Mesenchymal stem cell and the method of use thereof

Abstract

Demyelinated axons were remyelinated in the demyelinated rat model by collecting bone marrow cells from mouse bone marrow and transplanting the mononuclear cell fraction separated from these bone marrow cells.

Claims

1. A method for treating neurodegeneration, comprising injecting an effective amount of a cell population isolated from bone marrow or cord blood into a patient in need thereof, to treat neurodegeneration, wherein said isolated cell population comprises mesenchymal stem cells that are SH2(+), SH3(+), SH4(+), CD29(+), CD44(+), CD14(), CD34(), and CD45(), and does not exclude hematopoietic cells, and wherein cells within said injected cell population are capable of differentiating into neurons or glia cells.

2. A method for treating neurodegeneration in a patient with a neurodegenerative disorder, which comprises injecting into a patient in need thereof, a mononuclear cell fraction comprising cells capable of differentiating into neurons or glia cells, wherein said fraction is prepared by obtaining bone marrow cells or cord blood cells, diluting the bone marrow cells or cord blood cells, separating a mononuclear cell fraction, collecting said mononuclear cell fraction.

3. The method for treating neurodegeneration according to claim 2, wherein said neurodegeneration is caused by nerve demyelination.

4. The method for treating neurodegeneration according to claim 1, wherein said neurodegeneration is caused by nerve demyelination.

5. The method for treating neurodegeneration according to claim 1, wherein said cell population is isolated from cord blood.

6. The method for treating neurodegeneration according to claim 1, wherein said cell population is injected intra-arterially.

7. The method for treating neurodegeneration according to claim 1, wherein said cell population is injected intravenously.

8. The method for treating neurodegeneration according to claim 6, wherein said cell population is injected into the carotid artery.

9. The method for treating neurodegeneration according to claim 1, wherein said neurodegeneration is caused by ischemia.

10. The method for treating neurodegeneration according to claim 9, wherein said neurodegeneration caused by ischemia is stroke.

11. The method for treating neurodegeneration according to claim 1, wherein said neurodegeneration is caused by demyelination or trauma.

12. The method for treating neurodegeneration according to claim 11, wherein said trauma is caused by spinal cord injury or brain injury.

13. The method for treating neurodegeneration according to claim 11, wherein said demyelination is caused by spinal cord injury or multiple sclerosis.

14. The method for treating neurodegeneration according to claim 1, wherein said neurodegeneration is associated with Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1D show optical light micrographs of transfection of dorsal column of the spinal cord, which had been prepared at 1-mm intervals. (FIG. 1A), normal axon; and (FIG. 1C), damaged demyelinated axon. Patterns of dorsal column observed at higher magnification are shown in (B) for normal axon and in (FIG. 1D) for demyelinated axon. Scale bars: 250 m in (FIG. 1A) and (FIG. 1C); 10 m in (FIG. 1B) and (FIG. 1D).

(2) FIGS. 2A-2D show microphotographs demonstrating the remyelination of rat spinal cord (FIG. 2A), after transplantation of adult mouse bone marrow cells; and (FIG. 2C), after transplantation of Schwann cells. Photomicrographs demonstrating remyelinated axon observed at higher magnification are shown in (FIG. 2B), after transplantation of bone marrow cells; and (FIG. 2D), after transplantation of Schwann cells. Scale bars: 250 m in (FIG. 2A) and (FIG. 2C); 10 m in (FIG. 2B) and (FIG. 2D).

(3) FIGS. 3A-3B show an electron micrograph of remyelinated axon after the transplantation of bone marrow into the dorsal columns. The tissue was treated with substrate X-Gal to detect transplanted bone marrow cells containing the -galactosidase gene in the damaged tissues (the reaction product is indicated with arrow) (FIG. 3A). When observed at higher magnification (FIG. 3B), basal lamina was found around the fibers (wedge-shaped mark; scale bar 1 m). The presence of large cytoplasmic and nuclear regions and basal lamina in the transplanted cells indicates peripheral myelination.

BEST MODE FOR CARRYING OUT THE INVENTION

(4) The present invention will be described in more detail below with reference to examples based on specific experiments.

[Example 1] Preparation of Bone Marrow Cells and Schwann Cells

(5) (1) Bone Marrow Mononuclear Cells

(6) Mouse bone marrow cells (10 l) were obtained from the femur of adult LacZ (a structural gene of -galactosidase) transgenic mice (The Jackson Laboratory, Maine, USA). The collected sample was diluted in L-15 medium (2 ml) containing 3 ml Ficoll, and centrifuged at 2,000 rpm for 15 minutes. Cells were collected from the mononuclear cell fraction, and suspended in 2 ml serum-free medium (NPMM: Neural Progenitor cell Maintenance Medium). Following centrifugation (2,000 rpm, 15 min), the supernatant was removed, and precipitated cells were collected and re-suspended in NPMM.

(7) (2) Schwann Cells

(8) Primary Schwann cell cultures were established from the sciatic nerve of neonatal mouse (P1-3) according to the method of Honmou et al. (J. Neurosci., 16(10):3199-3208, 1996). Specifically, cells were released from sciatic nerve by enzymatic and mechanical treatment. 810.sup.5 cells per plate were plated onto 100-mm.sup.2 poly (L-lysine)-coated tissue culture plates and the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum.

[Example 2] Experimental Animal Preparation and Transplantation

(9) (1) Preparation of Demyelinated Rate Model

(10) Experiments were performed on 12 week old Wistar rats. A localized demyelinated lesion was created in the dorsal columns using X-ray irradiation and ethidium bromide injection (EB-X treatment). Specifically, rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) i.p., and a surface dose of 40 Grays of X-ray was irradiated using Softex M-150 WZ radiotherapy machine (100 kV, 1.15 mA, SSD 20 cm, dose rate 200 cGy/min) on the spinal cord caudal to the tenth thoracic spin level (T-10) through a 24 cm opening in a lead shield (4 mm thick). Three days after X-ray irradiation, rats were anesthetized as above, and aseptic laminectomy of the eleventh thoracic spine (T-11) was conducted. A demyelinating lesion was generated by the direct injection of ethidium bromide (EB) into the dorsal columns via a glass micropipette whose end was drawn. 0.5 l saline containing 0.3 mg/ml EB was injected at the depths of 0.7 and 0.4 mm.

(11) Transplantation of stem or progenitor cells that can differentiate or transform into the neural lineages.

(12) 3 days after the EB injection, 1 l of the cell suspension (110.sup.4 cells/l), which was obtained in Example 1, was injected into the middle of the EB-X-induced lesion. Transplanted rats were immunosuppressed with cyclosporin A (10 mg/kg/day).

[Example 3] Histological Examination

(13) Rats were deeply anesthetized by the administration of sodium pentobarbital (60 mg/kg, i.p.), and perfused through the heart cannula, first, with phosphate-buffer solution (PBS) and then with a fixative containing 2% glutaraldehyde and 2% paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4. Following in situ fixation for 10 minutes, the spinal cord was carefully excised, cut into 1 mm segments and kept in fresh fixative. The tissue was washed several times in Sorensen's phosphate buffer, post-fixed in 1% OSO.sub.4 for 2 hours at 25 C., dehydrated by elevating the concentration of the ethanol solution, passed through propylene oxide and embedded in EPON. Then, the tissue was cut into sections (1 m), counterstained with 0.5% methylene blue and 0.5% azure II in 0.5% borax, and examined under light microscope (Zeiss: Axioskop FS). Ultrathin sections were counterstained with uranyl and lead salts, and examined with JEOL JEM1200EX electron microscope (JEOL, Ltd., Japan) at 60 kV.

(14) A 5050 m standardized region in the central core of the dorsal columns in the spinal cords near the site wherein the cells were initially injected was used for morphometric analysis. The numbers of remyelinated axons and cell bodies associated with the axons were counted within this region; and the density to square millimeters was calculated. Furthermore, the diameters of the axons and cell bodies, the number of cells with multi-lobular nuclei, and cells showing myelination were examined in the same standardized region. Measurements and counts were obtained from five sections per rat, and five rats (n=5) were analyzed for each experimental condition. All variances represent standard error (SEM).

(15) The dorsal column in the spinal cord mostly consists of myelinated axons (FIGS. 1A, 1B). The proliferation of endogenous glial cells was inhibited by the irradiation of X-ray to the dorsal columns of the lumbar spinal cord. Further, by the administration of a nucleic acid chelator, ethidium bromide, glial cells such as oligodendrocytes were found damaged and local demyelination occurred. Such lesions generated according to this procedure are characterized by almost complete loss of endogenous glial cells (astrocytes and oligodendrocytes) and preservation of axons (FIG. 1C). Examination of the lesion with a light microscope under a higher magnification revealed that congested areas consisting of demyelinated axons are appositioned closely to one another separated by areas wherein the debris of myelin exist and wherein macrophages exist (FIG. 1D). The lesion occupied nearly the entire dorso-ventral extent of the dorsal columns 5 to 7 mm longitudinally. Almost none of the endogenous invasion of Schwann cells or astrocytes occurs till the sixth to eighth week. With the start of invasion, these glial cells begin to invade into the lesion from peripheral borders. Thus, a demyelinated and glial-free environment is present for at least 6 weeks in vivo.

(16) Three weeks after transplantation of LacZ transgenic mouse bone marrow cells (BM) into the central region of the lesion in immunosuppressed and demyelinated rat models, extensive remyelination of the demyelinated axons was observed (FIGS. 2A and 2B). Remyelination was observed across the entire coronal dimension of the dorsal columns and throughout the antero-posterior extent of the lesion. FIGS. 2C and 2D show the pattern of remyelination observed after transplantation of allogeneic Schwann cells (SC) into the EB-X lesion model. It is remarkable that large nuclear and cytoplasmic regions were found around the remyelinated axons, a characteristic of peripheral myelin, by both BM and SC transplantations.

[Example 4] Detection of -Galactosidase Reaction Products In Vivo

(17) Three weeks after transplantation, -galactosidase expressing myelin-forming cells were detected in vivo. Spinal cords were collected and fixed in 0.5% glutaraldehyde in phosphate-buffer for 1 h. Sections (100 m) were cut with a vibratome and -galactosidase expressing myelin-forming cells were detected by incubating the sections at 37 C. overnight with X-Gal (substrate which reacts with -galactosidase to develop color) at a final concentration of 1 mg/ml in X-Gal developer (35 mM K.sub.3Fe(CN).sub.6/35 mM K.sub.4Fe (CN).sub.63H.sub.2O/2 mM MgCl.sub.2 in phosphate-buffered saline) to form blue color within the cell. Sections were then fixed for an additional 3 h in 3.6% (vol/vol) glutaraldehyde in phosphate-buffer (0.14 M), and were examined with light microscope for the presence of blue reaction product (-galactosidase reaction product). Prior to embedding in EPON, the tissue was treated with 1% OSO.sub.4, dehydrated in a graded series of ethanol, and soaked in propylene oxide for a short period. Ultrathin sections were then examined under an electron microscope without further treatment.

(18) Under the electron microscope, most of the myelin-forming cells derived from donor cells retained the basal membrane (FIGS. 3A-3B: wedge-shaped mark). Additionally, the myelin-forming cells had relatively large nucleus and cytoplasm, which suggests the formation of a peripheral nervous system-type myelin sheath.

(19) It was confirmed that almost no endogenous remyelination by oligodendrocytes, or Schwann cells occurs for at least six weeks in the lesion model used in the present experiment. Furthermore, the donor cells that contained the reporter gene LacZ, i.e., X-Gal-positive cells, were observed to form myelin at the electron microscopic level (FIG. 3B: arrow).

(20) Differentiation into neurons and glial cells could be observed following the transplantation of bone marrow cells into the EB-X lesions, but not by SC transplantation. Five percent of lacZ-positive cells (transplanted bone marrow cells) in the EB-X lesions showed NSE (Neuron Specific Enolase)-immunoreactivity and 3.9% showed GFAP (Glial Fibrially Acidic Protein)-immunoreactivity, indicating that some of the bone marrow cells can differentiate into neurons or glial cells, respectively, in vivo.

(21) Furthermore, employing antibodies, the present inventors isolated mesenchymal stem cells with the characteristic of cell markers SH2(+), SH3(+), CD29(+), CD44(+), CD14(), CD34(), and CD45 () from the cell fraction obtained in Example 1(1). Furthermore, they discovered that transplantation of the cells into the demyelinated regions of rat spinal cord results in more efficient remyelination. It was also revealed that the cells survived favorably and differentiated into neurons or neuronal cells and glia cells when transplanted into cerebral infarction model rats.

(22) Further, the present inventors isolated stromal cells characterized by the cell surface markers Lin(), Sca-1(+), CD10(+), CD11D(+), CD44(+), CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+), CD127(+), CD164(+), fibronectin (+), ALPH(+), and collagenase-1(+) from the cell fraction obtained in Example 1(1). Transplantation of the cells into demyelinated regions of rat spinal cord also resulted in efficient remyelination.

(23) Further, the present inventors isolated cells characterized by cell surface marker AC133(+) from the cell fraction obtained in Example 1(1). Transplantation of the cells into demyelinated regions of rat spinal cord also resulted in efficient remyelination.

(24) In addition, the present inventors obtained a cell fraction containing AC133-positive cells capable of differentiating into neural cells from rat embryonic hepatic tissues by the following procedure. Specifically, first, liver tissues collected from rat fetuses were washed in L-15 solution, and then treated enzymatically in L-15 solution containing 0.01% DNaseI, 0.25% trypsin, and 0.1% collagenase at 37 C. for 30 minutes. Then, the tissue was dispersed into single cells by pipetting several times. The single-dispersed embryonic hepatic tissues were centrifuged as in Example 1(1) (preparation of mononuclear cell fraction from femur) to isolate a mononuclear cell fraction. The obtained mononuclear cell fraction was washed, and then, AC133(+) cells were recovered from the cell fraction using anti-AC133 antibody. The isolation of AC133-positive cells can be achieved using magnetic beads or a cell sorter (FACS or the like). Transplantation of the obtained AC133-positive cells into demyelinated regions of rat spinal cord also resulted in efficient remyelination.

INDUSTRIAL APPLICABILITY

(25) As described above, the present invention provides fractions of mononuclear cells isolated and purified by collecting bone marrow-derived bone marrow cells, cord blood-derived cells, or fetal liver-derived cells. Transplantation of such mononuclear cell fractions into a demyelination model animal was confirmed to result in remyelination of the demyelinated axon.

(26) Cells for transplantation can be relatively easily isolated from a small quantity of bone marrow cell fluid aspirated from bone marrow, and can be prepared for transplantation in several tens of minutes after the cells are being collected. Thus, these cells can serve as useful and regenerable cellular material for autotransplantation in the treatment of demyelinating diseases.

(27) This invention highlights development of the autotransplantation technique to treat demyelinating diseases in the central nervous system. Furthermore, the use of the present invention in transplantation and regeneration therapy for more general and diffuse damage in the nervous system is envisaged. In other words, this invention sheds light on autotransplantation therapy against ischemic cerebral damage, traumatic cerebral injury, cerebral degenerating diseases, and metabolic neurological diseases in the central and peripheral nervous systems.

(28) According to the present invention, cells in the hematopoietic system (bone marrow or cord blood) are used as donor cells. Thus, to treat neurological diseases, the cells may be transplanted into the vessels instead of directly transplanting them into neural tissues. Specifically, donor cells transplanted into a vessel can migrate to the neural tissues and thereby regenerate the neural tissues. Hence, the present invention is a breakthrough in developing a therapeutic method for relatively noninvasive transplantation.

(29) Furthermore, the present invention adds significantly to elucidate the mechanism underlying the differentiation of non-neural cells such as hematopoietic cells and mesenchymal cells into neural cells. When genes determining the differentiation are identified and analyzed, use of such genes will allow efficient transformation of a sufficient quantity of non-neural cells such as hematopoietic cells and mesenchymal cells in a living body to neural cells. Thus, the present invention is a breakthrough in the field of gene therapy for inducing regeneration of neural tissues.