DIFFERENTIATION OF TRANSPLANTED BONE MARROW CELLS IN THE... : Transplantation (original) (raw)
INTRODUCTION
Cell transplantation techniques have been used for the treatment of diseases of the central nervous system (CNS) (1–4). A previous study reported that Parkinson’s disease could be treated by inoculation of dopaminogenic neurons derived from human fetuses, although there are major ethical problems in using fetal tissue (5). Alternatively, fibroblasts (6), fetal astrocytes (7), and Sertoli’s cells (8) have been grafted into patient’s brains for treatment of Parkinson’s disease. A new possibility is the use of pluripotent embryonic stem cells (ES cells) (9). Until recently, ES cells could be obtained only from rodents (10), but the candidates of human ES cells have now been identified (11,12). In addition, human neural precursor cells were recently cloned and characterized (13,14). These findings suggest that stem cell therapy of CNS disorders may be possible in the near future, if the logical and ethical problems can be settled.
Bone marrow transplantation (BMT) has been the treatment of choice for patients with a variety of hematological disorders. Recently, histocompatible allogenic BMT has also been used for the treatment of various genetic diseases, including hematological and nonhematological disorders (15,16). In many cases, the engrafted hosts have shown a remarkably positive clinical improvement in response to normalization of previously deficient enzymatic activity. It has been speculated that normal enzymes synthesized in leukocytes from a donor will decrease substrate accumulation in the patient’s organs. Interestingly, BMT also effectively prevented the progression of neurological signs and symptoms in some clinical trials, if performed at a sufficiently early stage in the disease (17,18). Transplantation in globoid-cell leukodystrophy showed that CNS deterioration was reversed, and abnormalities in cerebrospinal fluid protein levels were corrected (19). Furthermore, BMT in metachromatic leukodystrophy (MLD) resulted in improved neurological function, electrophysiological parameters, and sulfatide metabolism in the CNS (20).
The mechanism of abnormality correction in the CNS after BMT is not yet clearly understood. One possible explanation is the transfer of normal enzymes from the donor hematopoietic cells to the patient’s neuronal cells (21). Another is that the donor monocytes cross the blood-brain-barrier and differentiate into microglia, given that at least some microglial cells are thought to be derived from circulating monocytes (22,23). Nervous system damage in many lysosomal storage diseases is caused by an accumulation of substrate in microglial cells. The donor microglia may functionally replace the defective cells of the patient, and they may also act as a reservoir that continuously supplies the deficient enzyme in the brain.
Recent research into stem cell biology suggests another possibility whereby bone marrow cells contain pluripotent precursor cells that are capable of differentiating into various brain cells. One study showed that systemically infused whole bone marrow cells made a small, but detectable, contribution to the development of both microglial cells and astrocytes in the recipient mouse brain (24). Another report demonstrated that directly injected bone marrow stromal cells could engraft, migrate, and survive in a manner similar to rat astrocytes (25). These findings suggest that bone marrow cells are a potential source of brain progenitor cells. This possibility is extremely important, because bone marrow cells can be easily isolated without major technical or ethical problems.
We are attempting to develop effective treatment of MLD (26). MLD is caused by deficiency of arylsulfatase A, which hydrolyzes cerebroside sulfate. Undigested sulfatide accumulates mainly in oligodendrocytes, resulting in massive demyelination of the white matter of the brain. If glial cells, such as oligodendrocytes, can be generated from bone marrow cells, BMT may represent an important option for the correction of CNS dysfunction in MLD. In this study, we compared the effectiveness of systemic infusion with direct injection of bone marrow cells as a method to reconstitute the neurological system.
MATERIALS AND METHODS
Production of Retroviral Vectors
A Moloney murine leukemic virus (MMLV)-based retroviral vector, G1GFP, which contains the eGFP cDNA (27) under the control of the long terminal repeat (LTR) enhancer/promoter of the MMLV, was generated by using an ecotropic virus-producing cell line, GP+E86 (28).
Gene Transfer into Bone Marrow Cells
Donor mice (C57BL/6J (Ly5.2)) were injected with 5-fluorouracil (5FU; Kyowa, Tokyo, Japan) at a dose of 150 mg/kg to kill the cells in the cell cycle. Bone marrow cells were harvested by flushing tibias and femurs of the mice, and red cells and debris were eliminated by density isolation using Ficoll-Paque (Amersham Pharmacia, Buckinghamshire). The isolated mononucleocytes were prestimulated on recombinant fibronectin fragment, CH296 (Takara, Otsu, Japan), coated dishes in Dulbecco’s modified essential medium (DMEM; Gibco BRL, Rockville, MD) containing 15% fetal bovine serum (FBS), 100 u/ml of penicillin, 100 μg/ml of streptomycin, and 10 mg/ml of glutamine supplemented with 100 ng/ml of human interleukin-6 (IL-6; Kirin Brewers Co., Tokyo, Japan) and 100 ng/ml of murine stem cell factor (PeproTech EC Ltd., London, England) for 48 hr. After prestimulation, bone marrow cells were incubated with G1GFP four times for 4 consecutive days in the presence of the same growth factors. Flow cytometric analysis was performed on FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) (29).
Bone Marrow Transplantation
All animal experiments were conducted according to the institutional guidelines of the Nippon Medical School.
Congenic mice (C57BL/6J-Ly5.1-Pep3b (Ly5.1)), 8 weeks old, were used as recipients in this study. Each mouse received 10 Gy of whole body irradiation (137Cs γ-Rays:1.0 Gy/min) for bone marrow ablation (30). Genetically marked bone marrow (BM) cells (1×107) were injected into the tail veins of recipients after irradiation. The recipient mice were maintained under pathogen-free conditions in our animal facility.
Direct Injection of Bone Marrow Cells
Recipient mice, 8 weeks old, received a sublethal dose of irradiation (5 Gy) to eliminate a significant fraction of the endogenous hematopoietic precursors without killing the hosts. After anesthetization with pentobarbiturate (10 mg/kg) and ketamine hydrochloride (6 mg/kg), the skin over the skull was incised. The injection site was 2-mm posterior and 2-mm lateral to the bregma. A small hole was drilled in the skull. Genetically marked bone marrow cells (2×105 cells in 2 μl of DMEM) were slowly injected through the hole into the striatum 2-mm below the dura mater by using a stereotaxic apparatus (31).
Immunohistochemical Staining of Cells
At least three animals were analyzed in each experimental condition. The bone marrow transplantation was repeated three times, whereas the direct injection experiment was repeated twice.
The animals were sacrificed under deep anesthesia by intracardiac perfusion via the left ventricle with cold 0.9% NaCl, followed by 4% paraformaldehyde in phophate buffered saline (PBS), pH 7.4. The brains were removed and fixed overnight at 4°C and then transferred to PBS solution containing 20% sucrose until equilibration. Approximately 200 slices of microsections (40–50 μm thick) were prepared with Coldtome (Sakura, Tokyo, Japan) for analysis from each mouse brain.
To identify cell types, the brain sections were stained immunohistochemically using specific antibodies. The primary antibodies used were as follows: rabbit anti-neuron specific enolase (NSE) (Polysciences, Warrington, PA) (diluted 1:2000 in PBS containing 0.3% Triton-X 100 and 10% donkey serum) for neurons, rabbit anti-glial fibrillary acidic protein (GFAP) (Dako, Copenhagen, Denmark) (diluted 1:500 in PBS containing 0.3% Triton-X 100 and 10% donkey serum) for astrocytes, sheep anti-carbonic anhydrase II (CAII) (Dako) (diluted 1:100 in PBS containing 0.3% Triton-X 100 and 10% rabbit serum) for oligodendrocytes, and rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1) (32) (Dako) (diluted 1:150 in PBS containing and 10% donkey serum) for microglia. The microsection samples were incubated with the primary antibodies overnight at 4°C, washed with PBS containing 0.3% Triton-X 100, and stained with the Texas Red-conjugated secondary antibodies that are donkey anti-rabbit IgG for GFAP, NSE, and Iba1, and rabbit anti-sheep IgG for CAII, used at 1:200 dilution with 2% mouse serum. After three cycles of washing, the sections were mounted and coverslipped. Fluorescent signals were imaged by a confocal, laser-scanning microscope (Leica TCSSP, Heidelberg, Germany).
RESULTS
Genetic Marking of Donor Bone Marrow Cells
Mononuclear cells from 5-FU treated bone marrow cells were transduced with G1GFP, a retroviral vector with the green fluorescence protein (GFP) gene as a marker. Approximately 80% of the mononuclear cells were strongly positive for GFP expression, as determined by FACS analysis (Fig. 1). GFP expression was also confirmed by fluorescence microscopy (data not shown).
Flowcytometric analysis of GFP expression in transduced bone marrow cells.
Bone Marrow Cells in the Mouse Brain after Systemic Infusion
To investigate whether bone marrow cells can reconstitute hematopoiesis and migrate into the mouse brain after systemic BMT, GFP positive bone marrow cells were injected into the tail veins of lethally irradiated mice. Twenty-four weeks after BMT, GFP positive cells reconstituted and differentiated into all hematopoietic lineages both in the peripheral blood and bone marrow of the recipient mice (Fig. 2). Examination of the brain sections (approximately 200 slices per mouse) under low magnification revealed that GFP positive cells were widely distributed in the brain, whereas examination under high magnification demonstrated that the majority of these remained in the blood vessels, even after extensive perfusion. Only a small fraction migrated into the cortex (data not shown).
Reconstitution of the hematopoietic system in a recipient mouse 24 weeks after BMT. Peripheral blood cells (PB) were examined after Wright-Giemsa staining (A) and by a fluorescence microscope (B) (×400). Bone marrow mononuclear cells (BM-MNC) were examined under a phase contrast microscope (C) and under a fluorescence microscope (D) (×1000).
To determine the phenotype of the GFP positive cells, immunohistochemical staining of the cell specific markers was performed. Some of the GFP positive cells in the cortex expressed Iba1, which is a specific marker for microglia. A few cells labeled for both GFP and Iba1 were detectable in each slice. We did not detect the GFP positive cells expressing NSE for neurons, CAII for oligodendrocytes, or GFAP for astrocytes (Fig. 3).
Confocal microscopic examination of bone marrow cells in the brain after systemic infusion. The brain slices were incubated with specific antibodies against neuronal cell markers and examined under a confocal microscope. Photomicrographs of GFP-positive cells (A, D, G, J). Immunohistochemical staining with anti-NSE (B), anti-CAII (E), anti-GFAP (H), and anti-Iba1 (K) antibodies. Overlay of GFP-positive cells and immunologically stained cells (C, F, I, and L) (Bar=20 um).
Bone Marrow Cells in the Mouse Brains after Direct Injection
In the second series of experiments, genetically marked bone marrow cells were directly injected into the striatum of the mouse brain by using a stereotaxic apparatus. We used both nonirradiated and sublethally irradiated animals. We found that transplantation was more efficient in sublethally irradiated animals. Twelve weeks after injection, the mice were killed and slices of brain were analyzed for expression of GFP and cell-specific markers. Numerous GFP positive cells were detected at the site of injection at the corpus striatum (approximately 10 slices). In addition, a few donor cells were found in multiple areas distant from the injected site, suggesting that some cells migrated through the layers of the brain.
Immunostaining revealed that some GFP-positive cells were labeled with anti-GFAP, anti-CAII, and anti-Iba1 antibodies, suggesting that donor cells differentiated into astrocytes, oligodendrocytes, and microglia (Fig. 4). Such double positive cells for GFP and specific markers were found consistently but only in one or two areas throughout the brain. We did not detect GFP-positive cells expressing NSE, a specific marker for neurons. These results suggest that bone marrow cells include cells that have the potential to differentiate into all glia lineages but not into neurons in the CNS.
Confocal microscopic examination of bone marrow cells in the brain after direct injection. Arrangement of the pictures are the same as for Figure 3 (Bar=20 um).
DISCUSSION
These findings demonstrated that genetically marked BM cells differentiate into oligodendroglia and astroglia after direct injection into the brain of sublethally irradiated adult mice. The results strongly suggest that mouse bone marrow contains a subset of neural stem cells that are capable of differentiating into glial cells in the adult brain environment.
Stem cells that have the capacity to self-renew and undergo multilineage differentiation have been identified in numerous kinds of adult tissues (33,34). Until recently, these cells have been thought to produce only the cell lineages characteristic of the tissues in which they reside. However, recent studies have suggested that some stem cells may have the potential for differentiation outside of their tissue of origin. Donor-derived cells have been found after BMT in multiple nonhematopoietic tissues, including astrocytes in the brain (24), skeletal muscle (35), and bone (36). Our findings demonstrate that all lineages of glia can be differentiated from stem cells in bone marrow. Conversely, stem cells derived from nonhematopoietic tissues, such as neural stem cells and muscle side population (SP) cells have been found to differentiate into hematopoietic cells (36–38). Taken together, these findings suggest the possibility of reconstituting tissue by using stem cells from a separate dermal origin. Stem cells derived from adult BM may possess a high degree of plasticity and their differentiation may be influenced by environmental factors.
Bone marrow reportedly contains hematopoietic stem cells and mesenchymal stem cells that may differentiate into mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue (36). However, a recent study showed that highly purified hematopoietic stem cells have the potential to differentiate into muscle (35). Therefore, the relationship between hematopoietic stem cells and mesenchymal stem cells remains to be clarified. These two subsets of cells may be interconvertible, or alternatively, there may be a common set of ancestor cells in the bone marrow. Given that we used whole bone marrow cells in this study, the fraction that contributed to the generation of glial cells is unclear. We are currently studying transplantation of selected cell populations.
After the systemic transfusion of gene modified bone marrow cells, we could detect neither GFAP-positive donor cells nor CAII-positive donor cells. The majority of the GFP-positive cells were observed in the blood vessels. The cells in the parenchyma were microglia-marker positive. These results are inconsistent with the report by Eglitis and Mezey (24), which showed that systemically administered bone marrow cells differentiated into both microglia and astrocytes in the adult mouse brain. We speculate that the donor cells could not penetrate the blood-brain barrier under the conditions used in our study. Osmotic disruption of the blood-brain barrier may effectively increase the reconstitution capability of bone marrow cells in the brain (39).
Bone marrow cells reportedly contain precursor cells that differentiate into astrocytes and microglia (24). The results from this study confirmed these findings and additionally suggest that oligodendrocytes can also be reconstituted by the bone marrow derived cells. Oligodendrocytes play essential roles in the synthesis and maintenance of myelin in the CNS (40). In lysosomal storage disorders, accumulation of unmetabolized substrates in lysosomes may be toxic to the myelin sheaths. The resultant demyelination of the white matter of the brain provides the pathophysiologic basis for the deterioration, especially in MLD, adrenoleukodystrophy, and globoid cell leukodystrophy. Remyelination by bone marrow derived oligodendrocytes may result in the clinical improvement of neurological symptoms of such disorders.
Bone marrow cells offer several advantages of use over other cells for cell and gene therapy for CNS disorders. The isolation procedures of neuronal stem cells from humans have yet to be established. Embryonic stem cells may spontaneously differentiate into unwanted types of tissues. Furthermore, the use of fetal embryonic and neuronal stem cells is problematic both from immunological and ethical standpoints. Conversely, the patient’s own bone marrow cells can be readily obtained, and therefore, they could circumvent the problems in host immunity and graft-versus-host disease. The patient’s bone marrow cells may become an important carrier for ex vivo gene therapy for a wide range of neurological disorders.
Acknowledgments.
The authors thank T. Tsuganesawa and K. Takahashi for technical assistance.
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