JP2008063290A - Therapeutic agent for parkinson's disease or parkinsonian syndrome containing cell relating to mesenchymal system and therapeutic method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent effective for the treatment of Parkinson's disease or parkinsonian syndrome and having high safety and provide a therapeutic method to use the therapeutic agent. <P>SOLUTION: The problem is solved by a therapeutic agent for the treatment of Parkinson's disease or parkinsonian syndrome and containing cells relating to the mesenchymal system and a therapeutic method to use the therapeutic agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for Parkinson's disease or Parkinson's syndrome comprising cells associated with the mesenchymal system, and a therapeutic method using the same.

  Parkinson's disease is a basal ganglia degenerative disease whose main symptoms are extrapyramidal functions such as tremor at rest, muscle stiffness, ataxia, and postural reflex disorder. It is known to be caused by a lack of dopamine based on degeneration of the sexual nerve. Currently, dopamine replacement therapy that administers dopamine precursors such as levodopa is the main treatment, but this dopamine replacement therapy has problems such as side effects such as dyskinesia and psychiatric symptoms. Although other anti-cholinergic drugs and dopamine receptor agonists are used in addition to dopamine precursors, all drug treatments are only symptomatic treatments. For patients, the effects of disease progression and long-term use of these drugs The decline is inevitable.

Therefore, in recent years, regenerative therapy has been attempted in which fetal substantia nigra cells are transplanted into the substantia nigra striatum, and cells having differentiation ability such as embryonic stem cells, neural stem cells, and bone marrow stromal cells are differentiated. Research for producing dopaminergic neurons useful for transplantation in vitro has progressed, and has attracted much attention (see Non-Patent Document 1). However, transplantation with fetal substantia nigra cells may cause dyskinesia in patients who have stopped taking levodopa after transplantation, and symptoms may worsen if they do not continue to use immunosuppressants. Because of the use of substantia nigra cells collected from aborted fetuses, there are problems in both ethical and supply aspects, and this is not a widely popular treatment. On the other hand, research for inducing differentiation of cells having the above-mentioned differentiation ability into dopaminergic neurons has been widely promoted, and neuronutrition such as GDNF (glial cell-derived neurotrophic factor) and BDNF (brain-derived neurotrophic factor). Methods for inducing differentiation more efficiently have been developed by various approaches such as the addition of growth factors such as factors and FGF (fibroblast growth factor) and the introduction of transcription factors such as Nurr-1.
However, all studies using this method are at the laboratory level, such as animal experiments, and further basic research is necessary from the viewpoint of ensuring safety. Although we are currently investigating whether induced dopaminergic neurons can be supplied in a sufficient number of cells that can actually be used for transplantation into the patient's brain, the prospects for practical clinical application are Still not standing. Therefore, it is desired to establish a safe regenerative therapy that can be applied to the clinic without ethical problems such as the use of fetal cells and tissues as well as the stable supply of cells necessary for transplantation.

  Parkinson's syndrome, on the other hand, refers to diseases other than Parkinson's disease among diseases that exhibit the same extrapyramidal symptoms as Parkinson's disease. In addition to Parkinson's syndrome caused by drugs and cerebrovascular disorders, strokes, brain tumors and brain degeneration Parkinson's syndrome caused by diseases or the like is included. Of these, for drug-induced Parkinsonian syndrome, symptoms often disappear by stopping the ingestion of the causative drug, but for Parkinsonian syndrome that appears due to other diseases, the cause of the disease When there is no root treatment for treatment, there are many cases where symptomatic treatment is not improved, and as with Parkinson's disease, there is no definitive treatment yet.

In contrast, the present inventors have developed a method for inducing differentiation of mesoderm stem cells or embryonic stem cells contained in bone marrow fluid or umbilical cord blood into neural stem cells or neurons, and transplanting these differentiation-induced cells. By discovering that the spinal cord injury site can be repaired in a rat spinal cord injury model (see Patent Document 1), on the other hand, by administering intravenously the mononuclear cell fraction of mesenchymal stem cells and bone marrow cells, In a rat cerebral infarction model, we succeeded in reducing the infarct volume, and found that administration of mesenchymal cells can obtain a protective effect on cranial nerves damaged by cerebral infarction and a cranial nerve regeneration effect (see Patent Document 2). We have been developing regenerative therapy using bone marrow-derived mesenchymal cells. However, even in Patent Documents 1 and 2, a study using mesenchymal cell transplantation has not yet been made for neurodegenerative diseases including Parkinson's disease.
Brian J. Snyder and C. Warren Olanow., Current Opinion in Neurology, 2005, 18: 376-385 WO2003 / 088075 WO2005 / 007176

  Therefore, an object of the present invention is to provide a highly safe therapeutic agent effective for the treatment of Parkinson's disease or Parkinson's syndrome, and a therapeutic method using the therapeutic agent.

  Accordingly, the inventors of the present invention have conducted intensive studies to solve the above-described problems, and have identified mesenchymal stem cells and cells associated with mesenchymal cells such as cells induced to differentiate from mesenchymal stem cells. It was found that by administering to a syndrome model rat, an extremely remarkable therapeutic effect can be obtained not only by local administration into the brain but also by intravenous administration, and the present invention was completed.

Accordingly, the present invention relates to a therapeutic agent for Parkinson's disease or Parkinson's syndrome comprising cells associated with the mesenchymal system.
The present invention also relates to the therapeutic agent, wherein the cells associated with the mesenchymal system are mesenchymal stem cells.
Furthermore, the present invention relates to the above therapeutic agent, wherein the cells related to the mesenchymal system are neural stem cells induced to differentiate from mesenchymal stem cells.

The present invention also relates to the aforementioned therapeutic agent, wherein the cells associated with the mesenchymal system are tyrosine hydroxylase positive neurons differentiated from mesenchymal stem cells.
The present invention further relates to the therapeutic agent described above, wherein an effective therapeutic effect can be obtained by intravenous administration.
The present invention also relates to the aforementioned therapeutic agent administered in the brain.
The invention also relates to a method of treatment for Parkinson's disease or Parkinson's syndrome, comprising administering to a subject a therapeutically effective amount of the therapeutic agent.
Furthermore, the present invention relates to the treatment method, wherein the cells associated with the mesenchymal system are autologous cells of the subject.

The present invention has an extremely excellent effect in the treatment of Parkinson's disease or Parkinson's syndrome, and this effect is caused by the substantia nigra in which cells associated with the administered mesenchymal system are degenerated and dropped from dopaminergic neurons. This is thought to be achieved by regenerating nerve tissue that has become dysfunctional in place of dopaminergic neurons that have migrated and settled at the striatum lesion site and have degenerated or dropped.
Thus, conventional therapeutic agents can only delay the progression of symptoms by supplementing mainly deficient dopamine, whereas the therapeutic agents of the present invention delay the progression of Parkinson's disease or Parkinson's syndrome In addition, it has an excellent therapeutic effect that could not be realized by conventional symptomatic therapy, in which lost nerve tissue can be reconstructed and its function can be restored.

  According to the present invention, cells related to the administered mesenchymal system and neural stem cells induced to differentiate from the cells are differentiated into nerve cells expressing tyrosine hydroxylase, which is a marker of dopaminergic neurons, at the lesion site. Since it can be established, it is possible to provide a simple regenerative therapy that does not require the step of differentiating cells having differentiation potential into tyrosine hydroxylase-positive neurons in vitro as in the prior art. In addition, according to the present invention, a cell having differentiation ability (for example, a mesenchymal stem cell or a neural stem cell induced to differentiate therefrom) is administered in an appropriate number of cells of a certain number or more, thereby relieving the lesion site. A sufficient number of cells are differentiated into tyrosine hydroxylase-positive neurons and settled at the lesion site, and an effective therapeutic effect can be obtained.

  In addition, surprisingly, the therapeutic agent of the present invention and the treatment method using the same can be directly administered to the substantia nigra striatum, even by intravenous administration of cells associated with the mesenchymal system. Equally superior therapeutic effects can be obtained, so it is not necessary to induce differentiation into dopaminergic neurons in vitro, and complicated and dangerous surgical operations such as direct administration to the brain of an individual It is an epoch-making thing that is extremely simple and safe.

  The administration of the therapeutic agent of the present invention makes it possible to treat Parkinson's disease or Parkinson's syndrome, which has been considered to have no radical cure, so great contributions can be expected in the medical and veterinary fields. In addition, since the cells related to the mesenchymal system used for the therapeutic agent of the present invention can be obtained from bone marrow, umbilical cord blood, peripheral blood or the like, ethical as in the case of using embryonic stem cells or fetal substantia nigra cells. There is no problem, and since it can be easily prepared from each patient's own autologous cells, the occurrence of undesirable biological reactions such as rejection can be avoided even during administration.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. In general, the terms and techniques used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genes and proteins and nucleic acid chemistry described herein are well known in the art. It shall be normally used. In general, unless otherwise indicated, the methods and techniques of the present invention are described in accordance with conventional methods well known in the art, as described in various general and more specific and cited herein. Performed as described in a specialized reference. Such references include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press (1989) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring. Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and 2000 supplement); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology -4 ^th Ed., Wiley & Sons (1999); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1990); and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1999) Etc.

The terms used herein for analytical chemistry, synthetic organic chemistry and medicinal chemistry and medicinal chemistry, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques shall be used for chemical synthesis, chemical analysis, drug manufacture, formulation and delivery, and treatment of subjects.
The term “subject” in the present invention means any living individual, preferably a vertebrate, more preferably a mammal, and still more preferably a human individual. In the present invention, the subject may be healthy or afflicted with any disease, but when treatment of Parkinson's disease or Parkinson's syndrome is intended, the subject afflicted with the disease or Experimentally affected subjects, for example rodents such as mice, rats, gerbils, guinea pigs, felines such as cats, pumas, tigers, deer animals such as deer and elk, minks, sheep, goats, cattle, Preferably, they are monkeys and humans.

In the present invention, a therapeutic agent for Parkinson's disease or Parkinson's syndrome comprising cells associated with the mesenchymal system is provided.
In the present invention, Parkinson's disease refers to a basal ganglia degenerative disease whose main symptom is abnormalities in extrapyramidal functions such as tremor at rest, muscle stiffness, ataxia, and posture reflex disorder.
Further, in the present invention, Parkinson's syndrome refers to all diseases other than Parkinson's disease, showing the extrapyramidal symptoms characteristic of Parkinson's disease, in addition to drug-related Parkinson's syndrome, cerebrovascular disorder Parkinson's syndrome, brain tumors, Includes Parkinsonian syndrome caused by basal ganglia degeneration, striatal nigra degeneration, progressive supranuclear paralysis, and the like.

In the present invention, cells related to the mesenchymal system are mesenchymal stem cells (MSC (mesenchymal stem cell)), mesenchymal cells, progenitor cells of mesenchymal cells, cells derived from mesenchymal cells, It means a cell derived from a leaf stem cell.
The mesenchymal stem cells can be obtained from an initial culture of various mesenchymal tissues such as bone marrow, peripheral blood, skin, fat, hair root, muscle tissue, endometrium, blood vessels, blood, and umbilical cord blood. It can be a stem cell.
A progenitor cell of a mesenchymal cell means a cell that is differentiated from a mesenchymal stem cell and is in the process of differentiation into a mesenchymal cell.
A mesenchymal cell is a cell that is generated by differentiation of a mesenchymal stem cell and does not have the ability to differentiate in many directions like a stem cell, but has a differentiation ability and a proliferation ability in a certain direction. It is a cell that has stopped in the G0 phase in a normal state but can enter the G1 phase (start of division) by stimulation. Mesenchymal cells include, for example, stromal cells and cells having the properties of stromal cells. Mesenchymal cells are present in all organs such as subcutaneous tissue, lungs, and liver, and are present in mesenchymal tissues such as bone, cartilage, fat, tendon, skeletal muscle, and bone interstitium.

  Cells derived from mesenchymal cells in the present invention include (1) cardiovascular cells such as endothelial cells or cardiomyocytes, or progenitor cells of cardiovascular cells, and cells having properties as these cells ( 2) Cells of any of bone, cartilage, tendon or skeletal muscle, and precursor cells of any of bone, cartilage, tendon, skeletal muscle, or adipose tissue, and cells having these cell properties, (3) Nervous system cells or progenitor cells of neural cells, further cells having properties as these cells, (4) endocrine cells or progenitor cells of endocrine cells, and cells having properties as these cells ( 5) Hematopoietic cells or progenitor cells of hematopoietic cells, further cells having properties as these cells, (6) Hepatocytes or progenitor cells of hepatocytes, and further cells having properties as these cells are included.

  The cells induced to differentiate from mesenchymal stem cells used in the present invention include all types of cells induced to differentiate from mesenchymal stem cells by various known methods. In addition to progenitor cells and mesenchymal cell precursor cells, neural stem cells, tyrosine hydroxylase positive neurons, adipocytes, osteoblasts, chondrocytes, skeletal muscle cells, ligament cells, cardiomyocytes and the like can be mentioned. The tyrosine hydroxylase-positive neuron in the present invention means a neuron expressing a tyrosine hydroxylase that is a marker of catecholamine-producing cells. ) Acting nerve cells are included. Preferred for use in the present invention as cells derived from mesenchymal stem cells are neural stem cells (particularly neural stem cells forming a neurosphere) and tyrosine hydroxylase positive neurons (particularly dopaminergic neurons). is there.

  Preferred examples of cells related to the mesenchymal system used in the present invention include bone marrow cells (particularly, mononuclear cell fraction components of bone marrow cells), cord blood cells, peripheral blood cells, fetal liver cells, endometrial cells, cartilage Cells, skin cells, adipocytes, vascular endothelial cells, amniotic membrane, skeletal muscle cells, etc., and more preferable examples are mesenchymal systems obtained from bone marrow cells (particularly, mononuclear cell fraction components of bone marrow cells). Stem cells, neural stem cells differentiated from such mesenchymal stem cells (particularly neural stem cells forming a neurosphere), tyrosine hydroxylase positive neurons (particularly dopaminergic neurons), and the like. It is desirable that the tyrosine hydroxylase-positive neuron induced to differentiate from mesenchymal stem cells is induced to differentiate via neural stem cells in the process of inducing differentiation from mesenchymal stem cells.

  In the bone marrow, hematopoietic stem cells and mesenchymal stem cells exist as stem cells. The term “stem cell” as used herein refers to an undifferentiated cell that has both the ability to self-proliferate and the ability to differentiate into a cell having a specific function in the process of physiological growth and differentiation of cells constituting a living body. That is. Hematopoietic stem cells are stem cells that differentiate into red blood cells, white blood cells, or platelets. When mesenchymal stem cells differentiate into nerves via neural stem cells, differentiate directly into neural cells without going through neural stem cells, differentiate into internal organs when differentiated into neural cells via stromal cells In some cases, it is known that the vascular system differentiates into bone, cartilage, fat, or muscle.

In the present invention, mainly mesenchymal stem cells and cells induced to differentiate therefrom are used, but hematopoietic stem cells and other stem cells and progenitor cells in the body can also be used. Mesenchymal stem cells can be obtained by separating from bone marrow cells collected from bone marrow, for example. In addition, bone marrow cells from which mesenchymal stem cells have not been separated can be used in the present invention in the same manner as mesenchymal stem cells, although their effectiveness is slightly inferior.
Mesenchymal stem cells can also be prepared from peripheral blood. In fact, the present inventors have succeeded in inducing cells cultured from cells existing in peripheral blood into cells that exhibit markers of neural stem cells and neural cells (neural cells and glial cells). . On the other hand, the present inventors have already cultivated mesodermal stem cells (mesenchymal stem cells) prepared from a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood, or embryonic stem cells in a basic culture solution, It has been found that the mesodermal stem cells (mesenchymal stem cells) or the embryonic stem cells induce differentiation into neural stem cells, neural cells or glial cells (see Patent Document 1). Therefore, by culturing cells in peripheral blood, mesenchymal stem cells and cells having the same function can be prepared and used in the present invention.

  In the present invention, a mesoderm stem cell means a cell having the ability to self-proliferate and the ability to differentiate into all the cells constituting a tissue that is developmentally classified as a mesoderm. Includes blood stem cells and bone marrow stem cells. Mesodermal stem cells are, for example, SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD11b (−), CD14 (−), CD34 (−), CD45 (− ), But is not particularly limited to these markers. Further, cells that are stem cells among so-called mesenchymal cells are also included in the mesodermal stem cells of the present invention.

Preferred embodiments of bone marrow cells, umbilical cord blood cells, peripheral blood cells, and fetal liver cells used in the present invention include a fraction of cells obtained from bone marrow, umbilical cord blood, peripheral blood, or fetal liver, Examples thereof include cell fractions containing cells that can differentiate into systemic cells (particularly neural stem cells and tyrosine hydroxylase positive neurons).
In one embodiment, the cell fraction is SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (−), CD34 (−), CD45 (−). It is a cell fraction containing the mesoderm stem cell which has the characteristics of these.
In the present invention, cell fractions other than those described above include Lin (−), Sca-1 (+), CD10 (+), CD11D (+), CD44 (+), CD45 (+), CD71 (+), Stromal cells having the characteristics of CD90 (+), CD105 (+), CDW123 (+), CD127 (+), CD164 (+), fibronectin (+), ALPH (+), collagenase-1 (+) A cell fraction containing cells having the characteristics of AC133 (+) or the like can be used.
In the present invention, the cells contained in the cell fraction are particularly preferably cells that can differentiate into neural cells (particularly neural stem cells or tyrosine hydroxylase positive neurons).

  The cell fraction used in the present invention is a mononuclear cell fraction obtained by separation from bone marrow cells, and can differentiate into neural cells (particularly neural stem cells and tyrosine hydroxylase positive neurons). A cell characterized by In another embodiment, for example, a mononuclear cell fraction obtained by separation from umbilical cord blood cells, peripheral blood cells, or fetal liver cells, and divided into neural cells (particularly neural stem cells or tyrosine hydroxylase positive neurons). Cells characterized by being capable of being transformed are included. In another embodiment, for example, mesenchymal stem cells in the bone marrow that have been released into the peripheral blood using an active substance or a drug, and divided into neural cells (particularly neural stem cells or tyrosine hydroxylase-positive neurons). Also included are cells characterized by being capable of being transformed. The differentiation of the cells contained in the cell fraction used in the present invention into neural cells is caused by transformation of so-called blood cells into neural cells, or bone marrow cells, umbilical cord blood cells, peripheral blood cells, etc. It is not clear whether this is due to the differentiation of immature cells that can differentiate into neurons, but the cells that differentiate into neural cells are mainly stem cells, that is, cells with self-proliferation ability and multipotency. It is believed that there is. In addition, cells that differentiate into nervous system cells can be stem cells that have differentiated to some degree in other germ layers.

  The cells contained in the cell fraction used in the present invention do not need to be grown by a trophic factor (can be grown by a trophic factor), are simple in terms of development of a nerve autograft technique, and It is highly realistic, and it can be said that its benefits in the medical industry are enormous. The bone marrow cells, umbilical cord blood cells or peripheral blood cells, or cell fractions thereof used in the present invention are generally derived from vertebrates. Preferably, it is derived from a mammal (eg, mouse, rat, rabbit, mink, cat, sheep, goat, cow, deer, pig, dog, monkey, human etc.), but is not particularly limited.

  The cell fraction used in the present invention is obtained by, for example, subjecting bone marrow cells collected from a vertebrate to density gradient centrifugation in a solution at a speed of 2000 revolutions for a time sufficient for separation according to the specific gravity. It can be obtained by collecting a cell fraction having a specific gravity contained in the range of 07 g / ml to 1.1 g / ml. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position according to the specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. The specific gravity of the cell fraction to be collected is preferably in the range of 1.07 g / ml to 1.08 g / ml (for example, 1.077 g / ml). As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto. Umbilical cord blood cells and peripheral blood cells collected from vertebrates can be prepared in the same manner as described above and used as a cell fraction.

  Specifically, first, bone marrow fluid (5 to 10 μl) collected from a vertebrate was mixed into a solution (culture medium L-15 (2 ml) + Ficol (3 ml)), centrifuged (2000 rpm for 15 minutes), Extract the mononuclear cell fraction (about 1 ml). This mononuclear cell fraction is mixed with a culture solution (NPBM 2 ml) for cell washing and centrifuged again (2000 rpm for 15 minutes). Next, after removing the supernatant, the precipitated cells are collected. In addition to the femur, the cell fraction used for the present invention can be collected from the sternum and the iliac bone forming the pelvis. Bone marrow fluid can be collected if it is a large bone other than these bones. Bone marrow fluid may be collected from bone marrow fluid or umbilical cord blood stored in a bone marrow bank.

  Another aspect of the cell fraction used in the present invention is a mononuclear cell fraction obtained by isolation and purification from bone marrow cells, umbilical cord blood cells, or peripheral blood cells, and is a neural cell (particularly, neural stem cell). It is a cell fraction containing mesodermal stem cells (mesenchymal stem cells) that can be differentiated into tyrosine hydroxylase positive neurons). As the cell fraction containing mesoderm stem cells, for example, a cell having a cell surface marker such as SH2 is selected from the above cell fraction obtained by centrifugation from bone marrow cells, umbilical cord blood cells, or peripheral blood cells. Can be obtained.

  In addition, a cell fraction containing mesodermal stem cells (mesenchymal stem cells) that can differentiate into neural cells is sufficient to separate bone marrow cells, umbilical cord blood cells, and peripheral blood cells collected from vertebrates according to specific gravity at 900 g. It can be prepared by performing density gradient centrifugation in a solution for a period of time, and recovering a cell fraction having a specific gravity within a range of 1.07 g / ml to 1.1 g / ml after centrifugation. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position according to the specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. The specific gravity of the cell fraction to be collected may vary depending on the type of animal from which the cells are derived (eg, human, rat, mouse). As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto.

Specifically, bone marrow fluid (25 ml), umbilical cord blood, or peripheral blood collected from vertebrates is first mixed with the same amount of PBS solution, centrifuged (900 g for 10 minutes), and the precipitated cells are mixed with PBS. Then, the blood component is removed by collecting (cell density is about 4 × 10 ⁷ cells / ml). Next, 5 ml of the mixture is mixed with Percol solution (1.073 g / ml) and centrifuged (900 g for 30 minutes) to extract the mononuclear cell fraction. For cell washing, the extracted mononuclear cell fraction is mixed with culture medium (DMEM containing 10% FBS and 1% anti-biotic-antimycotic solution) and centrifuged (2000 rpm for 15 minutes). Next, the supernatant after centrifugation is removed, and the precipitated cells are collected and cultured (37 ° C., 5% CO ₂ in air).

  Another aspect of the cell fraction used in the present invention is a mononuclear cell fraction obtained by separation from bone marrow cells and umbilical cord blood cells, and is a neural cell (particularly neural stem cell or tyrosine hydroxylase positive neuron cell). It is a cell fraction containing stromal cells that can be differentiated into). Stromal cells are, for example, Lin (−), Sca-1 (+), CD10 (+), CD11D (+), CD44 (+), CD45 (+), CD71 (+), CD90 (+), CD105 (+), CDW123 (+), CD127 (+), CD164 (+), fibronectin (+), ALPH (+), and collagenase-1 (+) cells. A cell fraction containing stromal cells can be obtained, for example, by selecting a cell having a cell surface marker such as Lin from the cell fraction obtained by centrifugation from bone marrow cells or cord blood cells. Can do.

  The cell fraction used in the present invention is obtained by subjecting bone marrow cells and umbilical cord blood cells collected from vertebrates to density gradient centrifugation in a solution for a time sufficient for separation according to the specific gravity at 800 g. It can also be prepared by collecting a cell fraction having a specific gravity contained in the range of 1.07 g / ml to 1.1 g / ml. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position according to the specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. The specific gravity of the cell fraction to be collected is preferably in the range of 1.07 g / ml to 1.08 g / ml (for example, 1.077 g / ml). As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto.

Specifically, first, bone marrow fluid or umbilical cord blood collected from a vertebrate is mixed with an equal amount of PBS solution (PBS containing 2% BSA, 0.6% sodium citrate and 1% penicillin-streptomycin). Then, 5 ml of the mixture is mixed with Ficol + Paque solution (1.077 g / ml) and centrifuged (800 g for 20 minutes) to extract the mononuclear cell fraction. This mononuclear cell fraction was used as a culture solution for washing cells (12.5% FBS, 12.5% horse serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol, 2 mM. (L-glutamine, αMEM containing 1 μM hydrocortisone and 1% anti-biotic-antimycotic solution) and centrifuged (2000 rpm, 15 minutes). Next, after removing the supernatant after centrifugation, the precipitated cells are collected and cultured (37 ° C., 5% CO ₂ in air).

Another embodiment of the cell fraction used in the present invention is a mononuclear cell fraction obtained by separation from bone marrow cells, umbilical cord blood cells, peripheral blood cells, or fetal liver cells, It is a cell fraction containing cells having the characteristics of AC133 (+) that can differentiate into stem cells and tyrosine hydroxylase positive neurons).
This cell fraction is obtained by, for example, selecting a cell having the cell surface marker of AC133 (+) from the cell fraction obtained by centrifugation from bone marrow cells, umbilical cord blood cells, or peripheral blood cells. Obtainable. As another embodiment, fetal liver cells collected from vertebrates are subjected to density gradient centrifugation in a solution at a rotation speed of 2000 for a time sufficient for separation according to the specific gravity. After centrifugation, the specific gravity is 1.07 g / ml. A cell fraction contained in the range of 1.1 g / ml can be collected, and a cell having the characteristics of AC133 (+) can be collected from the cell fraction. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position according to the specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. As a solution for density gradient centrifugation, Ficol liquid or Percol liquid can be used, but is not limited thereto.

  As a specific example, first, liver tissue collected from a vertebrate fetus is washed in an L-15 culture solution and treated with an enzyme (0.01% Dnase I, 0.25% trypsin and 0.1% collagenase). Incubate at 37 degrees for 30 minutes in the containing L-15 medium) and pipet to single cells. The fetal liver cells that have become single cells are centrifuged. The cells thus obtained are washed, and AC133 (+) cells are recovered from the washed cells using the AC133 antibody. As a result, cells capable of differentiating from fetal liver cells to nervous system cells can be prepared. Recovery of AC133 (+) cells using antibodies can be performed using magnetic beads or a cell sorter (such as FACS).

Cell fractions containing these mesodermal stem cells (mesenchymal stem cells), stromal cells, or AC133 positive cells can be efficiently obtained after direct transplantation into the substantia nigra-striatum or after intravenous administration. Differentiate into tyrosine hydroxylase positive dopaminergic neurons in the body. In particular, the cell fraction containing the mesodermal stem cells (mesenchymal stem cells) can be well engrafted and differentiated into dopaminergic neurons even when administered to Parkinson's disease / Parkinson syndrome model animals.
Examples of cells that can differentiate into neural cells contained in the cell fraction include neural stem cells, mesodermal stem cells (mesenchymal stem cells), stromal cells, and AC133 positive cells contained in the cell fraction. Although it is contained, as long as it can differentiate into a nervous system cell (especially tyrosine hydroxylase positive nerve cell), it is not restrict | limited to these.
Therefore, the therapeutic agent of the present invention may include not only bone marrow cells, umbilical cord blood cells, or peripheral blood cells, but also the above cell fraction.

Cells associated with the mesenchymal system used in the therapeutic agent of the present invention, for example, bone marrow cells, umbilical cord blood cells or peripheral blood cells, and mesenchymal stem cells or cells induced to differentiate from mesenchymal stem cells are used for administration as they are. However, various modifications may be made to improve the treatment efficiency. Thus, in another aspect of the invention, a therapeutic for Parkinson's disease or Parkinson's syndrome comprising cells associated with such a modified mesenchymal system may be provided.
Examples of the modification include introduction of a gene that improves the ability of the cell to produce dopamine.

  In the preparation of the therapeutic agent of the present invention, for example, (1) improving the proliferation rate or survival rate of the cells associated with the mesenchymal system or cells differentiated therefrom, or further adding to the nervous system cells Addition of substances that promote differentiation, introduction of genes having such effects, (2) addition of substances that extend the life of cells to be administered, or genes having such effects (telomerase genes, oncogenes, etc.) (3) Addition of a substance for the purpose of suppressing an immune reaction, or introduction of a gene expressing such an effect, (4) Parkinson's cells associated with the mesenchymal system Addition of substances for specific delivery to diseased or diseased sites of Parkinson's syndrome, introduction of genes having such effects, (5) possessing neuroprotective action Parkinson's disease or Parkinson's syndrome, such as addition of a substance or introduction of a gene expressing such an effect, (6) addition of a substance having an apoptosis-inhibiting effect at a lesion site, or introduction of a gene expressing such an effect Addition of a substance having an effect of enhancing the therapeutic effect and gene introduction may be performed. In addition, the therapeutic agent of the present invention may be added to those useful for research and diagnosis of Parkinson's disease or Parkinson's syndrome, such as labeling substances such as dyes, enzymes, fluorescent substances, and radioisotopes.

In a preferred embodiment of the present invention, the “cells induced to differentiate from mesenchymal stem cells” are obtained by culturing mesenchymal stem cells in a basic culture solution at a temperature of 33 ° C. to 38 ° C., preferably 37 ° C. Can be prepared. The basic culture solution is not particularly limited as long as it is a normal culture solution used for cell culture, but preferably DMEM (Dulbecco's Modified Eagle's Medium) or NPBM (Neural progenitor cell basic) Medium (Neural progenitor basal medium)) (Cambrex). The other components of the basic culture solution are not particularly limited, but preferably include F-12, FBS, neural cell survival factor (Cambrex) and the like. Concentrations in these culture solutions are, for example, 50% for F-12 and 10% for FBS. Further, the CO ₂ concentration in the culture solution is preferably 5%, but is not particularly limited. As other conditions, the cells may be cultured in a floating state (for example, a neurosphere state) or may be cultured in a state of being attached to a culture vessel. As the culture container, an uncoated dish (non-coated dish) may be used, or a coated dish may be used.

  In a preferred embodiment of the present invention, bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to the basic culture solution. In this case, these may be added alone or both may be added. Examples of the concentration of bFGF or EGF include 1 ng / ml to 100 ng / ml, preferably 10 ng / ml. Although there is no restriction | limiting in particular as an addition time and the addition method, Preferably, the method of adding every day, cultivating the said mesenchymal stem cell with this basic culture solution is mentioned. In addition, when performing differentiation induction of mesenchymal stem cells into neural stem cells, it is preferable to add bFGF and EGF to the basic culture solution.

  In the present invention, by culturing mesenchymal stem cells as described above under the above conditions, various types of cells that can ultimately differentiate into tyrosine hydroxylase-positive neurons (particularly dopaminergic neurons). Can be induced into the cells. Specific examples of such cells include neural stem cells, neural progenitor cells, and neural cells.

  Further, in a preferred embodiment of the present invention, “a tyrosine hydroxylase-positive neuron differentiated from a mesenchymal stem cell” is cultured from the mesenchymal stem cell by culturing as described above from the viewpoint of enhancing differentiation efficiency. In a dish in which differentiation-induced neural stem cells are further coated with poly-D-lysine / laminin, etc., FBS, L-glutamine, IL-1, penicillin-streptomycin, etc. are added to a mixed medium of DMEM and F-12. It is prepared by culturing for several days to one week after addition. In this case, the content of F-12 in the mixed medium is preferably 45% to 55%, more preferably 50%. The concentration of L-glutamine in the medium is preferably 1 mM to 5 mM, more preferably 2 mM, and the concentration of FBS is preferably 5% to 20%, more preferably 10%. The concentration of IL-1 is preferably 10 pg / mL to 200 pg / mL, more preferably 100 pg / mL, and the concentration of penicillin-streptomycin is preferably 0.1% to 2%, more preferably 1% .

For the preparation of the therapeutic agent of the present invention, cells (including modified ones) related to the mesenchymal system prepared as described above can be used as they are, but the prepared cells are proliferated as necessary. Then, it can be stored frozen and thawed for use in preparation of therapeutic agents.
The therapeutic agent of the present invention can be formulated by methods known to those skilled in the art. For example, if necessary, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection in a suspension. And also, for example, physiologically acceptable carriers or media, specifically sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives It can be formulated by combining with a binder or the like and mixing in a general unit dosage form.

In addition, a sterile composition for injection can be formulated according to a usual pharmaceutical method using a vehicle such as distilled water for injection.
As an aqueous solution for injection, for example, physiological saline or an isotonic solution containing physiologically acceptable substances (for example, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.) is used. As a suitable solubilizing agent, an alcohol such as ethanol, propylene glycol or polyethylene glycol, a nonionic surfactant such as polysorbate 80 or HCO-50, and the like may be appropriately used in combination.
When formulated as an oily liquid for injection, sesame oil, soybean oil and the like can be used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizer. In addition to a buffer such as a phosphate buffer and an anesthetic such as procaine hydrochloride, a stabilizer, an antioxidant and the like may be further blended. The prepared injection solution is usually filled in a container such as a suitable ampoule.

Administration of the therapeutic agent of the present invention to a subject may be performed by any route, but preferably intravenous administration, intraarterial administration, selective intraarterial administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, It is parenteral administration such as intracerebral administration, intrathecal fluid administration, more preferably intravenous administration or local intracerebral administration to the substantia nigra striatum, and most preferably intravenous administration.
The number of administration is preferably one time, but can be administered multiple times depending on the situation. Further, the administration time may be short or continuous. In the case of intravenous administration, it is possible to administer in the manner of normal blood transfusion, and it is not necessary to perform surgery on the subject, and there is no need for local anesthesia, thus reducing the burden on both the subject and the therapist. Can do. It is also advantageous in that the administration operation can be performed outside the operating room.

The present invention further relates to a method of treatment for Parkinson's disease or Parkinson's syndrome comprising administering (preferably intravenously) a therapeutically effective amount of the therapeutic agent of the present invention to a subject.
Cells associated with the mesenchymal system, such as bone marrow cells, umbilical cord blood cells, or peripheral blood cells, and mesenchymal stem cells or cells derived therefrom, included in the therapeutic agent used in the above therapeutic method are rejected by administration. In order to prevent this, unless a special operation such as immunosuppressive treatment is performed, it may be collected from the subject's own body (autologous cells) or derived from the subject (autologous cells derived from the subject) Preferred (autograft therapy). Such autotransplantation therapy is preferable in that it can avoid the combined use of immunosuppressive agents. However, when it is difficult to use autologous cells, cells derived from other subjects or other medical animals can be obtained by performing immunosuppressive treatment. It is also possible to use (other cells). As a therapeutic effect, an overwhelmingly better result can be expected when using autologous cells than when using other cells.

The cells used in the treatment method of the present invention may be frozen.
In addition, the autologous cells are those collected in an undifferentiated state from the subject's body, those obtained by performing genetic manipulation on cells collected in an undifferentiated state from the subject's body, or undifferentiated from the subject's body Any of those obtained by inducing differentiation of cells collected in a state may be used.
In the treatment method of the present invention, administration of the therapeutic agent of the present invention to a subject can be suitably performed, for example, according to the method described above. In addition, a doctor or veterinarian can appropriately modify the above method and administer the therapeutic agent of the present invention to a subject. In the treatment method of the present invention, the number of cells related to the mesenchymal system administered to the subject is not particularly limited as long as a good therapeutic effect is obtained, but at least 1 × 10 ⁴ to 10 ⁸ or more. For example, at least 1 × 10 ⁴ to 10 ⁶ or more in the case of intracerebral administration, and preferably at least 1 × 10 ⁶ to 10 ⁸ or more in the case of intravenous administration. As a specific example, when the subject is a human, at least 1 × 10 ⁶ or more when mesenchymal stem cells are administered into the brain, and at least 1 × 10 ⁸ or more when mesenchymal stem cells are administered intravenously. When neural stem cells or tyrosine hydroxylase positive neurons differentiated from leaf stem cells are administered in the brain, it is preferable that at least 1 × 10 ⁶ or more cells are contained in the therapeutic agent of the present invention.

  Further, the subject of the above-described treatment method of the present invention is not limited to only humans, but mammals other than humans (eg, rodents such as mice, rats, gerbils, guinea pigs, and felines such as cats, pumas, tigers, etc.) The method of the present invention can also be carried out in the same manner using cells related to the mesenchymal system in deer animals such as deer and deer, mink, sheep, goat, cow and monkey.

The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to these examples.
Example 1. Human Mesenchymal Stem Cell Culture and Differentiation Induction (1) Human Mesenchymal Stem Cell Culture Human mesenchymal stem cells consist of a mononuclear cell fraction obtained from human bone marrow, 10% MCGM (Mesenchymal Cell Growth Supplement), The cells were cultured in a tissue culture dish using SCBM (Mesenchymal Stem Cell Basal Medium) (Cambrex, USA) containing 2 mM L-glutamine and 0.1% penicillin-streptomycin. After growing about 70-80% of the dish area, the cells were subcultured.
The cells were photographed with an inverted microscope (CKX41, OLYMPUS) and a digital camera (C-5060 Wide Zoom OLYMPUS). A photographed image of human mesenchymal stem cells is shown in FIG. 1A (magnification magnification is 40 times).

(2) Differentiation induction from human mesenchymal stem cells to neurospheres (neurosphere) 2% NSF-1 in a non-coated dish without human mesenchymal stem cells (1 × 10 ⁶ cells) (NPBM (Neural Progenitor Basal Medium) containing Neural Cell Survival Factor) and 0.2% GA (gentamicin sulfate amphotericin-B) (Cambrex, USA) For 3 days, and continuously added trophic factors (EGF and bFGF, 10 ng / ml each).
The cells were photographed with an inverted microscope (CKX41, OLYMPUS) and a digital camera (C-5060 Wide Zoom, OLYMPUS). A photographed image of neural stem cells (neurosphere) induced to differentiate from human mesenchymal stem cells is shown in FIG. 1B (imaging magnification is 40 times).

(3) Differentiation induction from neural stem cells (neurosphere) to tyrosine hydroxylase-positive neurons Neural stem cells (neurosphere) (0.2 × 10 ⁶ cells) obtained in (2) above were converted into PDL (poly-D-lysine). / Laminin-coated dishes were cultured for 1 week in DMEM: F-12 (1: 1) supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin and IL-1 (100 pg / mL). The obtained cells were seeded in a 2-well chamber coated with PDL / laminin, and further cultured for 2 days. Cells were fixed with 4% paraformaldehyde, and rabbit anti-tyrosine hydroxylase polyclonal antibody (Chemicon, Cat. # AB151) (1000-fold dilution) as the primary antibody (Alexa Fluor as secondary antibody) The sample was stained with 594 goat anti-Rabbit IgG (Molecular Probes, Cat. # Z25307), and the stained image was observed with a confocal laser microscope (LSM5 Pascal, Carl Zeiss). The result is shown in FIG. 1C (imaging magnification is 630 times).

Example 2 Transplantation of cells associated with the mesenchymal system into Parkinson's disease / Parkinson syndrome model rats (1) Preparation of Parkinson's disease / Parkinson syndrome model rats
Sprague-Dawley female rats (body weight 200-230 g, 8-9 weeks old) were treated with 6-hydroxydopamine (6-OHDA, Sigma) in 12 μg / 4 μl (saline containing 0.2% ascorbic acid) in the brain. (Coordinates of Injection; AP-4.5 L-1.5 V-7.8 from Bregma). The injection into the brain was performed slowly at a rate of 0.5 μl / min, and the needle was left without removing the needle for 5 minutes or more after the injection, and then the needle was slowly removed.
Two weeks after 6-hydroxydopamine administration, D-methamphetamine (Dainippon Sumitomo Pharma Co., Ltd.) was administered intraperitoneally at 5 mg / kg, and a rotational exercise test was performed for 2 hours using Roto-Rat (MED, USA). A sample having an average rotational speed exceeding 360 times (clockwise) / hour was used in the following experiment.

(2) Introduction of LacZ gene into cells related to mesenchymal system Human mesenchymal stem cells prepared in Example 1 (using passage no. 4-6), human mesenchymal stem cells The LacZ gene was introduced into the induced neural stem cells (neurosphere) and further differentiated tyrosine hydroxylase positive neurons before transplantation into the model rat prepared in (1) above. Recombinant adenovirus Adex1CAlacZ was used for introduction of the LacZ gene into these cells. Details of the construction procedure are described in the following documents (I. Nakagawa, M. Murakami, K. Ijima, S. Chikuma, I. Saito, Y. Kanegae, H. Ishikura, T. Yoshiki, H. Okamoto, A. Kitabatake, T. Uede, Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG, Hum. Gene Ther. 9 (1998) 1739-1745. Y. Nakamura, H. Wakimoto, J. Abe, Y. Kanegae, I. Saito, M. Aoyagi, K. Hirakawa, H. Hamada, Adoptive immunotherapy with murine tumor-specific T lymphocytes engineered to secrete interleukin 2, Cancer Res. 54 (1994) 5757-5760. M. Takiguchi, M. Murakami, I. Nakagawa, I. Saito, A. Hashimoto, T. Uede, CTLA4IgG gene delivery prevents autoantibody production and lupus nephritis in MRL / lpr mice, Life Sci. 66 (2000) 991-1001.).

  This recombinant adenovirus has an adenovirus serotype-5 genome with deletions of the E1A, E1B and E3 regions to prevent viral replication. Instead of the E1A and E1B regions, it is a β-galactosidase gene of E. coli. A lacZ gene is a CAG promoter composed of a cytomegalovirus enhancer and a chicken β-actin promoter (H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene 108 ( 1991) 193-199.) And the rabbit β-globin polyadenylation signal. The recombinant adenovirus was grown and isolated in 293 cells and the virus solution was stored at -80 ° C until use. A virus solution was added to each of the three types of cells so as to give 3000 OPU / cell, and the mixture was allowed to stand at 37 ° C. for 3 hours in DMEM containing 10% fetal calf serum to infect a recombinant adenovirus in vitro.

(3) Transplantation of mesenchymal stem cells and cells derived therefrom into the striatum Within 24 hours from the infection with the recombinant adenovirus, the three types of cells were transferred for 1 week from the rotational movement test of (1) above. Each model rat was transplanted by direct administration to the striatum. All cells were transplanted at 1.5 × 10 ⁴ cells / 6 μl at two locations (Coordinates of Injection: AP 0.5 L-3.2 V-6.5 to −4.5 and AP 0.5 L -2.5 V-6.5 to -4.5) 3 μl each (total 6 μl), cell injection rate 0.5 μl / min, V from −6.5 to −4.5 1 mm / μl This was performed while slowly pulling up the needle at a rate of / 2 minutes, and the cell was left without removing the needle for 5 minutes or more after cell injection, and then the needle was slowly removed. As a control, rats were also prepared in which only medium (DMEM) (6 μl) was directly administered to the striatum in the same manner.

(4) Intravenous administration of mesenchymal stem cells Within 24 hours after infection with recombinant adenovirus, the human mesenchymal stem cells were transferred to the left femoral vein of a model rat in which one week had passed since the rotational movement test of (1) above. Was administered at 1 × 10 ⁶ cells / 500 μl.

(5) Immunohistochemical staining of striatal slices Approximately 1 week after administration of cells as described in (3) or (4) above, the rats were anesthetized and perfused and fixed with 4% paraformaldehyde and removed. The brains were fixed overnight with 4% paraformaldehyde. The next day, the fixed brain was dehydrated with PBS containing sucrose at 4 ° C. (4 hours with 10% sucrose solution, then 4 hours with 20% sucrose solution, and overnight with 30% sucrose solution). The next day, a frozen section having a thickness of 20 μm was prepared.

Rabbit Anti-Tyrosine hydroxylase polyclonal antibody (Chemicon, Cat. # AB151) (1000-fold diluted) and anti-β-galactosidase monoclonal antibody (Anti-β-) were used as primary antibodies for the prepared sections. Using galactosidase, Purified Monoclonal Antibody (Promega, Cat. # Z3781) (diluted 2000 times), Alexa Fluor 488 Anti-Rabbit IgG (Molecular Probes, Cat. # Z25302) and Alexa Fluor 594 Anti-Mouse as secondary antibodies, respectively Double staining was performed using IgG ₂ a (Molecular Probes, Cat. # Z25107), and the stained image was observed with a confocal laser microscope (LSM5 Pascal, Carl Zeiss). Fig. 2 shows a striatal staining image when human mesenchymal stem cells, neurospheres, and tyrosine hydroxylase positive neurons are directly transplanted into the striatum, and when human mesenchymal stem cells are administered intravenously. Each of the striatum stained images is shown in FIG. 3 (the photographing magnification is 200 times).

From the results shown in FIG. 2, human mesenchymal stem cells, neural stem cells (neurospheres) and tyrosine hydroxylase positive neurons that have been directly transplanted into the striatum and introduced with the lacZ gene are firmly established in the striatum. It is understood. In particular, in the striatum of rats transplanted with human mesenchymal stem cells and neural stem cells (neurosphere), since cells expressing lacZ also express tyrosine hydroxylase, these cells also become striatum. After establishment, it has been shown to differentiate into neurons that express tyrosine hydroxylase.
Similarly, from the results of FIG. 3, since cells expressing lacZ also express tyrosine hydroxylase, mesenchymal stem cells administered intravenously also colonize the striatum and express tyrosine hydroxylase. It was shown to differentiate into neurons.

(6) Evaluation of cytotherapeutic effect in model rat D-methamphetamine (large) after 1, 2, 4, 6, 8, 10 and 12 weeks after administration of the cells as described in (3) or (4) above Nippon Sumitomo Pharma Co., Ltd. was administered intraperitoneally at 5 mg / kg, and a rotational motion test was performed for 2 hours using Roto-Rat (MED, USA) to evaluate the inhibitory effect on rotational motion. The results are shown in FIGS.

From FIG. 4, in any group to which any cell was administered, rotational movement (clockwise) was induced by methamphetamine administration before cell administration, whereas methamphetamine was administered 6 weeks after cell administration. It is understood that the number of rotational movements after administration is reduced compared to before cell administration. (▲ in FIG. 4 indicates the counterclockwise rotation number after methamphetamine administration, and ■ indicates the clockwise rotation number after methamphetamine administration)
Further, from FIG. 5, as compared with the control group, the number of rotational movements started to decrease from one week after administration in all the groups administered with cells, and after 6 weeks reached about 20 to 50% before cell administration. It became clear that the rotational speed decreased. Furthermore, it was shown that intravenous administration can achieve the same effects as direct administration into the brain by administering a number of cells several tens of times greater than direct administration into the brain.

  As described above, human mesenchymal stem cells, neural stem cells (neurosphere) or tyrosine hydroxylase positive neurons were administered directly into the brain, and human mesenchymal stem cells were administered intravenously It was suggested that the cells settle in the striatum as tyrosine hydroxylase-positive neurons and improve the symptoms of Parkinson's disease / Parkinson syndrome.

  As described above, according to the present invention, it is possible to provide a therapeutic agent for Parkinson's disease or Parkinson's syndrome comprising cells associated with the mesenchymal system and a treatment method using the same. The invention of the present application is a mesenchymal stem cell or a cell induced to differentiate from a mesenchymal stem cell, for Parkinson's disease, for which there has been no fundamental therapeutic method and symptomatic treatment has existed. It provides a highly effective therapeutic agent and therapeutic method that enables regeneration of the striatum by administering cells related to the leaf system into the body, and it is easier not only for intracerebral administration but also for easier treatment. Since a good curative effect can be obtained even by intravenous administration, it is extremely revolutionary that is expected to be used in actual clinical settings. Therefore, similarly, clinical application is expected for Parkinson's syndrome that exhibits the same symptoms as Parkinson's disease.

It is a figure which shows the image of a human mesenchymal stem cell. It is a figure which shows the image of the neural stem cell (neurosphere) differentiation-induced from the human mesenchymal stem cell. It is a figure which shows the tyrosine hydroxylase positive nerve cell differentiation-induced from the human mesenchymal stem cell. Human mesenchymal stem cells transfected with the lacZ gene (first row from the left), neural stem cells (second row from the left), and tyrosine hydroxylase positive neurons (left) transplanted directly into the rat striatum (3rd column from the top): Stained image in the striatum (first column from the top: stained image with anti-tyrosine hydroxylase antibody, second column from the top: stained image with anti-β-galactosidase antibody, third column from the top: 2 above It is a figure which shows the superimposition of two dyeing | staining images. Stained images of striatum of human mesenchymal stem cells transfected with lacZ gene administered to rats intravenously (left: stained with anti-β-galactosidase antibody, right: stained with anti-tyrosine hydroxylase antibody) FIG. It is a figure which shows the rotational motion number before various cell administration and 6 weeks after administration. It is a figure which shows progress of the rotation rate after 1 week, 2 weeks, 4 weeks, and 6 weeks after various cell administration.

Claims (8)

  A therapeutic for Parkinson's disease or Parkinson's syndrome comprising cells associated with the mesenchymal system.   The therapeutic agent according to claim 1, wherein the cells associated with the mesenchymal system are mesenchymal stem cells.   The therapeutic agent according to claim 1, wherein the cells associated with the mesenchymal system are neural stem cells induced to differentiate from mesenchymal stem cells.   The therapeutic agent according to claim 1, wherein the cells associated with the mesenchymal system are tyrosine hydroxylase positive neurons differentiated from mesenchymal stem cells.   The therapeutic agent according to claim 1, wherein an effective therapeutic effect is obtained by intravenous administration.   The therapeutic agent according to claim 1, which is administered in the brain.   A therapeutic method for Parkinson's disease or Parkinson's syndrome, comprising administering to a subject a therapeutically effective amount of the therapeutic agent according to any one of claims 1 to 6.   The treatment method according to claim 7, wherein the cells associated with the mesenchymal system are autologous cells of the subject.

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