Pharmaceutical compositions comprising neural stem cells derived from three-dimensional hypothalamic organoids for preventing and treating geriatric diseases and uses of said compositions

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating geriatric diseases, comprising neural stem cells derived from hypothalamus-like organoids as an active ingredient. It was found that the decline in memory, cognitive ability, muscle strength, muscle tissue density, and bone mineral density in aged mice due to aging was restored when neural stem cells derived from hypothalamus-like organoids according to the present invention were transplanted into the aged mice. It was also found that transplantation of neural stem cells derived from hypothalamus-like organoids improves obesity by increasing energy metabolism. It was also found that the decline in memory, cognitive ability, and muscle strength in aged mice was restored when exosomes isolated from neural stem cells derived from hypothalamus-like organoids were transplanted into the aged mice. Therefore, it is expected that the neural stem cells derived from hypothalamus-like organoids according to the present invention are very useful as a drug for preventing and treating geriatric diseases.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing and treating geriatric diseases, comprising as an active ingredient neural stem cells obtained by isolating and separating hypothalamus-like organoids produced by 3D culture from human pluripotent stem cells.

[0002] [Background technology] The hypothalamus is a central organ for maintaining homeostasis in the human body, such as growth, puberty, metabolism, stress response, reproduction, lactation, parenting, and immunity. Neural stem / progenitor cells are known to reside in the adult hypothalamus and regulate hypothalamic function. Thus, neural stem cells have recently attracted attention as an alternative treatment for hypothalamic dysfunction caused by aging and other factors.

[0003] However, because the hypothalamus is located deep in the brain and is a small area, it is impossible to extract the actual tissue and use this tissue for research. Therefore, research is constantly being conducted to generate hypothalamic neural cells from pluripotent stem cells such as embryonic stem cells. For example, Wataya's team generated hypothalamic arginine vasopressin (AVP)-producing neural cells from mouse embryonic stem cells via 3D suspension culture by aggregating cells using gfCDM medium (Wataya T et al., Proc Natl Acad Sci USA, 2008, 105(33):11796-11801). In addition, Merkle's team succeeded in differentiating human embryonic stem cells and induced pluripotent stem cells into hypothalamic neural cells via 3D and 2D culture methods (Merkle FT et al., Development, 2015, 142(4):633-643). However, despite the above diverse research, there are still limitations in clinical applications.

[0004] [Detailed Description of the Invention] [Technical issues] Therefore, the present inventors have found that the neural stem cells obtained from the hypothalamus-like organoids cultured in 3D from human embryonic stem cells are similar to the neural stem cells present in the hypothalamus of real brain, and when transplanted into aged mice, improves the symptoms associated with aging.Based on the above, the present inventors have completed the present invention.

[0005] [Solution to the problem] In order to solve the above problems, one aspect of the present invention provides a pharmaceutical composition for preventing and treating geriatric diseases, comprising neural stem cells derived from hypothalamus-like organoids as an active ingredient.

[0006] In another aspect of the present invention, there are provided exosomes secreted from neural stem cells derived from hypothalamus-like organoids, and pharmaceutical compositions for preventing and treating geriatric diseases comprising the exosomes as an active ingredient.

[0007] In another aspect of the present invention, there is provided a method for producing exosomes, comprising the steps of i) culturing neural stem cells derived from hypothalamus-like organoids, and ii) recovering exosomes from the culture medium.

[0008] In another aspect of the present invention, there is provided a kit for preventing and treating geriatric diseases, comprising cells and / or exosomes selected from the group consisting of neural stem cells derived from hypothalamus-like organoids and exosomes secreted from neural stem cells derived from hypothalamus-like organoids.

[0009] In another aspect of the present invention, there is provided a use of neural stem cells derived from hypothalamus-like organoids, or exosomes secreted from neural stem cells derived from hypothalamus-like organoids, for the prevention or treatment of geriatric diseases.

[0010] In another aspect of the present invention, there is provided a method for preventing or treating geriatric diseases, comprising administering to a subject neural stem cells derived from hypothalamus-like organoids, or exosomes secreted from neural stem cells derived from hypothalamus-like organoids.

[0011] [Effects of the invention] It was found that the decline in memory, cognitive ability, muscle strength, muscle tissue density, and bone mineral density due to aging was restored when neural stem cells derived from hypothalamus-like organoids according to the present invention were transplanted into old mice. Furthermore, it was found that transplantation of neural stem cells derived from hypothalamus-like organoids improves obesity by increasing energy metabolism. It was also found that the decline in memory, cognitive ability, and muscle strength was similarly restored when exosomes isolated from the culture medium of neural stem cells derived from hypothalamus-like organoids were transplanted into old mice. Therefore, it is expected that neural stem cells derived from hypothalamus-like organoids and exosomes isolated from the culture medium of said neural stem cells are very useful as drugs for preventing and treating geriatric diseases. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of a method for generating human hypothalamic organoids from human embryonic stem cells (hESCs) or human dedifferentiated stem cells (hiPSCs) and for inducing differentiation of the generated hypothalamic organoids into hypothalamic neural stem cells (htNSCs). [Diagram 2] This is a graph showing the results of confirming the expression of hypothalamic marker genes (NKX2-1, RAX) or midbrain marker gene (EN1) by q-PCR after treating the culture medium of organoids produced by culturing hESCs with various hypothalamic patterning inducers. [Diagram 3] This figure shows the results of immunofluorescence staining confirming the expression of neural stem cell markers (SOX2, NESTIN, KI67) and hypothalamic region-specific marker genes (NKX2-1, RAX, ISL1) in organoid-derived human htNSCs. DAPI indicates cell nuclei. [Figure 4] 1 is a graph showing the results of confirming the expression of hypothalamic region-specific marker genes (RAX, NKX2.1, ISL1, OTP, SF-1) in organoid-derived human htNSCs by q-PCR. [Diagram 5] FIG. 1 shows the results of DGE (differential gene expression) analysis of transcripts from organoid-derived human htNSCs and various regions of the human fetal brain. [Figure 6] Graph showing population doubling level (PDL) and accumulated cell number by measuring the increase in cell number during five passages of organoid-derived human htNSCs in the presence of bFGF (basic fibroblast growth factor). [Figure 7] Figures and graphs showing the results of immunofluorescence staining of cryopreserved organoid-derived human htNSCs after replanking to observe the expression of proteins associated with cell death (ethidium homodimer I dye, EthD-1), cell survival (calcein), and neural stem cell (Rax, nestin) proteins. [Figure 8] FIG. 1 shows the results of immunofluorescence staining confirming the expression of neuronal markers (synapsin 1 (SYN1), TuJ1 (TUBB), MAP2, RBFOX3 (NeuN)), hypothalamic-specific neuropeptides (α-MSH, neuropeptide Y (NPY), secretagogin (SCGN), PMCH), and an astrocyte marker (GFAP) in differentiated htNSCs after inducing differentiation of organoid-derived human htNSCs into neurons. [Figure 9] This figure (top) shows the results of Western blot confirming the phosphorylation of STAT3 or FOXO1 by treatment with leptin or ghrelin after differentiation of organoid-derived human htNSCs into neurons, and a graph showing the quantification results (bottom). [Figure 10] FIG. 13 shows visualization of clustering results by single-cell RNA sequencing using uniform manifold approximation projection (UMAP) after induced differentiation of organoid-derived human htNSCs for 70 days. [Figure 11] FIG. 1 shows the results of the analysis of each cluster-specific gene. [Figure 12] FIG. 1 shows the results of visualizing the results of the analysis of cluster-specific genes by UMAP. [Figure 13] Figure 1 shows the results of comparing and analyzing the clustering results using Voxhunt with mouse brain transcripts (14 days old) from the Allen Developing Brain atlas. Comparison showed that all cell clusters differentiated from htNSCs were precisely located in the hypothalamus of 14 day old mouse brains, confirming that the cells differentiated by this established protocol are hypothalamus specific cells. [Figure 14] Pseudo-time series single-cell RNA sequencing results visualized using pseudo-time series analysis with Monocle3 after induced differentiation of organoid-derived human htNSCs for 70 days. [Figure 15] FIG. 1 shows that hypothalamic neural stem cells (htNSCs) differentiate from intermediate cells (Int1, Int2) to neural stem cells (NSCs) through pseudo-time series analysis, and that the expression of cell growth factor CDK1, proneural gene ASCL1, and neuronal factors TUBB3 and SNAP25 also change according to each developmental stage. [Figure 16] FIG. 13 is a graph showing the results of the intrinsic expression increase / decrease patterns of ROBO1, SLIT1, and SLIT2, known to be key factors in hypothalamic development, via pseudo-time series analysis when hypothalamic neural stem cells (htNSCs) differentiate from intermediate cells (Int1, Int2) to neural stem cells (NSCs). [Figure 17] Graph showing expression of master regulators (regulons) in each cell cluster (Neu1, Ast1, Tan) by performing gene regulatory network (GRN) analysis using pySCENIC. [Figure 18]FIG. 13 shows the visualization of the clustering results of neuronal clusters (Neu1 and Neu2) by reanalysing it using t-SNE (t-distributed stochastic neighbor embedding). [Figure 19] FIG. 1 shows the expression distribution of hypothalamus-related genes in each reanalyzed neuronal cluster. [Figure 20] FIG. 1 shows the gene expression patterns of single cells from neuronal cell clusters (Neu1, Neu2) by reanalysis of each cluster. [Figure 21] FIG. 1 shows the results of visualization of the clustering results of astrocyte precursor cell clusters (APC) by UMAP. [Figure 22] FIG. 1 shows the expression distribution of GFAP gene, SPARCL1 gene, and AQP4 gene in each cell cluster within an astrocyte precursor cell cluster (APC). [Figure 23] FIG. 1 shows the expression patterns of genes from single cells expressed in each cell cluster within the astrocyte cluster (Ast1, Ast2, Ast3), the astrocyte precursor cell cluster (APC), and the tanycyte cluster (Tan). [Figure 24] FIG. 1 shows an experimental schedule for confirming the effect of improving aging in mice transplanted with organoid-derived human htNSCs. [Diagram 25] FIG. 1 shows the results of immunofluorescence staining to observe the expression of human cell marker (hNCAM), hypothalamic neural stem cell marker (RAX), neuronal cell marker (RBFOX3(NeuN)), and human astrocyte marker (hGFAP) in transplanted cells in the hypothalamus of mice one month after transplantation of organoid-derived human htNSCs into the hypothalamus of 12-month-old mice. [Figure 26] 13 is a graph showing the results of measuring treadmill performance in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 27]1 is a graph showing the results of grip strength tests of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 28] 13 is a graph showing the results of rotarod testing of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 29] 1 is a graph showing the results of an open field test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 30] This is a graph showing the time required for spatial cognitive learning during hidden platform training in the Morris water maze test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 31] This is a graph showing the time spent in the target quadrant during the probe trial in the Morris water maze test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 32] 1 is a graph showing the results of a Y-maze test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 33] 13 is a graph showing the results of a novel object recognition test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 34] FIG. 1 shows a graph showing the results of measuring the body weight of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and the results of observing the external appearance of the mice. [Diagram 35]1 is a graph showing the results of measuring fat mass and lean mass of mice 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 36] FIG. 1 shows the results of measuring food intake in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 37] FIG. 13 is a graph showing the results of measuring locomotor activity in mice using metabolic cages 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 38] 1 shows the results of measuring the energy expenditure of mice using metabolic cages 1.5 months (13.5 months of age) and 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Figure 39] FIG. 1 shows the results of a glucose tolerance test in mice 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. [Diagram 40] This figure (left) shows the results of H&E staining of mouse inguinal white adipose tissue (iWAT) 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (right) shows the results of measuring the size of mouse adipocytes. [Diagram 41] This figure (left) shows the results of H&E staining of mouse epididymal white adipose tissue (eWAT) 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (right) shows the results of measuring the size of mouse adipocytes. [Diagram 42] This figure (left) shows the results of H&E staining of mouse interscapular brown adipose tissue (iBAT) 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (right) shows the results of measuring the size of mouse adipocytes. [Diagram 43]This is a diagram showing the results of H&E staining of liver tissue from mice 4 months (16 months old) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus. It was confirmed that fat accumulation in the liver tissue was reduced in the htNSC-transplanted mice. [Diagram 44] This figure (top) shows the results of Western blot analysis confirming the expression of β-actin and proteins associated with lipolysis, thermogenesis, and browning (MTCO1, SDHB, FGF21) in mouse inguinal white adipose tissue (iWAT) 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (bottom) shows the results of quantification of β-actin expression. [Diagram 45] This figure (top) shows the results of Western blot analysis confirming the expression of β-actin and proteins associated with lipolysis, thermogenesis, and browning (MTCO1, SDHB, FGF21) in mouse epididymal white adipose tissue (eWAT) 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (bottom) shows the results of quantification of β-actin expression. [Figure 46] This figure (top) shows the results of Western blot analysis confirming the expression of β-actin and proteins related to lipolysis and stress (p-eIF2A, eIF2A, ATF4, FGF21, GDF15) in mouse liver tissue 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (bottom) shows the results of quantification of β-actin expression. [Figure 47] This figure (top) shows the results of Western blot analysis confirming the expression of p16 and p21 in mouse kidney tissue 4 months (16 months of age) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and a graph (bottom) showing the results of quantification of expression. [Figure 48]The figure (top) shows the results of X-ray imaging of the femur of a mouse 4 months (16 months old) after transplantation of organoid-derived human htNSCs into the mouse hypothalamus, and the graph (bottom) shows the results of bone index analysis of the mouse. BMD: bone mineral density, BV: bone mass, TV: bone tissue volume, Tb.Th: trabecular width, Tb.N: trabecular number. [Figure 49] FIG. 1 is a schematic diagram of the experimental schedule for confirming the anti-aging effect of exosomes derived from organoid-derived human htNSCs. [Figure 50] Graph showing the results of rotarod testing in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of exosomes derived from organoid-derived human htNSCs. [Figure 51] Graph showing the results of grip strength testing in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of exosomes derived from organoid-derived human htNSCs. [Figure 52] Graph showing the results of the passive avoidance test 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of exosomes derived from organoid-derived human htNSCs. [Diagram 53] 13 is a graph showing the time required for spatial cognitive learning during hidden platform training and the time spent in the target quadrant during the probe test in the Morris water maze test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of exosomes derived from organoid-derived human htNSCs.

[0013] [Best Mode for Carrying Out the Invention] Pharmaceutical compositions containing neural stem cells In one aspect of the present invention, a pharmaceutical composition is provided that comprises neural stem cells derived from hypothalamus-like organoid as an active ingredient.Furthermore, the pharmaceutical composition can be used for the prevention and treatment of geriatric diseases.

[0014] In this case, the pharmaceutical composition may contain, as an active ingredient, nerve cells derived from the hypothalamus-like organoid, or a cell population containing neural stem cells derived from the hypothalamus-like organoid and nerve cells derived from the hypothalamus-like organoid.

[0015] As used herein, the term "hypothalamus" refers to the organ located below the two thalamuses and functions to connect the nervous system and the endocrine system through the pituitary gland. Specifically, the hypothalamus controls metabolic processes and the activity of the autonomic nervous system, synthesizes and secretes neurohormones to regulate the pituitary gland, thereby regulating body temperature, hunger, thirst, fatigue, sleep, and circadian rhythms.

[0016] As used herein, the term "organoid" refers to a "small organ-like structure" created using stem cells to have minimal functions, characterized by being made in a three-dimensional (3D) structure and being able to create an environment similar to an actual body organ in a laboratory. In other words, organoid refers to cells having a 3D structure, and also refers to a model similar to organs such as nerves and intestines that is created through an artificial culture process that is not collected or obtained from animals, etc. In this case, the origin of the cells that constitute the organoid is not limited.

[0017] In addition, organoid can have an environment that can interact with the surrounding environment in the growth process of cells.Unlike 2D culture, in 3D cell culture, cells can grow in all directions ex vivo.Therefore, in the present invention, 3D organoid can be a good model for observing the development of therapeutic agents for diseases, etc., by almost completely mimicking the organ that actually interacts in vivo.

[0018] Usually, organoids can be produced by culturing pluripotent stem cells. At this time, pluripotent stem cells (PSCs) refer to stem cells that can be induced to differentiate into any type of cell that constitutes the body, and can include but are not limited to embryonic stem cells and induced pluripotent stem cells (iPSCs, dedifferentiated stem cells). Pluripotent stem cells can be isolated from mammals, but preferably from humans.

[0019] In the present invention, the organoids can be hypothalamus-like organoids that mimic the hypothalamus.

[0020] Specifically, in the present invention, hypothalamus-like organoid can be produced by growing and culturing human embryonic stem cell (hESC) to produce 3D nervous system organoid, and treating 3D nervous system organoid with a substance that induces patterning toward hypothalamic region.Patterned hypothalamus-like organoid can be separated to finally obtain hypothalamic neural stem cell.

[0021] As used herein, the term "patterning" refers to the generation of organoids such that the majority of the clusters contain cell clusters (enriched in target cells) that are destined to have characteristics of the tissue of origin of the cells that will ultimately be extracted from a particular tissue in the brain.

[0022] In the present invention, the patterning can be patterning towards the hypothalamic region.

[0023] In the present invention, the nervous system organoid can be generated and patterned from human pluripotent stem cells by culturing in the medium containing ascorbic acid, ALK inhibitor and BMP inhibitor.Preferably, ALK inhibitor can be SB431542, and BMP inhibitor can be Noggin.

[0024] Additionally, the material that induces pattern formation can further include a material that activates the Hedgehog signaling pathway. The material that activates the Hedgehog signaling pathway can be a Sonic Hedgehog (SHH) and SMO (Smoothened) agonist.

[0025] As used herein, the term "sonic hedgehog (SHH)" has a very important role in cell proliferation and morphogenesis in the development of various body tissues such as the skeletal system, the central nervous system, and the face. In particular, sonic hedgehog is a protein that plays an important role in the development of the central nervous system such as the spinal cord, cerebrum, and cerebellum, as well as in the proliferation and differentiation of neural stem cells. When SHH binds to the cell membrane receptor PTCH1 (Patched1), the SMO receptor is activated, and this activated SMO receptor inhibits SUFU (suppressor of fused), a negative regulator of hedgehog, thereby activating the transcription factor GLI1 and promoting the proliferation of nerve cells. In the present invention, the SMO agonist can be palmorfamine.

[0026] The hypothalamic patterned organoids can be further cultured in medium containing insulin, ascorbic acid, and bFGF.

[0027] As used herein, the term "culture medium" refers to the culture medium that can support the growth, survival and differentiation of organoid in vitro, and includes all conventional culture mediums suitable for the culture and differentiation of organoids used in related fields.The type of culture medium and culture conditions can be appropriately selected according to the type of cell.In the present invention, the culture medium can be the culture medium suitable for inducing the differentiation of human stem cells into neural progenitor cells.

[0028] As used herein, the term "neural stem cell (NSC)" refers to an immature cell capable of self-renewal, continuing to proliferate in an undifferentiated state, and having the ability to differentiate into neural cells. Neural stem cells are present in various anatomical parts throughout the fetal nervous system of mammals, including humans, and recently it has been discovered that neural stem cells are not only present in fetuses, but also in certain parts of the adult nervous system, and throughout life, neural stem cells can continue to proliferate in certain parts of the brain and generate new neural cells. Neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes. Neural stem cells can proliferate in vitro, be transplanted in vivo, and can migrate, engraft, and integrate into the host nervous system, secrete therapeutically useful substances, and differentiate into appropriate neural cells with respect to cell structure and function.

[0029] In the present invention, the neural stem cells (hypothalamic NSCs, htNSCs) derived from the hypothalamus-like organoids may be characterized by expressing any one of the neural stem cell markers selected from the group consisting of SOX2, NESTIN, and KI67, and expressing any one of the hypothalamic markers selected from the group consisting of RAX, NKX2-1, ISL1, OTP, and SF-1. At this time, the neural stem cells derived from the hypothalamus-like organoids may be characterized by expressing the neural stem cell marker and the hypothalamic marker in about 84% or more of the entire DAPI-positive cell population. At this time, the neural stem cell marker and the hypothalamic marker may be expressed in about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% of the entire DAPI-positive cell population.

[0030] SOX2 (SRY-box transcription factor 2) is a transcription factor that regulates embryonic stem cell pluripotency and has a role in maintaining the self-renewal or pluripotency of embryonic stem cells and in regulating the differentiation process into specific cells.

[0031] Nestin is a type VI intermediate filament (IF) protein that is associated with radial growth of axons and is expressed primarily in neuronal cells. Nestin is expressed in the early stages of development in the central nervous system, peripheral nervous system, muscle, and other tissues, and in adults, nestin expression is induced in pathological situations such as the formation of glial scars after central nervous system injury and regeneration of damaged muscle tissue. Nestin is known to play a role in regulating the polymerization and depolymerization of intermediate filaments involved in cell remodeling in mitotic cells.

[0032] KI67 is a nuclear protein associated with cell proliferation, and the expression of KI67 increases significantly during the S phase of the cell cycle, therefore KI67 is used as a cell proliferation marker.

[0033] Furthermore, RAX (retinal and anterior neural fold homeobox) is a transcription factor with paired homeodomains that is expressed in the anterior neural plate during early embryogenesis and in the retina, hypothalamus, and pineal gland during late embryogenesis.

[0034] NKX2-1 is a transcription factor, also known as thyroid transcription factor 1 (TTF-1), that regulates the expression of thyroid-, lung-, and diencephalon-specific genes.

[0035] ISL1 (ISL LIM homeobox 1) is a transcription factor with two LIM domains (N-terminus) and one homeodomain (C-terminus). ISL1 is known to regulate the development of islets of Langerhans during embryogenesis and to play an important role in the differentiation of motor neurons in the neural tube of mouse embryos.

[0036] OTP (orthopedia homeobox) is a transcription factor that contains a homeodomain and is known to be involved in the differentiation and development of hypothalamic neuroendocrine cells.

[0037] SF-1 (steroidogenic factor 1) is known to be an essential factor for the development and function of the adrenal gland and reproductive organs. In the brain, SF-1 is known to be expressed exclusively in the ventromedial nucleus of the hypothalamus (VMH), to regulate the development and function of the VMH, and to be involved in the regulation of energy metabolism homeostasis.

[0038] Furthermore, neural stem cells derived from hypothalamus-like organoids can maintain their morphology even after freezing and thawing and can exhibit high viability.

[0039] Furthermore, the neural stem cells derived from the hypothalamus-like organoid can be passaged. Specifically, the neural stem cells derived from the hypothalamus-like organoid can be passaged 1 to 10 times, 2 to 8 times, 3 to 7 times, or 4 to 6 times.

[0040] The neural stem cells derived from the hypothalamus-like organoids according to the present invention can differentiate into hypothalamus-specific neural cells.

[0041] Specifically, the neural stem cells derived from the hypothalamus-like organoids according to the present invention can be differentiated into neural cells that express at least one neural cell marker selected from the group consisting of synapsin 1 (SYN1), TuJ1 (TUBB), MAP2, and RBFOX3 (NeuN), and express at least one hypothalamic neuropeptide selected from the group consisting of α-MSH, neuropeptide Y (NPY), secretagogin (SCGN), and PMCH.

[0042] Tuj1 is known as neuron-specific class III β-tubulin (TUBB3) and is the main component of microtubules. Tuj1 is mainly expressed in neurons and is involved in neurogenesis and axon guidance and maintenance.

[0043] MAP2 (microtubule-associated protein 2) is a cytoskeletal protein known to be involved in microtubule polymerization, an essential step in neurogenesis. During the developmental stages of mouse neurons, MAP2 is known to be mainly located in dendrites and to be involved in stabilizing the shape of dendrites.

[0044] Synapsin is a member of the synapsin family and functions in the regulation of synapse formation and neurotransmitter release.

[0045] NeuN, also known as hexaribonucleotide-binding protein-3 or Fox-3, is a protein found in the nuclei of neurons that is commonly used as a neuronal biomarker.

[0046] α-MSH (α-melanocyte-stimulating hormone) is an endogenous peptide hormone and a neuropeptide of the melanocortin family, which is one of the melanocyte-stimulating hormones that promotes melanin production. α-MSH is also known to be involved in feeding behavior, energy homeostasis, sexual behavior, and protection against ischemia and reperfusion injury.

[0047] NPY (neuropeptide Y) is a 36 amino acid neuropeptide involved in various physiological and homeostatic regulation in the central and peripheral nervous systems. NPY is the most abundant peptide in the mammalian central nervous system, which consists of the brain and spinal cord. In the autonomic nervous system, NPY is mainly produced by neurons of the sympathetic nervous system, has a role in vasoconstriction, and is also involved in the growth of adipose tissue. In addition, NPY is known to be produced in various locations in the brain, including the hypothalamus, to increase food intake and energy storage, reduce anxiety, stress, and pain sensitivity, and participate in the regulation of circadian rhythms.

[0048] Secretagogin (SCGN) is a calcium-binding protein expressed in the cytoplasm and is known to be involved in potassium chloride-induced calcium influx and cell proliferation. Furthermore, secretagogin is known to be involved in the release of the stress hormone corticotropin-releasing hormone (CRH) and stress activation of the brain.

[0049] PMCH (pro-melanin-concentrating hormone) is the precursor protein of MCH (melanin-concentrating hormone), a neuropeptide that regulates hunger, energy homeostasis, reproductive function, and sleep. Dysfunction of PMCH is known to be associated with frontometaphyseal dysplasia2 and Huntington's disease.

[0050] As used herein, the term "differentiation" refers to the phenomenon in which cells divide and grow, and the structure or function of cells becomes specialized during the growth of the whole organism. In other words, differentiation refers to the process by which cells, tissues, and the like of living organisms change to have morphology and functions suitable for performing their respective roles. In the present invention, the above term may include the process by which pluripotent stem cells transform into cells that constitute the hypothalamus, and may also include the process by which progenitor cells express specific differentiation characteristics. In the present invention, when neural stem cells (htNSCs) derived from hypothalamus-like organoids differentiate into neural cells, astrocytes (GFAPs) are differentiated into neural cells. + ) and glial cells can also differentiate together. Differentiated neurons and astrocytes can then sense the body's nutritional status by responding to the peptides leptin and ghrelin, which are produced in peripheral organs.

[0051] Specifically, the neural stem cells (htNSCs) derived from the hypothalamus-like organoids according to the present invention can differentiate and cluster into neural stem / progenitor cell clusters (NSCs), intermediate progenitor cell clusters (IPCs), neuronal cell clusters (Neu1 and Neu2), astrocyte precursor cell clusters (APCs), astrocyte clusters (Ast1, Ast2, and Ast3), tanycyte clusters (Tan), interneuron precursor cell clusters (Int1 and Int2), radial glia clusters (RGs), and proteoglycan-secreting glial clusters.

[0052] A progenitor cell is a cell that differentiates into a specific type of cell, and is more specific than a stem cell in differentiating into a "target cell". Stem cells can divide themselves infinitely, whereas progenitor cells can divide only a limited number of times. In the present invention, the progenitor cell can be a neural progenitor cell, an astrocyte progenitor cell, or an interneuron progenitor cell.

[0053] A nerve cell is a cell that constitutes the nervous system and can transmit signals in an electrical manner using ion channels such as sodium channels and potassium channels. In addition, nerve cells receive and store various information by exchanging chemical signals with other adjacent nerve cells through structures called synapses. In this specification, nerve cells can be used interchangeably with "neurons" or "neuron cells".

[0054] A neural progenitor cell refers to a cell that can differentiate into a neural cell.

[0055] Furthermore, astrocytes, also called astroglia, are present in the brain and spinal cord. Astrocytes provide biochemical support to cells in the blood-brain barrier (BBB) ​​and have processes attached to blood vessel walls to supply nutrients to nerve cells. Astrocytes also perform various roles such as regulating ion concentrations in nerve cells, supporting nerve cells, removing waste products, and phagocytosis. Furthermore, when nerve tissue is damaged, astrocytes proliferate and fill the area with processes called gliomas, and play a role in repairing or destroying the damaged tissue. Hydrogen peroxide, which is generated during the process of astrocytes breaking down toxins, has been shown to be the cause of nerve cell destruction, suggesting the possibility of treating brain diseases such as dementia.

[0056] Astrocyte precursor cells refer to cells that can differentiate into astrocytes.

[0057] Tanycytes are ependymal cells present in the third and fourth ventricles of the hypothalamus, and they sense nutritional status and regulate appetite.

[0058] Interneurons are nerve cells that connect two different areas of the brain, and are a type of nerve cell that is neither a sensory neuron nor a motor neuron. Sensory neurons transmit stimuli received by sensory organs to interneurons, which integrate and determine these stimuli and transmit them to motor neurons. Interneurons are included in the central nervous system and play an important role in neurogenesis in the mammalian adult brain, including reflexes and neural oscillations.

[0059] Interneuron clusters (interneuron progenitor cells) refer to cells that differentiate into interneurons.

[0060] Radial glia are a type of glial cell that are precursors of neurons and astrocytes. They also function as support for guiding neurons to their desired location.

[0061] Proteoglycan-secreting glia are glial cells that secrete proteoglycans. When the nervous system is damaged, astrocytes and mesenchymal cells secrete proteoglycans and extracellular matrix proteins to repair the damaged nervous system. This has the positive function of isolating the damaged area and preventing the damage from spreading, but the substances secreted by astrocytes during the process of forming this glial scar also inhibit the growth of neurites.

[0062] Among the clustered cell clusters, the neuronal cell clusters (Neu1 and Neu2) may express genes related to the development of the hypothalamus. The astrocyte precursor cell cluster (APC) may include cells differentiated into a cell cluster (Ast2) expressing at least one gene selected from the group consisting of SLC1A3, SLCO1C1, CRYM, and SCG2; a cell cluster (Ast1) expressing at least one gene selected from the group consisting of RSAD2, TRAIL, ISG5, IFIT, and STAT1; and a cell cluster (Ast3) expressing at least one gene selected from the group consisting of SOX9, ID3, Meis2, CLDN5, ALCAM, and COL1A2. In this case, the Ast2 cluster may be involved in the nutritional supply and neuroendocrine function of the neuronal cells. The Ast1 cluster may be involved in the immune function. The Ast3 cluster may regulate the activity of the neuronal cells. Tanycyte clusters (Tan) can be cell clusters that express at least one gene selected from the group consisting of RAX, FGF10, COL25A1, CRYM, and SCN7A.

[0063] SLC1A3 (solute carrier family 1 member 3) is the gene encoding the excitatory amino acid transporter 1.

[0064] SLCO1C1 (solute carrier organic anion transporter family member 1C1) is a gene that encodes a membrane transport protein (organic anion transporting polypeptide) that mediates the transport of organic anions and is expressed in the brain and testis.

[0065] CRYM is a gene encoding the crystallin mu homolog, also known as thyroid hormone-binding protein (THBP), which is regulated by NADP.

[0066] SCG2 is the gene encoding secretogranin II, a member of the chromogranin / secretogranin family of neuroendocrine proteins. In its full-length form before cleavage, secretogranin II functions to select and package neuropeptides and peptide hormones into secretory bodies, and after cleavage, secretogranin II functions as the active form of secretoneurin.

[0067] RSAD2 is the gene encoding radical s-adenosylmethionine domain-containing protein 2, also known as viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). Viperin is activated by interferon (IFN) and inhibits infection by DNA or RNA viruses.

[0068] TRAIL is a gene that encodes the TNF-related apoptosis-inducing ligand, which is a ligand that induces apoptosis.

[0069] IFIT is a gene that encodes an interferon-inducible antiviral protein (interferon-inducible protein with tetratricopeptide repeats).

[0070] STAT1 is a gene that encodes a protein (signal transducer and activator of transcription 1) that mediates cell signaling of interferons, cytokines, and growth factors.

[0071] SOX9 is a gene that encodes a transcription factor that regulates chondrocyte differentiation and skeletal development.

[0072] ID3 is a gene encoding a transcription factor that lacks a DNA-binding site and forms a heterodimer to inhibit DNA binding and transcriptional activity. The transcription factor ID3 regulates various cellular processes, including cell proliferation, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation.

[0073] Meis2 is a gene that encodes the homeobox protein Meis2, which binds to HOX proteins or PBX proteins to form dimers, or binds to DNA-binding dimers of PBX proteins and HOX proteins to stabilize homoprotein-DNA complexes, thereby regulating transcription.

[0074] CLDN5 is the gene that encodes claudin-5, which specifically removes intracellular tight junctions.

[0075] ALCAM is the gene encoding CD166, which mediates adhesion between thymic epithelial cells and CD6+ cells during T cell development in the thymus.

[0076] COL1A2 is the gene that codes for the collagen alpha-2 (I) chain, a type of type 1 collagen.

[0077] FGF10 is a gene that encodes fibroblast growth factor 10, which is involved in embryonic development, cell proliferation, regulation of cell differentiation, and wound healing.

[0078] COL25A1 is a gene encoding the collagen alpha-1 (XXV) chain, a membrane-bound collagen protein that is specifically expressed in the brain and has a role in inhibiting fibril formation of amyloid-β peptide (Aβ).

[0079] SCN7A is the gene encoding the sodium channel protein type 7 subunit alpha. SCN7A plays a role in mediating the voltage-dependent sodium ion permeability of excitable membranes.

[0080] Use of pharmaceutical compositions containing neural stem cells The pharmaceutical composition comprising the neural stem cells derived from the hypothalamus-like organoid according to the present invention as an active ingredient can be used for the purpose of preventing or treating geriatric diseases.Furthermore, the pharmaceutical composition can further comprise the neural cells derived from the hypothalamus-like organoid, or a cell population comprising neural stem cells and neural cells.At this time, the hypothalamus, organoid, neural stem cells, and neural cells are the same as above.

[0081] In the present invention, the geriatric disease may be selected from the group consisting of neurodegenerative diseases, osteoporosis, and age-related diseases of muscle.

[0082] In this case, the neurodegenerative disease may be selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, corticobasal degeneration, diffuse Lewy body disease, and Pick's disease.

[0083] In this case, the age-related muscle disease may be selected from the group consisting of muscle atrophy, disuse muscle atrophy, and senile sarcopenia.

[0084] As used herein, the term "prevention" refers to any action of administering a pharmaceutical composition to inhibit or delay the onset of a geriatric disease. The term "treatment" refers to any action of administering a pharmaceutical composition to improve or favorably alter the symptoms of a geriatric disease.

[0085] In the pharmaceutical composition for preventing and treating geriatric diseases of the present invention, neural stem cells, nerve cells, or cell populations containing these cells may be contained in any amount (effective amount) depending on the purpose, formulation, purpose of mixing, etc., as long as they can exhibit anti-aging activity. The pharmaceutical composition of the present invention contains about 2 × 10 5 ~about 2×10 10 cells, preferably about 2 x 10 6 ~about 2×10 9 cells, more preferably about 1×107 ~about 2×10 8 Preferably, the pharmaceutical composition contains 2×10 cells per mL. 8 The cell may include cells.

[0086] Here, the term "effective amount" refers to the amount of an active ingredient capable of inducing an anti-aging effect. Such an effective amount can be determined experimentally within the scope of ordinary skill in the art.

[0087] The composition of the present invention can be used in an unfrozen state or frozen for later use.When freezing, standard cryopreservation agents (e.g., DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, Epilife® cell freezing medium (Cascade Biologics)) can be added to cell population before freezing.

[0088] The pharmaceutical composition of the present invention can be applied in the form of a cell therapy agent, and can be formulated by further including a pharma- ceutically acceptable carrier.

[0089] In this case, a "cell therapy" is a drug used for therapeutic, diagnostic, and preventive purposes using cells and tissues prepared through isolation from a subject, culture, and special manufacturing (US FDA regulations). A cell therapy refers to a drug used for therapeutic, diagnostic, and preventive purposes through a series of actions, such as expanding or selecting viable autologous, allogeneic, or xenogeneic cells ex vivo, or otherwise changing the biological properties of cells so that the function of the cells or tissue is restored. Cell therapy is broadly classified into somatic cell therapy and stem cell therapy depending on the degree of cell differentiation. In the present invention, the cell therapy can be a stem cell therapy.

[0090] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier or diluent that does not significantly stimulate an organism and does not inhibit the biological activity and properties of the administered component. In the present invention, pharmaceutical acceptable carriers that can be included in a cell therapy agent, such as buffers, preservatives, analgesics, solubilizers, isotonicity agents, stabilizers, bases, excipients, lubricants, etc., can be used without limitation as long as the carrier is known in the art. The pharmaceutical composition of the present invention can be prepared in various formulations according to commonly used techniques.

[0091] The pharmaceutical composition of the present invention can be administered in a pharmacologic effective amount, and can be administered via any route as long as it can induce migration to diseased sites.Here, "administration" means introducing a predetermined substance into a subject by a suitable method.In some cases, it can be considered that the pharmaceutical composition of the present invention is loaded into a medium that contains a means for directing neural stem cells to lesions.

[0092] Thus, the pharmaceutical composition of the present invention can be administered via several routes, including topical administration (including oral, sublingual, dermal, and ocular administration), parenteral administration (including subcutaneous, intradermal, intramuscular, infusion, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid), or transdermal administration, preferably directly to the disease site. In one embodiment, the neural stem cells can be administered to the subject suspended in a suitable diluent, which is used to protect and maintain the cells and to facilitate the use of the cells when injected into the target tissue. The diluent can include buffer solutions such as saline, phosphate buffered saline, and HBSS, cerebrospinal fluid, and the like. Furthermore, the composition can be administered by any device that allows the active substance to be transferred to the target cells.

[0093] The preferred mode of administration and formulation is injection. Injection can be prepared using aqueous solutions such as physiological saline, Ringer's solution, Hank's solution, or sterile aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleate, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol, or glycerin, and the like. For mucosal penetration, impermeable agents known in the art suitable for the barrier of passage can also be used, which can further include pharmaceutical carriers, stabilizers to prevent deterioration, such as ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, and the like, emulsifiers, buffers to adjust pH, and preservatives to inhibit the growth of microorganisms, such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol. Methods for formulating pharmaceutical compositions are known in the art, and specific reference may be made to documents such as Remington's Pharmaceutical Sciences (19th Edition, 1995), which are considered part of the present specification.

[0094] As used herein, the term "pharmaceutical effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio that is applicable to medical treatment and does not cause side effects. Effective dose levels can be easily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of disease, drug activity, drug sensitivity, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0095] The preferred dosage of the pharmaceutical composition of the present invention is about 1.0×10 cells / day, depending on the condition, weight, sex, and age of the patient, the severity of the patient, and the route of administration. 3 ~Approx. 1.0×10 10 Individual cells / kg, approximately 1.0×10 4 ~Approx. 1.0×10 9Individual cells / kg, approximately 1.0×10 5 ~Approx. 1.0×10 8 cells / kg, or approximately 1.0 x 10 6 ~Approx. 1.0×10 7 It may be cells / kg.

[0096] However, the dosage can be formulated in various ways depending on factors such as the formulation method, the mode of administration, the age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and those skilled in the art can appropriately adjust the dosage taking these factors into consideration. The frequency of administration can be one or more than one time within the range of clinically acceptable side effects. With regard to the site of administration, the dosage can be administered at one site or two or more sites. In animals other than humans, the dosage can be administered at the same dosage as in humans per kg or per subject, or the dosage can be administered at an amount converted from the dosage above, for example, by the volume ratio (e.g., average value) of organs (such as the heart) between the target animal and humans. Such dosage should not be interpreted as limiting the scope of the present invention in any way.

[0097] As used herein, the term "subject" refers to a subject to which the composition of the present invention can be applied (prescribed), and may be a mammal, such as a rat, a mouse, or a livestock animal, including a human. Preferably, the subject may be a human, but is not limited to a human. In addition to the neural stem cells derived from hypothalamus-like organoids, the pharmaceutical composition of the present invention may further include any compound or natural extract that has already been confirmed to be safe and has anti-aging activity in order to increase or enhance the neural cells or anti-aging activity derived from hypothalamus-like organoids. In this case, the neural stem cells, neural cells, or cell populations containing these cells, and the compound or natural extract having anti-aging activity may be administered simultaneously or sequentially.

[0098] In another aspect of the present invention, the use of the pharmaceutical composition comprising the neural stem cell derived from hypothalamus-like organoid as an active ingredient for the prevention and treatment of geriatric disease is provided.At this time, the pharmaceutical composition can further comprise the neural cell derived from hypothalamus-like organoid.Here, the hypothalamus, organoid, neural stem cell, neural cell, geriatric disease, prevention and treatment are the same as above.

[0099] In another aspect of the present invention, a method for preventing and treating geriatric diseases is provided, comprising administering to a subject a pharmaceutical composition comprising neural stem cells derived from hypothalamus-like organoid as an active ingredient.At this time, the pharmaceutical composition can further comprise neural cells derived from hypothalamus-like organoid.Here, the hypothalamus, organoid, neural stem cells, geriatric diseases, administration, prevention and treatment are the same as above.

[0100] The subject may be a mammal, such as a rat, mouse, or livestock, including a human. Preferably, the subject may be a human.

[0101] The dosage is approximately 1.0×10 cells / day depending on the condition, weight, sex, and age of the patient, the severity of the patient, and the route of administration. 3 ~Approx. 1.0×10 10 Individual cells / kg, approximately 1.0×10 4 ~Approx. 1.0×10 9 Individual cells / kg, approximately 1.0×10 5 ~Approx. 1.0×10 8 cells / kg, or approximately 1.0 x 10 6 ~Approx. 1.0×10 7 However, the dosage can be formulated in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and those skilled in the art can appropriately adjust the dosage taking these factors into account.

[0102] Exosomes derived from neural stem cells In another aspect of the present invention, there is provided an exosome secreted from neural stem cells derived from hypothalamus-like organoids, wherein the hypothalamus, organoid, and neural stem cells are the same as those described above.

[0103] As used herein, the term "exosome" refers to extracellular vesicles, also known as microvesicles, secreted by cells, having a size of 50 to 150 nm. Exosomes are secreted from cells when multivesicular bodies fuse with the cell membrane or are secreted directly through the cell membrane. Exosomes contain a complex mixture of factors related to cell differentiation, proliferation, migration, and signal transduction, such as intracellular proteins, cell membrane proteins, lipids, and nucleic acids, such as RNA, miRNA, and DNA, and are therefore known to have important roles in various processes such as coagulation, intercellular signal transduction, and management of metabolic waste. The diameter of exosomes can be about 30 nm to about 500 nm, about 30 nm to about 400 nm, about 30 nm to about 300 nm, about 30 nm to about 200 nm, about 50 nm to about 200 nm, about 50 nm to about 180 nm, about 75 nm to about 180 nm, or about 50 nm to about 150 nm. Exosomes may originate from the plasma membrane or multivesicular bodies (MVBs) and may be released or secreted to the outside of the cell.

[0104] In the present invention, exosomes can be isolated from cultures of neural stem cells, neural cells, or cell populations comprising hypothalamus-like organoids.

[0105] The exosomes derived from neural stem cells derived from hypothalamus-like organoids according to the present invention can prevent or treat geriatric diseases, where the hypothalamus, organoids, neural stem cells, nerve cells, prevention and treatment are the same as above.

[0106] The geriatric disease may be a neurodegenerative disease and an age-related disease of muscle, where the neurodegenerative disease and the age-related disease of muscle are the same as described above.

[0107] Pharmaceutical compositions comprising exosomes derived from neural stem cells In another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating geriatric diseases, comprising an exosome as an active ingredient, wherein the exosome, prevention, and treatment are the same as those described above. Furthermore, the geriatric diseases can be neurodegenerative diseases and muscular aging-related diseases, and the neurodegenerative diseases and muscular aging-related diseases are the same as those described above.

[0108] In the pharmaceutical composition for preventing and treating geriatric diseases of the present invention, exosomes may be contained in any amount (effective amount) depending on the purpose, formulation, purpose of mixing, etc., as long as they can exhibit anti-aging activity. The pharmaceutical composition of the present invention may contain about 1 μg / mL to 1 mg / mL, 10 μg / mL to 100 μg / mL, 10 μg / mL to 50 μg / mL, or 10 μg / mL to 20 μg / mL per mL. Here, the effective amount is the same as above.

[0109] The compositions of the present invention can be administered to a subject in a pharma- ceutically effective amount, where the pharma- ceutically effective amount, administration, and subject are the same as described above.

[0110] A preferred dosage of the pharmaceutical composition comprising the exosome of the present invention as an active ingredient can be administered at a dosage of 0.001 mg / kg to 100 mg / kg of body weight, 0.005 mg / kg to 50 mg / kg of body weight, 0.02 mg / kg to 20 mg / kg of body weight, or 0.1 mg / kg to 10 mg / kg of body weight per day, depending on the condition, body weight, sex, and age of the patient, the severity of the patient, and the route of administration.

[0111] Administration can be once a day or divided into several times a day. However, the dosage can be formulated in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and those skilled in the art can appropriately adjust the dosage taking these factors into account. The frequency of administration can be one or more than one time within the range of clinically acceptable side effects. In terms of the administration site, the dosage can be administered at one site or two or more sites. In animals other than humans, the dosage can be administered at the same dosage as in humans per kg or per subject, or the dosage can be administered at an amount converted from the dosage above, for example, by the volume ratio (e.g., average value) of organs (such as the heart) between the target animal and humans. Such dosages should not be interpreted as limiting the scope of the present invention in any way.

[0112] In some cases, it may be considered to load the pharmaceutical composition of the present invention into a medium that includes a means for directing exosomes to lesions.Therefore, the pharmaceutical composition of the present invention may be administered via several routes, including topical administration (including oral, sublingual, dermal, and intraocular administration), parenteral administration (including subcutaneous, intradermal, intramuscular, drip, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid), or transdermal administration, and may be preferably administered directly to the disease site.However, the administration method is not limited thereto, and any method suitable for treatment may be used.

[0113] The preferred mode of administration and formulation of the pharmaceutical composition of the present invention is injection.Methods for formulating pharmaceutical compositions are known in the art, and specific reference can be made to the literature [Remington's Pharmaceutical Sciences (19th edition, 1995)], etc. The above literature is considered as part of this specification.

[0114] Further, the composition of the present invention may include a pharma- ceutically acceptable carrier. The composition and the pharma- ceutically acceptable carrier are the same as above. The pharma- ceutically acceptable carrier means an excipient, a diluent, or an adjuvant. Examples of the carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, saline, buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, and the like.

[0115] In addition to exosomes as an active ingredient, the anti-aging composition of the present invention may further comprise a cell therapy agent (e.g., neural stem cells, neural cells, or cell populations containing these cells), any compound or natural extract that has already been confirmed to be safe and has anti-aging activity for increasing or enhancing the anti-aging activity. In this case, the exosomes and the cell therapy agent, the compound or natural extract having anti-aging activity may be administered simultaneously or sequentially.

[0116] In another aspect of the present invention, there is provided a use of exosomes isolated from neural stem cells derived from hypothalamus-like organoids for the prevention and treatment of geriatric diseases, wherein the hypothalamus, organoid, neural stem cells, neural cells, exosomes, geriatric diseases, prevention, and treatment are the same as above.

[0117] In another aspect of the present invention, there is provided a method for preventing and treating geriatric diseases comprising administering exosomes to a subject, wherein the exosomes, subject, administration, geriatric disease, prevention, and treatment are the same as above.

[0118] A preferred dosage of exosomes may be administered at a dosage of 0.001 mg / kg to 100 mg / kg of body weight, 0.005 mg / kg to 50 mg / kg of body weight, 0.02 mg / kg to 20 mg / kg of body weight, or 0.1 mg / kg to 10 mg / kg of body weight per day, depending on the condition, weight, sex, and age of the patient, the severity of the patient, and the route of administration.

[0119] Administration can be once a day or divided into several times a day. However, dosage can be formulated in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and those skilled in the art can appropriately adjust the dosage taking these factors into account.

[0120] Methods for producing exosomes derived from neural stem cells In another aspect of the present invention, a method for producing exosomes is provided, comprising culturing neural stem cells from hypothalamus-like organoids and recovering exosomes from the culture, wherein the hypothalamus, organoids, neural stem cells and exosomes are the same as above.

[0121] Kits Comprising Exosomes Derived from Neural Stem Cells and / or Neural Stem Cells In another aspect of the present invention, a kit for preventing and treating geriatric diseases is provided, comprising neural stem cells derived from hypothalamus-like organoids and / or exosomes secreted from neural stem cells derived from hypothalamus-like organoids, wherein the hypothalamus, organoids, neural stem cells, exosomes, prevention and treatment are the same as above.

[0122] The geriatric disease may be a neurodegenerative disease, osteoporosis, and an age-related disease of muscle, where the neurodegenerative disease and the age-related disease of muscle are the same as described above.

[0123] [Mode for carrying out the invention] The present invention will now be described in more detail with reference to the following examples. However, the following examples are merely for illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0124] Experimental example 1. Cell culture Human embryonic stem cell line H9 (H9 hESC, WiCell) was cultured in mTeSR-1 medium (Stem Cell Technology) on Matrigel-coated plates in the absence of feeder cells. Human hypothalamic-derived neural stem cells (ntNSCs) were generated from H9 hESCs using a 3D culture method.

[0125] Specifically, hESCs were suspended, seeded, and cultured for 24 hours in neural induction medium at a density of 10,000 cells per well in low-adhesion 96-well round-bottom plates using a 1:1 mixture of Neurobasal medium (Gibco) supplemented with 10 μM SB431542 (Tocris), 200 ng / mL Noggin (Peprotech), and 20 μM Y27632 (ROCK inhibitor, Sigma) and N2 medium containing vitamin A-free B27 (Invitrogen), 200 μM ascorbic acid (Sigma), and 1.25 mg / L insulin.

[0126] For hypothalamic patterning of H9 hESCs, H9 hESCs were cultured in neural induction medium containing 10 μM SB431542 (Tocris) and 200 ng / mL noggin (Peprotech) for 5 days, then cultured for 6 days with 100 ng / mL sonic hedgehog (SHH, Peprotech) and 2 μM palmorfamine (Calbiochem) added to the medium. On day 12 after the start of differentiation, the formed spheres were transferred to low-adhesion 6-well plates and cultured for 8 days (until day 20 after the start of differentiation) in neural proliferation medium using an orbital shaker (80 rpm). The neural proliferation medium used was N2 medium containing 1.25 mg / L insulin, 200 μM ascorbic acid, 20 ng / L bFGF (Peprotech), and penicillin / streptomycin. The spheres were then chopped, suspended in neural proliferation medium, and seeded on poly-L-ornithine / fibronectin-coated plates. The neural stem cells obtained through the above process were cultured in N2 medium (neuron differentiation medium) containing 1.25 mg / L insulin, 200 μM ascorbic acid, 10 ng / mL BDNF (Peprotech), 10 ng / mL GDNF (Peprotech), 125 μM db-cAMP (Sigma), and penicillin / streptomycin to differentiate into neural cells.

[0127] Experimental example 2: Animal experiment All procedures for animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, Hanyang University (approval numbers 2018-0217A and 2020-0142A) and the IACUC and Institutional Biosafety Committee (IBC) of the Korea Institute of Science and Technology (KIST) (approval number KIST-2019-048). For animal experiments, 11-month-old middle-aged C57BL / 6 male mice were purchased from the Institute of Basic Science Research or Janvier Lab. Mice were housed in a vivarium with constant temperature (23 ± 1 °C), constant humidity (50 ± 10%), and a 12-h light / dark cycle and had free access to food and water.

[0128] Experimental example 3. Cell transplantation experiment For cell transplantation, 12-month-old male C57BL / 6J mice were used. First, the mice were anesthetized with Zoletil (37.5 mg / kg) and Rompun (5.83 mg / kg), and then human hypothalamic-derived NSCs (1 μL, 2 × 10 5 100 cells / μL) were injected into the medial basal hypothalamus (MBH) at a rate of 250 nL / min for 2 min (AP: -1.5 mm, ML: ±0.4 mm, DV: -5.6 mm). After injection, the syringe was not removed but left in place for an additional 5 min.

[0129] Experimental Example 4. Histological analysis Mice were anesthetized and perfused intracardially with 30 mL of PBS (pH 7.4) to remove blood, then perfused intracardially with 4% paraformaldehyde (PFA, 30 mL) diluted in PBS to fix the tissue. Fixed brain tissue was extracted and further fixed overnight in 4% PFA (diluted in PBS) at 4°C. Fixed brain tissue was reacted with 30% sucrose for 7 days to remove water in the tissue (cryoprotection). Dehydrated brain tissue was molded in TissueTek OCT compound (Sakura Finetech Japan Co., Ltd.), frozen, and then sectioned at a thickness of 30 μm using a cryostat (Leica) to prepare brain tissue sections.

[0130] For peripheral tissues, white adipose tissue (WAT), brown adipose tissue (BAT), liver, and femur extracted after dissection were separated and preserved for protein analysis and immunostaining. For tissue staining, each tissue was fixed in 4% formaldehyde (PFA) at 4°C overnight, then embedded in paraffin, and sectioned at a thickness of 4 μm to prepare tissue sections. Tissue staining was performed in the same manner as the known H&E method.

[0131] Experimental example 5. Immunostaining Cells and cryosectioned brain tissue sections were fixed in 4% paraformaldehyde (PFA) diluted in PBS, then treated with 0.3% Triton X-100 containing 1% BSA and incubated for 1 hour. These were then incubated with anti-nestin antibody (1:500, BioLegend, 65680), anti-Rax antibody (1:500, Takara Bio Inc., M228), anti-Sox2 antibody (1:200, ThermoFisher, MA5-13961), anti-Nkx2-1 antibody (1:10, DSHB, 40.2D6), anti-KI67 antibody (1:500, Abcam, ab15580), anti-TUJ1 antibody (1:200, BioLegend, 801202), anti-MAP2 antibody (1:200, Sigma-Aldrich, M1406), anti-RBFOX3 antibody (1:200, Millipore, MAB377), anti-SYN1 antibody (1:200, Millipore, AB1543), and anti-αMSH antibody (1:2000, Phoenix) as primary antibodies. The cells were treated with anti-NPY antibody (1:1000, ImmunoStar, 22940), anti-SCGN antibody (1:500, Sigma-Aldrich, HPA006641), anti-PMCH antibody (1:500, Sigma-Aldrich, M8440), anti-GFAP antibody (1:500, Agilent, Z0334), anti-hNCAM antibody (1:500, Santa Cruz Biotechnology, sc-106), anti-hGFAP antibody (1:400, BioLegend, 837201), or anti-huMt antibody (1:500, Abcam, ab92824) and incubated overnight at 4°C in the dark. For visualization, anti-mouse IgG conjugated with Cy3 or Alex Flour 488 (1:1000, Jackson Immunoresearch Laboratories, 111-165-003 or A-11001) was used as secondary antibody.

[0132] Stained cells and brain tissue sections were mounted using VECTASHIELD mounting solution containing DAPI (Vector Laboratories) and observed using a fluorescence microscope (Leica).

[0133] Experimental Example 6. Quantitative RT-PCR analysis RNA was extracted using TRIzol™ Reagent (Invitrogen) according to the manufacturer's protocol, and cDNA was then synthesized using the SuperScript™ Kit (Invitrogen). Quantitative RT-PCR (q-PCR) was performed on a CFX96™ Real-Time System using iQTM SYBR® Green Supermix (Bio-Rad), and gene expression levels were quantified as GAPDH or beta-actin expression levels. The primers used are shown in Table 1.

[0134] [Table 1]

[0135] Experimental Example 7. Western Blot RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing protease inhibitors and kinase inhibitors was added to the cells, and the cells were lysed by sonication. The cell lysate was centrifuged at 4°C and 15000g for 5 minutes to obtain the supernatant, which was then quantified using Pierce BCA analysis (Thermo Fisher). Proteins separated by SDS-PAGE electrophoresis were transferred to a nitrocellulose membrane, and then TBST containing 5% BSA was added and reacted for 1 hour to block. After blocking, the proteins were treated with the primary antibodies shown in Table 2 and reacted at 4°C overnight. After the primary antibody reaction, goat anti-rabbit-HRP conjugate or goat anti-mouse-HRP conjugate (Cell Signaling Technology) as the secondary antibody was diluted 1:1000 in TBST containing 5% BSA and applied to the membrane and reacted for 1 hour. Protein expression of each antibody was confirmed using West-Pico chemiluminescence substrate (Thermo Fisher). Activity analysis of leptin and ghrelin was performed by treating the differentiated cells with leptin (100 ng / mL) or ghrelin (10 nM), respectively, and then reacting for 1 hour.

[0136] [Table 2]

[0137] Experimental Example 8. Exosome isolation and treatment of mice The exosomes in the cell culture medium were purified using an ultracentrifuge. Specifically, the cell culture medium was centrifuged to obtain a supernatant, which was then centrifuged at 300g for 10 minutes, 2000g for 10 minutes, 10000g for 30 minutes, and 100,000g for 1 hour in an ultracentrifuge, filtered, and then centrifuged at 100,000g for 1 hour in an ultracentrifuge to obtain exosomes.

[0138] The exosomes isolated through the above process were finally resuspended in PBS and prepared.

[0139] In all mice, a 26-gauge guide cannula was implanted into the third ventricle (3V) of the hypothalamus at midline coordinates 2.0 mm dorsal to bregma and 5.0 mm inferior to bregma using a precision small animal stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA) in the same manner as in Example 2. Among these mice, the vehicle (PBS)-treated group and the exosome-treated group were injected with PBS or exosomes via the cannula into the 3V three times a week for three months.

[0140] Experimental example 9. Mouse behavioral experiment All behavioral experiments were performed in a behavioral laboratory, and all behavioral assessments were recorded using a computerized ANY-maze video tracking system (Stoelting). Furthermore, all experiments were performed in accordance with the ARRIVE guidelines.

[0141] Experimental Example 9.1. Evaluation of muscle strength (grip strength test) The mouse was placed on a square grid (1 cm mesh size), then the grid was turned over and the time the mouse hung by its feet was measured and recorded for 2 minutes. The experiment was repeated three times, and each mouse was given at least 10 minutes of rest.

[0142] Experimental Example 9.2. Evaluation of motor coordination (rotarod test) Mice were trained on a rotarod (BS Technolab INC.) moving at a speed of 5 rpm for 60 seconds, and then rested for 10 minutes. Each mouse was then placed on the rotarod moving at a speed of 5 rpm, and the mouse's behavior was recorded while the speed was increased by 5 rpm per minute for 5 minutes. The experiment was repeated three times, and each mouse was given a 30-minute rest.

[0143] Experimental Example 9.3. Evaluation of endurance (treadmill test) Endurance assessment was performed using a treadmill (Jeung Do Bio & Plant Co., Ltd.) that can assess up to eight mice in individual lanes. Before the experiment, the mice were acclimated to the treadmill at a speed of 5 rpm for 5 minutes to prevent injury and failure during running. Then, the speed was increased by 2 rpm per minute for 10 minutes, and the time until the mice stopped running was recorded.

[0144] Experimental Example 9.4. Novel Object Recognition Test First, before the experiment, the mouse was placed in an open field box (40 cm wide x 40 cm long x 50 cm high) in a free state for 10 minutes. Then, the mouse was allowed to freely recognize two objects of the same shape (first session). After that, one of the two objects was replaced with a novel object, and the mouse was then exposed for 10 minutes (second session). Then, the time that the mouse recognized each object was recorded. The orientation index was expressed as the ratio of the time that the mouse recognized one of the two objects (including the novel object) to the total time that the mouse recognized the two objects.

[0145] Experimental Example 9.5. Morris Water Maze Test The Morris water maze test was performed in the following manner.

[0146] First, the water tank was filled with water at 24°C, and the water tank was painted with Crayola non-toxic paint to make the background of the water tank milky white.The water tank was then placed in the center of a room with extramaze cues (various black shapes on a white background).The water tank was then divided into four quadrants (northwest, northeast, southwest, and southeast) with a diameter of 90 cm.A circular platform with a diameter of 10 cm was then placed 25 cm away from the wall of the tank, and the time, distance, and speed for the mouse to move from the same starting position to the platform in each experiment were measured.

[0147] The experiment was carried out in two stages: hidden platform training and probe test. Hidden platform training was carried out as follows. Mice were first trained to swim to a visible platform attached 0.5 cm above the water surface and allowed to stay on the platform for 10 seconds. If the mice did not find the platform within 60 seconds, a glass stirrer was used to guide the mice to the platform. Training was carried out four times a day for four consecutive days using different access positions (the access positions were north, south, east, and west). After training, the platform was attached 1 cm below the water surface to prevent the platform from being visible. Then, the mouse's latency to reach the platform, movement distance, path efficiency, time and movement distance spent in each quadrant, total movement distance, and swimming speed were measured. In the probe test, the platform was removed on the fifth day after the start of training, and the trained mice were started in the opposite position to where the platform was attached (target quadrant). The time that the mouse spent in all quadrants, the distance and number of times that the mouse crossed the target quadrant, the total distance traveled, and the swimming speed were recorded during 60 seconds. Learning and memory of the mouse were assessed by the total time spent in the target quadrant during the probe test.

[0148] Experimental Example 9.6. Y-maze test The measurement device used a Y-maze consisting of three identical branches (40 cm long, 4 cm wide, 15 cm high), each of which was bent at an angle of 120°. The three branches were called A, B, and C, and the mouse was placed at the end of one branch and moved to the other branch for 10 minutes. The number of times the experimental animal entered the branch with its tail and the number of times the experimental animal entered each branch in turn were counted and 1 point (actual alternation) was given. Alternation behavior was defined as the presence of all three without overlap (e.g., ABC, BCA, CAB, but not ABA), and spontaneous alternation was calculated using the following mathematical equation 1. <Mathematical equation 1> Spontaneous alternations = [(number of alternations) / (total items - 2)] x 100

[0149] Experimental Example 9.7. Open field test Mice were placed individually in the center of an open-field box (length 40 cm × width 40 cm × height 50 cm) and allowed to acclimate for 20 min. The movement of the mice was recorded for 10 min and then analyzed using a digital camera connected to Any-Maze animal tracking system software (Stoeling, Wood Dale). The total distance traveled (meters) and the time spent in the center and the inner edge of the open-field box (seconds) were measured.

[0150] Experimental Example 9.8. Passive Avoidance Test There was a gate in the center of the measurement apparatus (length 48 cm × width 23 cm × height 28 cm), and the area in the measurement apparatus was equally divided into a lighted section (light section) and a dark section. On the first day, the mouse was allowed to freely recognize the two sections for 10 minutes. On the next day (training), the mouse was placed in the lighted section for 60 seconds, and then the next gate was opened to allow the mouse to freely enter the dark section. When the mouse entered the dark section, the door was closed and an electric shock (0.3 mA, 3 seconds) was given to the mouse's foot. At this time, the latency in each stage, that is, the time it took for the mouse to enter the dark section, was recorded. On the third day (probe test), the mouse was placed in the lighted section, and the gate was opened after 60 seconds, and the time it took for the mouse to move to the dark section within the gate was measured. The latency in each stage was measured for 540 seconds.

[0151] Experimental Example 10. Body composition analysis As in Experimental Example 3, the percentages of fat and lean tissue in the cell-transplanted mice were measured using Minispec LF50 (Bruker) at Korea Mouse Phenotyping Center (KMPC).

[0152] Experimental Example 11. Glucose Tolerance Test As in Experimental Example 3, the cell-transplanted mice were fasted for 12 hours, and then glucose was administered intraperitoneally at a concentration of 2 g / kg. Blood was collected from the tail at 0, 30, 60, and 120 minutes after glucose administration, and blood glucose levels were measured using a blood glucose meter.

[0153] Experimental Example 12. Metabolic cage and behavioral analysis To measure metabolic rates, metabolic parameters of mice were analyzed by indirect calorimetry 1-2 using a CLAMS (comprehensive laboratory animal monitoring system) system (Columbus Instruments, Columbus, OH, USA) at room temperature (22 °C). Two to three days before the experiment, mice were placed in one measurement cage (length 20.5 cm × width 10.5 cm × height 12.5 cm) and fed the same diet (normal diet or high-fat diet) to acclimate them to the diet. O 2 consumption, CO 2 The production, energy expenditure, heat production, and ambulatory activity were measured for 24 h. The above measurements were analyzed using the CLAX software (version 2.2.15, Columbus Instruments) to calculate the respiratory quotient, i.e., CO 2 / O 2 The exchange ratio and Δ heating value were calculated.

[0154] Experimental example 13. Bulk RNA sequencing transcriptome analysis Total RNA (RIN>8) was isolated using TRIzol (approximately 2 × 10 6A cDNA library was prepared using TruSeq Stranded mRNA LT (Illumina). Sequencing was performed using NovaSeq 6000 (Macrogen). Paired-end libraries were analyzed by aligning with the human reference UCSC hg19 using the HISAT2 pipeline. The DESeq2 method was used to analyze differentially expressed genes (DEGs), which indicate differences in expression levels between the control and sample groups. In this study, genotype-tissue expression (GTEx) data were used by randomly selecting four samples from the hypothalamus, hippocampus, cerebral cortex, and substantia nigra of postmortem donors from dbGaP (The database of Genotypes and Phenotypes) (study accession phs000424.v7. p2, https: / / www.ncbi.nlm.nih.gov / projects / gap / cgi-bin / study.cgi?study_id=phs000424.v7.p2).

[0155] Experiment 14. Analysis of the nucleotide sequence of single-cell RNA htNSCs were cultured in neurospheres in neuronal differentiation medium for 70 days.

[0156] The cultured hypothalamus-like organoids were treated with a 20:1 ratio of papain solution (PAP2, Worthington) and DNAse solution (D2, Worthington), reacted at 37°C for 20 minutes, and centrifuged at 500g for 5 minutes to separate the cells. The cell pellet obtained by the above method was resuspended in neuronal differentiation medium containing 0.004% BSA, and the viability of the cells was measured. For the separated cells, a single-cell cDNA library was generated using the Chromium Next GEM Single Cell 3'RNA Library v3.1 Reagent Kit (10X Genomics, USA). The single-cell cDNA library was sequenced with a NextSeq 500 system (Illunimia) to obtain 91bp and 28bp paired-end reads. The above results were demultiplexed and quantified based on UMI (Unique Molecule Identifier) ​​using Cell Ranger version 5.0.0 (10X Genomics, USA).

[0157] A matrix of counts was generated for each sample using default parameters and genes were mapped based on the human reference genome GRCh38.

[0158] Experimental Example 15. Cell proliferation analysis test Cell proliferation was confirmed through population doubling level (PDL) calculated by mathematical equation 2. <Mathematical equation 2> Population doubling level (PDL) = log(N / N 0 ) / log2 Here, N refers to the number of cells at the end of each passage, and N 0 refers to the number of cells inoculated at the beginning of the culture.

[0159] Experimental Example 16. Cell viability analysis Cell viability was measured using the LIVE / DEAD Viability / Cytotoxicity kit (ThermoFisher no. L3224).

[0160] Experimental Example 17. Micro-CT Analysis of Bone The right femur of each mouse was extracted and fixed overnight in 4% formaldehyde. Fixed femurs were placed in a 12.3 mm diameter scan tube and images were acquired using a desktop micro-CT (μCT) scanner (SkyScan 1076, Bruker), and the scanned images were merged into a 3D image. Total bone tissue volume, bone mineral density (BMD), bone volume (BV) / bone tissue volume (TV), trabecular number (Tb.N), and trabecular width (Tb.Th) were analyzed in 100 slides taken from a region of interest (ROI) approximately 0.5 mm–1.6 mm from the growth plate.

[0161] Example 18. Statistical analysis Statistical analyses were performed using two-tailed unpaired samples t-tests, one-way analysis of variance (ANOVA), or Mann-Whitney U-tests using Prism software version 6 (GraphPad Software) or IBM SPSS Statistics 26 (IBM). All results were considered statistically significant if p<0.05. Error bars are expressed as the mean ± standard error of the mean (SEM) or median, and p-values ​​are * p<0.05, ** p<0.01, *** p<0.001, **** Expressed as p<0.0001.

[0162] Experimental results 1. Generation of htNSCs from hypothalamus-like organoids derived from hPSCs To provide a stable source of human hypothalamic neural and non-neuronal cells for cell therapy, we developed a protocol for differentiation of hPSCs into hypothalamic NSCs (htNSCs). hESCs (H9) were cultured in 3D and induced to differentiate into NSCs via orbital shaking in the presence of substances that induce patterning into neuroepithelial and hypothalamic regions (Figure 1). Based on the expression of RAX, NKX2-1, and ILS1, which are known to be highly expressed in the hypothalamus, and Engrailed-1 (EN1), which is known to be expressed at a low level, the concentration and treatment time of the hypothalamic patterning inducer were optimized (Figure 2). Specifically, when treated simultaneously with sonic hedgehog (SHH) and palmorfamine, the expression of hypothalamic neural stem cell markers NKX2.1 and RAX was increased, whereas the expression of midbrain marker Engrailed-1 (EN1) was decreased. Furthermore, treatment with other small molecules known to be involved in hypothalamic patterning (CHR, CNTF, IWP-2, XAV, FGF8, NOG) reduced the expression of hypothalamic markers and concomitantly increased the expression of the midbrain marker EN1. Therefore, in the present invention, hypothalamic patterning was performed using only SHH and palmorfamine.

[0163] Organoids patterned under the above conditions displayed gene expression similar to the hypothalamic differentiation pattern by day 20 of culture.

[0164] In particular, fibroblast growth factor (bFGF) expanded htNSCs in early hypothalamus-like organoids. On day 20 (D20) of culture, cells were isolated from the organoids and subcultured on fibronectin-coated plates using N2 medium containing bFGF. As a result, expression of NSC markers (SOX2, NESTIN, KI67) and hypothalamic region-specific markers (RAX, NKX2-1, ISL1, OTP, SF-1) was observed in 84-97% of the cultured cells (Figures 3 and 4). Comparative analysis of the transcripts of differentiated htNSCs and human fetal brain (GTEX) by region confirmed that the transcripts of differentiated htNSCs clustered with the human fetal hypothalamus and had similar characteristics to the hypothalamic region (Figure 5).

[0165] Furthermore, htNSCs maintained cell proliferation even after multiple passages (approximately 5 passages) (Figure 6). htNSCs were cryopreserved and then recultured to confirm the expression of marker proteins for cell death (ethidium heterodimer-1, EthD-1), cell survival (calcein), and neural stem cell (Rax, nestin). As shown in Figure 7, it was confirmed that most of the thawed cells survived, and the expression levels of hypothalamic neural stem cell marker proteins were similar to those of the cells before freezing. Based on the above results, it was confirmed that htNSCs can be cryopreserved and can maintain the characteristics of htNSCs even after thawing.

[0166] Experimental results 2. Analysis of the differentiation phenotype of organoid-derived human htNSCs We induced differentiation of htNSCs into neurons. The induced neurons expressed neuronal markers (synapsin 1 (SYN1), TuJ1 (TUBB), MAP2, and RBFOX3 (NeuN)), and a subset of cells expressing hypothalamic-specific neuropeptides (α-MSH, neuropeptide Y (NPY), secretagogin (SCGN), and PMCH) was also observed (Figure 8).

[0167] In particular, GFAP +Astrocytes were also found in differentiated htNSC cultures, confirming the expansion of glial cells at the end of differentiation. Furthermore, expression of leptin and ghrelin receptors was confirmed in cells differentiated from htNSCs (Figure 8), and treatment of cells with leptin or ghrelin increased phosphorylation of STAT3 or FOXO1, downstream signaling factors of the respective receptors (Figure 9).

[0168] More specifically, to analyze the phenotype of cells differentiated from htNSCs, RNA sequencing (sc-RNA-Seq) analysis was performed on day 70 of differentiation. A total of 11,813 cells were sequenced using the Smart-seq2 method, read values ​​were filtered to remove doublets and low confidence values, and a total of 6,394 transcripts were reanalyzed. First, the clustering results using the Seurat algorithm were divided into 13 clusters, and these clusters were visualized by uniform manifold approximation projection (UMAP) maps. Based on the expression levels of known marker genes within the above clusters (Artegiani et al., 2017; Hochgerner et al., 2018; Zeisel et al., 2018), the clusters were divided into neural stem / progenitor cells (NSCs), intermediate progenitor cells (Int1 and Int2), astrocyte precursor cells (APCs), astrocytes (Ast1, Ast2, and Ast3), tanycytes (Tan), interneuron precursor cells (Int1 and Int2), radial glia (RG), and proteoglycan-secreting glia (Figure 10). At this time, specific genes were confirmed to be expressed by each cluster (Figures 11 and 12).

[0169] To confirm the above analysis results again, Voxhunt was used to compare and analyze mouse brain transcripts (14 days old) from the Allen developed brain atlas. The analysis based on voxel-based spatial resolution confirmed that all cell clusters differentiated from htNSCs were precisely located in the hypothalamus of 14-day old mouse brains. Furthermore, it was confirmed that progenitor / intermediate cell clusters (Int1, Int2, IPC, APC, and NSC) were enriched in the hypothalamic region adjacent to the ventricular region where adult neuronal differentiation occurs (Figure 13). Based on the above results, it was confirmed that the neural stem cells by the method of the present invention are hypothalamus specific.

[0170] Furthermore, similar to the differentiation of neurons and astrocytes from the same progenitor cells, the results of UMAP clustering confirmed the dual differentiation of intermediate progenitor cell clusters (Int1 and Int2) into neuronal clusters (Neu1 and Neu2) and astrocyte progenitor cell clusters (APC) (Figure 14). Using a pseudochronological ordering method using Monocle3, the results of the analysis of progenitor / intermediate cell clusters (Int1, Int2, IPC, APC, and NSC) confirmed that neural stem cells differentiated in the order of intermediate cell clusters (Int1, Int2) and neurons (Neu1, Neu2). Furthermore, as differentiation proceeded, it was confirmed that the expression of the cell proliferation marker CDK1 was rapidly reduced at the early progenitor cell stage, the expression of the progenitor neuronal gene ASCL1 was increased at the late progenitor cell stage, and the expression of the neuronal cell-specific genes TUBB3 and SNAP25 was increased at the late neuronal cell stage (Figure 15).

[0171] SLIT-ROBO signaling was recently reported to be a key regulator of hypothalamus-specific brain development (Romanov et al., 2020). In cells induced to differentiate from the htNSCs of the present invention, a SLIT-ROBO signaling gene expression pattern similar to in vivo hypothalamic brain development was confirmed (Figure 16). Based on the above results, it was confirmed that the differentiation pattern of organoid-derived human htNSCs was similar to in vivo hypothalamic development.

[0172] Next, to search for the top regulators (regulons) of the cluster genes, we performed gene regulatory network (GRN) analysis using pySCENIC. The results confirmed that SOX4 / SMARCA4, STAT1 / 2, and THRB / RAX were the top regulator genes (master regulators) in the neuronal cluster (Neu1), astrocyte cluster (Ast1), and tanycyte-like cell cluster (Tan) (Figure 17).

[0173] To further separate the above neuronal clusters (Neu1 and Neu2), additional clustering was performed. First, the above neuronal clusters (Neu1 and Neu2) were clustered again to distinguish each cell cluster based on the most expressed genes in each cell cluster (Figure 18). As a result, a total of 13 cell clusters were clustered, and it was confirmed that most of the genes expressed in each cell cluster were related to hypothalamic development. For example, PITX2 is a gene that is specifically expressed during hypothalamic development and is essential for hypothalamic development (PMID:18206388). SCGN neurons are hypothalamic-specific neurons that function as gatekeepers for CRH-mediated stress responses (Figure 19). Furthermore, the transcripts, transcriptional regulators (regulons), and neurotransmitters (NTs) expressed in each neuronal cluster were identified (Figure 20).

[0174] The astrocyte precursor cell cluster (APC) was further clustered into three astrocyte clusters (Figures 21 to 23). As shown in Figure 23, it was confirmed that the Ast2 cluster was a cluster of astrocytes involved in homeostasis in which SLC1A3 (GLAST) was expressed, and that the Ast2 cluster may function primarily to supply nutrients to nerve cells. Furthermore, the Ast2 cluster was found to be related to neuroendocrine function based on the expression of genes such as SLCO1C1 (thyroid hormone transporter), CRYM (crystallin mu, thyroid hormone binding protein), and SCG2 (secretogranin II, neuroendocrine protein). It was confirmed that genes related to immune function such as RSAD2 and TRIL, as well as genes induced by interferon (ISG5, IFIT, and STAT1) were specifically expressed in the Ast1 cluster. The Ast3 cluster, which was differentiated from astrocyte precursor cells (APC), expressed astrocyte-related transcription factors such as SOX9 and ID3, but did not express GFAP. It was confirmed that the Ast3 group expressed Meis2, which is related to the rest or activation of NCS in the subependymal layer (PMID:31304985). In addition, the Ast3 cluster expressed genes required for tight junction formation, such as CLDN5, ALCAM, and COL1A2 (Figure 24). A recently reported study confirmed that COL1A2 was expressed by a subset of astrocytes in the SVZ (PMID:34413515, PMID:30625322). Therefore, based on the above results, the inventors hypothesized that the Ast3 cluster is a "B-type" astrocyte that interacts with brain parenchymal cells and / or pericytes. Furthermore, since it has been recently reported that qNSCs exist in the subventricular layer of the third ventricle of the hypothalamus, it was hypothesized that the Ast3 cluster is a qNSC.

[0175] Until now, the origin of tanycytes has not been identified. However, similar to a recent report that tanycytes are derived from astrocyte populations (Romanov et al., 2020), in the present invention, based on the results of single-cell RNA sequencing analysis, we confirmed that the tanycyte-like cell (Tan) cluster is derived from the Ast2 cluster (Figures 14 and 23).

[0176] In the case of tanycyte (Tan) clusters, the expression of tanycyte-specific marker genes such as RAX, FGF10, COL25A1, CRYM, and SCN7A was confirmed. Furthermore, the expression of LEPR (leptin receptor) was confirmed, suggesting that tanycytes may be involved in metabolic homeostasis through leptin signaling.

[0177] Through the above single-cell RNA sequencing analysis, it was confirmed that the neural differentiation method of organoid-derived human htNSCs resembled the main hypothalamic cell population in vivo.Furthermore, it was confirmed that tanycytes and hypothalamic qNSCs can be generated through the differentiation method of the present invention.

[0178] Experimental result 3. Confirmation of the effect of improving aging in mice transplanted with organoid-derived human htNSCs Previous studies have reported that transplantation of mouse-derived htNSCs into aged mice reduced the rate of physiological changes associated with aging (Zhang et al., 2017). Therefore, to confirm the anti-aging effect of human htNSCs derived from hypothalamus-like organoids made from hESCs of the present invention, htNSCs were transplanted into 12-month-old aged mice (Figure 24).

[0179] Human htNSCs were transplanted twice into the medial basal hypothalamus (MBH) region and followed for 1 to 4 months. Mice injected with vehicle were used as the control group. As a result, cells expressing the human-specific marker hNCAM (hNCAM+) were observed in the hypothalamus of mice 1 month after transplantation. Expression of the htNSC-specific marker protein RAX was observed in some of the hNCAM+ cells, and expression of RBFOX3 protein or human GFAP (hGFAP) protein was also observed (Figure 25).

[0180] According to the results of the mouse behavioral experiment, the endurance (treadmill performance) (Figure 26) of the group transplanted with human htNSCs was improved compared to the control group 4 months after transplantation. In addition, the muscle endurance (grip strength) (Figure 27), motor coordination (rotarod) (Figure 28), and activity (ambulatory activity, open field test) (Figure 29) of the group transplanted with human htNSCs were improved compared to the control group 1.5 and 4 months after transplantation. In addition, the long-term and spatial memory and cognitive ability (Morris water maze test, Figures 30 and 31) (Y-maze test, Figure 32) (novel object recognition test, Figure 33) of the group transplanted with human htNSCs were also improved compared to the control group.

[0181] The hypothalamus regulates whole-body metabolism by regulating endocrine substances such as orexin and TRH, as well as neurotransmission via the sympathetic autonomic nervous system. It has been reported that this regulatory function of the hypothalamus is impaired with age (Waterson et al., 2015; Saper et al., 2014; Gonzalez-Garcia et al., 2020). The human htNSC-transplanted group of the present invention suppressed aging-related whole-body metabolism and tissue aging compared to the control group. As shown in FIG. 34, when the body weight of the mice was observed 1.5 months (13.5 months old) and 4 months (16 months old) after transplantation of human htNSC, a significant weight loss was observed in the human htNSC-transplanted group compared to the body weight of the control group (FIG. 34).

[0182] Furthermore, fat % was decreased compared to the control group, while lean % was significantly increased (Figure 35). Conversely, there was no significant difference in food intake between the control group and the group transplanted with human htNSCs. As shown in Figure 36, the results of the metabolic cage experiment showed that the ambulatory activity (nighttime, 1.5 months after transplantation (13.5 months old)) of the group transplanted with human htNSCs was significantly increased compared to the control group (Figure 37). Furthermore, at 1.5 months (13.5 months old) or 4 months (16 months old) after transplantation, energy expenditure (EE) was significantly increased in the group transplanted with human htNSCs compared to the control group, both at night and during the day (Figure 38). Furthermore, 4 months after transplantation of human htNSCs, glucose tolerance of the group transplanted with human htNSCs was significantly improved compared to the control group (Figure 39).

[0183] It is known that as age progresses, not only does body fat increase, but the amount and activity of brown adipose tissue (BAT) decreases. To confirm the effect of the htNSCs of the present invention on the above-mentioned aging phenomenon, inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and interscapular brown adipose tissue (iBAT) were stained with H&E 4 months after transplantation of human htNSCs (16 months of age), and each tissue was observed.

[0184] As a result, the size of adipocytes in white and brown adipose tissues in the group transplanted with human htNSCs was significantly reduced compared to the control group (Figures 40 to 42). Furthermore, although lipid accumulation in liver tissue increases with age, lipid accumulation in liver tissues in the group transplanted with htNSCs was reduced compared to the control group (Figure 43).

[0185] In white adipose tissue (iWAT and eWAT), the expression of mitochondrial homeostasis genes MTCO1 and SDHB, which are associated with browning, and the expression of FGF21, a key regulator of brown adipocyte differentiation and energy metabolism, were significantly increased (Figures 44 and 45). Furthermore, the expression of FGF21 and GDF15, as well as the phosphorylation of the stress-induced transcription factor eIF2A, and the expression of ATF4 were increased in liver tissue of the transplanted group (Figure 46). Furthermore, the expression of p16 and p21 proteins, which are associated with cellular senescence, was reduced in the kidneys of the htNSC-transplanted group compared to the control group (Figure 47).

[0186] The hypothalamus is known to regulate not only whole body metabolism but also bone metabolism (remodeling and homeostasis) (Sharan et al., 2014). In the present invention, X-ray examination of femurs 4 months (16 months old) after transplantation of htNSCs into 12-month-old mice revealed that osteoporosis was observed in the control group, while the indices of bone mineral density (BMD), bone mass (BV) / bone tissue volume (TV) ratio, trabecular thickness (Tb.Th), and trabecular number (Tb.N) were significantly increased in the htNSC-transplanted group compared to the control group (Figure 48).

[0187] Experimental result 4. Confirmation of anti-aging effect via intraventricular transplantation of htNSC-derived exosomes Recent studies have reported that exosomes secreted from transplanted NSCs show therapeutic effects (Zhang et al., 2017). Therefore, to confirm the anti-aging effect of exosomes derived from human htNSCs, exosomes were isolated from the culture medium of human htNSCs derived from human hypothalamic organoids and injected into the third ventricle of 12-month-old mice using an osmotic pump (Figure 49). As a result, similar to the results of the human htNSC transplantation experiment described above, injection of exosomes derived from htNSCs showed increased motor coordination (rotarod) and muscle strength (grip strength) compared to the control group at 1.5 and 4 months after injection, respectively (Figures 50 and 51). In addition, it was confirmed that the memory [(passive avoidance test, Figure 52) and (Morris water maze test, Figure 53)] of mice injected with exosomes derived from htNSCs was improved compared to the control group. Based on the above results, it was determined that exosomes derived from human htNSCs may potentially be utilized as a method for preventing and treating aging.

Claims

1. A pharmaceutical composition for preventing and treating geriatric diseases, comprising neural stem cells derived from hypothalamus-like organoids as an active ingredient.

2. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the neural stem cells derived from the hypothalamus-like organoid express any one neural stem cell marker selected from the group consisting of SOX2, NESTIN, and KI67, and express any one hypothalamic marker selected from the group consisting of RAX, NKX2-1, ISL1, OTP, and SF-1.

3. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 2, wherein the neural stem cells derived from the hypothalamus-like organoid express neural stem cell markers and hypothalamic markers in 84% or more of the total DAPI-positive cell population.

4. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the neural stem cells derived from the hypothalamus-like organoid can be proliferated for 1 to 10 passages.

5. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the neural stem cells derived from the hypothalamus-like organoid are differentiated into neurons, and the neurons express at least one neuronal marker selected from the group consisting of synapsin 1 (SYN1), TuJ1 (TUBB), MAP2, and RBFOX3 (NeuN), and express at least one hypothalamic neuropeptide selected from the group consisting of α-MSH, neuropeptide Y (NPY), secretagogin (SCGN), and PMCH.

6. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 5, wherein the neuronal cells are responsive to leptin and ghrelin.

7. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the neural stem cells derived from the hypothalamus-like organoid differentiate and cluster into neural stem / progenitor cell clusters (NSC), intermediate progenitor cell clusters (IPC), neuronal cell clusters (Neu1 and Neu2), astrocyte progenitor cell clusters (APC), astrocyte clusters (Ast1, Ast2, and Ast3), tanycyte clusters (Tan), interneuron progenitor cell clusters (Int1 and Int2), radial glia clusters (RG), and proteoglycan-secreting glial clusters.

8. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 7, wherein the neuronal cell clusters (Neu1 and Neu2) are characterized by increased expression of genes related to hypothalamic development.

9. The astrocyte precursor cell clusters A cell cluster expressing at least one gene selected from the group consisting of SLC1A3, SLCO1C1, CRYM, and SCG2 (Ast2); A cell cluster expressing at least one gene selected from the group consisting of RSAD2, TRAIL, ISG5, IFIT, and STAT1 (Ast1); and A cell cluster (Ast3) expressing at least one gene selected from the group consisting of SOX9, ID3, Meis2, CLDN5, ALCAM, and COL1A2. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 7, characterized in that the cellular components of the cellular components are differentiated into:

10. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 7, wherein the tanycyte cluster (Tan) expresses at least one gene selected from the group consisting of RAX, FGF10, COL25A1, CRYM, SCN7A, and LEPR.

11. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the geriatric disease is any one selected from the group consisting of neurodegenerative diseases, osteoporosis, and aging-related muscle diseases.

12. 12. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 11, wherein the neurodegenerative disease is any one selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, corticobasal degeneration, diffuse Lewy body disease, and Pick's disease.

13. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 11, wherein the aging-related muscle disease is selected from the group consisting of muscle atrophy, disuse muscle atrophy, and senile sarcopenia.

14. Exosomes secreted from neural stem cells derived from hypothalamus-like organoids.

15. The exosome of claim 14, wherein the neural stem cells derived from the hypothalamus-like organoid express any one neural stem cell marker selected from the group consisting of SOX2, NESTIN, and KI67, and express any one hypothalamic marker selected from the group consisting of RAX, NKX2-1, ISL1, OTP, and SF-1.

16. The exosome of claim 14, wherein the neural stem cells derived from the hypothalamus-like organoid express neural stem cell markers and hypothalamic markers at 84% or more of the total DAPI positive cell population.

17. A pharmaceutical composition for preventing and treating geriatric diseases, comprising the exosome according to claim 14 as an active ingredient.

18. The pharmaceutical composition according to claim 17, wherein the geriatric diseases are neurodegenerative diseases and aging-related diseases of muscle.

19. 19. The pharmaceutical composition of claim 18, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, corticobasal degeneration, diffuse Lewy body disease, and Pick's disease.

20. The pharmaceutical composition according to claim 18, wherein the age-related muscle disease is selected from the group consisting of muscle atrophy, disuse muscle atrophy, and senile sarcopenia.

21. i) culturing neural stem cells derived from hypothalamus-like organoids; and ii) Recovering exosomes from the culture medium A method for producing exosomes comprising:

22. A kit for preventing and treating geriatric diseases, comprising neural stem cells derived from the hypothalamic organoid of claim 1 and / or the exosome of claim 14.

23. The kit according to claim 22, wherein the neural stem cells derived from the hypothalamus-like organoid express any one neural stem cell marker selected from the group consisting of SOX2, NESTIN, and KI67, and express any one hypothalamic marker selected from the group consisting of RAX, NKX2-1, ISL1, OTP, and SF-1.

24. The kit of claim 22, wherein the neural stem cells derived from the hypothalamus-like organoid express neural stem cell markers and hypothalamic markers at 84% or more of the total DAPI-positive cell population.

25. Use of neural stem cells derived from the hypothalamic organoid of claim 1 or the exosome of claim 14 for preventing or treating geriatric diseases.

26. A method for preventing or treating geriatric diseases, comprising a step of administering neural stem cells derived from the hypothalamic organoid described in claim 1 or the exosome described in claim 14 to a subject.

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