000 | 90 | 0 |
Eye | 34 000 | 40 | 0 |
Intestine | 17 000 | 106 | 0 |
Lung | 28 000 | 55 | 0 |
Stomach | 42 000 | 61 | 0 |
Tissues . | Average number of cells per section . | Number of sections analyzed . | Number of transdifferentiated cells . |
---|---|---|---|
Kidney | 77 000 | 588 | 0 |
Muscle | 2 200 | 100 | 0 |
Muscle (regenerated)a | 2 200 | 80 | 1 |
Liver | 44 000 | 180 | 1 |
Heart | 19 000 | 280 | 8 |
Pancreas | 16 000 | 170 | 6 |
Brain | 52 000 | 90 | 0 |
Eye | 34 000 | 40 | 0 |
Intestine | 17 000 | 106 | 0 |
Lung | 28 000 | 55 | 0 |
Stomach | 42 000 | 61 | 0 |
Frozen sections of transplanted mito-mice (n=8) were analyzed for the number of transdifferentiated cells. We used primary antibody to Laminin-α2 for detection of skeletal muscle myofibers, albumin for detection of hepatocytes, α-actinin for detection of cardiac myocytes and cytokeratin AE1/AE3 for detection of acinous cells in the pancreas.
aLeft tibialis anterior muscles were regenerated by injection of cardiotoxin (10 mg/kg) at 3 months after bone marrow transplantation.
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