Pharmaceutical composition for treating degenerative brain diseases containing neural progenitor cells derived from pluripotent stem cells

Abstract

The present invention relates to a pharmaceutical composition for treating degenerative brain diseases, comprising neural progenitor cells derived from pluripotent stem cells. In one embodiment, neural progenitor cells differentiated from pluripotent stem cells are injected into the cerebroventricular ventricles of an amyloid-β-injected animal model, and have the effect of maintaining the efficacy of enhancing cognitive function for a long period of time, and can be usefully used in treating degenerative brain diseases, including Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating degenerative brain diseases, which contains neural progenitor cells derived from pluripotent stem cells.

Background Art

[0002] Degenerative brain diseases are diseases that show degenerative changes in nerve cells of the central nervous system and induce various symptoms such as damage to motor and sensory functions and inhibition of higher-order causal functions such as memory, learning, and arithmetic reasoning. Representative diseases include Alzheimer's disease, Parkinson's disease, and memory loss.

[0003] Alzheimer's disease is a variety of complex cognitive impairments such as regression of cognitive function accompanied by memory loss, personality changes, and behavioral abnormalities, which results in irreversible functional disorders such as nerve cell death and ultimately causes permanent brain damage. Regarding how Alzheimer's disease occurs, a complete investigation has not been made. There were treatment candidates for dementia based on the amyloid-β hypothesis of dementia, but most of them failed clinically. Recently, various dementia treatment target studies such as tau have been actively conducted. However, even if amyloid-β deposits and neurofibrillary tangles caused by tau are removed in Alzheimer's disease patients, it is difficult to expect effects such as nerve regeneration, and there is a need for radical treatment through complete nerve regeneration. Accordingly, dementia therapeutic agents using stem cells have been developed (International Publication Patent WO2016088930A1), but one of the main problems of cell therapy agents is the low cell survival rate (less than 5%) of transplanted cells, and it has been reported that transplanted cells undergo significant cell death immediately after in vivo injection. However, research on effective administration routes that can maintain the long-term therapeutic effect without the death of transplanted cells is very limited.

Summary of the Invention

Problems to be Solved by the Invention

[0004] One aspect provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising as an active ingredient neural progenitor cells differentiated from pluripotent stem cells.

[0005] Another aspect provides the use of neural progenitor cells differentiated from pluripotent stem cells for the manufacture of a pharmaceutical composition for preventing or treating a degenerative brain disease.

[0006] Still another aspect provides a method for preventing or treating a degenerative brain disease, comprising administering an effective amount of neural progenitor cells differentiated from pluripotent stem cells to an individual in need thereof.

Means for Solving the Problems

[0007] One aspect provides a pharmaceutical composition for preventing or treating a degenerative brain disease, comprising as an active ingredient neural progenitor cells differentiated from pluripotent stem cells.

[0008] The term "pluripotent stem cell" may mean a cell having self-renewal ability (the ability to pass through a number of cell division cycles while maintaining an undifferentiated state) and capable of exhibiting one or more types of pluripotency (the ability to differentiate into one or more specialized cells). The pluripotent stem cell may also be a nuclear transfer pluripotent stem cell (NT-hPSC), a pluripotent stem cell derived from a cell arising from parthenogenesis (pn-hPSC), an induced pluripotent stem cell (iPSC), or an embryonic stem cell (ESC). In one specific example, it is also an embryonic stem cell (ESC), and the embryonic stem cell may mean an inner cell mass extracted from a blastocyst-stage embryo immediately before a fertilized egg implants in the uterus of a mother and cultured in vitro.

[0009] The pluripotent stem cell may also be a cell derived from a mammal, such as a human, a mouse, a rat, an ape, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

[0010] The term "neural precursor cell (NPC)" is a cell that is capable of self-renewal, is a nervous system cell, and also has the ability to differentiate. The said neural precursor cell is also called a neural progenitor cell (NPC) or a neural stem cell (NSC). The said neural precursor cell can also differentiate into a neuron, an astrocyte, or an oligodendrocyte. The said neural precursor cell is also a cell that expresses SOX1.

[0011] The term "differentiation" may mean a phenomenon in which the structure and function of a cell are specialized while the cell divides and grows. A pluripotent stem cell can be completely differentiated into a specific cell after passing through a neural precursor cell of a specific form. The said embryonic stem cell differentiates into the said neural precursor cell, and the said neural precursor cell also differentiates into a neuron, an astrocyte, or an oligodendrocyte, etc.

[0012] The term "neurodegenerative disease" may mean a disease that occurs when the structure or function of brain tissue or brain cells degenerates due to the progression of aging.

[0013] In one specific example, the degenerative brain disease is any one disease selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloidosis, Dutch type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette’s syndrome, Friedreich’s ataxia, Machado-Joseph’s disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0014] The term "Alzheimer’s disease" can be used interchangeably with senile dementia and can mean a disease accompanied by mental degradation associated with a specific degenerative brain disease characterized by senile plaques, neuroinflammatory tangles, and progressive nerve loss.

[0015] The composition is also administered parenterally. In one specific example, the composition is for administration intravenously (IV), intrahippocampally (IH), intracerebrally, intracranially, intraspinally, intracerebrospinally, intracerebroventricularly (ICV) or intrathecally. The terms “administer,” “infuse,” “inject,” and “transplant” are also used interchangeably and can mean the placement of a composition according to one specific example into an individual by a method or route that results in at least partial localization of the composition according to one specific example to a desired site. At least a portion of the cells or cellular components of a composition according to one specific example are also administered by any suitable route that delivers them to a desired location within a living individual. In one specific example, the composition is injected intracerebroventricularly or intrathecally and reaches the cerebrospinal fluid. Conventionally, most attempts at administration routes for cell therapy agents have been methods of direct injection into the brain and intravenous injection, and significant cell death has been shown within several days after in vivo injection. However, if the composition is administered intracerebroventricularly or intrathecally, the survival period of the cells is maintained for a long time, and there is an effect that the cognitive function enhancing effect can be sustained for a long time.

[0016] In one specific example, the neural progenitor cells are also those differentiated by two-dimensional culture of embryoid bodies (EBs) formed by three-dimensional culture of the pluripotent stem cells in a medium containing a protein kinase C-β (PKC-β) inhibitor and a bone morphogenetic protein (BMP) inhibitor.

[0017] In one specific example, the pluripotent stem cells are also those cultured under feeder cell-free conditions.

[0018] The term "protein kinase C (PKC)" may mean one of the protein phosphorylation enzymes that phosphorylate the hydroxy groups of serine and threonine of proteins and regulate the functions of proteins.

[0019] The above-mentioned "PKC-β inhibitor" also suppresses the expression or activity of PKC-β. In one specific example, the PKC-β inhibitor is 2-[1-(3-dimethylaminopropyl)-5-methoxy indol-3-yl]-3-(1H-indol-3-yl)maleimide, 3-[1-(3-imidazol-1-yl propyl)-1H-indol-3-yl]-4-anilino-1H-pyrrole-2,5-dione, 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione, 3-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate, 13-hydroxyoctadecadienoic acid, bisindolylmaleimide, 2,6-diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide, 4’-demethylamino-4’-hydroxystaurosporine and 3-(13-methyl-5-oxo-6,7-dihydro-5H-indolo[2,3-a]pyrrolo[3,Any one selected from the group consisting of 3-(13-methyl-5-oxo-6,7-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol-12(13H)-yl)propanenitrile. The PKC-β inhibitor is also included at a concentration of 5 μM to 25 μM, 5 μM to 20 μM, 6 μM to 18 μM, 8 μM to 15 μM, or 8 μM to 12 μM.,

[0020] The term "bone morphogenetic protein (BMP)" can mean a growth factor that induces the formation of bone and cartilage.,

[0021] The "BMP inhibitor" is also a low molecular weight compound or polypeptide that inhibits the cell signaling of BMP. In one specific example, the BMP inhibitor is any one selected from the group consisting of dorsomorphin (6-[4-[2-(1-piperinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine)), dorsomorphin homolog 1 (DMH1: dorsomorphin homolog 1) (4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), K02288 (3-[(6-amino 5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol), LDN 212854 (5-(6-(4-(1-piperazinyl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone) and Noggin polypeptide, for example, DMH1. The BMP inhibitor is also included at a concentration of 5 μM to 25 μM, 5 μM to 20 μM, 6 μM to 18 μM, 8 μM to 15 μM, or 8 μM to 12 μM.,

[0022] In one specific example, the three-dimensional culture may be performed for 1 to 10 days, for example, 2 to 6 days, or 3 to 5 days, and is also used to differentiate towards the neuroectodermal side.

[0023] In one specific example, the two-dimensional culture may be performed for 1 to 15 days, for example, 2 to 8 days, 3 to 7 days, or 4 to 6 days, and is also used to form a rosette structure.

[0024] The term "prevention" may mean any act of suppressing or delaying a degenerative brain disease by administering the pharmaceutical composition.

[0025] The term "treatment" refers to or includes the alleviation, suppression of progression, or prevention of a disease, disorder, or pathological condition, or one or more symptoms thereof. The "active ingredient" or "pharmaceutically effective amount" may mean any amount of the composition utilized in the process of practicing the invention provided herein that is sufficient for the alleviation, suppression of progression, or prevention of a disease, disorder, or pathological condition, or one or more symptoms thereof.

[0026] The pharmaceutical composition according to one specific example may also contain a pharmaceutically acceptable carrier and / or additive. For example, it may contain sterilized water, physiological saline, conventional buffers (such as phosphoric acid, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspension agents, isotonic agents, or preservatives. When the pharmaceutical composition according to one specific example is formulated into a dosage form suitable for injection, the neural progenitor cells may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.

[0027] The dosage of the pharmaceutical composition according to one specific example, based on neural progenitor cells, is 1.0×10 3 to 1.0×10 10 cells / kg (body weight) or individual, or 1.0×10 7 to 1.0×10 8It is also in cells / kg (body weight) or per individual. However, the dosage is variably formulated according to factors such as the formulation method, administration method, the patient's age, weight, gender, pathological condition, diet, administration time, administration route, excretion rate, and reactivity, and those skilled in the art will be able to appropriately adjust the dosage considering such factors. The number of administrations can be once or more than twice within the range of clinically acceptable side effects, and the administration site can also be administered at one location or two or more locations. For animals other than humans, the same dosage as that for humans can be administered per kg or per individual, or, for example, an amount obtained by converting the aforementioned dosage can be administered according to the volume ratio (e.g., average value) of organs (such as the heart) between the target animal and humans. Examples of target animals for treatment by a specific example include humans and other target mammals, specifically including humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc.

[0028] The pharmaceutical composition according to a specific example is manufactured in a unit dosage form or can also be manufactured by being placed in a multi-dose container by formulating using a pharmaceutically acceptable carrier and / or excipient by a method that can be easily implemented by those skilled in the art in the technical field to which the invention belongs. At this time, the dosage form is in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or can also be in the form of a powder, granule, tablet, or capsule type. In addition, the cell therapy agent is also formulated into an injectable dosage form. In that case, known general components for formulation are used and it is also formulated by conventional methods.

[0029] In one specific example, the method for differentiating the pluripotent stem cells into neural progenitor cells includes culturing the isolated pluripotent stem cells under feeder cell-free conditions, three-dimensionally culturing the cultured pluripotent stem cells in a medium containing a PKC-β inhibitor and a BMP inhibitor to obtain embryonic bodies (EBs), and differentiating the obtained EBs by two-dimensional culture to form a rosette structure. Regarding the aforementioned pluripotent stem cells, neural progenitor cells, differentiation, PKC-β inhibitor, BMP inhibitor, three-dimensional culture, and two-dimensional culture, it is as described above.

[0030] In one specific example, the method further includes subculturing the cultured neural progenitor cells.

[0031] Another aspect provides the use of neural progenitor cells differentiated from pluripotent stem cells for the manufacture of a pharmaceutical composition for preventing or treating a degenerative brain disease. Regarding the aforementioned degenerative brain disease, prevention, treatment, pharmaceutical composition, pluripotent stem cells, differentiation, and neural progenitor cells, it is as described above.

[0032] Still another aspect provides a method for preventing or treating a degenerative brain disease, including administering an effective amount of neural progenitor cells differentiated from pluripotent stem cells to an individual in need thereof. Regarding the pluripotent stem cells, differentiation, neural progenitor cells, administration, degenerative brain disease, prevention, and treatment in the method, it is as described above.

[0033] The term "effective amount" can be readily determined by one of ordinary skill in the art based on factors well known in the medical field, such as the type of disease, the age, weight, health, and gender of the patient, the patient's sensitivity to the drug, the route of administration, the method of administration, the frequency of administration, the treatment period, the formulation, or the drugs used concomitantly.

[0034] The individual is also a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The individual is also an individual in need of an effect of improving, preventing or treating a degenerative brain disease, such as Alzheimer's disease.

Advantages of the Invention

[0035] Neural progenitor cells differentiated from pluripotent stem cells according to one aspect have the effect of being able to be injected into the cerebral ventricle in an amyloid-β injected animal model and maintaining the effect of enhancing cognitive function for a long period of time, and can be usefully used for the treatment of degenerative brain diseases including Alzheimer's disease.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Mode for Carrying Out the Invention

[0037] Hereinafter, the present invention will be described in more detail through examples. However, those examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to those examples.

[0038] Example 1. Differentiation of Human Embryonic Stem Cells into Neural Progenitor Cells 1.1. Culture of Human Embryonic Stem Cells In order to culture human embryonic stem cells (hESC: human embryonic stem cell) without feeder cells, the following was carried out.

[0039] Specifically, CTS Cellstart, which is a cell adhesion function enhancer TM , Matrigel, vitronectin or laminin was treated in a 6-well tissue culture dish with a surface area of 9.6 cm 2 at a concentration of 1,000 μl / well at 4°C for 24 hours. Then, after removing all the remaining cell adhesion function enhancer at room temperature from the tissue culture dish, mTeSR TM (STEMCELL Technologies, Canada), Essential 8 TM (Gibco, USA), StemFit (AJINOMOTO, Japan), TeSR TM -E8 TM (STEMCELL Technologies, Canada) culture medium was placed in the tissue culture dish at a concentration of 2,000 μl / well and then placed in an incubator at 37°C and 5% CO2.

[0040] Next, human embryonic stem cells (CHA-hES NT 18, CHA University) grown on feeder cells were cut into small clumps by a mechanical sub-passage method using a microchip (Micro-tip, Axygen, USA), and then dispensed into the tissue culture dish placed in the incubator so that there were 40 to 50 clumps / well. Then, human embryonic stem cells were grown without feeder cells while alternating with 500 μl / well of fresh embryonic stem cell culture medium (mTeSR TM ) every day for 5 days.

[0041] 1.2. Induction of differentiation of human embryonic stem cells into neural progenitor cells The human embryonic stem cells cultured under the feeder cell-free conditions of Example 1.1 described above were treated with collagenase to form embryonic bodies (EBs), which are three-dimensional aggregates of pluripotent stem cells. In a xenopathogen-free culture medium condition, a protein kinase C inhibitor (PKC-β inhibitor) and a BMP inhibitor (DMH1) were added, and differentiation toward the neuroectoderm side was induced through three-dimensional culture for 4 days. Thereafter, the three-dimensionally cultured EBs were placed in a cell culture dish and differentiated by two-dimensional culture for 5 days to form a rosette structure with a specific morphology, physically acquire the rosette site, and isolate neural precursor cells (NPCs). To confirm the NPCs obtained in this way, flow cytometry was used to confirm the expression of SOX1 (SRY-Box Transcription Factor 1), a transcription factor that is a protein specific to neural precursor cells. Hereinafter, neural precursor cells are referred to as "NPCs".

[0042] Comparative Example 1. Differentiation of human embryonic stem cells into mesenchymal stem cells 1.1. Induction of differentiation of human embryonic stem cells into ectodermal cells In Example 1.1 described above, the human embryonic stem cells grown without feeder cells were treated with a TGF-β inhibitor and a BMP inhibitor to differentiate them into ectodermal cells.

[0043] Specifically, TGF-β inhibitor (SB431542) or PKC-β Inhibitor and DMH1 (BMP inhibitor) were dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA) at stack concentrations of 10 mM, 5 mM, and 0.5 mM, respectively. Then, they were diluted in embryonic stem cell differentiation medium (DMEM / F12 (20% (v / v) SR, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 1% (v / v) penicillin-streptomycin + SB341542 10 μM / ml + 0.5 μM / ml), DMEM / F12 (20% (v / v) SR, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 1% (v / v) penicillin-streptomycin + PKC-β Inhibitor 5 μM / ml + 0.5 μM / ml), DMEM / F12 (insulin 10 μM / ml, transferrin 14 μM / ml, selenium 9 μM / ml + SB341542 10 μM / ml, or DMEM / F12 (insulin 10 μM / ml, transferrin 14 μM / ml, selenium 9 μM / ml + PKC-β Inhibitor 5 μM / ml)) so that the final concentrations were 10 μM, 5 μM, and 0.5 μM. Subsequently, in Example 1.1 described above, human embryonic stem cells cultured for 5 days in a proliferative manner were treated in the above-mentioned embryonic stem cell differentiation medium containing SB431542, PKC-β Inhibitor, and DMH1 in a floating state for 4 days to differentiate into ectodermal cells.

[0044] 1.2. Pretreatment of Ectodermal Cells The floating ectodermal cells in Comparative Example 1.1 described above were treated with Matrigel or vitronectin, which are cell adhesion function enhancers, at room temperature for 1 hour. Then, the remaining cell adhesion function enhancers were removed from the tissue culture dish at room temperature, and the floating cells were allowed to adhere and cultured in an ectoderm induction medium (DMEM / F12 + insulin 25 μM / ml + bFGF 20 μM / ml) for 5 days.

[0045] 1.3. Induction of Differentiation into Mesenchymal Stem Cells In the aforementioned Comparative Example 1.2, after examining whether p75, a Neural Crest marker, is expressed via a flow cytometer, ectodermal cells were differentiated and subcultured in a mesenchymal stem cell medium (DMEM / F12 (10% (v / v) FBS, 4 ng / ml bFGF, 1% (v / v) NEAA, 0.1 mM β-mercaptoethanol, 1% (v / v) penicillin-streptomycin, a-MEM (10% (v / v) FBS, 4 ng / ml bFGF), Cellartis(R) MSC Xeno-Free Culture Medium, StemMACS TM MSC Expansion Media Kit, MSC NutriStem(R) XF Medium, StemXVivo Xeno-Free Human MSC Expansion Medium, or CellCor Serum Free Chemically Defined Medium) to obtain mesenchymal stem cells.

[0046] Specifically, on day 9 of differentiation, after removing all culture media, the cells were washed with PBS mixed with 1% (v / v) penicillin-streptomycin. Then, StemPro TM Accutase TM Cell Dissociation Reagent was treated at 37 °C and 5% CO2 for 5 minutes to make the cells single-celled, then neutralized in a mesenchymal stem cell medium and centrifuged at 1,000 rpm for 5 minutes. Next, the cells obtained by centrifugation were dispensed into a 1-well plate / 12-well plate pre-coated with CTS Cellstart TM , gelatin, and fibronectin, and then subcultured each time the cell confluency reached 80 - 90%. The cell confluency was 1x10^ 4 cells / cm 2Subculture was performed, and the size was changed in the order of 12-well plate (passage 0) → 6-well plate (passage 1) → T-25 flask (passage 2) → T-75 flask (passage 3). 0.05% trypsin was used to treat at room temperature, and continuous subculture was carried out by the method of making single cells. In this process, non-mesenchymal stem cells were shed, and mesenchymal stem cells were obtained.

[0047] Hereinafter, mesenchymal stem cells derived from embryonic stem cells are referred to as "ES-MSC".

[0048] Experimental Example 1. Confirmation of the dementia treatment effect of NPC 1.1. Preparation of animal model Male B6 mice with a body weight of about 23 - 25 g at 6 weeks after birth were used, and 5 mice were housed in each cage. They were maintained under conditions of 12-hour light and dark at 21 °C, and food and drinking water were provided. Amyloid β, a substance that causes memory deficit, 1-42 was dissolved in 10% dimethyl sulfoxide (DMSO) saline, cultured at 37 °C for 1 week, and prepared at a concentration of 100 μM. After the mice were anesthetized by inhalation using 3% isoflurane, a respiratory anesthesia device was used to maintain 1.5% isoflurane during the surgery. After preparing a stereotaxic fixation device, the head of the experimental animal was fixed, and 5 μl of amyloid β 1-42 was injected over 10 minutes with a 10 μl Hamilton microsyringe with a 26-gauge needle at a position 0.9 mm posterior, 1.7 mm lateral, and 2.2 mm deep from bregma.

[0049] 1.2. Administration of NPC and evaluation of behavior The NPC prepared in Example 1 above was p3 (passage 3) subcultured 3 times or p12 (passage 12) subcultured 12 times. Administration of NPC was performed on the 7th day after the production of the amyloid β-injected animal model, and all animal model experiments were carried out blindly. Each cell was frozen at 3×10 6 cells per vial, and at the time of use, 3×10 5The cells were dissolved in 300 μl of physiological saline and injected into the tail vein. In addition, for intracerebral injection and intraventricular injection, they were dissolved in 5 μl and injected using a stereotaxic apparatus, and the control group was injected with the same volume of physiological saline.

[0050] Before the surgery, the evaluation of the Y-maze and NORT cognitive ability was trained and scored, and then the behavioral evaluation was carried out. On the 5th day after the surgery, a lesion model was selected to proceed with the experiment. Figure 2 is a drawing showing the treatment schedule for an animal model of amyloid-β injection of NPC according to a specific example. Five days after the surgery, the cell line was treated, and whether the cognitive function was improved was measured for 7 days, 14 days, 21 days, and 28 days.

[0051] For the evaluation of behavior, the following Y-maze evaluation and NORT cognitive ability evaluation were carried out.

[0052] Y-maze test: The apparatus extends into three passages (arms) and is in the shape of the alphabet Y. The various lengths are 35 cm, the height is 15 cm, and the width is 5 cm, and they are positioned at the same angle. The movement of the experimental animal is indicated by the number of crossings. The number of crossings is defined as one crossing (spontaneous alteration) when passing through three passages sequentially in a row. Each experimental animal started from one maze and was made to go to the other maze, and the number of successful correct crossings was scored and expressed as a percentage. After allowing it to move freely for 2 minutes, it was measured for 5 minutes.

[0053] Novel object recognition test (NORT): It is a cognitive experimental technique that stimulates the curiosity of rodents. During the existing adaptation training period, memory of two identical objects is implanted, and at the same time as the start of the measurement, by replacing them with different objects and objects of different hues, instinctive curiosity is induced and memory is measured. After allowing it to move freely among the familiar objects for 2 minutes and replacing them with novel objects, it was measured for 5 minutes, and the time spent staying on the novel object was divided by the sum of the time spent staying on each object and then expressed as a percentage.

[0054] Figures 3A and 3B are graphs showing the Y-maze evaluation results and NORT cognitive ability evaluation results on the 7th, 14th, 21st, and 28th days for the groups in which ES-MSCs were administered to the amyloid-β injected animal model by intravenous injection (IV), intrahippocampal injection (IH), and intracerebroventricular injection (ICV), respectively.

[0055] Figures 4A and 4B are graphs showing the Y-maze evaluation results and NORT cognitive ability evaluation results on the 7th, 14th, 21st, and 28th days for the groups in which NPCs were administered to the amyloid-β injected animal model by intravenous injection (IV), intrahippocampal injection (IH), and intracerebroventricular injection (ICV), respectively.

[0056] As a result, as shown in Figures 3A and 3B, when ES-MSCs were administered on the 7th day after the establishment of the amyloid-β injected animal model, it was effective by intravenous injection (IV), but a decrease in function was observed from the 3rd week. For all other injection methods, an increase was observed in the 1st to 2nd week, and then a tendency to decrease was confirmed. In contrast, in the case of NPCs, as shown in Figures 4A and 4B, when NPCs were administered on the 7th day after the establishment of the amyloid-β injected animal model, both intravenous injection (IV) and intrahippocampal injection (IH) seemed to show a cognitive function enhancing effect for 2 weeks, but both decreased. On the other hand, intracerebroventricular injection (ICV) was confirmed to maintain the cognitive function enhancing effect for 4 weeks.

[0057] Figures 5A and 5B are graphs showing the Y-maze evaluation results and NORT cognitive ability evaluation results on the 7th, 14th, 21st, and 28th days for the groups in which NPCs cultured up to early passage (p2) and late passage (p12) were administered to the amyloid-β injected animal model by intracerebroventricular injection (ICV), respectively.

[0058] As a result, as shown in FIGS. 5A and 5B, it was confirmed that the cognitive function enhancing effect was observed in both of the two cell groups by administering NPCs cultured up to early passage (p2) and late passage (p12) by intracerebroventricular injection (ICV).

[0059] FIGS. 6A and 6B are graphs showing the results of Y-maze evaluation and NORT cognitive ability evaluation confirmed by long-term observation for 6 weeks in the group administered by intracerebroventricular injection (ICV) of NPCs in an amyloid-β injected animal model.

[0060] As a result, as shown in FIGS. 6A and 6B, it was confirmed that the cognitive function was enhanced at a level similar to that of the normal group in the group administered by intracerebroventricular injection (ICV) of NPCs compared to the group not administered with NPCs after amyloid-β injection, and it was confirmed that such a cognitive function enhancing effect persists during a long period of 6 weeks.

[0061] Overall, the above results mean that NPCs are superior to ES-MSCs in the persistent effect of the cognitive function enhancing effect, and in particular, when NPCs are injected into the cerebral ventricle (ICV), long-term persistence of the cognitive function enhancing effect is possible compared to other administration routes, so that a significant therapeutic effect can be obtained in degenerative brain diseases such as Alzheimer's disease.

Claims

1. A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising, as an active ingredient, neural progenitor cells differentiated from embryonic stem cells (ESCs), wherein the neural progenitor cells are obtained by two-dimensionally culturing and differentiating embryoid bodies (EBs) formed by three-dimensionally culturing the embryonic stem cells in a medium containing a protein kinase C-β (PKC-β) inhibitor and a bone morphogenetic protein (BMP) inhibitor, and is for administration into the intracerebroventricular (ICV).

2. The composition according to claim 1, wherein the degenerative brain disease is any one selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloidosis, Dutch-type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette’s syndrome, Friedreich’s ataxia, Machado-Joseph’s disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

3. The composition according to claim 1, wherein the embryonic stem cells are cultured under feeder cell-free conditions.

4. The PK C-β inhibitor is 2-[1-(3-dimethylaminopropyl)-5-methoxy indol-3-yl]-3-(1H-indol-3-yl)maleimide, 3-[1-(3-imidazol-1-ylpropyl)-1H-indol-3-yl]-4-anilino-1H-pyrrole-2,5-dione, 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione, 3-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide methane sulfonate, 13-hydroxyoctadecadienoic acid, bisindolylmaleimide, 2,6-diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide, 4’-demethylamino-4’-hydroxystaurosporine and 3-(13-methyl-5-oxo-6,7-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12(13H)-yl)propanenitrileThe composition according to claim 1, which is any one selected from the group consisting of [[3-a]pyrrolo[3,4-c]carbazol-12(13H)-yl)propanenitrile]].

5. The BMP inhibitor is any one selected from the group consisting of dorsomorphin (6-[4-[2-(1-piperinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), dorsomorphin homolog 1 (DMH1: 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), K02288 (3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol), LDN 212854 (5-(6-(4-(1-piperazinyl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone), and Noggin polypeptide, the composition according to claim 1.

6. The composition according to claim 1, wherein the three-dimensional culture is performed for 1 to 10 days.

7. The composition according to claim 1, wherein the two-dimensional culture is performed for 1 to 15 days.

Images