Pharmaceutical composition for preventing and treating senile diseases comprising three-dimensional hypothalamic organ-derived neural stem cells and use thereof

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating senile diseases, which contains neural stem cells derived from hypothalamic organs as an active ingredient. When the neural stem cell derived from the hypothalamic organ according to the present invention is transplanted into the body of an aged mouse, the memory, cognitive ability, muscle strength, muscle tissue density, and bone mineral density of the aged mouse are restored due to aging. In addition, transplantation of hypothalamic organ-derived neural stem cells has also been found to ameliorate obesity by increasing energy metabolism. It is found that when the exosome separated from the neural stem cells derived from the hypothalamic-like organ is transplanted into the body of an aged mouse, the reduced memory, cognitive ability and muscle strength of the aged mouse are also recovered. Therefore, the neural stem cell derived from the hypothalamic organ according to the present invention is expected to be a very useful agent for preventing and treating senile diseases.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing and treating senile diseases, which comprises neural stem cells obtained by isolating and dissociating hypothalamus-like organoids generated by 3D culture from human pluripotent stem cells as active ingredients. Background Art

[0002] The hypothalamus is a central organ for the body to maintain homeostasis, such as growth, puberty, metabolism, stress response, reproduction, lactation, childcare, and immunity. Neural stem cells / progenitor cells exist in the hypothalamus of adults and are known to regulate the function of the hypothalamus. Therefore, neural stem cells have attracted much attention in recent years as a replacement therapy for hypothalamic dysfunction caused by aging and other factors.

[0003] However, since the hypothalamus is located deep in the brain and has a very small area, it is impossible to extract actual tissue and use it for research. Therefore, research on the generation of hypothalamic neurons from pluripotent stem cells such as embryonic stem cells has been steadily carried out. For example, the Wataya team aggregated cells using gfCDM culture medium and produced neurons that can produce hypothalamic arginine vasopressin (AVP) from mouse embryonic stem cells through 3D suspension culture (Wataya T et al., Proc Natl Acad Sci USA, 2008, 105 (33): 11796-11801). In addition, the Merkle team successfully differentiated human embryonic stem cells and induced pluripotent stem cells into hypothalamic neurons through 3D and 2D culture methods (Merkle FT et al., Development, 2015, 142 (4): 633-643). However, despite the various studies mentioned above, there are still limitations in clinical applications. Summary of the invention

[0004] Technical issues

[0005] Therefore, the inventors found that neural stem cells obtained from hypothalamic-like organoids cultured in 3D from human embryonic stem cells are similar to neural stem cells existing in the hypothalamus of the actual brain, and when transplanted into aged mice, symptoms associated with aging are improved. Based on the above, the inventors completed the present invention.

[0006] Solution

[0007] In order to solve the above problems, in one aspect of the present invention, a pharmaceutical composition for preventing and treating geriatric diseases is provided, which contains hypothalamic-like organoid-derived neural stem cells as an active ingredient.

[0008] In another aspect of the present invention, provided are exosomes secreted by neural stem cells derived from hypothalamic-like organoids; and a pharmaceutical composition for preventing and treating geriatric diseases comprising the exosomes as an active ingredient.

[0009] In another aspect of the present invention, a method for producing exosomes is provided, comprising the steps of: i) culturing hypothalamic-like organoid-derived neural stem cells; and ii) collecting exosomes from the culture medium.

[0010] In another aspect of the present invention, a kit for preventing and treating geriatric diseases is provided, comprising cells and / or exosomes, wherein the cells and / or exosomes are selected from a group consisting of neural stem cells derived from hypothalamic-like organoids; and exosomes secreted by neural stem cells derived from hypothalamic-like organoids.

[0011] In another aspect of the present invention, provided is a use of hypothalamic-like organoid-derived neural stem cells; or exosomes secreted by hypothalamic-like organoid-derived neural stem cells for preventing or treating geriatric diseases.

[0012] In another aspect of the present invention, a method for preventing or treating geriatric diseases is provided, comprising the following steps: administering to a subject hypothalamic-like organoid-derived neural stem cells; or exosomes secreted by hypothalamic-like organoid-derived neural stem cells.

[0013] Effects of the Invention

[0014] It was found that when the neural stem cells derived from hypothalamic-like organoids according to the present invention were transplanted into aged mice, the memory, cognitive ability, muscle strength, muscle tissue density and bone mineral density that had declined due to aging were restored. In addition, it was found that the transplantation of neural stem cells derived from hypothalamic-like organoids improved obesity by increasing energy metabolism. It was also found that when the exosomes isolated from the culture medium of neural stem cells derived from hypothalamic-like organoids were transplanted into aged mice, the declined memory, cognitive ability and muscle strength were also similarly restored. Therefore, neural stem cells derived from hypothalamic-like organoids and exosomes isolated from their culture medium are expected to be very useful agents for preventing and treating geriatric diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of a method for generating human hypothalamic organoids from human embryonic stem cells (hESCs) or human dedifferentiated stem cells (hiPSCs) and inducing the generated hypothalamic organoids to differentiate into hypothalamic neural stem cells (htNSCs).

[0016] Figure 2The graph shows the results of confirming the expression of hypothalamic (NKX2-1, RAX) or midbrain (EN1) marker genes by q-PCR after treating the culture medium of hESC-derived organoids with various hypothalamic pattern-inducing substances.

[0017] Figure 3 This is a diagram showing the results of immunofluorescence staining to confirm 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. In this case, DAPI indicates the cell nucleus.

[0018] Figure 4 The graph shows 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.

[0019] Figure 5 is a diagram showing the results of DGE (differential gene expression) analysis of transcripts of organoid-derived human htNSCs and transcripts of various regions in the human fetal brain.

[0020] Figure 6 is a graph showing population doubling level (PDL) and cumulative cell number measured by proliferation of cell number during five subcultures of organoid-derived human htNSCs in the presence of basic fibroblast growth factor (bFGF).

[0021] Figure 7 are diagrams and graphs showing the results of observing the expression of proteins related to cell death (ethidium homodimer I dye, EthD-1), cell survival (calcein), and neural stem cells (Rax, Nestin) by immunofluorescence staining after reculturing cryopreserved organoid-derived human htNSCs.

[0022] Figure 8 This is a graphical representation of the results of confirming the expression of neural cell markers (synaptophysin 1 (SYN1), TuJ1 (TUBB), MAP2, RBFOX3 (NeuN)), hypothalamus-specific neuropeptides (α-MSH, neuropeptide Y (NPY), secretagogue (SCGN), PMCH) and astrocyte marker (GFAP) in differentiated htNSCs by immunofluorescence staining when organoid-derived human htNSCs were induced to differentiate into neural cells.

[0023] Fig. 9A diagram showing the results of confirming the phosphorylation of STAT3 or FOXO1 induced by leptin or ghrelin treatment after organoid-derived human htNSCs were differentiated into neural cells by Western blotting (upper), and a diagram showing the quantitative results (lower).

[0024] Fig.10 is a graphic representation showing the results of single-cell RNA sequencing after induced organoid-derived human htNSC differentiation for 70 days, visualizing clustering results by UMAP (Uniform Manifold Approximation and Projection).

[0025] Fig.11 is a diagram showing the results of analyzing each cluster-specific gene.

[0026] Fig.12 is a diagram showing the visualization of the results of analyzing specific genes in each cluster by UMAP.

[0027] Fig.13 This is a graphic representation of the results of comparing and analyzing the clustering results with the Allen Developmental Brain Atlas mouse brain transcriptome (14 days old) using Voxhunt. The comparison results show that all cell clusters differentiated from htNSCs are precisely located in the hypothalamus of the 14-day-old mouse brain, confirming that the cells differentiated by this established protocol are hypothalamus-specific cells.

[0028] Fig.14 Figure 3. Graphical representation of single-cell RNA sequencing results visualized according to pseudo-time using Monocle3 after 70 days of differentiation of organoid-derived human htNSCs.

[0029] Fig.15 It was shown that hypothalamic neural stem cells (htNSCs) differentiated from intermediate cells (Int1, Int2) into neural stem cells (NSCs) by pseudo-time analysis, and the expression of cell proliferation factor CDK1, neurogenic gene ASCL1, and neural cell factors TUBB3 and SNAP25 also changed according to each developmental stage.

[0030] Fig.16 This is a result diagram of the unique expression increase / decrease pattern of ROBO1, SLIT1, and SLIT2 obtained by pseudo-temporal analysis when hypothalamic neural stem cells (htNSC) differentiate from intermediate cells (Int1, Int2) into neural stem cells (NSC). These three genes are considered to be key factors in hypothalamic development.

[0031] Fig.17 This is a diagram showing the expression of master regulatory factors (regulators) of each cell cluster (Neu1, Ast1, Tan) by performing gene regulatory network (GRN) analysis using pySCENIC.

[0032] Fig.18 is a diagram showing results of reanalyzing the neural cell clusters (Neu1 and Neu2) by using t-SNE (t-Distributed Stochastic Neighbor Embedding) to visualize the results of clustering the neural cell clusters (Neu1 and Neu2).

[0033] Fig.19 is a diagram showing the expression distribution of hypothalamus-related genes for each reanalyzed neural cell cluster.

[0034] Fig. 20 It is a graphic representation showing the gene expression patterns of single cells of each cluster by reanalyzing neural cell clusters (Neu1, Neu2).

[0035] Fig.21 is a diagram showing the results of visualizing the clustering results of astrocyte progenitor cell clusters (APC) by UMAP.

[0036] Fig. 22 It is a diagram showing the expression distribution of GFAP, SPARCL1 and AQP4 genes in each cell cluster within astrocyte progenitor cell cluster (APC).

[0037] Fig.23 It is a diagram showing the expression pattern of single-cell genes expressed in each cell cluster within the astrocyte cluster (Ast1, Ast2, Ast3), the astrocyte progenitor cell cluster (APC), and the elongated cell cluster (Tan).

[0038] Fig.24 is a diagram showing the experimental plan for confirming the aging-improving effect of mice transplanted with organoid-derived human htNSCs.

[0039] Fig.25 The figure shows the expression results of human cell marker (hNCAM), hypothalamic neural stem cell marker (RAX), neural cell marker (RBFOX3 (NeuN)), and human astrocyte marker (hGFAP) in the transplanted cells in the hypothalamus of 12-month-old mice observed by immunofluorescence staining one month after organoid-derived human htNSCs were transplanted into the hypothalamus of 12-month-old mice.

[0040] Fig.26 The graph shows the results of measuring the treadmill performance of mice 1.5 months (13.5 months old) and 4 months (16 months old) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0041] Fig. 27is a graph showing the results of grip strength test of mice 1.5 months (13.5 months old) and 4 months (16 months old) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0042] Fig.28 is a graph showing the results of the rotarod test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0043] Fig.29 is a graph showing the results of the open field test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0044] Fig.30 is a graph showing the time required for spatial perception learning during hidden platform training in the results of Morris water maze test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0045] Fig.31 is a graph showing the time spent in the target quadrant during the exploration trial in the results of the Morris water maze test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0046] Fig.32 Graphs showing the results of a Y-maze test of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0047] Fig.33 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 organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0048] Fig.34 The graphs show the results of measuring the weight of mice 1.5 months (13.5 months old) and 4 months (16 months old) after transplantation of organoid-derived human htNSCs into the hypothalamus of mice and the results of observation of the external appearance of the mice.

[0049] Fig.35 The graph shows the results of measuring the fat mass and lean mass of mice 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0050] Fig.36is a graphical representation showing the results of measuring food intake in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0051] Fig.37 Graphs showing the results of measuring the motor activity of mice using metabolic cages 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0052] Fig.38 Graphs showing the results of measuring energy expenditure of mice using metabolic cages 1.5 months (13.5 months of age) and 4 months (16 months of age) after organoid-derived human htNSCs were transplanted into the hypothalamus of mice.

[0053] Fig.39 is a graphical representation showing 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.

[0054] Fig.40 A graphic representation showing the results of H&E staining of mouse inguinal white adipose tissue (iWAT) 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (left) and a graph showing the results of measuring the size of mouse adipocytes (right).

[0055] Fig.41 A graphic representation showing the results of H&E staining of mouse epididymal white adipose tissue (eWAT) 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (left) and a graph showing the results of measuring the size of mouse adipocytes (right).

[0056] Fig.42 A graphic representation showing the results of H&E staining of mouse interscapular brown adipose tissue (iBAT) 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (left) and a graph showing the results of measuring the size of mouse adipocytes (right).

[0057] Fig.43 The figure shows the results of H&E staining of mouse liver tissue 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months old). It confirms that fat accumulation in the liver tissue of mice transplanted with htNSCs is reduced.

[0058] Fig.44A diagram showing the results of Western blotting to confirm the expression of β-actin and proteins (MTCO1, SDHB, FGF21) related to lipolysis, thermogenesis, and browning in mouse inguinal white adipose tissue (iWAT) 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (top), and a figure showing the results of quantification of expression using β-actin (bottom).

[0059] Fig.45 A diagram showing the results of Western blotting to confirm the expression of β-actin and proteins (MTCO1, SDHB, FGF21) related to lipolysis, thermogenesis, and browning in mouse epididymal white adipose tissue (eWAT) 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (top), and a figure showing the results of quantification of expression using β-actin (bottom).

[0060] Fig.46 A diagram showing the results of Western blotting to confirm the expression of β-actin and proteins (p-eIF2A, eIF2A, ATF4, FGF21, GDF15) related to lipolysis and stress in mouse liver tissue 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (top) and a figure showing the results of quantification of expression using β-actin (bottom).

[0061] Fig.47 A diagram showing the results of Western blotting to confirm the expression of p16 and p21 in mouse kidney tissue 4 months after organoid-derived human htNSCs were transplanted into the mouse hypothalamus (16 months of age) (top) and a diagram showing the results of quantification of the expression (bottom).

[0062] Fig.48 A graphic representation showing the results of X-ray imaging of the femur of mice 4 months after organoid-derived human htNSCs were transplanted into the hypothalamus of mice (16 months of age) (top), and a graphic representation showing the results of bone index analysis of mice (bottom). BMD: bone mineral density, BV: bone volume, TV: tissue volume, Tb.Th: trabecular thickness, Tb.N: trabecular number.

[0063] Fig.49 Schematic diagram of the experimental plan to confirm the anti-aging effects of organoid-derived human htNSC-derived exosomes.

[0064] Fig.50 is a graph showing the results of the rotarod test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of organoid-derived human htNSC-derived exosomes.

[0065] Fig.51 is a graph showing the results of grip strength testing of mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of organoid-derived human htNSC-derived exosomes.

[0066] Fig.52 is a 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 organoid-derived human htNSC-derived exosomes.

[0067] Fig.53 is a graph showing the time required for spatial perception learning during hidden platform training and the time spent in the target quadrant during the exploration trial in the results of the Morris water maze test in mice 1.5 months (13.5 months of age) and 4 months (16 months of age) after injection of organoid-derived human htNSC-derived exosomes. DETAILED DESCRIPTION

[0068] Pharmaceutical composition comprising neural stem cells

[0069] In one aspect of the present invention, a pharmaceutical composition comprising hypothalamic-like organoid-derived neural stem cells as an active ingredient is provided. In addition, the pharmaceutical composition can also be used for preventing and treating geriatric diseases.

[0070] At this time, the pharmaceutical composition may contain hypothalamic-like organoid-derived neural cells; or a cell population containing hypothalamic-like organoid-derived neural stem cells and hypothalamic-like organoid-derived neural cells as active ingredients.

[0071] The term "hypothalamus" as used herein is located below the two thalamus and its function is 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, and synthesizes and secretes neurohormones to regulate the pituitary gland, thereby regulating body temperature, hunger, thirst, fatigue, sleep, and circadian rhythms.

[0072] The term "organoid" used in this article refers to a "miniature organ-like structure" with minimal functions produced using stem cells, and its characteristic is that it is made in a three-dimensional (3D) structure, and an environment similar to an actual body organ can be created even in a laboratory. In other words, organoids refer to cells with a 3D structure, and are models similar to organs such as nerves and intestines that are produced through an artificial culture process without being collected or obtained from animals, etc. At this time, the source of the cells that make it up is not limited.

[0073] In addition, organoids can have an environment that interacts with the surrounding environment during cell growth. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Therefore, in the present invention, 3D organoids can become an excellent model for observing the development of disease therapeutic agents, etc. by almost completely simulating the organs that actually interact in vivo.

[0074] Generally speaking, organoids can be produced by culturing pluripotent stem cells. At this time, pluripotent stem cells (PSC) refer to stem cells that can be induced to differentiate into any type of cells that constitute the body, and may include embryonic stem cells and induced pluripotent stem cells (iPSC, dedifferentiated stem cells), but are not limited thereto. Pluripotent stem cells can be isolated from mammals, but are preferably isolated from humans.

[0075] In the present invention, the organoid may be a hypothalamus-like organoid that simulates the hypothalamus.

[0076] Specifically, in the present invention, 3D nervous system organoids can be generated by proliferating and culturing human embryonic stem cells (hESCs), and treating them with substances that induce patterning toward the hypothalamic region to generate hypothalamic-like organoids. The patterned hypothalamic-like organoids can be isolated to ultimately generate hypothalamic neural stem cells.

[0077] The term "patterning" as used herein refers to the generation of organoids such that a certain fate somatic cell cluster having the characteristics of the tissue of origin of cells to be ultimately extracted from a specific tissue in the brain is included as the majority of all clusters (enriched for target cells).

[0078] In the present invention, the patterning may be patterning toward the hypothalamus region.

[0079] In the present invention, the production and patterning of nervous system organoids from human pluripotent stem cells can be achieved by culturing in a culture medium containing ascorbic acid, an ALK inhibitor and a BMP inhibitor. Preferably, the ALK inhibitor can be SB431542, and the BMP inhibitor can be noggin.

[0080] In addition, the material inducing patterning may also include a material activating the hedgehog signaling pathway. The material activating the hedgehog signaling pathway may be a sonic hedgehog (SHH) and SMO (Smoothened) agonist.

[0081] The term "sonic hedgehog (SHH)" as used herein plays a very important role in cell proliferation and morphogenesis in the development of various body tissues such as the skeletal system, central nervous system and face. In particular, it is a protein that plays an important role in the development of the central nervous system such as the spinal cord, brain, cerebellum, and the proliferation and differentiation of neural stem cells. When SHH binds to the cell membrane receptor PTCH1 (Patched1), the SMO receptor is activated, inhibiting the hedgehog negative regulatory factor SUFU (suppressor of fused), thereby activating the transcription factor GLI1 and promoting the proliferation of nerve cells. In the present invention, the SMO agonist can be purmorphamine.

[0082] Hypothalamic patterned organoids can be further cultured in medium containing insulin, ascorbic acid, and bFGF.

[0083] The term "culture medium" as used herein refers to a culture medium that allows the support of organoid proliferation, survival and differentiation in vitro, including all conventional culture media 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 cells. In the present invention, the culture medium can be a culture medium suitable for inducing human stem cells to differentiate into neural progenitor cells.

[0084] The term "neural stem cell (NSC)" used herein refers to immature cells that can self-renew and continue to proliferate in an undifferentiated state, and has the potential to differentiate into nervous system cells. Neural stem cells are present in various anatomical parts of the fetal nervous system of mammals including humans, and in recent years, neural stem cells are not only present in fetuses, but also in specific parts of the adult nervous system, and throughout the life process, neural stem cells can continuously proliferate and produce new nerve cells in specific parts of the brain. Neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes. Neural stem cells can proliferate in vitro and transplant in vivo, and can migrate, implant and integrate into the host nervous system to secrete therapeutic substances and differentiate into appropriate nerve cells in cell structure and function.

[0085] In the present invention, the neural stem cells derived from hypothalamic-like organoids (hypothalamic NSC, htNSC) 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 hypothalamic-like organoids may be characterized by expressing neural stem cell markers and hypothalamic markers at about 84% or more in the entire DAPI-positive cell population. At this time, neural stem cell markers and hypothalamic markers may be expressed at 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%, and about 99% in the entire DAPI-positive cell population.

[0086] SOX2 (SRY-box transcription factor 2) is a transcription factor that regulates the pluripotency of embryonic stem cells. It plays a role in maintaining the self-renewal or pluripotency of embryonic stem cells and regulating their differentiation into specific cells.

[0087] Nestin is a type VI intermediate filament (IF) protein that is primarily expressed in neurons associated with radial axon growth. It is expressed in the early stages of development of the central nervous system, peripheral nervous system, muscle, and other tissues, and in adulthood, its expression is induced in pathological conditions such as glial scar formation after central nervous system injury and regeneration of damaged muscle tissue. Nestin is known to play a role in regulating the assembly and disassembly of intermediate filaments associated with cell remodeling in mitotic cells.

[0088] KI67 is a nuclear protein associated with cell proliferation. Its expression increases significantly during the S phase of the cell cycle and is therefore used as a cell proliferation marker.

[0089] In addition, RAX (retinal and anterior neural fold homeobox) is a paired homeodomain-containing transcription factor that is expressed in the anterior neural plate during early embryogenesis and in the retina, hypothalamus, and pineal gland during late embryogenesis.

[0090] NKX2-1 is a transcription factor, also known as thyroid transcription factor 1 (TTF-1), which regulates the expression of specific genes in the thyroid, lung, and diencephalon.

[0091] ISL1 (ISL LIM homeobox 1) is a transcription factor that contains two LIM domains (N-terminus) and one homeodomain (C-terminus). ISL1 is known to regulate the development of pancreatic islets during embryogenesis and play an important role in the differentiation of motor neurons in the neural tube of mouse embryos.

[0092] OTP (orthopedia homeobox) is a homeodomain-containing transcription factor known to be involved in the differentiation and development of hypothalamic neuroendocrine cells.

[0093] SF-1 (steroidogenic factor 1) is considered to be an important factor in the development and function of the adrenal glands and reproductive organs. In the brain, it is known to be expressed only in the ventromedial nucleus of the hypothalamus (VMH), regulating the development and function of the VMH and participating in the regulation of energy metabolism homeostasis.

[0094] Furthermore, hypothalamic-like organoid-derived neural stem cells can maintain their morphology even after freezing and thawing and can show high survival rate.

[0095] In addition, the hypothalamic-like organoid-derived neural stem cells can be passaged. Specifically, the hypothalamic-like organoid-derived neural stem cells can be passaged 1 to 10 times, 2 to 8 times, 3 to 7 times, or 4 to 6 times.

[0096] The hypothalamus-like organoid-derived neural stem cells of the present invention can be differentiated into hypothalamus-specific neural cells.

[0097] Specifically, the neural stem cells derived from the hypothalamic-like organoids of the present invention can differentiate into neural cells, which express at least one neural cell marker selected from the group consisting of synaptophysin 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), secretagogue (SCGN), and PMCH.

[0098] Tuj1, known as neuron-specific class III β-tubulin (TUBB3), is a major component of microtubules. It is mainly expressed in neurons and is involved in neurogenesis and axon guidance and maintenance.

[0099] MAP2 (microtubule-associated protein 2) is a cytoskeletal protein known to be involved in microtubule assembly, an important step in neurogenesis. During the development stage of mouse neurons, MAP2 is mainly located in dendrites and is known to be involved in stabilizing the shape of dendrites.

[0100] Synapsin is a member of the synapsin family and plays a role in synapse formation and the regulation of neurotransmitter release.

[0101] NeuN, also known as hexaribonucleotide-binding protein-3 or Fox-3, is a protein present in the nuclei of neurons and is often used as a biomarker for neurons.

[0102] α-MSH (α-melanocyte stimulating hormone) is an endogenous peptide hormone, a neuropeptide belonging to the melanocortin family, and is one of the melanocyte stimulating hormones that promotes melanin production. It is also said to be involved in feeding behavior, energy homeostasis, sexual behavior, and protection against ischemia and reperfusion injury.

[0103] NPY (neuropeptide Y) is a neuropeptide composed of 36 amino acids that is involved in various physiological and homeostatic regulation of the central and peripheral nervous systems. NPY is the most abundant peptide in the mammalian central nervous system consisting of the brain and spinal cord. In the autonomic nervous system, it is mainly produced by neurons of the sympathetic nervous system, plays a vasoconstrictive role, and is also involved in the growth of adipose tissue. In addition, it is known to be produced in various locations of 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.

[0104] Secretin (SCGN) is a calcium-binding protein expressed in the cytoplasm, which is known to be involved in potassium chloride-induced calcium influx and cell proliferation. In addition, it is known to be involved in the release of corticotropin-releasing hormone (CRH), a stress hormone, and stress activation of the brain.

[0105] PMCH (promelanin-concentrating hormone) is the precursor protein of MCH (melanin-concentrating hormone), a neuropeptide that regulates hunger, energy homeostasis, reproductive function, and sleep. PMCH dysfunction is known to be associated with frontal epiphyseal dysplasia 2 and Huntington's disease.

[0106] The term "differentiation" as used herein refers to the phenomenon that the structure or function of cells becomes specialized while cells divide and proliferate and the whole organism grows. In other words, it refers to the process in which cells, tissues, etc. of an organism change into a morphology and function suitable for performing their respective roles. In the present invention, the above terms may include the process in which pluripotent stem cells are converted into cells that constitute the hypothalamus, and may also include the process in which progenitor cells exhibit specific differentiation traits. In the present invention, when hypothalamic-like organoid-derived neural stem cells (htNSC) differentiate into neurons, astrocytes (GFAP+) and glial cells can also differentiate together. At this time, the differentiated neurons and astrocytes can sense the nutritional status of the body by responding to leptin and ghrelin (peptides produced in peripheral organs).

[0107] Specifically, the hypothalamic-like organoid-derived neural stem cells (htNSCs) according to the present invention can differentiate and cluster into neural stem / progenitor cell clusters (NSCs), intermediate progenitor cell clusters (IPCs), neural cell clusters (Neu1 and Neu2), astrocyte progenitor cell clusters (APCs), astrocyte clusters (Ast1, Ast2 and Ast3), elongated cell clusters (Tan), interneuron progenitor cell clusters (Int1 and Int2), radial glial cell clusters (RG), and proteoglycan-secreting glial cell clusters.

[0108] Progenitor cells are cells that differentiate into specific types of cells, and refer to cells that differentiate into "target cells" more specifically than stem cells. Stem cells can divide themselves indefinitely, while progenitor cells can only divide a limited number of times. In the present invention, the progenitor cells can be neural progenitor cells, astrocyte progenitor cells, or interneuron progenitor cells.

[0109] Nerve cells are cells that make up the nervous system and can transmit signals electrically using ion channels such as sodium channels and potassium channels. In addition, they exchange chemical signals with other adjacent nerve cells through structures called synapses to receive and store various information. In this specification, nerve cells can be used interchangeably with "neurons" or "neuronal cells".

[0110] Neural progenitor cells are cells that have the ability to differentiate into neurons.

[0111] In addition, astrocytes, also called astroglial cells, are present in the brain and spinal cord. It biochemically assists cells within the blood-brain barrier (BBB) ​​and has protrusions that attach to the walls of blood vessels to provide nutrition to nerve cells. It also plays various roles, such as regulating ion concentrations in nerve cells, supporting nerve cells, removing waste products, and phagocytosis. In addition, when nerve tissue is damaged, it proliferates into a type of protrusion called glioma to fill the area and plays a role in repairing or destroying damaged tissue. It has been revealed that hydrogen peroxide produced during the decomposition of toxins by astrocytes is the cause of nerve cell destruction, suggesting the possibility of treating brain diseases such as dementia.

[0112] Astrocyte progenitor cells are cells that are capable of differentiating into astrocytes.

[0113] Elongocytes are ependymal cells present in the third and fourth ventricles of the hypothalamus that sense nutritional status to regulate appetite.

[0114] Interneurons are nerve cells that connect two different areas of the brain. They 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 judge these stimuli and transmit them to motor neurons. Interneurons are included in the central nervous system and play a key role in neurogenesis in the adult mammalian brain, including reflexes and neural oscillations.

[0115] Interneuron clusters (interneuron progenitor cells) refer to cells that differentiate into interneurons.

[0116] Radial glial cells are a type of glial cell, which are the progenitor cells of nerve cells and astrocytes. Radial glial cells also serve as supports to guide nerve cells to necessary locations.

[0117] Proteoglycan-secreting glial cells are a type of glial cell that secretes 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 a positive effect on isolating the damaged area and preventing the spread of damage, but in the process of forming this glial scar, the substances secreted by astrocytes also inhibit the growth of neurites.

[0118] In the above-mentioned clustered cell clusters, the neural cell clusters (Neu1 and Neu2) can express hypothalamus development-related genes. The astrocyte progenitor cell cluster (APC) can include cells that differentiate into the following cell clusters: 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, IFITs 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. At this time, the Ast2 cluster can be responsible for the nutritional supply and neuroendocrine function of neural cells. The Ast1 cluster can be responsible for immune function. The Ast3 cluster can regulate the activity of neural cells. The elongated cell cluster (Tan) can be a cell cluster expressing at least one gene selected from the group consisting of RAX, FGF10, COL25A1, CRYM and SCN7A.

[0119] SLC1A3 (solute carrier family 1 member 3) is a gene encoding excitatory amino acid transporter 1.

[0120] SLCO1C1 (solute carrier organic anion transporter family member 1C1) is a gene encoding a membrane transporter (organic anion transporting polypeptide) that mediates the transport of organic anions and is expressed in the brain and testis.

[0121] CRYM is a gene encoding a homolog of Crystallin Mu, also known as NADP-regulated thyroid hormone binding protein (THBP).

[0122] SCG2 is a 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 sort and package neuropeptides and peptide hormones into the secretosome, whereas after cleavage, it functions as an activated secretory neuropeptide.

[0123] RSAD2 is a gene encoding free S-adenosylmethionine domain-containing protein 2, also known as Viperin (interferon-induced endoplasmic reticulum-associated viral inhibitory protein). Viperin is activated by interferon (IFN) to inhibit infection by DNA or RNA viruses.

[0124] TRAIL is a gene encoding TNF-related apoptosis-inducing ligand, a ligand that induces apoptosis.

[0125] IFIT is a gene encoding interferon-induced antiviral protein (interferon-induced protein with tetratricopeptide repeats).

[0126] STAT1 is a gene that encodes a protein that mediates cellular signaling of interferons, cytokines, and growth factors (signal transducer and activator of transcription 1).

[0127] SOX9 is a gene that encodes a transcription factor that regulates chondrocyte differentiation and bone development.

[0128] ID3 is a gene encoding a transcriptional regulator that lacks a DNA binding site and forms heterodimers to inhibit DNA binding and transcriptional activity. Transcription factor ID3 regulates various cellular processes, including cell growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation.

[0129] Meis2 is a gene encoding the homeobox protein Meis2, which binds to HOX or PBX proteins to form dimers or to the DNA-binding dimers of PBX and HOX proteins to stabilize the homeobox protein-DNA complex, thereby regulating transcription.

[0130] CLDN5 is a gene that encodes Claudin-5, which specifically removes tight junctions within cells.

[0131] ALCAM is a gene that encodes CD166, which mediates adhesion of thymic epithelial cells and CD6+ cells during T cell development in the thymus.

[0132] COL1A2 is a gene encoding collagen α-2(I) chain, which is one type of type 1 collagen.

[0133] FGF10 is a gene encoding fibroblast growth factor 10, which is involved in embryonic development, cell proliferation, cell differentiation regulation and wound healing.

[0134] COL25A1 is a gene encoding collagen alpha-1 (XXV) chain, a membrane-bound collagen specifically expressed in the brain and plays a role in inhibiting the fibrillation of amyloid-β peptide (Aβ).

[0135] SCN7A is a gene encoding the sodium channel protein type 7 subunit alpha. SCN7A plays a role in mediating voltage-dependent sodium ion permeability of excitable membranes.

[0136] Use of a pharmaceutical composition comprising neural stem cells

[0137] The pharmaceutical composition comprising the neural stem cells derived from the hypothalamic-like organoids of the present invention as an active ingredient can be used for the purpose of preventing or treating senile diseases. In addition, the pharmaceutical composition may also comprise: neural cells derived from hypothalamic-like organoids, or a cell population containing neural stem cells and neural cells. In this case, the hypothalamus, organoids, neural stem cells, and neural cells are the same as described above.

[0138] In the present invention, the geriatric disease may be selected from the group consisting of neurodegenerative diseases, osteoporosis and muscle aging-related diseases.

[0139] At this time, the neurodegenerative disease can 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, cortical basal degeneration, diffuse Lewy body disease, and Pick's disease.

[0140] At this time, the muscle aging-related disease can be selected from the group consisting of muscle atrophy, muscle disuse atrophy, and senile sarcopenia.

[0141] The term "prevention" as used herein refers to any action to inhibit or delay the onset of geriatric diseases by administering a pharmaceutical composition. The term "treatment" refers to any action to improve or beneficially alter the symptoms of geriatric diseases by administering a pharmaceutical composition.

[0142] In the pharmaceutical composition for preventing and treating geriatric diseases of the present invention, any amount (effective amount) of neural stem cells, neural cells or cell groups containing them can be included according to the purpose, dosage form, mixing purpose, etc., as long as they can show anti-aging activity. The pharmaceutical composition of the present invention can contain about 2×10 5 About 2×10 10 cells, preferably about 2×10 6 About 2×10 9 cells, more preferably about 1×10 7 About 2×10 8 Preferably, the pharmaceutical composition may contain 2×10 8 cells.

[0143] Here, "effective amount" refers to the amount of active ingredients that can produce anti-aging effects. Such effective amount can be determined by experiments within the normal capabilities of ordinary technicians in the field.

[0144] The compositions of the present invention may be used without freezing or may be frozen for later use. If frozen, a standard cryopreservative (e.g., DMSO, glycerol, polyvinyl pyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, Cell freezing medium (Cascade Biologics)) was added to the cell population.

[0145] The pharmaceutical composition of the present invention can be used in the form of a cell therapeutic agent, and can be formulated by further comprising a pharmaceutically acceptable carrier.

[0146] At present, "cell therapeutic agent" refers to a drug used for treatment, diagnosis and prevention using cells and tissues prepared by separation, cultivation and special manufacturing from a subject (US FDA regulations). It refers to a series of actions such as in vitro proliferation, sorting of living autologous, allogeneic or xenogeneic cells, or otherwise changing the biological properties of cells to restore the function of cells or tissues, and used for treatment, diagnosis or prevention purposes. Depending on the degree of cell differentiation, cell therapeutic agents are roughly divided into somatic cell therapeutic agents and stem cell therapeutic agents. In the present invention, the cell therapeutic agent can be a stem cell therapeutic agent.

[0147] The term "pharmaceutically acceptable carrier" as used herein 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, the pharmaceutically acceptable carrier that can be included in the cell therapy agent can be used without restriction as long as it is known in the art, such as a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, etc. The pharmaceutical composition of the present invention can be prepared into various dosage forms according to commonly used techniques.

[0148] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount and can be administered by any route as long as it can induce migration to the diseased site. Here, "administering" refers to introducing a predetermined substance into a subject by an appropriate method. In some cases, it is contemplated that the pharmaceutical composition of the present invention is loaded into a carrier containing a means for guiding neural stem cells to the lesion.

[0149] Therefore, the pharmaceutical composition of the present invention can be applied by multiple approaches, including topical application (including buccal application, sublingual application, skin application and intraocular application), parenteral application (including subcutaneous application, intradermal application, intramuscular application, instillation application, intravenous application, intraarterial application, intraarticular application and cerebrospinal fluid application) or transdermal application, and preferably, can be applied directly to the affected part. In one embodiment, neural stem cells can be suspended in a suitable diluent and applied to the object, and the diluent is used to protect and maintain cells and is convenient for them to use when injected into the target tissue. The diluent can include a buffer solution, such as normal saline, phosphate buffered solution, HBSS, cerebrospinal fluid, etc. In addition, the composition can be applied by any device to allow the active substance to migrate to the target cell.

[0150] The preferred mode of administration and preparation is injection. The injection can be prepared using aqueous solutions such as physiological saline, Ringer's solution, Hanks solution or sterile aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleate, non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerol, etc. For mucosal penetration, non-permeation agents suitable for passing through barriers known in the art can be used, and drug carriers can be further included, for example, stabilizers for preventing deterioration (such as ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for adjusting pH, and preservatives for inhibiting microbial growth (such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol and benzyl alcohol). Methods for preparing pharmaceutical compositions are known in the art, and specific reference can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is regarded as part of this specification.

[0151] The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects. The effective dosage level can be easily determined by one of ordinary skill in the art based on factors including the patient's sex, age, weight, health status, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, duration of treatment, combined or concurrently used drugs, and other factors well known in the medical field.

[0152] The preferred dosage of the pharmaceutical composition of the present invention can be about 1.0×10 cells per day. 3 About 1.0×10 10 cells / kg, about 1.0×10 4 About 1.0×10 9 cells / kg, about 1.0×10 5 About 1.0×10 8 cells / kg, or about 1.0×10 6 About 1.0×10 7 cells / kg, depending on the patient's condition, weight, sex and age, severity of the patient's condition and route of administration.

[0153] However, the dosage may be specified in various ways according to factors such as the formulation method, the mode of administration, the patient's age, body weight, sex, pathological condition, diet, administration time, route of administration, excretion rate and reaction sensitivity, and a person of ordinary skill in the art can appropriately adjust the dosage by considering these factors. Within the range of clinically acceptable side effects, the frequency of administration may be once or twice or more times. With regard to the site of administration, it may be administered to one site or to two or more sites. For animals other than humans, it may be administered in the same dosage as humans per kg or per object, or it may be administered in the amount converted to the above dosage, for example, according to the volume ratio (e.g., mean value) of organs (heart, etc.) between the target animal and humans. This dosage should not be construed as limiting the scope of the present invention in any way.

[0154] As used herein, the term "subject" refers to a subject to which the composition of the present invention can be applied (prescribed), and can be a mammal including a human, such as a rat, a mouse, or a livestock. Preferably, the subject can be a human, but is not limited thereto. In addition to comprising hypothalamic-like organoid-derived neural stem cells, the pharmaceutical composition of the present invention may also comprise any compound or natural extract that has been proven to be safe and known to have anti-aging activity to increase or enhance hypothalamic-like organoid-derived neural cells or anti-aging activity. At this time, the neural stem cells, neural cells, or cell populations comprising the same; and the compound or natural extract having anti-aging activity can be administered simultaneously or sequentially.

[0155] In another aspect of the present invention, there is provided a pharmaceutical composition comprising a hypothalamic-like organoid-derived neural stem cell as an active ingredient for the prevention and treatment of senile diseases. In this case, the pharmaceutical composition may also comprise a hypothalamic-like organoid-derived neural cell. Here, the hypothalamus, organoid, neural stem cell, neural cell, senile disease, prevention, and treatment are the same as described above.

[0156] In another aspect of the present invention, a method for preventing and treating senile diseases is provided, comprising administering to a subject a pharmaceutical composition comprising hypothalamic-like organoid-derived neural stem cells as an active ingredient. In this case, the pharmaceutical composition may also comprise neural cells derived from hypothalamic-like organoids. Here, the hypothalamus, organoids, neural stem cells, senile diseases, administration, prevention, and treatment are the same as described above.

[0157] The subject may be a mammal including a human, such as a rat, a mouse or a livestock. Preferably, the subject may be a human.

[0158] The dosage can be about 1.0×10 cells per day. 3 About 1.0×10 10 cells / kg, about 1.0×10 4 About 1.0×10 9 cells / kg, about 1.0×10 5 About 1.0×10 8 cells / kg, or about 1.0×10 6 About 1.0×10 7 The dosage may be specified in various ways according to factors such as the formulation method, administration mode, age, weight, gender, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, and those skilled in the art may appropriately adjust the dosage by considering these factors.

[0159] Exosomes derived from neural stem cells

[0160] In another aspect of the present invention, an exosome secreted by a neural stem cell derived from a hypothalamic-like organoid is provided. In this case, the hypothalamus, the organoid and the neural stem cell are the same as described above.

[0161] The term "exosome" used herein refers to an extracellular vesicle of 50 to 150 nm secreted by a cell, also referred to as a microvesicle. Exosomes are secreted from cells when multivesicular bodies fuse with cell membranes, or are directly secreted through cell membranes. Exosomes are known to contain complex mixed factors related to cell differentiation, growth, movement and signal transduction, such as intracellular proteins, cell membrane proteins, lipids, and nucleic acids such as RNA, miRNA and DNA, and therefore play an important role in various processes such as coagulation, intercellular signal transduction and metabolic waste management. The diameter of an exosome 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 can be derived from plasma membranes or multivesicular bodies (MVBs) and released or secreted outside the cell.

[0162] In the present invention, exosomes can be isolated from a culture of hypothalamic-like organoid-derived neural stem cells, neural cells, or a cell population comprising the same.

[0163] The exosomes derived from the neural stem cells derived from the hypothalamus-like organoids of the present invention can prevent or treat senile diseases. In this case, the hypothalamus, organoids, neural stem cells, neural cells, prevention and treatment are the same as above.

[0164] Geriatric diseases may be neurodegenerative diseases and muscle aging-related diseases. Here, neurodegenerative diseases and muscle aging-related diseases are the same as described above.

[0165] Pharmaceutical composition comprising exosomes derived from neural stem cells

[0166] In another aspect of the present invention, a pharmaceutical composition for preventing and treating geriatric diseases is provided, comprising exosomes as active ingredients. Here, exosomes, prevention, and treatment are the same as described above. In addition, geriatric diseases may be neurodegenerative diseases and muscle aging-related diseases, which are the same as described above.

[0167] In the pharmaceutical composition for preventing and treating geriatric diseases of the present invention, any amount (effective amount) of exosomes may be included according to the purpose, dosage form, mixing purpose, 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 1 mL. The effective amount here is the same as above.

[0168] The composition of the present invention can be administered to a subject in a pharmaceutically effective amount. Here, the pharmaceutically effective amount, administration, and subject are the same as described above.

[0169] The preferred dosage of the pharmaceutical composition comprising the exosomes of the present invention as an active ingredient can be 0.001 mg / kg to 100 mg / kg body weight, 0.005 mg / kg to 50 mg / kg body weight, 0.02 mg / kg to 20 mg / kg body weight, or 0.1 mg / kg to 10 mg / kg body weight per day, depending on the patient's condition, weight, sex and age, the severity of the patient, and the administration route.

[0170] Administration can be once a day or divided into several times a day. However, the dosage may be specified in various ways according to factors such as formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate and reaction sensitivity, and those of ordinary skill in the art can appropriately adjust the dosage by considering these factors. Within the scope of clinically acceptable side effects, the frequency of administration may be once or twice or more times. With regard to the site of administration, it may be administered to 1 site, or it may be administered to 2 or more sites. For animals other than humans, it may be administered in the same dosage as humans per kg or per object, or it may be administered by, for example, the volume ratio (e.g., mean value) of the organ (heart, etc.) between the target animal and the human to the amount converted to the above dosage. This dosage should not be construed as limiting the scope of the present invention in any way.

[0171] In some cases, it is contemplated that the pharmaceutical composition of the present invention is loaded into a carrier comprising a means for directing exosomes to the lesion. Therefore, the pharmaceutical composition of the present invention can be administered by a variety of routes, including topical administration (including buccal administration, sublingual administration, skin administration, and intraocular administration), parenteral administration (including subcutaneous administration, intradermal administration, intramuscular administration, instillation administration, intravenous administration, intraarterial administration, intraarticular administration, and intracerebrospinal fluid administration) or transdermal administration, and preferably, can be directly administered to the diseased site. However, the method of administration is not limited thereto, and a method suitable for treatment can be used.

[0172] The preferred administration mode and dosage form of the pharmaceutical composition of the present invention is injection. The method of preparing the pharmaceutical composition is known in the art, and specific reference may be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is regarded as a part of this specification.

[0173] In addition, the composition of the present invention may include a pharmaceutically acceptable carrier. The composition and the pharmaceutically acceptable carrier are the same as above. A pharmaceutically acceptable carrier refers to an excipient, a diluent or an adjuvant. The example of a carrier may be selected from the group consisting of: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, saline, buffer (e.g., PBS), methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, etc.

[0174] In addition to exosomes as active ingredients, the anti-aging composition of the present invention may also contain cell therapy agents (e.g., neural stem cells, neural cells, or cell populations containing them), any compounds or natural extracts that have been proven to be safe and known to have anti-aging activity, to increase or enhance the anti-aging activity. In this case, the exosomes and the cell therapy agents, compounds or natural extracts with anti-aging activity may be administered simultaneously or sequentially.

[0175] In another aspect of the present invention, there is provided a use of exosomes isolated from neural stem cells derived from hypothalamic-like organoids for preventing and treating geriatric diseases. Here, the hypothalamus, organoids, neural stem cells, neural cells, exosomes, geriatric diseases, prevention, and treatment are the same as described above.

[0176] In another aspect of the present invention, a method for preventing and treating geriatric diseases is provided, comprising administering exosomes to a subject. Here, exosomes, subjects, administration, geriatric diseases, prevention, and treatment are the same as described above.

[0177] The preferred dosage of exosomes can be 0.001 mg / kg to 100 mg / kg body weight, 0.005 mg / kg to 50 mg / kg body weight, 0.02 mg / kg to 20 mg / kg body weight, or 0.1 mg / kg to 10 mg / kg body weight per day, depending on the patient's condition, weight, sex and age, the severity of the patient, and the administration route.

[0178] Administration can be performed once a day or divided into several times a day. However, the dosage may be specified in various ways according to factors such as the formulation method, administration mode, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity, and a person of ordinary skill in the art can appropriately adjust the dosage by considering these factors.

[0179] Methods for producing exosomes derived from neural stem cells

[0180] In another aspect of the present invention, a method for producing exosomes is provided, comprising the steps of: culturing neural stem cells derived from hypothalamic-like organoids; and collecting exosomes from the culture medium. Here, the hypothalamus, organoids, neural stem cells, and exosomes are the same as described above.

[0181] Kit comprising exosomes derived from neural stem cells and / or neural stem cells

[0182] In another aspect of the present invention, a kit for preventing and treating geriatric diseases is provided, comprising neural stem cells derived from hypothalamic-like organoids; and / or exosomes secreted by neural stem cells derived from hypothalamic-like organoids. The hypothalamus, organoids, neural stem cells, exosomes, prevention, and treatment are the same as those described above.

[0183] Geriatric diseases may be neurodegenerative diseases, osteoporosis, and muscle aging-related diseases. Here, neurodegenerative diseases and muscle aging-related diseases are the same as above.

[0184] Embodiments of the present invention

[0185] Below, the present invention will be described in more detail by the following examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0186] Experimental Example 1. Cell Culture

[0187] The human embryonic stem cell line H9 (H9 hESC, WiCell) was cultured on Matrigel-coated plates in mTeSR-1 medium (Stem Cell Technology) without feeder cells. Human hypothalamus-derived neural stem cells (ntNSC) were generated from H9 hESC using a 3D culture method.

[0188] Specifically, hESCs were suspended at a density of 10,000 cells / well in a neural induction medium in a low-adhesion 96-well round-bottom plate, seeded and cultured for 24 hours. The neural induction medium used at this time was a 1:1 mixed medium of Neurobasal medium (Gibco) and N2 medium, containing B27 without vitamin A (Invitrogen), 200 μM ascorbic acid (Sigma) and 1.25 mg / L insulin, supplemented with 10 μM SB431542 (Tocris), 200 ng / mL noggin (Peprotech) and 20 μM Y27632 (ROCK inhibitor, Sigma).

[0189] For hypothalamic patterning of H9 hESCs, they were cultured in neural induction medium containing 10 μM SB431542 (Tocris) and 200 ng / mL noggin (Peprotech) for 5 days, and then cultured for 6 days by adding 100 ng / mL sonic hedgehog (SHH, Peprotech) and 2 μM purmorphamine (Calbiochem) to the medium. On day 12 after the start of differentiation, the formed spheres were transferred to low-adhesion 6-well plates and cultured in neural expansion medium for 8 days (until day 20 after the start of differentiation) using an orbital shaker (80 rpm). The neural expansion medium used was N2 medium containing 1.25 mg / L insulin, 200 μM ascorbic acid, 20 ng / L bFGF (Peprotech) and penicillin / streptomycin. Afterwards, the spheres were minced and suspended in neural expansion medium and seeded into poly-L-ornithine / fibronectin-coated plates. The neural stem cells obtained by the above method were cultured in N2 medium (neuronal 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.

[0190] Experimental Example 2. Animal Experiment

[0191] All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University College of Medicine (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 for Basic Science or Janvier Laboratory, Korea. Mice were housed in a constant temperature (23 ± 1 °C), constant humidity (50 ± 10%), and 12-h day-night cycle housing room with free access to food and water.

[0192] Experimental Example 3. Cell transplantation experiment

[0193] 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 hypothalamus-derived NSCs (1 μL, 2 × 10 5Cells / μL) were injected into the medial basal hypothalamus (MBH) at a rate of 250 nL / min for 2 minutes (AP: -1.5 mm, ML: ±0.4 mm, DV: -5.6 mm). After injection, the syringe was left in place for another 5 minutes before being removed.

[0194] Experimental Example 4. Histological Analysis

[0195] After the mice were anesthetized, they were perfused intracardially with 30 mL PBS (pH 7.4) to remove blood, and then perfused intracardially with 4% paraformaldehyde (PFA, 30 mL) diluted in PBS to fix the tissue. The fixed brain tissue was extracted and further fixed overnight at 4 ° C with 4% PFA (diluted in PBS). The fixed brain tissue was reacted with 30% sucrose for 7 days to remove moisture from the tissue (cryoprotection). The dehydrated brain tissue was shaped with TissueTek OCT compound (Sakura Finetek), frozen, and then cut into 30 μm thick slices using a cryostat (Leica) to prepare brain tissue slices.

[0196] For peripheral tissues, white adipose tissue (WAT), brown adipose tissue (BAT), liver and femur extracted after dissection were separated and stored 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 cut into 4 μm thick sections to prepare tissue sections. Tissue staining was performed in the same manner as the known H&E method.

[0197] Experimental Example 5. Immunostaining

[0198] Cells and frozen brain tissue sections were fixed with 4% paraformaldehyde (PFA) diluted in PBS, and then treated with 0.3% TritonX-100 containing 1% BSA and reacted for 1 hour. Subsequently, anti-Nestin antibody (1:500, BioLegend, 65680), anti-Rax antibody (1:500, Takara, 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), anti-αMSH antibody (1:2000, Phoenix Pharmaceuticals, H-043-01), 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) was used as the primary antibody and reacted overnight at 4°C in a light-proof environment. For visualization, anti-mouse IgG conjugated with Cy3 or AlexFlour 488 (1:1000, Jackson Immunoresearch Laboratories, 111-165-003 or A-11001) was used as a secondary antibody.

[0199] The stained cells and brain tissue sections were mounted using VECTASHIELD mounting solution containing DAPI (Vector Laboratories) and observed using a fluorescence microscope (Leica).

[0200] Experimental Example 6. Quantitative RT-PCR analysis

[0201] TRIzol was used according to the manufacturer's protocol. TMReagent (Invitrogen) to extract RNA, and then use SuperScript TM cDNA was synthesized using the iQTM Green Supermix (Bio-Rad) in CFX96 TM Quantitative RT-PCR (q-PCR) was performed on a real-time system, and the gene expression level was quantified according to the expression level of GAPDH or β-actin. The primers used at this time are shown in Table 1.

[0202] [Table 1]

[0203]

[0204] Experimental Example 7. Western blotting

[0205] RIPA buffer (50mM Tris-HCl pH7.4, 150mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, and 1mM EDTA) containing protease inhibitors and phosphorylase inhibitors was added to the cells and sonicated to lyse the cells. The cell lysate was centrifuged at 4°C and 15,000g for 5 minutes to obtain the supernatant, which was then quantified using Pierce BCA Assay (Thermo Fisher). The proteins separated by SDS-PAGE electrophoresis were transferred to a nitrocellulose membrane, and then TBST containing 5% BSA was added and reacted for 1h for blocking. After blocking, the primary antibodies shown in Table 2 were treated and reacted overnight at 4°C. After the primary antibody reaction was completed, goat anti-rabbit HRP conjugate or goat anti-mouse HRP conjugate (Cell Signaling Technology) was used as a secondary antibody, diluted 1:1,000 in TBST containing 5% BSA, treated on the membrane, and reacted for 1 hour. Protein expression of each antibody was confirmed using West-Pico chemiluminescent substrate (Thermo Fisher). Leptin and ghrelin activity analysis was performed by treating differentiation-induced cells with leptin (100 ng / mL) or ghrelin (10 nM), respectively, followed by reacting for 1 hour.

[0206] [Table 2]

[0207]

[0208]

[0209] Experimental Example 8. Exosome Isolation and Mouse Treatment

[0210] Exosomes in the cell culture medium were purified using an ultracentrifuge. Specifically, the cell culture fluid was centrifuged to obtain a supernatant, which was then centrifuged in an ultracentrifuge at 300 g for 10 minutes, 2000 g for 10 minutes, 10,000 g for 30 minutes, and 100,000 g for 1 hour, filtered, and then centrifuged again in an ultracentrifuge at 100,000 g for 1 hour to obtain exosomes.

[0211] The exosomes separated through the above process were finally suspended in PBS and prepared.

[0212] For all mice, a 26-gauge catheter was implanted into the third ventricle (3V) of the hypothalamus at the midline coordinates of 2.0 mm posterior and 5.0 mm below the bregma using an ultra-precision small animal stereotaxic apparatus (Kopf Instruments, Tujunga, CA, USA) in the same manner as in Experimental Example 2. Among them, the vehicle (PBS) and exosome-treated groups were injected with PBS or exosomes into the 3V through the catheter three times a week for three months.

[0213] Experimental Example 9. Mouse behavior experiment

[0214] All behavioral experiments were conducted in a behavioral laboratory, and all behavioral assessments were recorded using a computerized ANY-maze video tracking system (Stoelting). In addition, all experiments were performed in accordance with the ARRIVE guidelines.

[0215] Experimental Example 9.1. Muscle strength assessment (grip strength test)

[0216] The mice were placed on a square grid (grid size 1 cm), then the grid was turned over, and the time the mice spent hanging by their feet for 2 minutes was measured and recorded. The experiment was repeated three times for each mouse, with at least 10 minutes of rest.

[0217] Experimental Example 9.2. Motor coordination assessment (rotarod test)

[0218] The mice were trained on a rotarod (BS Technolab INC.) for 60 seconds at 5 rpm, after which they were allowed to rest for 10 minutes. Each mouse was then placed on a rotarod moving at 5 rpm, and the behavior of the mice was recorded while increasing the speed by 5 rpm per minute for 5 minutes. The experiment was repeated three times for each mouse, followed by a 30-minute rest.

[0219] Experimental Example 9.3. Endurance Assessment (Treadmill Test)

[0220] Endurance assessment was performed using a treadmill (Jeung Do Bio&Plant Co., Ltd.) that can assess up to 8 mice on a single track. Before the experiment, mice were allowed to adapt to the treadmill at a speed of 5 rpm for 5 minutes to prevent running injuries and failure. Afterwards, the speed was increased by 2 rpm per minute for 10 minutes, and the time until the mice gave up running was recorded.

[0221] Experimental Example 9.4. New Object Recognition Test

[0222] First, before the experiment, the mice were placed freely in an open field box (40 cm wide × 40 cm long × 50 cm high) for 10 minutes. Then, the mice were allowed to freely recognize two objects of the same shape (first stage). After that, one of the two objects was replaced with a new object, and the mice were then exposed for 10 minutes (second stage). Then, the time it took the mice to recognize each object was recorded. The preference index was expressed as the ratio of the time spent recognizing one of the two objects (including the new object) to the total time spent recognizing the two objects.

[0223] Experimental Example 9.5. Morris Water Maze Test

[0224] The Morris water maze test was performed in the following manner.

[0225] First, fill the water tank with water at 24°C and paint the tank background opaque white with Crayola non-toxic paint. Then, place the tank in the center of the room with additional maze cues (various black shapes on a white background). At this point, the tank is divided into four quadrants (northwest, northeast, southwest, southeast) with a diameter of 90 cm. Then, place a circular platform with a diameter of 10 cm at a distance of 25 cm from the wall of the box, and measure the time, distance, and speed required for the mouse to move from the same starting position to the platform in each experiment.

[0226] The experiment was conducted in two stages: hidden platform training and exploration test. Hidden platform training was conducted as follows. First, mice were trained to swim to a visible platform installed 0.5 cm above the water surface and stay there for 10 seconds. At this point, if the mice did not find the platform within 60 seconds, they were guided to the platform with a glass stirring rod. The training was conducted for four consecutive days, four times a day, with different water entry locations (the water entry locations were north, south, east, and west). After the training, the platform was installed at a depth of 1 cm below the water surface to make it invisible. Subsequently, the delay time for mice to reach the platform, the distance moved, the path efficiency, the time spent in each quadrant and the distance moved, the total moving distance, and the swimming speed were measured. For the exploration test, on the fifth day after the training started, the platform was removed and the trained mice were started at the position facing the platform installation location (target quadrant). The time spent in all quadrants, the distance and number of times of crossing the target quadrant, the total moving distance, and the swimming speed of the mice were recorded within 60 seconds. The learning and memory of mice was evaluated by the total time spent in the target quadrant during the exploration test.

[0227] Experimental Example 9.6. Y-maze test

[0228] The measuring device uses a Y-maze, which consists of three identical branches (40 cm long, 4 cm wide, and 15 cm high), each with a bending angle of 120 °. The three branches are designated A, B, and C, respectively, and the mouse is placed at the end of one branch and allowed to move to another branch for 10 minutes. The number of times the experimental animal enters the branch until its tail and the number of times it enters each branch in sequence are counted, and 1 point is awarded (actual alternation). Alternating behavior is defined as three (e.g., ABC, BCA, CAB, but not ABA) that do not overlap, and spontaneous alternation is calculated using the following mathematical equation 1.

[0229] <Mathematical equation 1>

[0230] Spontaneous alternation = [(number of alternations) / (total number of events - 2)] × 100

[0231] Experimental Example 9.7. Open Field Test

[0232] Mice were placed one by one in the center of an open field box (40 cm long × 40 cm wide × 50 cm high) and allowed to acclimate for 20 min. The movements of the mice were recorded for 10 min and then analyzed using a digital camera connected to the Any-Maze animal tracking system software (Stoeling, Wood Dale). At this point, the total distance moved (meters) and the time spent in the center and periphery of the open field box (seconds) were measured.

[0233] Experimental Example 9.8. Passive avoidance test

[0234] A door is provided in the center of the measuring device (48 cm long × 23 cm wide × 28 cm high), which equally divides the area within the measuring device into an illumination chamber (bright chamber) and a dark chamber. On the first day, the mice were allowed to freely identify the two chambers for 10 minutes. On the second day (training), the mice were left in the illumination chamber for 60 seconds, and then the next door was opened to allow the mice to freely enter the dark chamber. When the mice entered the dark chamber, the door was closed and an electric shock (0.3 mA, 3 seconds) was applied to the feet of the mice. At this time, the waiting time for each stage or the time required for the mice to enter the dark chamber was recorded. On the third day (exploratory test), the mice were placed in the illumination chamber, the door was opened after 60 seconds, and the time it took for the mice to move to the dark area inside the door was measured. The waiting time for each stage was measured to be 540 seconds.

[0235] Experimental Example 10. Body Composition Analysis

[0236] As in Experimental Example 3, the fat and lean percentages of the cell-transplanted mice were measured using minispec LF50 (Bruker) at the Korean Mouse Phenotyping Center (KMPC).

[0237] Experimental Example 11. Glucose tolerance test

[0238] As in Experimental Example 3, the cell-transplanted mice were fasted for 12 hours and then intraperitoneally administered with glucose at a concentration of 2 g / kg. Blood was collected from the tail at 0, 30, 60, and 120 minutes after the glucose administration, and the blood glucose level was measured using a glucometer.

[0239] Experimental Example 12. Metabolic Cages and Behavioral Analysis

[0240] To measure metabolic rate, metabolic parameters of mice were analyzed by indirect calorimetry1-2 using the 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 housed in individual measurement cages (20.5 cm long × 10.5 cm wide × 12.5 cm high) and fed the same diet (normal diet or high-fat diet) to acclimate them to the environment. O2 consumption, CO2 production, energy expenditure, heat, and locomotor activity of the experimental animals were measured for 24 hours. The above measurement data were expressed by calculating the respiratory quotient or CO2 / O2 exchange ratio and Δ calorific value using CLAX software (version 2.2.15, Columbus Instruments).

[0241] Experimental Example 13. Batch RNA Sequencing Transcriptome Analysis

[0242] Total RNA (RIN>8) was isolated using TRIzol (approximately 2×10 6cells), and cDNA libraries were prepared using TruSeq StrandedmRNA LT (Illumina). Sequencing was performed using NovaSeq 6000 (Macrogen). Paired-end libraries were analyzed by comparison (alignment) with the human reference UCSC hg19 using the HISAT2 pipeline. The differentially expressed genes (DEGs) showing differences in expression levels between the control and sample groups were analyzed using the DESeq2 method. At this time, genotype-tissue expression (GTEx) data were used by randomly selecting four samples of hypothalamus, hippocampus, cortex, and substantia nigra from postmortem donors from dbGaP (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).

[0243] Experimental Example 14. Single-cell RNA nucleotide sequence analysis

[0244] htNSCs were cultured in neurosphere state for 70 days in neuronal differentiation medium.

[0245] The cultured hypothalamic 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 cell viability was determined. A single-cell cDNA library was prepared for the isolated cells using the Chromium Next GEM Single Cell 3'RNA Library v3.1 Reagent Kit (10X Genomics, USA). The single-cell cDNA library was sequenced using the NextSeq 500 system (Illunimia) to obtain double-end reads of 91bp and 28bp. The above results were decomposed and quantified according to UMI (unique molecular identifier) ​​using Cell Ranger version 5.0.0 (10XGenomics, USA).

[0246] Count matrices were generated for each sample using default parameters and genes were mapped based on the human reference genome GRCh38.

[0247] Experimental Example 15. Cell proliferation analysis test

[0248] Cell proliferation was confirmed by the population doubling level (PDL) calculated by mathematical equation 2.

[0249] <Mathematical equation 2>

[0250] Population doubling level (PDL) = log(N / N0) / log2

[0251] Here, N refers to the number of cells at the end of each generation, and N0 refers to the number of cells inoculated at the beginning of culture.

[0252] Experimental Example 16. Cell Viability Analysis

[0253] Cell viability was measured using the LIVE / DEAD viability / cytotoxicity kit (ThermoFisher #L3224).

[0254] Experimental Example 17. Bone Micro-CT Analysis Test

[0255] The right femur of each mouse was removed and fixed overnight in 4% formaldehyde. The fixed femur was placed in a scanning tube with a diameter of 12.3 mm, and images were taken using a desktop micro-CT (μCT) scanner (SkyScan 1076, Bruker), and the scanned images were merged into 3D images. 100 slices were taken as the target region (ROI) within a range of about 0.5 mm to 1.6 mm from the growth plate to analyze total tissue volume, bone mineral density (BMD), bone volume (BV) / tissue volume (TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th).

[0256] Experimental Example 18. Statistical Analysis

[0257] Two-tailed unpaired sample t-test, one-way analysis of variance (ANOVA), or Mann-Whitney U test were used for statistical analysis using Prism software version 6 (GraphPad Software) or IBM SPSS Statistics 26 (IBM). All results were considered statistically significant at p < 0.05. Error bars are expressed as mean ± standard error (SEM) or median, and p values ​​are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0258] Experimental Results 1. Generation of htNSCs from hPSC-derived Hypothalamic Organoids

[0259] To provide a stable source of human hypothalamic neural and non-neural cells for cell therapeutics, a protocol for differentiating hPSCs into hypothalamic NSCs (htNSCs) was developed. hESCs (H9) were cultured in 3D and induced to differentiate into NSCs (htNSCs) by orbital oscillation in the presence of agents that induce patterning into neuroepithelial and hypothalamic regions. Figure 1). Based on the expression of RAX, NKX2-1, ILS1, which are known to be highly expressed in the hypothalamus, and Engrailed-1 (EN1), which is known to be lowly expressed, the concentration and treatment time of the hypothalamic patterning inducing substance were optimized ( Figure 2 ). Specifically, it was confirmed that when sonic edgehog (SHH) and purmophamine were treated simultaneously, the expression of hypothalamic neural stem cell markers NKX2.1 and RAX increased, while the expression of midbrain marker Engrailed-1 (EN1) decreased. In addition, treatment with other small molecules known to be associated with hypothalamic patterning (CHR, CNTF, IWP-2, XAV, FGF8, NOG) reduced the expression of hypothalamic markers while increasing the expression of midbrain marker EN1. Therefore, in the present invention, only SHH and purmophamine were used for hypothalamic patterning.

[0260] Organoids patterned under the above conditions showed gene expression similar to the hypothalamic differentiation pattern at day 20 of culture.

[0261] In particular, fibroblast growth factor (bFGF) increased htNSCs in early hypothalamic-like organoids. On day 20 (D20) of culture, cells were isolated from organoids and subcultured using N2 medium containing bFGF on fibronectin-coated plates. The results showed that the 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 ( Figure 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 those of the human fetal hypothalamus and had characteristics similar to those of the hypothalamus region ( Figure 5 ).

[0262] In addition, htNSCs can maintain cell growth even after multiple passages (about 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 cells (Rax, nestin). Figure 7 As shown, it was confirmed that most of the cells survived after thawing, and the expression level of hypothalamic neural stem cell marker proteins was similar to that of the cells before freezing. Based on the above results, it was confirmed that htNSCs can be cryopreserved and the characteristics of htNSCs can be maintained even after thawing.

[0263] Experimental Results 2. Differentiation Phenotypic Analysis of Organoid-Derived Human htNSCs

[0264] htNSCs were induced to differentiate into neural cells. The induced neural cells expressed neuronal markers (synaptophysin 1 (SYN1), TuJ1 (TUBB), MAP2, RBFOX3 (NeuN)), and a subset of cells expressing hypothalamus-specific neuropeptides (α-MSH, neuropeptide Y (NPY), secretagogue (SCGN), PMCH) were also observed ( Figure 8 ).

[0265] In particular, GFAP was observed in differentiated htNSC cultures. + Astrocytes were identified and the number of glial cells increased at the end of differentiation. In addition, the expression of leptin receptor and ghrelin receptor was observed in cells differentiated from htNSCs ( Figure 8 ) and confirmed that when cells were treated with leptin or ghrelin, phosphorylation of STAT3 or FOXO1, which are downstream signaling factors of each receptor, increased ( Fig. 9 ).

[0266] More specifically, to analyze the phenotype of cells differentiated from htNSCs, RNA sequencing (sc-RNA-Seq) analysis was performed on differentiation day 70. A total of 11,813 cells were sequenced using the Smart-seq2 method, and the read values ​​were filtered to remove duplicates and unreliable values, and a total of 6,394 transcripts were analyzed. First, the results of clustering using the Seurat algorithm were divided into 13 clusters, which were visualized by a uniform manifold approximation (UMAP) plot. Based on the expression levels of well-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 (NSC), intermediate progenitor cells (Int1 and Int2), astrocyte progenitor cells (APC), astrocytes (Ast1, Ast2, and Ast3), elongating cells (Tan), interneuron progenitor cells (Int1 and Int2), radial glial cells (RG), and proteoglycan-secreting glial cells ( Fig.10 ). At this point, confirm that each cluster expresses a specific gene ( Fig.11 and 12 ).

[0267] To reconfirm the above analysis results, Voxhunt was used to compare and analyze the mouse brain transcripts (14 days old) of the Allen Developmental Brain Atlas. The analysis results based on voxel spatial resolution confirmed that all cell clusters differentiated from htNSCs were precisely located in the hypothalamus of the 14-day-old mouse brain. In addition, it was confirmed that the progenitor / intermediate cell clusters (Int1, Int2, IPC, APC, and NSC) were concentrated in the hypothalamic area adjacent to the ventricular area where adult neuronal differentiation occurs ( Fig.13 ). Based on the above results, it was confirmed that the neural stem cells obtained by the method of the present invention have hypothalamus specificity.

[0268] In addition, similar to the differentiation of neurons and astrocytes from the same progenitor cells, UMAP clustering results confirmed that the intermediate progenitor cell clusters (Int1 and Int2) dually differentiated into neural cell clusters (Neu1 and Neu2) and astrocyte progenitor cell clusters (APC) ( Fig.14 ). The results of using Monocle3 to analyze the progenitor / intermediate cell clusters (Int1, Int2, IPC, APC, NSC) by pseudo-time sorting confirmed that neural stem cells differentiated in the order of intermediate cell clusters (Int1, Int2) and neurons (Neu1, Neu2). In addition, it was confirmed that as differentiation progressed, the expression of the cell proliferation marker CDK1 rapidly decreased in the early progenitor stage, the expression of the progenitor neuron gene ASCL1 increased in the late progenitor stage, and the expression of the neuron-specific genes TUBB3 and SNAP25 increased in the late neuron stage ( Fig.15 ).

[0269] SLIT-ROBO signaling has recently been reported to be an important regulator of hypothalamic-specific brain development (Romanov et al. 2020). In the cells induced to differentiate from htNSCs of the present invention, a SLIT-ROBO signaling gene expression pattern similar to that of hypothalamic brain development in vivo was confirmed ( Fig.16 Based on the above results, it was confirmed that the differentiation pattern of organoid-derived human htNSCs was similar to the in vivo hypothalamic development.

[0270] Next, gene regulatory network (GRN) analysis was performed using pySCENIC to find the top regulators (regulators) of cluster genes. The results confirmed that SOX4 / SMARCA4, STAT1 / 2, THRB / RAX were the top regulator genes (master regulators) in the neuronal cluster (Neu1), astrocyte cluster (Ast1), and elongated cell-like cell cluster (Tan) ( Fig.17 ).

[0271] To further subdivide the above neural cell clusters (Neu1 and Neu2), additional clustering was performed. First, the above neural cell clusters (Neu1 and Neu2) were re-clustered to distinguish each cell cluster based on the most expressed genes in the respective cell clusters ( Fig.18 ). As a result, 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 specifically expressed during hypothalamic development and is an essential gene for hypothalamic development (PMID: 18206388). SCGN neurons are hypothalamic-specific neurons that serve as gatekeepers for CRH-mediated stress responses ( Fig.19 In addition, transcripts, transcriptional regulators (regulators), and neurotransmitters (NTs) expressed in each neuronal cluster were identified ( Fig. 20 ).

[0272] Astrocyte progenitor cell clusters (APCs) were further clustered into three astrocyte clusters ( Figures 21 to 23 ).like Fig.23 As shown, it was confirmed that the Ast2 cluster is an astrocyte cluster involved in homeostasis, in which SLC1A3 (GLAST) is expressed, which can mainly play the function of supplying nutrients to nerve cells. In addition, based on the expression of genes such as SLCO1C1 (thyroid hormone transporter), CRYM (Crystallin mu, a thyroid hormone binding protein) and SCG2 (secretin granulin II, a neuroendocrine secretory protein), the Ast2 cluster was found to be associated with neuroendocrine function. In the Ast1 cluster, genes related to immune function such as RSAD2 and TRIL, as well as interferon-induced genes (ISG5, IFIT, STAT1) were specifically expressed. The Ast3 cluster differentiated from astrocyte progenitor cells (APC) expresses astrocyte-related transcription factors such as SOX9 and ID3, but does not express GFAP. In the Ast3 group, the expression of Meis2, which is associated with the quiescence or activation of NCS in the subventricular zone, was confirmed (PMID: 31304985). In addition, the Ast3 cluster expresses genes required for tight junction formation, such as CLDN5, ALCAM, and COL1A2 ( Fig.24 ). In a recently reported study, COL1A2 was demonstrated to be expressed by a subset of astrocytes in the SVZ (PMID: 34413515, PMID: 30625322). Therefore, based on the above results, we hypothesized that the Ast3 cluster would be a “type B” astrocyte that interacts with brain parenchymal cells and / or pericytes. In addition, since qNSCs have recently been reported to exist in the subventricular layer of the third ventricle of the hypothalamus, it is speculated that the Ast3 cluster is a qNSC.

[0273] So far, the developmental origin of the elongated cells has not been confirmed. However, similar to recent reports that elongated cells are derived from astrocytes (Romanov et al., 2020), the present invention confirms that the elongated cell-like cell (Tan) cluster is derived from the Ast2 cluster ( Fig.14 and 23 ).

[0274] In the case of the elongated cell (Tan) cluster, the expression of elongated cell-specific marker genes such as RAX, FGF10, COL25A1, CRYM, SCN7A, etc. was confirmed. In addition, since the expression of LEPR (leptin receptor) was confirmed, it can be expected that elongated cells can participate in metabolic homeostasis through leptin signaling.

[0275] The above single-cell RNA sequencing analysis confirmed that the neural cell differentiation method of organoid-derived human htNSCs is similar to the main hypothalamic cell population in vivo. In addition, it was confirmed that elongated cells and hypothalamic qNSCs can be generated by the differentiation method of the present invention.

[0276] Experimental Results 3. Confirmation of the Aging-Ameliorating Effect in Mice Transplanted with Organoid-Derived Human htNSCs

[0277] Previous studies have reported that when mouse-derived htNSCs were transplanted into aged mice, the rate of physiological changes associated with aging was reduced (Zhang et al., 2017). Therefore, to confirm the anti-aging effects of the human htNSCs derived from the hESC-generated hypothalamic-like organoids of the present invention, htNSCs were transplanted into 12-month-old aged mice ( Fig.24 ).

[0278] Human htNSCs were transplanted twice into the medial basal hypothalamus (MBH) region and followed up for 1 to 4 months. At this time, mice injected with the vehicle served as a control group. The results showed that one month after transplantation, cells expressing the human-specific marker hNCAM (hNCAM+) were observed in the mouse hypothalamus. Expression of the htNSC-specific marker protein RAX was observed in some hNCAM+ cells, and expression of RBFOX3 or human GFAP (hGFAP) protein was also observed ( Fig.25 ).

[0279] According to the results of the mouse behavioral experiment, 4 months after transplantation, the endurance (treadmill performance) of the human htNSC transplanted group ( Fig.26 ) was improved compared with the control group. In addition, at 1.5 and 4 months after transplantation, the muscle endurance (grasping) ( Fig. 27 ), motor coordination (rotarod) ( Fig.28) and activity (locomotor activity, open field test) ( Fig.29 ) were improved compared with the control group. In addition, the long-term and spatial memory and cognitive ability (Morris water maze test, Fig.30 and 31 )(Y-maze test, Fig.32 )(New object recognition test, Fig.33 ) were also improved compared with the control group.

[0280] The hypothalamus regulates neurotransmission through endocrine substances such as orexin and TRH and the sympathetic autonomic nervous system, thereby regulating systemic metabolism. It is reported that this regulatory function of the hypothalamus is impaired with aging (Waterson et al., 2015; Saper et al., 2014; Gonzalez-Garcia et al., 2020). Compared with the control group, the human htNSC transplantation group of the present invention inhibited systemic metabolism and tissue aging caused by aging. Fig.34 As shown in Figure 2, when the body weight of mice was observed 1.5 months (13.5 months old) and 4 months (16 months old) after transplantation of human htNSC, it was observed that the body weight of mice in the human htNSC transplantation group was significantly decreased compared with that of mice in the control group ( Fig.34 ).

[0281] In addition, fat % decreased and lean % increased significantly compared with the control group ( Fig.35 ). In contrast, there was no significant difference in food intake between the control group and the human htNSC transplantation group. Fig.36 As shown in the results of the metabolic cage experiment, the motor activity of the mice in the human htNSC transplantation group (1.5 months after transplantation (13.5 months old), at night) was significantly higher than that in the control group ( Fig.37 In addition, at 1.5 months (13.5 months of age) or 4 months (16 months of age) after transplantation, energy expenditure (EE) was significantly increased in the htNSC transplanted group compared with the control group, both during the night and during the day ( Fig.38 In addition, 4 months after human htNSC transplantation, glucose tolerance in the human htNSC transplantation group was significantly improved compared with the control group ( Fig.39 ).

[0282] It is known that with the progress of aging, not only the body fat increases, but also the amount and activity of brown adipose tissue (BAT) decrease. In order to confirm the effect of htNSCs of the present invention on the above-mentioned aging phenomenon, 4 months after transplantation of human htNSCs (16 months old), inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and interscapular brown adipose tissue (iBAT) were stained with H&E, and each tissue was observed.

[0283] As a result, the size of adipocytes in white adipose tissue and brown adipose tissue of the human htNSC transplantation group was significantly reduced compared with that of the control group ( Figures 40 to 42 In addition, although the accumulation of lipids in liver tissue increases with aging, the accumulation of lipids in liver tissue of the htNSC transplantation group was reduced compared with the control group ( Fig.43 ).

[0284] In white adipose tissue (iWAT and eWAT), the expression of mitochondrial homeostasis genes MTCO1 and SDHB, which are associated with browning, and FGF21, a major regulator of brown adipocyte differentiation and energy metabolism, were significantly increased ( Fig.44 and 45 In addition, the expression of FGF21 and GDF15, the phosphorylation of stress-induced transcription factor eIF2A, and the expression of ATF4 in the liver tissue of the transplant group were increased ( Fig.46 In addition, the expression of p16 and p21 proteins associated with cellular senescence was decreased in the kidneys of the htNSC transplantation group compared with the control group ( Fig.47 ).

[0285] It is known that the hypothalamus regulates not only systemic metabolism but also bone metabolism (remodeling and homeostasis) (Sharan et al., 2014). In the present invention, 4 months after htNSCs were transplanted into 12-month-old mice (16 months old), the femur was examined by X-ray, showing that osteoporosis was observed in the control group, while the bone mineral density (BMD), bone volume (BV) / tissue volume (TV) ratio, trabecular thickness (Tb.Th), and trabecular number (Tb.N) in the htNSC transplantation group were significantly increased compared with the control group ( Fig.48 ).

[0286] Experimental results 4. Confirmation of the anti-aging effect of htNSC-derived exosomes via intraventricular transplantation

[0287] Recent studies have reported that exosomes secreted by transplanted NSCs exhibit therapeutic effects (Zhang et al., 2017). Therefore, to confirm the anti-aging effects of human htNSC-derived exosomes, 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 ( Fig.49 As a result, similar to the results of the human htNSC transplantation experiment described above, injection of htNSC-derived exosomes showed an increase in motor coordination (rotarod) and muscle strength (grip) at 1.5 and 4 months after injection, respectively, compared with the control group ( Fig.50 and 51 In addition, it was confirmed that the injection of htNSC-derived exosomes [(passive avoidance test, Fig.52 ) and (Morris water maze test, Fig.53 The memory of mice treated with htNSCs was improved compared with that of the control group. Based on the above results, it was confirmed that human htNSC-derived exosomes can potentially be used as a method for preventing and treating aging.

Claims

1. A pharmaceutical composition for preventing and treating geriatric diseases, comprising hypothalamic-like organoid-derived neural stem cells as active ingredients.

2. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the hypothalamic-like organoid-derived neural stem cells 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 hypothalamic-like organoid-derived neural stem cells express neural stem cell markers and hypothalamic markers at 84% or more in the entire DAPI-positive cell population.

4. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 1, wherein the hypothalamic-like organoid-derived neural stem cells are capable of proliferating for 1 to 10 generations.

5. A pharmaceutical composition for the prevention and treatment of geriatric diseases according to claim 1, wherein the neural stem cells derived from the hypothalamic-like organoids differentiate into neurons, and the neurons express at least one neural cell marker selected from the group consisting of synaptophysin 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), secretagogue (SCGN), and PMCH.

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

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

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

9. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 7, wherein the astrocyte progenitor cell cluster is characterized in that it differentiates 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, IFITs 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.

10. The pharmaceutical composition for preventing and treating geriatric diseases according to claim 7, wherein the elongated cell 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 muscle aging-related diseases.

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, cortical basal 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 muscle aging-related disease is selected from the group consisting of muscle atrophy, muscle disuse atrophy, and senile sarcopenia.

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

15. The exosome according to claim 14, wherein the hypothalamic-like organoid-derived neural stem cells 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 according to claim 14 , wherein the hypothalamic-like organoid-derived neural stem cells express a neural stem cell marker and a hypothalamic marker at 84% or more in the entire DAPI-positive cell population.

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

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

19. The pharmaceutical composition according to claim 18, 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, cortical basal degeneration, diffuse Lewy body disease, and Pick's disease.

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

21. A method for producing exosomes, comprising the following steps: i) Cultivating hypothalamic-like organoid-derived neural stem cells; as well as ii) Collecting exosomes from the culture medium.

22. A kit for preventing and treating geriatric diseases, comprising the hypothalamic organoid-derived neural stem cells according to claim 1; and / or the exosomes according to claim 14.

23. The kit according to claim 22, wherein the hypothalamic-like organoid-derived neural stem cells 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 according to claim 22, wherein the hypothalamic-like organoid-derived neural stem cells express a neural stem cell marker and a hypothalamic marker at 84% or more in the entire DAPI-positive cell population.

25. The hypothalamic organoid-derived neural stem cell according to claim 1; or the exosome according to claim 14 for preventing or treating geriatric diseases.

26. A method for preventing or treating geriatric diseases, comprising administering to a subject the hypothalamic organoid-derived neural stem cells according to claim 1; or the exosomes according to claim 14.

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