Pharmaceutical composition for preventing or treating alzheimer's disease, comprising neural crest-derived nasal turbinate stem cells expressing SSEA3 and CD105 as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease, the composition comprising, as an active ingredient, neural crest-derived nasal turbinate stem cells (NTSCs) expressing SSEA3 and CD105. Treatment with the NTSCs expressing SSEA3 and CD105 or with an NTSC cell line including at least a predetermined proportion of the NTSCs was found to result in remarkably good therapeutic activity against Alzheimer's disease. Therefore, the present invention is expected to be effectively used not only as a composition for preventing or treating Alzheimer's disease in which the composition includes, as an active ingredient, NTSCs expressing SSEA3 and CD105 or an NTSC cell line including at least a predetermined proportion of the NTSCs, but also for uses such as screening of NTSC formulations that can be used to treat Alzheimer's disease, or prediction of the therapeutic efficacy thereof.

Description

Pharmaceutical composition for preventing or treating Alzheimer's disease comprising neural crest-derived nasal stem cells expressing SSEA3 and CD105 as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising neural crest-derived nasal stem cells expressing SSEA3 and CD105 as an active ingredient.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2024-0036787, filed March 15, 2024, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Dementia is a complex symptom characterized by a decline in cognitive functions, such as intelligence, learning, and language, as well as higher mental functions, due to damage or destruction of the normally mature brain caused by external factors such as acquired trauma or disease. With the rapid increase in the aging population in modern society, the incidence of age-related dementia is emerging as a serious social problem. Despite its high incidence, there is currently no effective treatment or preventative treatment for dementia, resulting in enormous socioeconomic losses.

[0005] Among dementias, Alzheimer's disease is the most common form of dementia. It is a degenerative brain disease that causes serious impairments in memory and cognitive function as brain tissue loses function during the aging process. Therefore, it is necessary to identify and study important factors that affect rapid cognitive decline in Alzheimer's disease in order to develop the mechanism of Alzheimer's disease and new treatments.

[0006] Although much research has been conducted on the mechanism of Alzheimer's disease, it has not yet been clearly identified. According to research reports on Alzheimer's disease, amyloid-β peptide (Aβ) is considered an important causative agent in the progression of the disease, and it is formed from amyloid precursor protein (APP). The β-pleated sheet form is insoluble and accumulates by forming amyloid fibril aggregates. These amyloid aggregates themselves are known to be neurotoxic and induce an inflammatory response, causing brain cell damage.

[0007] Meanwhile, with the remarkable advancements in academic and technological advancements in stem cell-based treatment of neurodegenerative diseases, the number of cases applying stem cell therapy in clinical practice has increased, solidifying its position as a crucial field for treating neurodegenerative diseases. While research into stem cell-based treatment of neurodegenerative diseases has focused on human embryonic stem cells and induced pluripotent stem cells, which possess high proliferative and pluripotent capabilities, there are still few stem cell therapies that have demonstrated clear efficacy.

[0008] Under this technical background, the inventors of the present invention invented a stem cell treatment for preventing or treating Alzheimer's disease using neural crest-derived nasal stem cells (hereinafter referred to as NTSC).

[0009]

[0010] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises at least one active ingredient selected from the group consisting of:

[0011] i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and

[0012] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0013] Another object of the present invention is to provide a method for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps:

[0014] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0015] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in neural crest-derived nasal stem cells.

[0016] Another object of the present invention is to provide a method for predicting the effect of a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps:

[0017] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0018] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

[0019] Another object of the present invention is to provide a kit for preventing or treating Alzheimer's disease, comprising a composition comprising at least one active ingredient selected from the group consisting of:

[0020] i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and

[0021] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0022] Another object of the present invention is to provide a kit for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising a composition comprising as an active ingredient an agent measuring at least one selected from the group consisting of:

[0023] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0024] ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more based on the total number of neural crest-derived nasal stem cells.

[0025]

[0026] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0027]

[0028] The present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising at least one active ingredient selected from the group consisting of:

[0029] i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and

[0030] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0031] In one embodiment of the present invention, the neural crest-derived nasal stem cell line may include SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in an amount of 16% or more based on the total number of neural crest-derived nasal stem cells, but is not limited thereto.

[0032] In one embodiment of the present invention, the stem cells may be derived from inferior turbinate tissue, but are not limited thereto.

[0033] In one embodiment of the present invention, the composition can express at a high level at least one selected from the group consisting of Nestin, β-Ⅲ tubulin, and Microtubule-associated protein 2 (MAP2), but is not limited thereto.

[0034] In one embodiment of the present invention, the composition may secrete one or more cytokines or chemokines selected from the group consisting of, but not limited to:

[0035] Brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), angiogenin, C-X-C motif chemokine ligand 1 (CXCL1), leptin, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinase-1 / 2, and osteoprotegerin.

[0036] In one embodiment of the present invention, the composition may exhibit a protective effect against the toxicity of amyloid beta by inhibiting the cell death effect caused by amyloid beta, but is not limited thereto.

[0037] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0038] a) Reduced expression of 6E10, Iba-1, pTau, and CD11b;

[0039] b) Increases the expression of β-Ⅲ tubulin, NeuN, and GFAP; and

[0040] c) Reduces the expression of CCL4, CCL5, CXCL10, and OPN.

[0041] In one embodiment of the present invention, the composition may inhibit amyloid beta deposition or improve learning and memory ability, but is not limited thereto.

[0042] The present invention provides a method for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps:

[0043] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0044] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

[0045] In one embodiment of the present invention, when measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105,

[0046] The above screening method may additionally include, but is not limited to, a step of determining that the neural crest-derived nasal stem cell preparation is a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease if the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

[0047] In one embodiment of the present invention, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in neural crest-derived nasal stem cells,

[0048] The above screening method may additionally include, but is not limited to, a step of determining that the neural crest-derived nasal stem cell preparation is for the prevention or treatment of Alzheimer's disease when the level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more based on the total number of neural crest-derived nasal stem cells.

[0049] The present invention provides a method for predicting the Alzheimer's prevention or treatment effect of a neural crest-derived nasal stem cell preparation for Alzheimer's prevention or treatment, comprising the following steps:

[0050] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0051] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

[0052] In one embodiment of the present invention, when measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105,

[0053] The above prediction method may additionally include, but is not limited to, a step of determining that the Alzheimer's prevention or treatment effect of the neural crest-derived nasal stem cell preparation for Alzheimer's prevention or treatment is likely to be high when the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

[0054] In one embodiment of the present invention, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line,

[0055] The above prediction method may additionally include, but is not limited to, a step of determining that the effect of preventing or treating Alzheimer's disease by a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease is high when the level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more based on the total number of neural crest-derived nasal stem cells.

[0056] In one embodiment of the present invention, if it is determined that the therapeutic effect is high in the above step, a step of treating Alzheimer's disease with a neural crest-derived nasal stem cell preparation may be additionally included, but is not limited thereto.

[0057] The present invention provides a kit for preventing or treating Alzheimer's disease, comprising a composition comprising at least one active ingredient selected from the group consisting of the following, and an instruction manual:

[0058] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0059] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0060] The present invention provides a kit for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising a composition comprising as an active ingredient an agent measuring at least one agent selected from the group consisting of:

[0061] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0062] ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 20% or more based on the total number of neural crest-derived nasal stem cells.

[0063] In addition, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one active ingredient selected from the group consisting of:

[0064] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0065] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0066] In addition, the present invention provides a use of a composition comprising at least one selected from the group consisting of the above as an active ingredient for use in preventing or treating Alzheimer's disease.

[0067] In addition, the present invention provides a use for preparing one or more Alzheimer's prevention or treatment agents selected from the group consisting of the above.

[0068] In addition, the present invention provides a pharmaceutical composition comprising at least one selected from the group consisting of the following as an active ingredient:

[0069] i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and

[0070] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0071] In addition, the present invention provides a use for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising as an active ingredient a preparation measuring at least one selected from the group consisting of:

[0072] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0073] ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more based on the total number of neural crest-derived nasal stem cells.

[0074] In addition, the present invention provides a use for predicting the effect of preventing or treating Alzheimer's disease of a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising as an active ingredient a preparation measuring at least one agent selected from the group consisting of the above.

[0075]

[0076] According to the pharmaceutical composition for preventing or treating Alzheimer's disease, etc., comprising neural crest-derived nasal stem cells expressing SSEA3 and CD105 of the present invention as an active ingredient, significantly superior Alzheimer's disease treatment activity was confirmed when neural crest-derived nasal stem cells (NTSC) expressing SSEA3 and CD105 or an NTSC cell line containing a certain ratio or more of the NTSC were treated, and therefore, it is expected to be useful not only for a composition for preventing or treating Alzheimer's disease, which comprises NTSC expressing SSEA3 and CD105; or an NTSC cell line containing a certain ratio or more of the NTSC as an active ingredient, but also for screening NTSC preparations that can be used for treating Alzheimer's disease, and for predicting therapeutic effects.

[0077]

[0078] Figure 1a shows hNTSCs isolated and cultured from human nasal tissue obtained through frequent rhinitis surgery.

[0079] Figure 1b shows the results of an in vivo toxicity analysis of hNTSC-administered mice confirmed through histopathological experiments such as weight changes.

[0080] Figures 1c and 1d show the results of in vivo toxicity analysis of hNTSC-administered mice, confirmed at the serum level and the whole blood cell level, respectively.

[0081] Figure 2a shows the results of a water maze escape experiment in Alzheimer's mice administered hNTSC.

[0082] Figure 2b shows the probe test results of Alzheimer's mice administered hNTSC.

[0083] Figures 2c and 2d show that chronically activated microglia directly damage brain neurons in Alzheimer's disease individuals by secreting inflammatory cytokines around amyloid beta plaques.

[0084] Figure 2e shows that the level of amyloid beta plaque deposition and inflammatory microglia expression in microglia of Alzheimer's mice administered hNTSC was reduced.

[0085] Figures 2f and 2g show the results of SDS-PAGE gel Western blot analysis of brain extracts from Alzheimer's mice administered hNTSC, showing that the level of amyloid beta decreases with hNTSC administration (Figure 2f) and the level of amyloid beta deposition and the expression level of inflammatory microglia decreases (Figure 2g).

[0086] Figures 3a and 3b show the experimental results confirming the SSEA3-CD105 double positivity ratio of hNTSCs obtained from different donors.

[0087] Figure 4a shows the results of culture immunostaining, confirming SSEA3-CD105 double-positive cells (hNTSC-M) from hNTSC.

[0088] Figures 4b and 4c show the results of analyzing the expression markers of hNTSC and hNTSC-M.

[0089] Figure 4d shows the results of hNTSC and hNTSC-M staining analysis using Alizarin Red S, Oil red O, and Alcian blue.

[0090] Figure 4e shows the results of analyzing the growth rates of hNTSC and hNTSC-M.

[0091] Figure 4f shows the results of analyzing the cytokine secretion profiles of hNTSC and hNTSC-M, and Figure 4g shows the results of quantifying Figure 4f.

[0092] Figure 4h shows the results of analyzing the amyloid beta toxicity of hNTSC and hNTSC-M.

[0093] Figure 5a shows the results of confocal microscopy analysis of organoids co-cultured with hNTSC and hNTSC-M, showing a decrease in Alzheimer's disease-related markers.

[0094] Figure 5b shows the results of a Western blot experiment showing a reduction in the amount of Aβ plaques and microglial cells in organoids co-cultured with hNTSCs and hNTSC-Ms, and Figure 5c shows the quantification thereof.

[0095] Figure 6a shows the results of immunohistochemical analysis of neuronal and glial cell markers in Alzheimer's organoids, confirming that the expression levels of β-III tubulin, a neural progenitor cell marker, NeuN, a mature neuron marker, and GFAP, a glial cell marker, were significantly increased in hNTSC-M compared to hNTSC. In addition, Figures 6b and 6c confirm this through Western blot analysis.

[0096] Figure 7a confirms that the level of Alzheimer's disease pathogenesis-related molecules is higher in Alzheimer's organoids compared to normal brain organoids.

[0097] Figure 7b shows the results of analyzing the levels of molecules related to the pathogenesis of Alzheimer's disease, confirming that hNTSC-M significantly reduced the levels of CCL4, CCL5, CXCL10, and OPN. Furthermore, Figure 7c is a graph showing the quantitative results.

[0098] Figure 8a shows the results of immunohistochemical staining analysis performed by transplanting hNTSC-M, etc. into Alzheimer's organoids, and it was confirmed that the level of amyloid beta plaque deposition was significantly reduced by hNTSC-M, and Figure 8b shows the quantification of this.

[0099] Figure 8c shows Aβ by transplanting hNTSC-M etc. into Alzheimer's organoids. 42 ELISA analysis was performed on hNTSC-M to determine the availability of Aβ 42 It was confirmed that the level of was significantly reduced.

[0100] Figure 8d shows that learning ability was improved by hNTSC-M after transplanting hNTSC-M and other substances into Alzheimer's organoids and performing water maze training.

[0101] Figures 8e and 8f show the results of a probe test performed by transplanting hNTSC-M, etc. into Alzheimer's organoids, confirming that memory ability was improved by hNTSC-M.

[0102] Figure 9a shows the results of confocal microscopy image analysis performed after transplanting hNTSC-M and other cells into Alzheimer's disease organoids, showing that hNTSC-M reduces microglia exhibiting an inflammatory response. Figure 9b is a graph quantifying this.

[0103] Figure 9c shows the results of Western blot analysis on proteins related to neuroinflammation and pathology by transplanting hNTSC-M and others into Alzheimer's organoids, confirming that the expression of CD11b and OPN was reduced by hNTSC-M, and the level of NEP was increased.

[0104] Figures 9d to 9f are graphs quantifying the expression levels of CD11b, OPN, and NEP confirmed in Figure 9c.

[0105]

[0106] The present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising at least one active ingredient selected from the group consisting of:

[0107] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0108] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0109] In one embodiment of the present invention, the neural crest-derived nasal stem cell line may include SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in an amount of 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more based on the total number of neural crest-derived nasal stem cells, but is not limited thereto.

[0110] The term “neural crest” used in the present invention refers to a group of ectodermal cells that temporarily appear immediately after embryogenesis during the development of vertebrates, and is the region corresponding to the final part that separates from the outer epidermis when the neural tube is formed. Neural crest stem cells migrate from the posterior part of the neural fold during the migratory stage of development, and neural crest stem cells migrate and are distributed to a wide range of tissues and organs within the body. After the migratory stage, neural crest stem cells are known to differentiate into peripheral nervous system nerve cells, glial cells, melanocytes of the skin, endocrine cells, and various mesenchymal cells, and are distributed to non-neural tissues and organs in adults.

[0111] In the present invention, “stem cell” refers to a cell that is the basis of cells or tissues that constitute an individual, and has the ability to self-renew through repeated division and has the ability to differentiate into cells with specific functions depending on the environment. It is produced in all tissues during fetal development, and is also found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissues, even in adults. Depending on the type of cell capable of differentiation, stem cells are classified into totipotent stem cells that are formed when a fertilized egg begins its first division, pluripotent stem cells in the blastocyst lining that are created through continuous division of these cells, and multipotent stem cells that exist in mature tissues and organs. At this time, pluripotent stem cells are cells that can differentiate only into cells specific to the tissues and organs in which they are contained. They are involved in the growth and development of each tissue and organ in the fetal, neonatal, and adult stages, as well as in maintaining homeostasis in adult tissues and inducing regeneration in the event of tissue damage. These tissue-specific pluripotent cells are collectively referred to as adult stem cells.

[0112] Among adult mesenchymal stem cells, bone marrow-derived and adipose tissue-derived mesenchymal stem cells (BMMSCs) require surgical procedures that are extremely painful and time-consuming. Furthermore, the yield of these cells is very small. Culturing clinically sufficient quantities is time-consuming and expensive, and carries a high risk of infection and cell loss. Furthermore, umbilical cord blood-derived MSCs are difficult to obtain when needed and require long-term storage.

[0113] Mesenchymal stem cells, classified as adult stem cells, are well known as repair cells of various connective tissues, and these cells can differentiate into various types of mesenchymal cells. In addition, because they are easy to obtain and can be applied clinically, they can complement the shortcomings of neural stem cells, which are located deep in the brain and are difficult to obtain in sufficient quantities and have a risk of brain damage, and are thus attracting attention as a material for the development of treatments for nervous system diseases and regenerative medicine. They can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord, and unlike blood stem cells, they have the ability to differentiate into cells that constitute various human tissues, such as adipocytes, osteocytes, chondrocytes, nerve cells, and cardiomyocytes. In one embodiment of the present invention, the stem cells may be at least one selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In the present invention, mesenchymal stem cells isolated from human nasal turbinate tissue were used, but are not limited thereto.

[0114] The above “inferior turbinate tissue” is an independent small bone that is shaped like a shell and is located on the lower outer side of the nasal cavity on both the left and right sides, and is attached to the maxilla and the palatine bone. It is a tissue rich in submucosal tissue with a large number of reticular venous sinuses distributed inside and a large number of mesenchymal stromal cells surrounding them. It can be obtained during a partial inferior turbinate resection performed in the otolaryngology field to improve nasal obstruction symptoms. In addition, it can be used by easily biopsying the tissue in a short period of time without pain to the patient in an otolaryngology outpatient clinic rather than in an operating room. That is, in one embodiment of the present invention, the stem cell may be derived from inferior turbinate tissue, but is not limited thereto.

[0115] Therefore, in the case of the human neural crest-derived nasal stem cells of the present invention, the surgery to obtain them requires very little bleeding and pain and takes less time, and stem cells can be continuously secured through recycling of mesenchymal stem cells isolated from discarded inferior turbinate tissue during inferior turbinate surgery (rhinitis surgery), which is the most frequently performed procedure in the field of otolaryngology, and the proliferative capacity of the stem cells is higher than that of the bone marrow-derived and adipose tissue-derived mesenchymal stem cells.

[0116] In addition, the present invention can provide materials for autologous donation and transplantation by utilizing tissues removed and discarded during many surgical procedures performed to improve the anatomical structure of the human body for the purpose of alleviating symptoms, and has the characteristic of contributing to the recycling of discarded tissues and the establishment of a patient-tailored tissue and cell bank using the same.

[0117] The term “differentiation” used in the present invention refers to the process by which the stem cells in the initial stage acquire the characteristics of each tissue, and in the present invention, it was confirmed that some of the human neural crest-derived nasal stem cells transplanted into the brain of an Alzheimer’s disease mouse differentiated into neural cells.

[0118] In the present invention, “SSEA3 and CD105 double positive stem cells” can be used interchangeably with “Muse cells (Multi-lineage differentiating stress enduring cells, muse cells).” Muse cells are endogenous non-cancerous pluripotent stem cells that express both stage-specific embryonic antigen 3 (SSEA3), a pluripotent marker, and the MSC marker CD105. Muse cells exist in the connective tissues of almost all organs, including the umbilical cord, bone marrow, and peripheral blood, and can be obtained from commercially available mesenchymal cells such as human fibroblasts, bone marrow-derived mesenchymal stem cells, and adipose-derived stem cells. In one embodiment of the present invention, the stem cells are obtained from neural crest-derived nasal stem cells, but are not limited thereto. Muse cells have the ability to repair or replace damaged cells through differentiation into damaged cell types, immune regulation, and migration to damaged sites.

[0119] In one embodiment of the present invention, the composition can express at a high level at least one selected from the group consisting of Nestin, β-Ⅲ tubulin, and Microtubule-associated protein 2 (MAP2), but is not limited thereto.

[0120] In the present invention, nestin and β-Ⅲ tubulin may be markers of neural progenitor cells, and MAP2 may be a marker of mature neurons.

[0121] “Nestin (neuroepithelial stem cell protein, nestin)” is a type VI intermediate filament (IF) protein encoded by the NES gene in humans. This intermediate filament protein is known to be expressed mainly in nerve cells involved in the radial growth of axons.

[0122] Additionally, "β-Ⅲ tubulin" is a major protein that constitutes the microscopic tubular structures present in almost all cells of living organisms. Alpha tubulin and beta tubulin polymerize to form microtubules, which make up a large portion of the eukaryotic cytoskeleton.

[0123] Meanwhile, “MAP2 (Microtubule-associated protein 2),” a human protein encoded by the MAP2 gene, is a key cytoskeletal regulator within neuronal dendrites and is abundant and specific enough to serve as a potent somatodendritic marker. Furthermore, similar to the closely related MAP Tau, it is known to influence microtubule dynamics and microtubule / actin interactions to control neurite outgrowth and synaptic function.

[0124] In the present invention, “expressing at a high level” can be used interchangeably with “expression level is increased.” “Increased” means that something that was not detectable is detected, or that the detection amount is relatively greater than the normal level. For example, an “increased” level means that the level of the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more higher than that of the control group, and / or 0.5 times, 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more higher. Specifically, it may mean an increase of 1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or more than 10 times compared to that of the control group, but is not limited thereto. In addition, in the present invention, “the expression level is increased” may have a broad meaning including a statistically significant level, but is not limited thereto. The meaning of the opposite term can be understood by those skilled in the art to have the opposite meaning according to the above definition.

[0125] In one embodiment of the present invention, the composition may secrete one or more cytokines or chemokines selected from the group consisting of, but not limited to:

[0126] Brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), angiogenin, C-X-C motif chemokine ligand 1 (CXCL1), leptin, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinase-1 / 2, and osteoprotegerin.

[0127] The above cytokines or chemokines may be, but are not limited to, mesenchymal stem cells (MSC)-related cytokines or chemokines.

[0128] In one embodiment of the present invention, the composition may exhibit a protective effect against the toxicity of amyloid beta by inhibiting the cell death effect caused by amyloid beta, but is not limited thereto.

[0129] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0130] a) Reduced expression of 6E10, Iba-1, pTau, and CD11b;

[0131] b) Increases the expression of β-Ⅲ tubulin, NeuN, and GFAP; and

[0132] c) Reduces the expression of CCL4, CCL5, CXCL10, and OPN.

[0133] According to one embodiment of the present invention, the composition of the present invention can reduce the expression level of Alzheimer's disease-related markers by suppressing the expression of 6E10, Iba-1, pTAU, and CD11b, thereby reducing the expression amount of Aβ protein, Tau protein, and inflammatory microglia. In addition, the composition of the present invention can increase the expression levels of β-Ⅲ tubulin, a neural progenitor cell marker, NeuN, a mature neuron marker, and GFAP, a glial cell marker, thereby inhibiting progressive neuronal loss, and thus producing an effect of increasing neuronal survival. In addition, the composition can suppress cytokine secretion caused by Alzheimer's disease by reducing the expression levels of CCL4, CCL5, CXCL10, and OPN.

[0134] In one embodiment of the present invention, the composition may inhibit amyloid beta deposition or improve learning and memory ability, but is not limited thereto.

[0135] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0136] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0137] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0138] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0139] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0140] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0141] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0142] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0143] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0144] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0145] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0146] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0147] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0148] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0149] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0150] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight, and severity of the disease of the patient.

[0151] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0152] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0153] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0154]

[0155] The present invention provides a method for screening a neural crest-derived turbinate stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps:

[0156] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0157] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

[0158] In one embodiment of the present invention, when measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105,

[0159] The above screening method may additionally include, but is not limited to, a step of determining that the neural crest-derived nasal stem cell preparation is a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease if the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

[0160] In one embodiment of the present invention, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line,

[0161] The above screening method may additionally include, but is not limited to, a step of determining that the neural crest-derived nasal stem cell preparation is for the prevention or treatment of Alzheimer's disease when the level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more based on the total number of neural crest-derived nasal stem cells.

[0162] In the present invention, “screening” may mean selecting a substance having a specific desired property from a candidate group consisting of various substances through a specific manipulation or evaluation method.

[0163] For the purpose of the present invention, the screening method of the present invention may mean a series of processes including, but not limited to, the steps of measuring whether a neural crest-derived nasal stem cell preparation is double positive for SSEA3 and CD105, or measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line, in order to identify a neural crest-derived nasal stem cell treatment agent that produces the best therapeutic effect on an Alzheimer's disease subject, and determining the neural crest-derived nasal stem cell or neural crest-derived nasal stem cell line as a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease when the neural crest-derived nasal stem cell preparation or neural crest-derived nasal stem cell line is double positive for SSEA3 and CD105, or the neural crest-derived nasal stem cell line comprises 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more of the total number of neural crest-derived nasal stem cells.

[0164] The above measuring step may be repeated several times, and may additionally include steps used in the art as a general screening method, such as adding additional substances or steps for treatment response and accurate confirmation, but is not limited thereto.

[0165] In the present invention, "confirmation" may correspond to the meaning of "analysis," but is not limited thereto. In the present invention, analysis may preferably mean "measurement," and the qualitative analysis may mean measuring and confirming the presence or absence of a target substance for confirming a therapeutic response, and the quantitative analysis may mean measuring and confirming changes in the presence level (expression level) or amount of a target substance. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and may be performed as quantitative measurement.

[0166] In the present invention, “neural crest-derived nasal stem cell preparation” includes all stem cell therapeutic agents that contain neural crest-derived nasal stem cells as a main component and are obtained and developed through any formulation, method, etc. that can be used for the treatment of Alzheimer’s disease.

[0167]

[0168] The present invention provides a method for predicting the Alzheimer's prevention or treatment effect of a neural crest-derived nasal stem cell preparation for Alzheimer's prevention or treatment, comprising the following steps:

[0169] A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or

[0170] Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

[0171] In one embodiment of the present invention, when measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105,

[0172] The above prediction method may additionally include, but is not limited to, a step of determining that the Alzheimer's prevention or treatment effect of the neural crest-derived nasal stem cell preparation for Alzheimer's prevention or treatment is likely to be high when the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

[0173] In one embodiment of the present invention, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line,

[0174] The above prediction method may additionally include, but is not limited to, a step of determining that the effect of preventing or treating Alzheimer's disease by a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease is high when the level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more based on the total number of neural crest-derived nasal stem cells.

[0175] In one embodiment of the present invention, if it is determined that the therapeutic effect is high in the above step, a step of treating Alzheimer's disease with a neural crest-derived nasal stem cell preparation may be additionally included, but is not limited thereto.

[0176] In the present invention, it was confirmed that the effects of preventing or treating Alzheimer's disease were completely different when neural crest-derived nasal stem cells isolated from different donors were treated. In particular, since no preventive or therapeutic effect was observed even when neural crest-derived nasal stem cells were treated, it can be proven that, according to the prediction method of the present invention, the effects of preventing or treating Alzheimer's disease were very excellent when not all neural crest-derived nasal stem cells were treated, but SSEA3 and CD105 double-positive neural crest-derived nasal stem cells; or when a neural crest-derived nasal stem cell line containing 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells based on the total number of neural crest-derived nasal stem cells was treated.

[0177]

[0178] The present invention provides a kit for preventing or treating Alzheimer's disease, comprising a composition comprising at least one active ingredient selected from the group consisting of the following, and an instruction manual:

[0179] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0180] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0181] In the present invention, a "kit" refers to a tool that can prevent or treat Alzheimer's disease using a composition comprising at least one active ingredient selected from the group consisting of the above-described components of the present invention. In addition to the above-described substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for storage and processing methods thereof. As a specific example, each component may be applied at least once without limitation on the number of times, there is no limitation on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0182] The present invention provides a kit for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising a composition comprising as an active ingredient an agent measuring at least one agent selected from the group consisting of:

[0183] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0184] ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more of the total number of neural crest-derived nasal stem cells.

[0185] In the present invention, the term "kit" refers to a tool that enables screening of a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease using a composition comprising, as an active ingredient, at least one agent for measuring a substance selected from the group consisting of the above-described agents of the present invention. In addition to the above-described substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for methods of storing and processing them. As a specific example, in this case, each component may be applied once or more without limitation in the number of times, there is no limitation on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0186] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0187]

[0188] In addition, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one active ingredient selected from the group consisting of:

[0189] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0190] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0191] In addition, the present invention provides a use of a composition comprising at least one selected from the group consisting of the above as an active ingredient for use in preventing or treating Alzheimer's disease.

[0192] In addition, the present invention provides a use for preparing one or more Alzheimer's prevention or treatment agents selected from the group consisting of the above.

[0193] In addition, the present invention provides a pharmaceutical composition comprising at least one selected from the group consisting of the following as an active ingredient:

[0194] i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and

[0195] ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

[0196] In addition, the present invention provides a use for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising as an active ingredient a preparation measuring at least one selected from the group consisting of:

[0197] i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and

[0198] ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more of the total number of neural crest-derived nasal stem cells.

[0199] In addition, the present invention provides a use for predicting the effect of preventing or treating Alzheimer's disease of a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising as an active ingredient a preparation measuring at least one agent selected from the group consisting of the above.

[0200]

[0201] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0202]

[0203] [Example]

[0204]

[0205] Acquisition of neural crest-derived nasal stem cells

[0206] To obtain neural crest-driven tubinate stem cells, the tissues were first isolated from the subturbinate tissue of patients who underwent partial turbinate resection and washed with saline and phosphate-buffered saline (PBS). Then, 1 mm 3The tissue was cut into small pieces, plated on a culture dish, and covered with a sterile glass cover slide. The tissue was cultured in α-minimum essential medium (α-MEM) containing 1% (v / v) penicillin / streptomycin (antibiotics) and 10% (v / v) fetal bovine serum (FBS) at 37°C in an incubator with 5% (v / v) CO2. The culture medium was changed every two days during the three-week culture period, and after removing the glass cover slide, neural crest-derived nasal stem cells were isolated from the tissue with a 0.25% trypsinin 1 mM EDTA solution and used for the experiment (Fig. 1a).

[0207]

[0208] Example 1. Confirmation of long-term toxicity of hNTSC

[0209] To evaluate the long-term safety of human neural stem cells (hNSCs), an in vivo toxicity analysis was performed on nude mice administered hNTSCs. Specifically, mice (treatment group) were administered 3×10 6 hNTSCs were intravenously injected at a dose of 1 cell / head. Mice administered saline were used as controls.

[0210]

[0211] As a result, the in vivo toxicity analysis of mice following hNTSC administration was confirmed through survival experiments, as shown in Table 1. Specifically, both the treatment and control groups were confirmed to survive until the experimental point for toxicity analysis (Table 1). Furthermore, no changes in body weight or histopathological findings were observed between the control and treatment groups during the 13 weeks following injection (Figure 1b).

[0212]

[0213]

[0214] Additionally, liver function tests were performed to assess toxicity. At 13 weeks post-injection, no statistically significant differences were found in serum levels of the parameters AST, ALT, ALP, ALB, GLU, CRE, and TP between the control and treatment groups (Fig. 1c). Similar to the serum analysis results, at 13 weeks post-injection, no statistically significant differences were found in complete blood counts for WBC, RBC, HGB, HCT, MCV, MCH, MCHC, and RDW between the control and treatment groups (Fig. 1d).

[0215]

[0216] Example 2. Confirmation of the different effects of hNTSCs of different origins on Alzheimer's disease.

[0217] To investigate whether hNTSC transplantation from different donors differentially modulates Alzheimer's disease neuropathology, the effects of transplantation on Alzheimer's disease pathology and cognitive impairment in 5 x FAD mice were analyzed. Specifically, PBS or hNTSCs from five different donors (hNTSC#1 to 5) were injected into the brains of 16-week-old 5 x FAD transgenic (Tg) mice. The effects of hNTSCs on the protection of learning and memory abilities were then analyzed through Morris water maze training in Tg mice 6 weeks after transplantation. For the spatial learning memory test, Tg mice injected with PBS (Tg-Sham) or hNTSCs (Tg-NTSC) and PBS-injected wild-type age-matched mice (WT-Sham) were trained to learn the location of the target platform for 8 days.

[0218]

[0219] As a result (Fig. 2a), the average escape latency times of WT-Sham, Tg-Sham, Tg-NTSC#1, Tg-NTSC#2, Tg-NTSC#3, Tg-NTSC#4, and Tg-NTSC#5 mice were confirmed to be 21.0 (11.6 sec), 56.9 (21.7 sec), 32.0 (19.4 sec), 45.9 (15.9 sec), 48.0 (18.5 sec), 46.0 (16.9 sec), and 25.3 (13.1 sec).

[0220]

[0221] Accordingly, WT-Sham and Tg-NTSC showed gradual cognitive improvements in a time-dependent manner during the training period to find the platform, but no cognitive effects were observed in Tg-Sham. In particular, among the five donor-derived hNTSCs, Tg-NTSC#1 to Tg-NTSC#5, the escape latency was significantly improved on the 8th day of the training period. Among them, Tg-NTSC#5 was confirmed to show more significant improvements on the 7th and 8th days of the training period compared to Tg-Sham. On the other hand, compared to WT-Sham, Tg-NTSC#2, Tg-NTSC#3, and Tg-NTSC#4 did not show significant improvements in escape latency during the 8-day training period.

[0222]

[0223] Additionally (Fig. 2b), in the probe test, WT-Sham, Tg-NTSC#1, and Tg-NTSC#5 mice showed a significant increase in the time spent in the target quadrant area 4 of the memory stage compared to Tg-Sham mice. On the other hand, Tg-NTSC#2, Tg-NTSC#3, and Tg-NTSC#4 did not show a significant difference compared to Tg-Sham, confirming that they were at a similar level.

[0224]

[0225] In addition, the results of the water maze test were analyzed in depth through analysis of amyloid beta (Aβ) plaque deposition and inflammation in microglia. Specifically, 7 weeks after cell administration, Aβ plaque deposition or inflammation in the brain was analyzed through immunostaining using the Aβ-specific antibody 6E10 or the microglial marker Iba-1.

[0226]

[0227] As a result, chronically activated microglia were found to directly damage brain neurons of Alzheimer's disease patients by secreting inflammatory cytokines around Aβ plaques (Fig. 2c and d). Confocal microscopy images showed that Aβ plaque burden and the expression of inflammatory microglia were reduced in the hippocampus or cortex regions of Tg-NTSC compared to Tg-Sham, confirming that Tg-NTSC reduced and treated neuronal damage in Alzheimer's disease subjects (Fig. 2e).

[0228]

[0229] Western blot experiments of brain extract SDS-PAGE gels revealed a strong expression level of Aβ-specific bands representing monomeric or oligomeric forms in Tg-Sham, whereas they were reduced in brain extracts from five different Tg-NTSC mice (Fig. 2f). In particular, among the five different Tg-NTSC#1-5, Tg-NTSC#1 and Tg-NTSC#5 showed greater reductions in not only Aβ fragment levels but also Aβ plaque burden and the expression of inflammatory microglia.

[0230] Also (Fig. 2g), availability of Aβ in brain homogenates 42Levels were found to be significantly reduced in Tg-NTSC compared to Tg-Sham. In particular, Tg-NTSC#1 and Tg-NTSC#5 showed significantly higher availability of Aβ compared to other Tg-NTSCs, Tg-NTSC#2, Tg-NTSC#3, and Tg-NTSC#5. 42 It was confirmed that this indicates a decrease in level.

[0231]

[0232] These results from the water maze test experiment confirmed that hNTSC transplantation protects learning and memory abilities, and in particular, some of the five donor-derived hNTSCs, Tg-NTSC#1 and Tg-NTSC#5, significantly improved cognitive function.

[0233]

[0234] In conclusion, it was confirmed that even if they are the same hNTSC, their individual characteristics differ depending on their origin, and their effects on treating Alzheimer's disease are also completely different.

[0235]

[0236] Example 3. Identification of a SSEA3-CD105 double-positive cell population in hNTSCs and establishment of a relationship with Alzheimer's disease therapeutic activity.

[0237] To investigate the SSEA3 and CD105 double-positive cell population (muse cells) in hNTSCs obtained from five different donors, hNTSCs were cultured in vitro in proliferation medium for 4 to 5 passages, followed by immunostaining and flow cytometry using anti-SSEA3 and CD105 antibodies.

[0238]

[0239] As a result (Figs. 3a and 3b), the proportion of SSEA3-CD105 double-positive cells (Muse cells) present in hNTSCs from five different donors was found to be different, confirming that hNTSCs obtained from each donor were cell populations exhibiting different characteristics. Specifically, hNTSC#1 and hNTSC#5 were found to contain approximately 20% and 23% Muse cell populations, respectively. This level was found to be 4-5 times higher than that of the Muse cell population in hNTSC#2.

[0240]

[0241] The experimental results of Example 2 confirmed that among the hNTSCs obtained from five different donors, hNTSC#1 and hNTSC#5 showed the best therapeutic activity in terms of reducing neuropathology and improving cognitive impairment in an Alzheimer's disease mouse model, suggesting that this effect is due to the level of the SSEA3-CD105 double positive cell population of hNTSC#1 and hNTSC#5.

[0242]

[0243] Example 4. Characterization of SSEA3-CD105 double-positive cells isolated from hNTSCs.

[0244] We analyzed the characteristics of SSEA3-CD105 double-positive cells isolated from hNTSCs. Specifically, hNTSC#5, which was confirmed to have the best Alzheimer's disease treatment effect among hNTSCs in Examples 1 to 3, was cultured to isolate SSEA3-CD105 double-positive cells (hereinafter referred to as hNTSC-M). Thereafter, the cultured hNTSC#5 (hereinafter referred to as hNTSC) was analyzed for its differentiation ability into multilineage tissues, proliferation capacity, and cytokine secretion characteristics, using it as a control.

[0245]

[0246] Immunostaining hNTSCs and hNTSC-Ms using anti-SSEA3 and anti-CD105 antibodies confirmed the presence of a SSEA3-CD105 double-positive cell population (Fig. 4a). Therefore, the hNTSCs used in Example 4 and below include various cells, including SSEA3-CD105 double-positive hNTSCs.

[0247]

[0248] Both hNTSC and hNTSC-M strongly expressed Nestin and β-Ⅲ tubulin, which are markers of neural progenitor cells, in the proliferative state, but MAP2, a marker of mature neurons, was not expressed in hNTSC, but was expressed only in hNTSC-M (Fig. 4b).

[0249]

[0250] After 4 weeks of neurogenesis by culturing under neural differentiation conditions, hNTSCs and hNTSC-Ms were found to express high levels of differentiation and mature neural markers, β-Ⅲ tubulin and MAP2 (Fig. 4c).

[0251]

[0252] In addition, when hNTSC and hNTSC-M were cultured under osteogenic differentiation conditions, adipogenic differentiation conditions, and chondrogenic differentiation conditions, hNTSC-M showed greater staining with Alizarin Red S, Oil red O, and Alcian blue compared to hNTSC at 2-3 weeks after induction (Fig. 4d). In addition, hNTSC-M showed a growth rate approximately 1.5 times faster than hNTSC during 5 days of cell culture (Fig. 4e).

[0253]

[0254] To determine the cytokine secretion profiles of hNTSCs and hNTSC-Ms, a human cytokine array kit consisting of a total of 80 cytokines and chemokine antibodies was used to analyze the basal cytokines secreted from hNTSCs or hNTSC-M cultures.

[0255] As a result (Fig. 4f), both hNTSC-conditioned medium and hNTSC-M-conditioned medium were confirmed to exhibit major hybridization signals known as MSC-associated cytokines or chemokines, such as brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), angiogenin, C-X-C motif chemokine ligand 1 (CXCL1), leptin, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinase-1 / 2, and osteoprotegerin.

[0256]

[0257] To facilitate analysis, the expression levels of the markers were quantified by averaging the intensity values ​​across three different membranes for all points on the array membrane, and the results showed that hNTSC-M showed approximately 1.5 to 3 times higher secretion levels of some cytokines, including BDNF, angiogenin, and leptin, which are known to be involved in the pathogenesis of neurological disorders, than hNTSC (Fig. 4g).

[0258]

[0259] To investigate the protective effect of hNTSC or hNTSC-M against Aβ toxicity in culture of hNSCs, Aβ 1-42 hNSCs were co-cultured with hNTSCs or hNTSC-M in the presence of oligomers. As a result (Fig. 4h), Aβ 1-42Treatment with oligomers induced cell death, which was inhibited by co-culture with hNTSCs and hNTSC-M. In particular, co-culture with hNTSC-M was found to have a greater protective effect against Aβ toxicity than hNTSCs.

[0260]

[0261] Example 5. Confirmation of the effect of reducing the expression of Alzheimer's disease-related markers by SSEA3-CD105 double-positive cells or hNTSC cell lines containing the cells.

[0262] We analyzed whether the hNTSC-M and hNTSC of the present invention could modulate the Alzheimer's disease phenotype of brain organoids derived from Alzheimer's disease patients. In this case, hNTSC-M and hNTSC were derived from hNTSC#5 of Examples 1 to 3, as in Example 4. First, three individual brain organoid types were generated for 60 days based on three independent iPSC lines from the blood of each Alzheimer's disease patient (hereinafter referred to as AD#1-3). AD#1-3 were then co-cultured with hNTSC-M and hNTSC for 5 to 6 days, and then immunohistochemical analysis was performed using Alzheimer's disease-related markers.

[0263]

[0264] As a result (Fig. 5a), confocal microscopy images of AD#1-3 for Aβ deposition marker 6E10 and microglial cell marker Iba-1 revealed that microglia were clustered around Aβ aggregates in AD#1-3. Immunostaining for the Alzheimer's disease marker p-Tau (Ser202 / Thr205) revealed that the expression level of P-Tau-positive cells was significantly higher in AD#1-3, which was confirmed to be reduced by co-culture with hNTSC-M and hNTSC.

[0265]

[0266] Additionally, the amount of Aβ plaques and microglia was significantly reduced in AD#1-3 co-cultured with hNTSC-M and hNTSC compared to the control organoids of AD#1-3 (Fig. 5b). Western blot analysis using the 6E10 antibody demonstrated the expression of multiple Aβ-specific bands in AD#1-3, and the levels were reduced by approximately 5-40% in AD#1-3 co-cultured with hNTSC-M and hNTSC (Fig. 5c).

[0267] In addition, the Western blot results of the SDS-PAGE gel showed that the levels of P-Tau and microglial marker CD11b in AD#1-3 organoids were reduced by approximately 10 to 45% compared to the control group by co-culture of hNTSC-M and hNTSC (Fig. 5c).

[0268]

[0269] These results demonstrate that treatment of hNTSC-M and hNTSC reduces Alzheimer's-related markers, including Aβ protein levels, p-Tau protein levels, and the number of inflammatory microglia.

[0270]

[0271] Example 6. Confirmation of the effect of increasing neural survival by SSEA3-CD105 double-positive cells or hNTSC cell lines containing the cells.

[0272] We analyzed whether the hNTSC-M and hNTSC treatments of the present invention protect against neuronal cell death in Alzheimer's disease patients. Specifically, AD#1-3 of Example 5 was co-cultured with hNTSC-M and hNTSC for 5 to 6 days, and then immunohistochemical analysis was performed for neuronal and glial cell markers such as β-III tubulin, NeuN, and GFAP.

[0273]

[0274] As a result, immunostaining analysis for β-Ⅲ tubulin, NeuN, and GFAP demonstrated the presence of neuronal and glial cell types in AD#1-3.

[0275] Specifically, confocal microscopy images (Fig. 6a) showed that co-culture of hNTSC-M and hNTSC significantly increased the levels of β-Ⅲ tubulin, a neural progenitor cell marker, NeuN, a mature neuron marker, and GFAP, a glial cell marker, in AD#1-3. Furthermore, quantitative analysis of Western blot experiments reconfirmed that co-culture of hNTSC-M and hNTSC increased the expression of these markers overall, despite differences in individual disease progression and the influence of repeated experiments, and this was statistically significant (Figs. 6b and 6c).

[0276]

[0277] These results suggest that the neuroprotective effect of hNTSC-M and hNTSC of the present invention is significantly superior, as hNTSC-M and hNTSC treatment inhibit progressive neuronal loss in Alzheimer's disease subjects.

[0278]

[0279] Example 7. Confirmation of the effect of reducing Alzheimer's disease-related cytokines by SSEA3-CD105 double-positive cells or hNTSC cell lines containing the cells.

[0280] It is known that various cytokines and chemokines, including Aβ expression and deposition, Tau tangle formation, and neuroinflammation, are involved in the pathogenesis of Alzheimer's disease, and peripheral levels of inflammatory cytokines change during the pathogenesis of Alzheimer's disease, which is significantly correlated with disease progression. In this example, we analyzed secreted factors associated with the regulation of Alzheimer's disease-related markers by hNTSC-M and hNTSC. Specifically, a cytokine array was constructed using conditioned media from AD#1-3 cultured with hNTSC-M and hNTSC using a human cytokine array kit.

[0281]

[0282] As a result, the array data confirmed that the levels of Alzheimer's disease-related molecules such as CCL2, CCL4, CCL5, CXCL10, and OPN were higher in AD#1-3 than in normal brain organoids (Fig. 7a).

[0283]

[0284] Additionally, for each sample, two different membranes were averaged to quantify all points on the array membrane. As a result, the intensity values ​​showed that the levels of CCL4, CCL5, and CXCL10 were approximately 20-50% lower in organoids AD#1-3 cultured with hNTSC-M and hNTSC than in the control organoid AD#1-3, and the OPN level was 30-50% lower (Fig. 7b and Fig. 7c).

[0285]

[0286] These results suggest a mechanism for the superior downregulation of cytokines in hNTSC-M and hNTSCs in Alzheimer's disease.

[0287]

[0288] Example 8. Confirmation of the therapeutic effect of Alzheimer's disease by transplantation of SSEA3-CD105 double-positive cells or hNTSC cell line containing the cells.

[0289] To confirm the therapeutic effects of hNTSC-M and hNTSC transplantation on Alzheimer's disease, we analyzed the regulation of neuropathology and cognitive impairment in Alzheimer's disease. At this time, the effect of hNTSC-M transplantation on the regulation of neuropathology and cognitive impairment in Alzheimer's disease was confirmed by reducing Aβ plaque deposition in the brain and improving cognitive impairment in 5 x FAD mice. Specifically, various experiments were performed after injecting PBS, hNTSC-M, or hNTSC into the brains of 16-week-old 5 x FAD Tg mice.

[0290]

[0291] First, immunohistochemical staining using the 6E10 antibody revealed Aβ plaque deposition in the hippocampus or cortex area of ​​the brain of Tg-Sham, but this result was confirmed to be significantly reduced in Tg mice 7 weeks after cell administration by injection of hNTSC (Tg-NTSC) or hNTSC-M (Tg-NTSC-M) (Fig. 8a).

[0292]

[0293] Quantitative data showed that the average Aβ plaque burden in Tg-Sham, Tg-NTSC, and Tg-NTSC-M brains was 6.6 (1.5%), 1.8 (0.3%), and 1.2 (0.3%), respectively (Fig. 8b).

[0294]

[0295] Aβ in brain homogenate 42 ELISA analysis results for available Aβ 42 The levels were significantly reduced by about 2.0 to 2.5 times in Tg-NTSC or Tg-NTSC-M compared to Tg-Sham (Fig. 8c).

[0296]

[0297] Furthermore, the learning and memory enhancement effects of hNTSC-M and hNTSC transplantation were analyzed through Morris water maze training in WT-Sham, Tg-Sham, Tg-NTSC, and Tg-NTSC-M mice 6 weeks after transplantation. All mice used for the spatial learning memory test were trained to learn the location of the target platform for 7 days.

[0298]

[0299] As a result (Fig. 8d), the average escape latency times of WT-Sham, Tg-Sham, Tg-NTSC, and Tg-NTSC-M on the 7th day of training were 14.9 (3.4 s), 51.9 (6.4 s), 26.5 (10.7 s), and 22.4 (8.1 s), respectively. According to this, WT-Sham, Tg-NTSC, and Tg-NTSC-M showed greater cognitive improvements in platform finding than Tg-Sham. In particular, compared to Tg-NTSC, Tg-NTSC-M showed greater improvements in escape latency on the 7th day of training.

[0300]

[0301] Additionally, in the probe test, WT-Sham, Tg-NTSC, and Tg-NTSC-M significantly increased the time spent in the target quadrant area 4 of the memory stage compared to Tg-Sham. Tg-NTSC-M took more time, which was confirmed to be significantly better than Tg-NTSC (Figs. 8e and 8f).

[0302] According to these results, it was confirmed that hNTSC-M and hNTSC transplantation containing hNTSC-M had a significantly up-regulating effect on neuropathology and cognitive impairment modulation activity in Alzheimer's disease.

[0303]

[0304] Example 9. Confirmation of the effect of reducing brain inflammation following transplantation of SSEA3-CD105 double-positive cells or hNTSC cell lines containing the cells.

[0305] Activated microglia directly damage neurons and secrete inflammatory cytokines that can cross the blood-brain barrier and cause systemic inflammation. Therefore, in this study, the effects of hNTSC-M and hNTSC transplantation on the regulation of brain inflammation in Alzheimer's disease were investigated using immunohistochemical staining analysis. Brain Aβ plaque deposition or inflammation was assessed using the Aβ-specific antibody 6E10 or the microglial marker Iba-1. Specifically, PBS, hNTSC-M, or hNTSC were injected into the brains of 16-week-old 5 × FAD Tg mice and used in the experiments.

[0306]

[0307] As a result, confocal microscopy images showed that a large number of microglia were concentrated around Aβ plaques in the Tg-Sham brain, whereas both Aβ plaques and Iba-1-positive microglia were significantly reduced in the Tg-NTSC or Tg-NTSC-M brain (Fig. 9a). Quantitative analysis revealed that the average percentages of Iba-1-positive cells in brain sections treated with Tg-Sham, Tg-NTSC, and Tg-NTSC-M were 10.6 (2.2%), 5.2 (1.5%), and 3.2 (0.9%), respectively (Fig. 9b). At this time, Iba-1-positive cells were counted in the hippocampus and cortical areas of brain tissue, and the data were expressed as the average percentage of positive cells.

[0308]

[0309] Additionally, Western blot was performed on brain extracts from WT-Sham, TG-Sham, TG-NTSC, and TG-NTSC-M to investigate protein levels associated with neuroinflammation and pathology after stem cell transplantation in Alzheimer's disease. As a result, Western blots on SDS-PAGE gels showed that the level of CD11b, a microglial marker, was approximately 2.0-fold higher in TG-Sham than in WT-Sham, and significantly decreased by approximately 1.4-fold or 1.5-fold in TG-NTSC and TG-NTSC, respectively (Figures 9c and 9d). In particular, TG-NTSC-M showed a greater decrease in CD11b levels than TG-NTSC.

[0310]

[0311] Meanwhile, OPN is known as a multifunctional inflammatory cytokine associated with cell-mediated immunity, inflammation, and neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, and frontotemporal dementia. Analysis of this revealed that the OPN level was approximately 3.0-fold higher in TG-Sham than in WT-Sham in Western blot of SDS-PAGE gel, and the OPN level was significantly reduced by approximately 1.4-fold or 1.5-fold in TG-NTSC and TG-NTSC-M, respectively (Figures 9c and 9e). In particular, the OPN level was found to be reduced more significantly in TG-NTSC-M than in TG-NTSC.

[0312]

[0313] Defects in the neuronal clearance pathway due to a dysfunctional cleavage enzyme are known to be key mechanisms that propagate the neuropathology of Alzheimer's disease by accumulating Aβ and increasing neuroinflammation. Our analysis revealed no significant differences in the levels of neprilysin (NEP), a key regulator of Aβ degradation and clearance, between WT-Sham and TG-Sham brains in Western blot analysis of SDS-PAGE gels. In contrast, NEP levels were significantly increased approximately 1.4-fold in TG-NTSC or TG-NTSC-M brains compared to TG-Sham brains (Figures 9c and 9f).

[0314]

[0315] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0316]

[0317] According to the pharmaceutical composition for preventing or treating Alzheimer's disease, etc., comprising neural crest-derived nasal stem cells expressing SSEA3 and CD105 of the present invention as an active ingredient, significantly superior Alzheimer's disease treatment activity was confirmed when neural crest-derived nasal stem cells (NTSC) expressing SSEA3 and CD105 or an NTSC cell line containing a certain ratio or more of the NTSC were treated, and therefore, not only the composition for preventing or treating Alzheimer's disease comprising NTSC expressing SSEA3 and CD105; or an NTSC cell line containing a certain ratio or more of the NTSC as an active ingredient, but also the use for screening NTSC preparations that can be used for treating Alzheimer's disease, the use for predicting the therapeutic effect, etc., has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising at least one active ingredient selected from the group consisting of: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

2. In paragraph 1, A pharmaceutical composition, wherein the neural crest-derived nasal stem cell line comprises SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in an amount of 16% or more based on the total number of neural crest-derived nasal stem cells.

3. In either paragraph 1 or paragraph 2, A pharmaceutical composition wherein the stem cells are derived from inferior turbinate tissue.

4. In any one of paragraphs 1 to 3, A pharmaceutical composition wherein the composition expresses at a high level at least one selected from the group consisting of Nestin, β-Ⅲ tubulin, and Microtubule-associated protein 2 (MAP2).

5. In any one of paragraphs 1 to 4, The pharmaceutical composition secretes at least one cytokine or chemokine selected from the group consisting of: Brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), angiogenin, C-X-C motif chemokine ligand 1 (CXCL1), leptin, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinase-1 / 2, and osteoprotegerin.

6. In any one of paragraphs 1 to 5, A pharmaceutical composition characterized in that the composition exhibits a protective effect against the toxicity of amyloid beta by inhibiting the cell death effect caused by amyloid beta.

7. In any one of paragraphs 1 to 6, A pharmaceutical composition characterized in that the composition comprises at least one selected from the group consisting of: a) Reduced expression of 6E10, Iba-1, pTau, and CD11b; b) Increases the expression of β-Ⅲ tubulin, NeuN, and GFAP; and c) Reduces the expression of CCL4, CCL5, CXCL10, and OPN.

8. In any one of paragraphs 1 to 7, A pharmaceutical composition characterized in that the composition inhibits amyloid beta deposition or improves learning and memory ability.

9. A method for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps: A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

10. In the 9th paragraph, when measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105, The above screening method further comprises a step of determining that the neural crest-derived nasal stem cell preparation is for the prevention or treatment of Alzheimer's disease, if the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

11. In any one of paragraphs 9 and 10, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line, The above screening method further comprises a step of determining that the neural crest-derived nasal stem cell preparation is for the prevention or treatment of Alzheimer's disease, when the level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more based on the total number of neural crest-derived nasal stem cells.

12. A method for predicting the effect of a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising the following steps: A step of measuring whether neural crest-derived nasal stem cells are double positive for SSEA3 and CD105; or Step of measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line.

13. In the 12th paragraph, when measuring whether the neural crest-derived nasal stem cells are double positive for SSEA3 and CD105, The above prediction method further comprises a step of determining that the effect of preventing or treating Alzheimer's disease by the neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease is high when the neural crest-derived nasal stem cell is double positive for SSEA3 and CD105.

14. In any one of paragraphs 12 and 13, when measuring the level of inclusion of SSEA3 and CD105 double positive neural crest-derived nasal stem cells in a neural crest-derived nasal stem cell line, The above prediction method further includes a step of determining that the effect of preventing or treating Alzheimer's disease by a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease is high when the level of inclusion of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells in the neural crest-derived nasal stem cell line is 16% or more based on the total number of neural crest-derived nasal stem cells.

15. In any one of paragraphs 12 to 14, A prediction method, which additionally includes a step of treating Alzheimer's disease with a neural crest-derived nasal stem cell preparation, if the treatment effect is determined to be high in the above step.

16. A kit for preventing or treating Alzheimer's disease, comprising a composition comprising at least one active ingredient selected from the group consisting of the following, and an instruction manual: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

17. A kit for screening a neural crest-derived nasal stem cell preparation for preventing or treating Alzheimer's disease, comprising a composition comprising as an active ingredient a preparation measuring at least one selected from the group consisting of the following, and an instruction manual: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more based on the total number of neural crest-derived nasal stem cells.

18. A method for preventing or treating Alzheimer's disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one active ingredient selected from the group consisting of: i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

19. Use of a composition comprising at least one active ingredient selected from the group consisting of the following for the prevention or treatment of Alzheimer's disease: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

20. Use for manufacturing one or more Alzheimer's prevention or treatment agents selected from the group consisting of: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

21. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: i) SSEA3 and CD105 double positive neural crest-driven tubinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line comprising SSEA3 and CD105 double positive neural crest-derived nasal stem cells.

22. Use of a composition comprising as an active ingredient a preparation measuring at least one agent selected from the group consisting of the following: Screening use of a preparation of neural crest-derived nasal stem cells for preventing or treating Alzheimer's disease: i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more based on the total number of neural crest-derived nasal stem cells.

23. Predicting the effect of preventing or treating Alzheimer's disease of a neural crest-derived nasal stem cell preparation for the prevention or treatment of Alzheimer's disease, comprising as an active ingredient a preparation measuring at least one agent selected from the group consisting of: i) SSEA3 and CD105 double-positive neural crest-derived turbinate stem cells; and ⅱ) A neural crest-derived nasal stem cell line having a level of SSEA3 and CD105 double-positive neural crest-derived nasal stem cells of 16% or more based on the total number of neural crest-derived nasal stem cells.

Images