JP7076709B2 - Mesenchymal stem cell activator - Google Patents

Description

[Related application]
This specification includes the contents described in the specification of Japanese Patent Application No. 2016-160268 (filed on August 18, 2016) which is the basis of the priority of the present application.
[Technical field]
The present invention uses β-carotene as a mesenchymal stem cell activator and a stem cell age-improving agent, and tissue repair by using the mesenchymal stem cell activator or the stem cell age-improving agent in combination with mesenchymal stem cells.・ Regarding regenerative medicine.

Mesenchymal stem cells (MSCs) are known to have a protective effect on the brain (parenchyma and blood vessels). It has been confirmed using an experimental infarction model that MSC administration after cerebral infarction reduces infarct volume and improves behavioral function (Non-Patent Documents 1 to 3 and Patent Document 1). In addition, many treatments for patients with cerebral infarction by intravenous administration of MSC have been carried out, and improvement of motor function and injured site has been reported (Non-Patent Document 4 and Patent Document 2).

In patients with SCI, intravenous administration of MSCs has been shown to restore function, promote axonal regeneration, and reduce the number of injured sites. Although many effects of MSCs have been reported in patients with spinal cord injury in the acute phase, studies on patients in the chronic phase are limited, and their effects have not been sufficiently confirmed.

Many mechanisms of action have been speculated for the therapeutic mechanism of MSC, and these are classified into three categories: neurotrophic / protective action by neurotrophic factors, angiogenic action (restoration of cerebral blood flow), and nerve regeneration. .. It is predicted that the neurotrophic / protective action is exerted through humoral factors such as BDNF (Brain Derived Neurotrophic Factor) and GDNF (Glial Derived Neurotrophic Factor), which are neurotrophic factors. There are two possible mechanisms for angiogenesis, one is that the MSC accumulated in the lesion secretes angiogenic factors and induces angiogenesis, and the other is that the administered MSC itself is a blood vessel. It differentiates into the endothelium and forms new blood vessels. There are two possible mechanisms for nerve regeneration, one is that MSCs accumulated in the lesion promote endogenous neurogenesis, and the other is that the administered MSCs themselves become nerve cells and glial cells. Is to differentiate with. However, all of the above mechanisms of action are only speculations from the observed phenomena, and the mechanism by which intravenous administration of MSC is used to treat cerebral infarction and spinal cord injury has not been demonstrated.

The profile of mesenchymal stem cells changes with age. It has been reported that telomerase activity of adipose-derived stem cells is improved by culturing them in a medium containing antioxidants such as selenium and β-carotene and cytokines (Patent Document 3). Beta-carotene is a type of carotenoid that has a strong antioxidant effect and is converted to vitamin A in the body as needed. Therefore, it is known that β-carotene has a function of maintaining normal mucous membrane, skin, immune function, and visual acuity, but its action on mesenchymal stem cells and its mechanism have not been elucidated.

WO2002 / 000849 WO2009 / 034708 Special table 2015-500018

Iihoshi S. et al., Brain Res. 2004; 1007: 1-9. Nomura T. et al., Neuroscience. 2005; 136: 161-169. Honma T. et al., Exp. Neurol. 2006; 199: 56-66. Honmou O. et al., Brain. 2011; 134: 1790-1807.

The subject of the present invention is to elucidate the action of physiologically active substances on the profile and therapeutic effect of mesenchymal stem cells (MSC), and based on the results, the homeostasis of stem cells with respect to the development, age-related changes, and injury healing of brain nerves. From the viewpoint, it is to provide a new treatment method for intractable neurological diseases.

The inventors have found that the number of mesenchymal stem cells in the bone marrow decreases with aging and the proliferative capacity also decreases, but the administration of β-carotene activates the mesenchymal stem cells and the mesenchymal stem cells in the bone marrow. It was confirmed that the number increased and it showed high proliferative ability. In addition, in a subacute cerebral infarction model, it was confirmed that administration of β-carotene improved motor function and increased plasticity.

The present invention has been completed based on the above findings, and relates to the following (1) to (11).
(1) Mesenchymal stem cell activator containing β-carotene as an active ingredient;
(2) The mesenchymal stem cell activator according to (1) above, wherein the mesenchymal stem cell is a mesenchymal stem cell derived from bone marrow or blood;
(3) The mesenchymal stem cell activator according to (2) above, wherein the bone marrow or blood is the bone marrow or blood to which the mesenchymal stem cells are administered;
(4) The mesenchymal stem cell activator according to any one of (1) to (3) above, wherein the mesenchymal stem cells are CD24 negative;
(5) Stem cell age improving agent for mesenchymal stem cells containing β-carotene as an active ingredient;
(6) The stem cell age improving agent according to (5) above, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow or blood;
(7) The stem cell age-improving agent according to (6) above, wherein the bone marrow or blood is the bone marrow or blood to which the mesenchymal stem cells are administered;
(8) The mesenchymal stem cell age-improving agent according to any one of (5) to (7) above, wherein the mesenchymal stem cells are CD24 negative;
(9) The mesenchymal stem cell activator according to any one of (1) to (4) above, or the above-mentioned mesenchymal stem cell activator, which is characterized by being used in combination with a tissue repair / regeneration drug containing CD24-negative mesenchymal stem cells. The stem cell age improving agent according to any one of (5) to (8);
(10) CD24 negative, which is used in combination with the mesenchymal stem cell activator according to any one of (1) to (4) above or the stem cell age improving agent according to any one of (5) to (8) above. Tissue repair / regeneration drug containing mesenchymal stem cells;
(11) The mesenchymal stem cell activator according to any one of (1) to (4) above or the stem cell age improving agent according to any one of (5) to (8) above, and a CD24-negative mesenchyme. A drug for tissue repair / regeneration in combination with a cell preparation containing stem cells. Examples of the above-mentioned "medicine for tissue repair / regeneration" include drugs for treating cerebral infarction, spinal cord injury, neurodegenerative disease, or dementia, improving higher-order functions, and extending lifespan.

According to the present invention, it was confirmed that administration (ingestion) of β-carotene can activate mesenchymal stem cells (MSC) and rejuvenate the age of stem cells. This will enable the establishment and rejuvenation of new treatment strategies for intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia caused by MSC.

FIG. 1 shows the bone marrow between the ginseng juice administration group (graph right) in which ginseng juice was orally ingested from 24 to 62 weeks of age and the control group at 8 weeks of age (graph left) and 62 weeks of age (center of the graph). It is the graph which compared the proliferative ability of the leaf stem cell. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passage days (passage number (P), passage days (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, day), P4Day6 (3rd passage). 4th generation, 6th day). FIG. 2 compares the proliferative capacity of bone marrow mesenchymal stem cells in the β-carotene-administered group (right graph) in which β-carotene was intraperitoneally administered from 33 to 43 weeks of age and the control group (left graph) of the same 43-week-old age. It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passage days (passage number (P), passage days (Day)). From the left, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, 5th day), P4Day6 (1st passage). 4th generation, 6th day). FIG. 3 compares the proliferative capacity of bone marrow mesenchymal stem cells in the ginseng juice administration group (graph right) in which ginseng juice was orally ingested from 33 to 43 weeks of age and the same 43-week-old control group (graph left). It is a graph. The vertical axis of the graph is the number of cells, and the horizontal axis is the number of passage days (passage number (P), passage days (Day)). From the left of the graph, P0Day9 (9th day of primary culture), P1Day9 (1st passage, 3rd day), P2Day4 (2nd passage, 4th day), P3Day5 (3rd passage, 5th day), P4Day6 ( 4th generation, 6th day). FIG. 4 shows an outline of the experimental protocol of Example 2. FIG. 5 is a graph comparing the improvement of motor function between the β-carotene-administered group and the control group in the cerebral infarction (subacute stage) model rat. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat. FIG. 6 is a graph comparing the improvement of motor function between the MSC group and the DMEM group (control group) in the cerebral infarction (chronic phase) model rat. The vertical axis of the graph is the maximum speed (m / min) that the rat can run, and the horizontal axis is the age of the rat.

[Mesenchymal stem cell activator]
The "mesenchymal stem cell activator" according to the present invention is a composition having an effect of restoring the cell number, proliferation ability and cell function of mesenchymal stem cells (MSC) that have decreased with aging or disease. Means.

The "mesenchymal stem cell activator" of the present invention contains β-carotene as an active ingredient, activates MSC, and has the effect of rejuvenating the stem cell age. When used in combination with MSC, the "mesenchymal stem cell activator" of the present invention can improve its tissue repair / regeneration ability and enhance the therapeutic effect on cerebral infarction, spinal cord injury, dementia and the like.

[Stem cell age improving agent]
The "stem cell age improving agent" according to the present invention is a composition that rejuvenates the stem cell age, and exhibits the cell number, proliferative ability, and cell function of mesenchymal stem cells (MSC) that have decreased with aging or disease. , Means a composition having a revitalizing effect.
It is known that the number of stem cells, their proliferative ability, their differentiation ability, and their associated stem cell functions such as tissue repair and regeneration decrease with aging. MSC exits the bone marrow into peripheral blood, supports systemic metabolism, and contributes to the maintenance of organs, including blood vessels and epidermis. When the number and function of MSCs declines, aging progresses and illness occurs (stem cell fileure). Even at the same age, motor function and health status are different, and life expectancy is also different for each person. It is considered that such individual differences in aging depend on the degree of activation of MSCs, and the main body of aging (age) of individual organisms is considered to be a decrease in MSC activity. The "stem cell age" according to the present invention is an index indicating a cell age different from the actual age, in other words, the youth of a living body or a cell.

[Mesenchymal stem cells]
The "mesenchymal stem cell" according to the present invention is a stem cell having pluripotency and self-renewal ability, which is present in a trace amount in the stromal cells of the mesenchymal tissue, such as bone cells, cartilage cells, and fat cells. It is known that it not only differentiates into bound tissue cells, but also has the ability to differentiate into nerve cells and myocardial cells.

Mesenchymal stem cells are present throughout the body such as bone marrow, peripheral blood, umbilical cord blood, fetal embryo, placenta, fat, and brain, but in the present invention, human bone marrow or blood-derived mesenchymal stem cells (bone marrow mesenchymal stem cells) In particular, human bone marrow mesenchymal stem cells are preferred. Bone marrow mesenchymal stem cells are 1) expected to have remarkable effects, 2) low risk of side effects, 3) expected supply of sufficient donor cells, and 4) non-invasive treatment and autologous transplantation possible. 5) Low risk of infectious disease, 6) No concern about immune rejection, 7) No ethical problems, 8) Easy to be socially accepted, 9) Easy to be widely established as general medical treatment There are advantages such as. Furthermore, bone marrow transplantation therapy is a treatment already used in clinical practice, and its safety has been confirmed. In addition, bone marrow-derived stem cells are highly migratory, and can be expected to have a therapeutic effect by reaching the target damaged tissue not only by local transplantation but also by intravenous administration.

The cell may be a cell differentiated from an ES cell or an induced pluripotent stem cell (iPS cell or the like), a cell that has been established, or a cell that has been isolated and proliferated from a living body. .. The cells may be derived from allogeneic cells or autologous cells, but mesenchymal stem cells derived from autologous cells (derived from the patient's own cells) are preferable.

As will be described later, when the mesenchymal stem cells are used as a pharmaceutical in combination with the mesenchymal stem cell activator or the stem cell age-improving agent (β-carotene) according to the present invention, the mesenchymal stem cells are CD24 negative, which is a differentiation marker. It is preferable that the cells maintain an undifferentiated state. CD24-negative mesenchymal stem cells are characterized by high proliferation rate and survival rate after introduction into the body, and can be further used in combination with a mesenchymal stem cell activator or a stem cell age-improving agent (β-carotene). It is expected that high MSC will establish a new treatment strategy for intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia, and improve the therapeutic effect. The inventors have also developed a method for obtaining such undifferentiated mesenchymal stem cells, the details of which are described in WO2009 / 00253.

When used in combination with the mesenchymal stem cell activator or stem cell age-improving agent according to the present invention, in addition to CD24, at least one of the mesenchymal stem cells selected from CD73, CD90, CD105, and CD200 is positive, and / Or at least one selected from CD19, CD34, CD45, CD74, CD79α, and HLA-DR is characterized by being negative. Preferably, the mesenchymal stem cells used in the present invention are positive for 2 or more of CD73, CD90, CD105, and CD200, and negative for 4 or more of CD19, CD34, CD45, CD74, CD79α, and HLA-DR. It is characterized by being. More preferably, the mesenchymal stem cells used in the present invention are characterized by being positive for CD73, CD90, CD105, and CD200 and negative for CD19, CD34, CD45, CD74, CD79α, and HLA-DR. Be done.

In the above method developed by the inventors, cells separated from bone marrow or the like under conditions that do not substantially come into contact with an anticoagulant (heparin or the like) contain human serum (preferably autologous serum) and anticoagulant. Proliferate using medium that does not contain agents (such as heparin) or contains very low concentrations.

The density of cells in the medium affects the nature of the cells and the direction of differentiation. In the case of mesenchymal stem cells, if the cell density in the medium exceeds 8,500 cells / cm ² , the cell properties will change, so subculture at a maximum of 8,500 cells / cm ² or less. Is preferable, and more preferably, subculture is performed when the number reaches 5,500 / cm ² or more.

Since the method developed by the inventors uses a human serum-containing medium, it is desirable that the medium is exchanged as few times as possible in consideration of the burden on the serum donor, for example, at least once a week, more preferably weekly. Change the medium once or twice.

For culturing, subculture is repeated until the total number of cells is ¹⁰⁸ or more. The number of cells required can vary depending on the intended use, for example, the number of mesenchymal stem cells required for transplantation for the treatment of cerebral infarction is thought to be ¹⁰⁷ or more. .. According to the method developed by the inventors, ¹⁰⁷ mesenchymal stem cells can be obtained in about 12 days.

Proliferated mesenchymal stem cells may be stored, if necessary, by a method such as cryopreservation (for example, in a deep freezer at −152 ° C.) until used. For cryopreservation, a medium containing serum (preferably human serum, more preferably autologous serum), dextran, DMSO (medium for mammalian cells such as RPMI) is used as the cryopreservation solution. For example, cells can be cryopreserved at −150 ° C. in a cryopreservation solution containing 20.5 mL of normal filtration-sterilized RPMI and 20.5 mL of autologous serum collected from a patient, 5 mL of dextran, and 5 mL of DMSO. For example, as DMSO, cryoserve manufactured by Nipro Co., Ltd. and dextran L injection manufactured by Otsuka Pharmaceutical Co., Ltd. can be used, but the DMSO is not limited thereto.

[Mesenchymal stem cell-containing tissue repair / regeneration drug]
The "medicine for tissue repair / regeneration containing mesenchymal stem cells" according to the present invention is a cell preparation containing the above-mentioned "mesenchymal stem cells (MSC)", and is a damaged site or an aged site due to aging (particularly). (Nervous system tissues such as brain and spinal cord) are repaired and regenerated (WO2009 / 034708). Furthermore, the inventors have also found that administration of MSCs improves synaptogenesis and plasticity (Japanese Patent Application No. 2016-091286, Japanese Patent Application No. 2016-091300).

Synapse formation means that axons extending from nerve cells appropriately extend to the vicinity of target cells and axons that establish nerve connections, reach the target, and are located between the axon terminal and the target cells or axons. It is the process of forming synapses and is an important process of forming correct neural circuits. When the MSC according to the present invention reaches the affected area, it differentiates into nerve cells and axons and forms synapses, thereby having an effect of reconstructing a neural circuit.

Brain plasticity is a phenomenon in which nerve cells and brain circuits create an optimal processing system according to the environment and needs. The MSC according to the present invention also has a function of promoting "brain plasticity" in which an undamaged site functions beyond the normal range so as to compensate for the function of the damaged site.

Furthermore, the inventors have also found that administration of "mesenchymal stem cells (MSCs)" has the effect of prolonging the lifespan of the administered subject.

It is preferable that the number of mesenchymal stem cells contained in the "medicine for tissue repair / regeneration containing mesenchymal stem cells" according to the present invention is large, but it is effective in consideration of the time of administration to the subject and the time required for culturing. It is practical that it is the minimum amount indicating. Therefore, in a preferred embodiment, the number of mesenchymal stem cells is 107 or more, preferably 5 × 10 ⁷ or more, more preferably ¹⁰⁸ or more, still more preferably 5 × ^{10 8 or} more.

The "medicine for tissue repair / regeneration containing mesenchymal stem cells" according to the present invention is preferably a parenteral administration preparation, more preferably a parenteral systemic administration preparation, particularly an intravenous administration preparation. Dosage forms suitable for parenteral administration include injections such as solution injections, suspension injections, emulsion injections, time-prepared injections, and implants. A pharmaceutical product for parenteral administration is in the form of an aqueous or non-aqueous isotonic sterile solution or suspension, eg, a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline. Media (particularly media used for culturing mammalian cells such as RPMI), physiological buffers such as PBS, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, preservatives, binding Agents and the like are appropriately combined to form an appropriate unit dosage form.

Examples of the aqueous solution for injection include physiological saline, medium, physiological buffer such as PBS, isotonic solution containing glucose and other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like. Can be used in combination with an appropriate solubilizing agent such as alcohol, specifically ethanol, polyalcohol, propylene glycol, polyethylene glycol or a nonionic surfactant such as polysorbitol 80, HCO-50 and the like.

[Combination of mesenchymal stem cell activator or stem cell age-improving agent with tissue repair / regeneration drug containing mesenchymal stem cells]
The "mesenchymal stem cell activator" or "stem cell age improving agent" (β-carotene) of the present invention can enhance the therapeutic effect of MSC by activating MSC and rejuvenating stem cell age. Specifically, the therapeutic effect of MSC administration is improved by using the mesenchymal stem cell activator or the stem cell age improving agent of the present invention in combination with "a tissue repair / regeneration drug containing mesenchymal stem cells". You can expect to do it. The effects are specifically described below.

(1) Treatment of cerebral infarction Cerebral infarction is a condition in which cerebral ischemia occurs due to obstruction or stenosis of cerebral arteries, and the brain tissue becomes necrotic or close to it. Mesenchymal stem cells have a protective effect on the brain (parenchymal and blood vessels), and in acute and subacute cerebral infarctions, administration of MSC reduces the infarct volume and improves behavioral function. Administration of a mesenchymal stem cell activator or a stem cell age-improving agent can be expected to further enhance the effect.

Necrotic cells and damaged nerve fibers do not recover in the chronic phase. Therefore, in the chronic phase of cerebral infarction, the main treatment is to prevent recurrence and to recover non-dead cells and cells that have stopped functioning around the necrotic cells to alleviate the condition. Has been considered. However, administration of MSCs promotes the reconstruction of neural circuits and the compensation of normal tissues, which enables recovery of motor function and brain function even in chronic cerebral infarction. Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the effect.

(2) Treatment of spinal cord injury Unlike peripheral nerves, the central nervous system including the spinal cord is not repaired or regenerated once it is injured. In particular, it is difficult to treat chronic spinal cord injury with advanced scarring, and clinical trials using ES cells have been tried but have not been successful. However, administration of mesenchymal stem cells promotes the reconstruction of neural circuits and the compensation of normal tissues, enabling recovery of motor and neural functions not only in the subacute phase but also in the chronic phase of cerebral infarction. Become. Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the effect.

(3) Treatment of Neurodegenerative Diseases The MSCs of the present invention include muscular atrophic lateral sclerosis (ALS), Parkinson's disease, progressive nuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), and black. It is also useful for neurodegenerative diseases such as plaque degeneration (SND), Shy-Drager syndrome, Olivopontocerebellar atrophy (OPCA), and spinal cord cerebral degeneration (SCD). Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the therapeutic effect.

(4) Treatment of dementia The inventors have demonstrated that intravenous administration of MSC improves cognitive function in spontaneously-onset rats with stroke-prone hypertension, and that vascular dementia can be treated with mesenchymal stem cells. .. Alzheimer-type dementia and vascular dementia have similar pathological conditions, and MSCs can be expected to improve cognitive function in Alzheimer-type dementia as well. Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the therapeutic effect of MSC on dementia.

(5) Treatment of psychiatric disorders In addition to the above-mentioned disorders, the MSC of the present invention includes schizophrenia, manic depression, personality disorders, mood disorders, psychological development disorders, stress-related disorders, autism, learning disorders, behaviors and It is also useful for mental illnesses such as emotional disorders, mental retardation, sleep disorders, feeding disorders, identity disorders, dissociative disorders, adaptation disorders, alcoholic disorders, and addictions. Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the therapeutic effect.

(6) Higher-order functions In addition to improving motor function and simple cognitive function, the MSC of the present invention improves higher-order functions such as attention disorder, memory disorder, aphasia, apraxia, apraxia, executive function, and emotional disorder. However, administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the effect.

(7) Life extension The MSC of the present invention has the effects of rejuvenation, physical strength enhancement, and life extension in healthy subjects, and in patients with dementia and intractable neurological diseases such as cerebral infarction and spinal cord injury, it has neurological function and In addition to improving motor function, it has the effect of prolonging life after treatment. Administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention can be expected to further enhance the effect.

The dose of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention is not particularly limited, and is appropriately determined according to the administration (ingestion) method, the age of the subject, and the health condition.

The administration route of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention is not particularly limited, but oral administration (ingestion) is convenient and preferable.

The form and shape of the mesenchymal stem cell activator or stem cell age-improving agent of the present invention is not particularly limited as long as it contains β-carotene, and may be a food or drink such as carrot juice or a supplement. .. Further, the mesenchymal stem cell activator of the present invention may be formulated in the form of a liquid, a tablet, a capsule or the like together with a pharmacologically acceptable carrier.

[Rehabilitation]
The administration of the mesenchymal stem cell activator or the stem cell age-improving agent of the present invention in combination with MSC is expected to significantly improve the effect by further using it in combination with rehabilitation. Although it is known that rehabilitation improves plasticity in patients with cerebral infarction and spinal cord injury, rehabilitation is used in combination with administration of the mesenchymal stem cell activator or stem cell age-improving agent (β-carotene) of the present invention and MSC. Thereby, the plasticity promoting function is further improved synergistically. The implementation method is not particularly limited, and rehabilitation by a robot or training by BMI (brain machine interface) may be used.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

Example 1. Effect of β-carotene on mesenchymal stem cells 1. Materials / Methods (1) Effect of ginseng juice (oral intake) SD rats (female) were randomly divided into a ginseng juice administration group (n = 5) and a control group (n = 5 at 8 and 62 weeks, respectively). In the carrot juice administration group, 100% carrot juice from Chiba prefecture (manufactured by Ikiiki Carrot Museum Sukoyaka) is approximately 100 ml / animal / day, and MF feed (manufactured by Oriental Yeast Co., Ltd.) is free feeding for 24 weeks. From to 62 weeks, bone marrow (2 femoral bones and 2 tibial bones) was collected at 62 weeks of age. The control group was bred with free feeding (feed: MF) and free water, and bone marrow (2 femurs and 2 tibias) was collected at 8 and 62 weeks of age.

The collected bone marrow was diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM) according to the previous report, and heat-inactivated 10% FBS, 2 mM l-glutamine, 100 U / ml penicillin, and 0.1 mg / ml streptomycin were added. Incubated at 37 ° C. in a 5% CO ₂ atmosphere for 3 days (Kim S. et al., Brain Res. 2006; 1123: 27-33. Ukai R. et al., J. Neurotrauma. 2007; 24: 508- 520.). The cells were subcultured four times by the above method and cultured, and the number of cells was counted at each subculture.

A similar experiment was also performed for ginseng juice administration from week 33 to week 43, and the presence or absence of administration was compared (carrot juice administration group (n = 5) and control group (n = 8)).
The evaluation was carried out by comparing the number of cultured cells per passage (average number of 5 cells) of the collected bone marrow up to the 4th passage (Passage = P4).

(2) Effect of β-carotene (intraperitoneal administration) SD rats (female) were randomly divided into a β-carotene administration group (n = 5) and a control group (n = 8). In the β-carotene administration group, water-soluble β-carotene (manufactured by Kyowa Hakko Bio Co., Ltd.) was intraperitoneally administered at about 0.1 μg / 0.5 ml / day from 33 weeks to 43 weeks, and the AIN76A diet (manufactured by Hokudo) was about 30 g. The animals were fed at / day and bred with free water supply, and bone marrow (2 femurs and 2 tibias) was collected. The control group was bred with free feeding (feed: MF) and free water, and bone marrow (2 femurs and 2 tibias) was collected at 8 and 62 weeks of age. The collected bone marrow cells were subcultured four times in the same manner as in (1) and cultured, and the number of cells was counted at the time of subculture.
The evaluation was carried out by comparing the number of cultured cells per passage (average number of 5 or 8 cells) of the collected bone marrow up to P4.

2. 2. Results (1) Ginseng juice (24-62 weeks oral administration: Fig. 1)
As can be seen from the data of the control group, at 62 weeks of age (center of the graph) compared to 8 weeks of age (left of the graph), a decrease in the number of cells is observed due to aging, but ginseng from 24 to 62 weeks of age. In the group in which the juice was orally ingested (right side of the graph), a marked increase in the number of cells was observed compared to the control group at 62 weeks of age (center of the graph), and the proliferation was close to that of young rats (8-week-old control group). It was confirmed that it has the ability.
(2) β-carotene (33-43 weeks intraperitoneal administration: Fig. 2)
As shown in FIG. 2, in the group in which β-carotene was intraperitoneally administered from 33 to 43 weeks of age, a significant increase in cell number (proliferative ability) was observed as compared with the control group of the same 43 weeks of age. .. This indicates that the effect of ginseng juice was due to β-carotene.
(3) Ginseng juice (33-43 weeks oral administration: Fig. 3)
As shown in FIG. 3, in the group in which ginseng juice was orally ingested from 33 to 43 weeks of age, a significant increase in cell number (proliferative ability) was observed as compared with the control group of the same 43 weeks of age. ..

3. 3. Discussion From the results of this example, it was confirmed that β-carotene has a significant MSC activating effect and a stem cell age rejuvenating effect. That is, it can be expected that ingestion of β-carotene improves the tissue maintenance / repair / regeneration effect of MSC, and improves the rejuvenation and the therapeutic effect of diseases (cerebral infarction, spinal cord injury, dementia, etc.).

Example 2. Effect of β-carotene administration on cerebral infarction (subacute stage) model rats 1. Materials / Methods (1) Cerebral infarction (subacute phase) model A rat middle cerebral artery occlusion (MCAO) model was used as a rat cerebral infarction model. According to the previous report, 9-week-old female SD rats (200-250 g) were anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg), and a 20.0-22.0 mm embolic thread (MONOSOF) was applied to the external neck. Inserted through an artery to perform permanent occlusion of the middle cerebral artery (Honma T. et al., Exp. Neurol. 2006; 199: 56-66. Sasaki M. et al., Methods Mol. Biol. 2009; 549: 187-195.). DWI-MRI was taken at 11 weeks of age to evaluate the initial infarct volume. Animals whose initial infarct volume was less than the standard (200 mm ³ ) were excluded from the experiment, and rats were randomly divided into a β-carotene-administered group (n = 2) and a control group (n = 2).

(2) Rehabilitation In the β-carotene administration group, 0.5 ml of physiological saline and 0.1 mg of β-carotene (manufactured by Kyowa Hakko Bio Co., Ltd.) were intraperitoneally administered every day. In the control group, 0.5 ml of physiological saline was intraperitoneally administered every day. All rats received daily cyclosporine A (10 mg / kg) intraperitoneally. For rehabilitation, I ran on the treadmill every day for 20 minutes from 12 weeks (treadmill angle 0 degrees, speed 8-12 m / min).

(3) Exercise function Exercise evaluation was performed by an exercise load test. The maximum running speed (m / min) was measured by forcibly running on a treadmill (angle 20 degrees) every week. FIG. 4 shows the protocol of this embodiment.

2. 2. Results As shown in FIG. 5, improvement in motor function was observed in the β-carotene-administered group, but no change in motor function was observed in the control group. It is considered that this is because the regeneration and plasticity of the injured tissue were enhanced by the administration of β-carotene.

Reference example 1. Effect of MSC administration on cerebral infarction (chronic phase) model rats 1. Materials and methods (1) Preparation of rat bone marrow-derived mesenchymal stem cells The experiments were carried out in accordance with the animal experiment management regulations of Sapporo Medical University. According to previously reported, bone marrow obtained from the femoral bones of mature SD rats was diluted to 25 ml with Dulbecco's modified Eagle's medium (DMEM) and heat-inactivated 10% FBS, 2 mM l-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin was added and incubated at 37 ° C. in a 5% CO ₂ atmosphere for 3 days (Kim S. et al., Brain Res. 2006; 1123: 27-33. Ukai R. et al., J. Neurotrauma. 2007. 24: 508-520.). The cells were cultured until they became confluent, and the adherent cells were exfoliated with trypsin-EDTA and subcultured three times at a density of 1 × 10 ⁴ cells / ml to obtain mesenchymal stem cells (MSC).

(2) Cerebral infarction (chronic phase) model rat A 9-week-old female SD rat (200-250 g) was anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg) and embolized at 20.0-22.0 mm. A thread (MONOSOF) was inserted through the external carotid artery for permanent middle cerebral artery occlusion (MCAO). Animals whose initial infarct volume was less than the standard (200 mm ³ ) were excluded from the experiment, and rats were randomly divided into MSC group (n = 8) and DMEM group (n = 8) as follows, 8 weeks after MCAO. Transplantation was performed in the chronic phase.
MSC group: MSC of SD rats 8 weeks after MCAO, DMEM containing ⁶ P2 1.0 × 10 in 1 ml was administered from the femoral vein.
DMEM group: 1 ml of DMEM was administered from the femoral vein.

(3) Rehabilitation From the day after administration, rehabilitation (treadmill angle 0 degrees, speed 8-12 m / min, 20 minutes) was performed, and cyclosporine administration (10 mg / kg) was administered every day for 1 week after transplantation, and then every other day. .. In all cases, daily rehabilitation (treadmill angle 0 degrees, speed 8-12 m / min, 20 minutes) was performed from the day after transplantation.

(4) Exercise function Exercise evaluation was performed by an exercise load test. The treadmill (angle 20 degrees) was forced to run every week, and the maximum running speed (m / min) was measured.

2. 2. Results As shown in FIG. 6, improvement in motor function was observed in the MSC group, but no change was observed in the DMEM group. From this, it was confirmed that administration of MSC in the chronic phase of cerebral infarction showed improvement in motor function. It is considered that this is because the regeneration and plasticity of the injured tissue were enhanced by the administration of MSC.

3. 3. Discussion The results of this reference example and the results of Examples 1 and 2 suggest that β-carotene administration (ingestion) improves the regeneration of damaged tissue and the enhancement of plasticity. In addition, it is suggested that the combined use of β-carotene administration (ingestion) with MSC administration further improves the regeneration and plasticity enhancement of damaged tissue by MSC.

INDUSTRIAL APPLICABILITY The present invention is useful for establishing a new therapeutic strategy and improving the therapeutic effect of intractable neurological diseases such as cerebral infarction, spinal cord injury, and dementia caused by MSC.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (2)

A mesenchymal stem cell activator containing β-carotene as an active ingredient, which is an agent for mesenchymal stem cells that is orally ingested by a subject to activate the bone marrow mesenchymal stem cells of the subject and decreases with age. A mesenchymal stem cell activator that improves cell number or proliferative capacity . The mesenchymal stem cell activator according to claim 1, which is used in combination with a tissue repair / regeneration drug containing CD24-negative mesenchymal stem cells.

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