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Title:  Method for remyelinating a demyelinized lesion due to injury in the brain or spinal cord
United States Patent: 
7,098,027
Issued: 
August 29, 2006

Inventors: 
Honmou; Osamu (Hokkaido, JP), Hashi; Kazuo (Hokkaido, JP), Uede; Teiji (Hokkaido, JP)
Assignee: 
Renomedix Institute, Inc. (Hokkaido, JP), Aska Pharmaceutical Co., Ltd. (Tokyo, JP), Mitsui Sumitomo Insurance Care Network Co., Ltd. (Tokyo, JP), Hitachi Ltd. (Tokyo, JP)
Appl. No.: 
10/330,963
Filed:
 December 23, 2002


 

Executive MBA in Pharmaceutical Management, U. Colorado


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.

DISCLOSURE OF THE INVENTION

The present inventors have previously isolated and cultured neural stem cells from adult human brain, and established some cell lines. By studying their functions, the inventors newly discovered that the neural stem cells have pluripotency and the ability to self-renewal. Specifically, single-cell expansion of neural progenitor (stem) cells obtained from adult human brain was conducted to establish cell lines; the established cells were then subjected to in vitro clonal analysis. The result showed that the cell lines had pluripotency (namely, differentiation into neuron, astroglia (or astrocyte), and oligodendroglia (i.e., oligodendrocyte)) and the ability to self-renewal (namely, proliferation potency). Thus, these cells were confirmed to possess the characteristics of neural stem cell.

Transplantation of these cells indeed resulted in very favorable graft survival, migration, and differentiation in cerebral ischemic model rats or injury model rats. Furthermore, transplantation of the cells was found to result in functional myelin sheath formation in spinal cord demyelination model rats. Thus, such transplantation allows remyelination of the demyelinated axon and restoration of the neural function in the rat spinal cord demyelination model. Effectiveness of such transplantation therapy using these cells was confirmed by histological, electrophysiological, and behavior studies.

Judging from the above-described findings, transplantation of cultured neural stem cells, which have been isolated from a small amount of neural tissue collected from the cerebrum of a patient, into the lesion of the brain or the spinal cord of the patient seems to be a widely applicable in autotransplantation therapy.

However, while not causing neurologic deficits, collecting tissues containing neural stem cells from cerebrum is relatively invasive. Thus, considering the need for establishing therapeutic methods for various complicated diseases in the nervous system today, it is crucial to establish a safer and simpler method for autotransplantation therapy.

Thus, an objective of the present invention is to provide cellular material that is useful in the treatment of neurological diseases, and which can be prepared safely and readily. Another objective of the present invention is to provide a method for treating neurological diseases, preferably a method for autotransplantation therapy, using the cellular material.

In view of the existing state as described above, to establish donor cells the present inventors focused on the technique of collecting bone marrow cells from bone marrow, a simpler technique as compared to the collection of neural stem cells and routinely used in today's medical practice. First, they collected bone marrow cells from mouse bone marrow, isolated mononuclear cell fraction, and then transplanted this fraction as donor cells into spinal cord demyelination model rats. Surprisingly, it was discovered that the demyelinated axon gets remyelination by the treatment. Hence, the present inventors newly revealed that the mononuclear cell fraction prepared from bone marrow cells have the ability to differentiate into neural cells. The present inventors also discovered that cell fractions containing mesodermal stem cells, mesenchymal stem cell, stromal cells, and AC133-positive cells, that were isolated from the mononuclear cell fraction had the ability to differentiate into neural cells. Besides bone marrow cells, these cell fractions can also be prepared from cord blood cells. Furthermore, AC133-positive cells can be prepared from embryonic hepatic tissues.

Thus, the present invention provides cell fractions containing cells capable of differentiating into neural cells, which are isolated from bone marrow cells, cord blood cells, and embryonic hepatic tissues.

In another embodiment, such cell fractions contain mesenchymal stem cells having the following character: SH2(+), SH3(+), SH4(+) CD29(+), CD44(+), CD14(-), CD34(-), and CD45(-).

In another embodiment, such cell fractions contain stromal cells having the following characteristics: Lin(-), Sca-1(+), CD10(+), CD11D(+), CD44(+), CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+), CD127(+), CD164(+), fibronectin (+), ALPH(+), and collagenase-1(+).

In another embodiment, such cell fractions contain cells having the character AC133(+).

In addition, the present invention provides cells capable of differentiating into neural cells, which are contained in the above-mentioned cell fraction.

Furthermore, the present invention provides compositions for treating neurological disease, which contain the above-mentioned mononuclear cell fractions or the above-mentioned cells. According to a preferred embodiment of the present invention, the neurological disease is selected from the group consisting of: central and peripheral demyelinating diseases; central and peripheral degenerative diseases; cerebral apoplexy (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage); brain tumor; dysfunction of higher function of the brain (the term "higher function of the brain" involves the cognitive function, short and long memory, speech, etc.); psychiatric diseases; dementia; infectious diseases; epilepsy; traumatic neurological diseases; and infarction of spinal cord diseases.

Furthermore, the present invention provides therapeutic methods for neurological diseases, which comprises transplanting of the above-mentioned mononuclear cell fractions, cells, or compositions. In preferred embodiments, the donor cells are derived from a recipient.

The present invention provides mononuclear cell fractions isolated from bone marrow cells, cord blood cells, or embryonic hepatic tissues, wherein the fractions contain cells capable of differentiating into neural cells. It is unclear whether the differentiation of cells contained in the cell fractions provided by the present invention into neural cells is caused by the transformation of so-called hematopoietic cells into neural cells, or, alternatively, by the differentiation of immature cells capable of differentiating into neural cells that are comprised in bone marrow cells, etc. However, the majority of the cells differentiating into neural cells are assumed to be stem or precursor cells, namely, cells having the self-renewal ability and pluripotency. Alternatively, the cells differentiating into neural cells may be stem or precursor cells which have differentiated to some extent into endoderm or mesoderm.

Cells in a cell fraction of the present invention do not have to be proliferated with any trophic factors (then again they can proliferate in the presence of trophic factors). Thus, these cells are simple and practical from the standpoint of the development of autotransplantation technique for the diseases in the neural, and are very beneficial in medical industry. In general, a cell fraction of the present invention is derived from vertebrate, preferably from mammal (for example, mouse, rat, human, etc.).

A cell fraction of the present invention can be prepared by subjecting bone marrow cells or cord blood cells collected from vertebrate to density-gradient centrifugation at 2,000 rpm in a solution for a sufficient time ensuring separation depending on specific gravity, and then recovering the cell fraction with a certain specific gravity within the range of 1.07 to 1.1 g/ml. Herein, the phrase "a sufficient time ensuring separation depending on specific gravity" refers to a time sufficient for the cells to shift to a position in the solution according to their specific gravity, which is typically about 10 to 30 minutes. The specific gravity of the cell fraction to be recovered is within the range of 1.07 to 1.08 g/ml (for example, 1.077 g/ml) Solutions, such as Ficoll solution and Percoll solution, can be used for the density-gradient centrifugation, but is not limited thereto.

Specifically, first, bone marrow (5 to 10 .mu.l) collected from a vertebrate is combined with a solution (2 ml L-15 plus 3 ml Ficoll), and then centrifuged at 2,000 rpm for 15 minutes to isolate a mononuclear cell fraction (approx. 1 ml). The mononuclear cell fraction is combined with culture solution (2 ml NPBM) to wash the cells, and then the cells are again centrifuged at 2,000 rpm for 15 minutes. Then, the precipitated cells are recovered after the removal of the supernatant. Besides femur, sources to obtain a cell fraction of the present invention include sternum, and ilium constituting the pelvis. Any other bone can serve as a source so long as it is large enough A cell fraction of the present invention can also be prepared from bone marrows and cord blood stored in bone marrow bank or cord blood bank.

Another embodiment of cell fractions of the present invention includes a mononuclear cell fraction isolated and purified from bone marrow cells or cord blood cells, which contains mesodermal (mesenchymal) stem cells capable of differentiating into neural cells. The term "mesodermal (mesenchymal) stem cell" refers to cells that can copy (divide and proliferate) cells with the same potential as themselves and that are capable of differentiating into any type of cells constituting mesodermal (mesenchymal) tissues. Mesodermal (mesenchymal) cells indicate cells constituting tissues that are embryologically categorized to the mesoderms, including blood cells. The mesodermal (mesenchymal) stem cell includes, for example, cells characterized by SH2(+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), CD34(-), and CD45(-). A cell fraction containing mesodermal (mesenchymal) stem cells can be obtained, for example, by selecting cells having a cell surface marker, such as SH2, as described above from the above-mentioned cell fraction obtained by centrifuging bone marrow cells or cord blood cells (the cell fraction according to claim 2).

Furthermore, a cell fraction containing mesodermal (mesenchymal) stem cells capable of differentiating into neural cells can be prepared by subjecting bone marrow cells or cord blood cells collected from vertebrate to density-gradient centrifugation at 900 G in a solution for a sufficient time ensuring separation depending on specific gravity, and then recovering the cell fraction with a certain specific gravity within the range of 1.07 to 1.1 g/ml. Herein, "a sufficient time ensuring separation depending on specific gravity" refers to a time sufficient for the cells to shift to a specific position corresponding to their specific gravity in the solution for density-gradient centrifugation, which is typically about 10 to 30 minutes. The specific gravity of a cell fraction to be recovered varies depending on the type of animal (for example, human, rat, and mouse) from which the cells have been derived. A solution to be used for density-gradient centrifugation includes Ficoll solution and Percoll solution, but is not limited thereto.

Specifically, first, bone marrow (25 ml) or cord blood collected from vertebrate is combined with an equal volume of PBS solution, and then centrifuge at 900 G for 10 minutes. Precipitated cells are mixed with PBS and then are recovered (cell density=approx. 4.times.10.sup.7 cells/ml) to remove blood components. Then, a 5-ml aliquot thereof is combined with Percoll solution (1.073 g/ml), and centrifuged at 900 G for 30 minutes to extract a mononuclear cell fraction. The extracted mononuclear cell fraction is combined with a culture solution (DMEM, 10% FBS, 1% antibiotic-antimycotic solution) to wash the cells, and is centrifuged at 2,000 rpm for 15 minutes. Finally, the supernatant is removed, precipitated cells are recovered and cultured at 37.degree. C. under 5% CO.sup.2 atmosphere.

Another embodiment of a cell fraction of the present invention is a fraction of mononuclear cells isolated from bone marrow cells or cord blood cells, which contains stromal cells capable of differentiating into neural cells. Examples of stromal cell include cells characterized by Lin(-); Sca-1(+), CD10(+), CD11D(+), CD44(+) CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+), CD127(+), CD164(+) fibronectin (+), ALPH(+), and collagenase-1 (+). A cell fraction containing stromal cells can be prepared, for example, by selecting cells having a cell surface marker, such as Lin as described above, from the above-mentioned cell fraction obtained by centrifuging bone marrow cells or cord blood cells (the cell fraction according to claim 2).

Furthermore, such a cell fraction can be prepared by subjecting bone marrow cells or cord blood cells collected from vertebrate to density-gradient centrifugation at 800 G in a solution for a sufficient time ensuring separation depending on specific gravity, and then recovering the cell fraction with a certain specific gravity within the range of 1.07 to 1.1 g/ml. Herein, "a sufficient time ensuring separation depending on the specific gravity" indicates a time sufficient for the cells to shift to a specific position corresponding to their specific gravity in the solution for density-gradient centrifugation, which is typically about 10 to 30 minutes. The specific gravity of a cell fraction to be recovered is preferably within the range of 1.07 to 1.08 g/ml (for example, 1.077 g/ml). A solution to be used for density-gradient centrifugation includes Ficoll solution and Percoll solution, but is not limited thereto.

Specifically, first, bone marrow or cord blood collected from vertebrate is combined with an equal volume of a solution (PBS, 2% BSA, 0.6% sodium citrate, and 1% penicillin-streptomycin). A 5-ml aliquot thereof is combined with Ficoll+Paque solution (1.077 g/ml) and centrifuged at 800 G for 20 minutes to obtain a mononuclear cell fraction. The mononuclear cell fraction is combined with a culture solution (Alfa MEM, 12.5% FBS, 12.5% horse serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1 .mu.M hydrocortisone, 1% antibiotic-antimycotic solution) to wash the cells, and then are centrifuged at 2,000 rpm for 15 minutes. The supernatant is removed after centrifugation. The precipitated cells are collected and then cultured at 37.degree. C. under 5% CO.sup.2 atmosphere.

Another embodiment of a cell fraction of the present invention is a mononuclear cell fraction containing cells characterized by AC133(+) capable of differentiating into neural cells, which is isolated from bone marrow cells, cord blood cells, or embryonic hepatic tissues. Such a cell fraction can be obtained, for example, by selecting cells having a cell surface marker including the above-mentioned AC133(+) from the cell fraction obtained, as described above, by centrifuging bone marrow cells or cord blood cells (the cell fraction according to claim 2).

Furthermore, the cell fraction can be obtained by subjecting embryonic hepatic tissues collected from vertebrate to density-gradient centrifugation at 2,000 rpm in a solution for a sufficient time ensuring separation depending on specific gravity, recovering a cell fraction within the range of a specific gravity of 1.07 to 1.1 g/ml, and then recovering cells with the characteristic of AC133(+) from the cell fraction. Herein, "a sufficient time ensuring separation depending on specific gravity" indicates a time sufficient for the cells to shift to a specific position corresponding to their specific gravity in the solution for density-gradient centrifugation, which is typically about 10 to 30 minutes. The solution to be used for density-gradient centrifugation includes Ficoll solution and Percoll solution, but is not limited thereto.

Specifically, first, liver tissue collected from vertebrate is washed in L-15 solution, and then treated enzymatically in an L-15 solution containing 0.01% DNaseI, 0.25% trypsin, and 0.1% collagenase at 37.degree. C. for 30 minutes. Then, the tissue is dispersed into single cells by pipetting. The single-dispersed embryonic hepatic cells are centrifuged by the same procedure as described for the preparation of the mononuclear cell fraction from femur in Example 1 (1). The cells thus obtained are washed, and then AC133 (+) cells are collected from the washed cells using an AC133 antibody. Thus, cells capable of differentiating into neural cells can be prepared from embryonic hepatic tissues. The antibody-based recovery of AC133(+) cells can be achieved using magnetic beads or a cell sorter (FACS, etc.).

Transplantation of any of these cell fractions containing mesodermal stem cells, mesenchymal stem cells, stromal cells, or AC133-positive cells into demyelinated spinal cord can lead to efficient remyelination of the demyelinated region. In particular, the above-mentioned cell fraction containing mesenchymal stem cells can engraft favorably and differentiate into neural cells such as neurons or glia when transplanted into a stroke model or a cerebral infarction model.

The present invention also provides cells capable of differentiating into neural cells, which are contained in the above-mentioned cell fraction. These cells include, for example, neural stem cells, mesodermal stem cells, mesenchymal stem cells, stromal cells, and AC133-positive cells which are contained in the above-mentioned cell fraction, but are not limited thereto so long as they can differentiate into neural cells.

The present invention also provides compositions for treating neurological diseases, which comprise a cell fraction or cells of the present invention. It is possible to transplant the cell fractions or cells of the present invention without modification. However, in order to improve the efficiency of therapy, they may be transplanted as compositions combined with various additives or introduced with genes. The preparation of compositions of the present invention may comprise, for example, (1) addition of a substance that improves the proliferation rate of cells included in a cell fraction of the present invention or enhances the differentiation of the cells into neural cells, or introducing a gene having the same effect; (2) addition of a substance that improves the viability of cells in a cell fraction of the present invention in damaged neural tissues, or introducing a gene having the same effect; (3) addition of a substance that, inhibits adverse effects of damaged neural tissue on the cells in a cell fraction of the present invention, or introducing a gene having the same effect; (4) addition of a substance that prolongs the lifetime of donor cells, or introducing a gene having the same effect; (5) addition of a substance that modulates the cell cycle, or introducing a gene having the same effect; (6) addition of a substance to suppress the immunoreaction or inflammation, or introducing a gene having the same effect; (7) addition of a substance that enhances the energy metabolism, or introducing a gene having the same effect; (8) addition of a substance that improves the migration of donor cells in host tissues, or introducing a gene having the same effect; (9) addition of a substance that improves blood flow (including inductions of angiogenesis), or introducing a gene having the same effect; (10) addition of a substance that cure the infectious diseases, or introducing a gene having the same effect, or (11) addition of a substance that cure the tumors, or introducing a gene having the same effect, but is not limited thereto.

It is considered that the cells according to the present invention are immobilized in the bone marrow by a distinct mechanism involving a certain type of substance (proteins, etc.) and do not normally move out into the peripheral blood. Therefore, to make these cells enter the peripheral blood circulation, conventionally, they are removed from the bone marrow, and then administered intravenously. However, the studies conducted by the present inventors gradually elucidated the mechanism of immobilization of these cells in the bone marrow. The discovery made by the present inventors showed that these cells, which had been localized in the bone marrow, could be made to move out into the peripheral blood by intravenous injection of active factors, such as an antibody, a cytokine, chemicals, or a growth factor. That is, therapeutic effect of bone marrow transplantation that is similar to that of the aforementioned method can be expected from intravenous injection of an active factor, such as an antibody, a cytokine, chemicals, or a growth factor.

A cell fraction, cell, and composition of the present invention can be used for treating neurological diseases. Target neurological diseases for the therapy include, for example, central and peripheral demyelinating diseases; central and peripheral degenerative diseases; cerebral apoplexy (includingcerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage); cerebral tumor; disorders of higher brain function including dementia; psychiatric diseases; epilepsy, traumatic neurological diseases (including head injury, cerebral contusion, and spinal cord injury); infectious diseases; and infarction of spinal cord, but are not limited thereto.

According to the present invention, cells derived from bone marrow cells of a recipient can be transplanted as donor cells (autotransplantation therapy) This has the following advantages: (1) low risk of rejection for the transplantation; and (2) no difficulty in using immunosuppressant. When autotransplantation therapy is arduous, then cells derived from other person or nonhuman animal may be used. Cells frozen for storage are also usable. The donor cells may be derived from cord blood.

Bone marrow can be collected, for example, by anesthetizing (by local or systemic anesthesia) an animal (including human) that serves as a source, put a needle into the sternum or iliac of the animal, and aspirating the bone marrow with a syringe. On the other hand, it is an established technique to collect cord blood at birth by putting needle directly into the umbilical cord, and aspirating the blood from the umbilical cord using syringe, and to store the blood.

Transplantation of cells into a patient can be performed, for example, by first filling a syringe with cells to be transplanted. Herein, the cells are suspended in an artificial cerebrospinal fluid or physiological saline. Then, the damaged neural tissue is exposed by surgery, and, with a needle, directly injecting the cells into the lesion. Due to high migrating potential of cells contained in a cell fraction of the present invention, they can migrate in the neural tissues. Hence, cells can be transplanted into a region adjacent to the lesion. Moreover, injection of the cells into the cerebrospinal fluid is also expected to be efficacious. In the case of the injection of the cells into the cerebrospinal fluid, the cells can be injected into a patient by typical lumbar puncture, without surgical operation only under local anesthetization. Thus, the patient can be treated in patient's sickroom (not in an operation room), which makes the method preferable. Alternatively, intravenous injection (including any systemic transplantations such as intravenous, intraarterial, selective intraarterial administration) of the cells is also expected to be effective. Thus, transplantation can be carried out by a procedure based on typical blood transfusion, which is advantageous in that the treatment can be performed in patient's sickroom.

Furthermore, due to their high migrating potential, cells in a cell fraction of the present invention can be used as a carrier (vector) for genes. For example, the cells are expected to be useful as a vector for gene therapy for various neurological diseases such as brain tumor.
 

Claim 1 of 2 Claims

1. A method of remyelinating a demyelinized lesion due to injury in the brain or spinal cord of a patient, comprising obtaining bone marrow cells from the patient, diluting the bone marrow cells, centrifuging the bone marrow cells, thereby separating a mononuclear cell fraction having a specific gravity of 1.07 to 1.1 g/mL, collecting said mononuclear cell fraction, suspending said mononuclear cell fraction in a serum-free medium to form a suspension, centrifuging said suspension to yield a centrifuged mononuclear cell fraction and a supernatant, removing said supernatant from the centrifuged mononuclear cell fraction, resuspending said centrifuged mononuclear cell fraction in a serum-free medium, thereby forming a resulting mononuclear cell fraction, and injecting said resulting mononuclear cell fraction directly into the lesion in the patient, whereby the injected mononuclear cell fraction migrates to neural tissue in the patient, differentiates into neural cells, and forms myelinated axons in the demyelinated lesion three weeks after injection.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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