Culture medium composition for neuromuscular organoids and method for producing neuromuscular organoids

Abstract

This invention relates to a culture medium composition for neuromuscular organoids and a method for producing neuromuscular organoids. The neuromuscular organoids of this invention, when cultured in a medium containing a composition with a sonic hedgehog signaling agonist as an active ingredient, not only is the process of differentiating nerve cells constituting the neuromuscular organoids into motor nerve cells accelerated, but the development and maturation of muscle fibers are also promoted. The interaction between motor nerve cells and muscle tissue through the neuromuscular junction is crucial to the pathogenesis and treatment of degenerative neuromuscular diseases. Therefore, the neuromuscular organoids of this invention can be used as a model of neuromuscular disease (specifically, a degenerative neuromuscular disease model). Consequently, it is expected to be applicable to mechanistic research and drug screening of nerve and muscle diseases.

Description

Technical Field

[0001] The present invention relates to a culture medium composition for culturing neuromuscular organoids, a method for producing neuromuscular organoids, and the like.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2023-0059675 filed on May 9, 2023 and Korean Patent Application No. 10-2024-0060292 filed on May 8, 2024, and all the contents disclosed in the specifications and drawings of the applications are incorporated herein by reference.

Background Art

[0003] Prior to conducting clinical trials in the process of new drug development, animal experiments on animals similar to humans are required. Animals such as mice and rabbits are used in animal experiments, but this raises ethical issues. In addition, animal experiment models have different genetic or biological characteristics from the human body, so existing drug tests have limitations in the reliability of drug test reactions, such as side effects not discovered in animal experiments being discovered in humans.

[0004] Various solutions have been studied to supplement such problems. However, the conventional 2D cell line culture method has the disadvantage of being unable to reproduce tissue-specific properties and characteristics. As an alternative, xenograft models derived from cancer patients are sometimes used, but they are not suitable for large-scale drug screening. Therefore, recently, research related to experimental methods using organoids has been actively conducted.

[0005] An organoid is an organoid formed by three-dimensionally culturing or recombining stem cells, which is derived from various internal organs, contains stem cells inside, and forms an aggregate that can differentiate into cell bodies. In addition, organoids derived from each internal organ in the body have a structure very similar to that of the organ and can be used as a model to replace cell and animal experiments.

[0006] On the other hand, organoid research is moving beyond basic research for development and is beginning to establish systems for its application in clinical research. However, compared to organoids such as the brain and gut, research on neuromuscular organoids, which are composed of two other tissues, is relatively insufficient (number of relevant papers published in the PubMed database over the past 10 years: brain organoids (1749), gut organoids (2295), neuromuscular organoids (66)). Furthermore, the Max Delbruck Center for Molecular Medicine in Germany induced the differentiation of human induced pluripotent stem cells into bidifferentiable neuromesodermal progenitors (NMPs) capable of differentiating into both nerve cells and skeletal muscle cells, and after forming aggregates, developed neuromuscular organoids composed of nerve tissue and skeletal muscle tissue through self-organization (Self-organizing 3D Human Trunk Neuromuscular Organoids, Cell Stem Cell, 2020). However, they confirmed that only about 6% of the nerve cells constituting the organoids differentiated into motor neurons.

[0007] Furthermore, motor neurons cause muscle tissue contraction through the neuromuscular junction, and the death of motor neurons is a typical symptom of degenerative neuromuscular disease. The interaction between motor neurons and muscle tissue through the neuromuscular junction is crucial to the pathogenesis and treatment of degenerative neuromuscular disease, and for a model of degenerative neuromuscular disease to be useful, both motor neurons and skeletal muscle tissue, as well as the neuromuscular junction between the two tissues, must be present.

[0008] Therefore, there is a need to optimize neuromuscular organoids composed of motor neurons so that they can be used for mechanistic research and drug screening of neuromuscular diseases (specifically, degenerative neuromuscular diseases). [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a culture medium composition for neuromuscular organoids for three-dimensional culture from pluripotent stem cells (PSCs) to neuromuscular organoids, comprising a sonic hedgehog (SHH) signaling activator as an active ingredient.

[0010] Another object of the present invention is to provide a method for producing neuromuscular organoids, comprising the following steps: (S0) The process involves culturing human pluripotent stem cells (hPSCs) derived from an individual in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate them into neuromesodermal progenitors (NMPs), and (S1) The step of diluting the bifacial mesoderm precursor and dispensing it to form an embryoid body, (S2) A step of culturing the embryo in a culture medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0011] Another object of the present invention is to provide a three-dimensional neuromuscular organoid manufactured by the manufacturing method described above.

[0012] A further object of the present invention is to provide a neuromuscular disease model characterized by including the neuromuscular organoids.

[0013] Another object of the present invention is to provide a method for screening neuromuscular disease treatment agents, comprising the following steps: (1) A step of treating neuromuscular organoids derived from individuals with neuromuscular disease produced by the above method with a candidate substance for the treatment of neuromuscular disease, (2) A screening method comprising the step of comparing the morphological characteristics of neuromuscular organoids treated with the candidate substance.

[0014] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and any other problems not mentioned will be clearly understood by those with ordinary skill in the art to which this invention belongs from the description below. [Means for solving the problem]

[0015] The present invention provides a culture medium composition for neuromuscular organoids for three-dimensional culture of pluripotent stem cells (PSCs) into neuromuscular organoids, comprising a sonic hedgehog (SHH) signaling activator as an active ingredient.

[0016] In one embodiment of the present invention, the sonic hedgehog signaling activator is one or more selected from the group consisting of sonic hedgehog signaling agonists and sonic hedgehog proteins, but is not limited thereto.

[0017] In other embodiments of the present invention, the sonic hedgehog signaling agent is one or more selected from the group consisting of purmorphamine and smoothed agonist (SAG).

[0018] In yet another embodiment of the present invention, the culture medium composition may further contain, but is not limited to, one or more selected from the group consisting of GSK-3 (Glycogen synthase kinase 3) inhibitors, FGF (Fibroblast Growth Factor), Y-27632, IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

[0019] In yet another embodiment of the present invention, the neuromuscular organoid may include, but is not limited to, one or more selected from the group consisting of nerve tissue, skeletal muscle tissue, and neuromuscular junctions.

[0020] In yet another embodiment of the present invention, the pluripotent stem cell (PSC) is also, but is not limited to, a human induced pluripotent stem cell (hiPSC).

[0021] The present invention provides a method for producing neuromuscular organoids, comprising the following steps: (S0) The stage in which human pluripotent stem cells (hPSCs) derived from an individual are cultured in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate into bipolar neuromesodermal progenitors (NMPs); (S1) Diluting the bifacial mesoderm precursor and dispensing it in order to form an embryoid body; (S2) A step of culturing the embryo in a culture medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0022] In one embodiment of the present invention, at step (S0), the human pluripotent stem cells are in a state in which colonies have formed, but are not limited to that state.

[0023] In other embodiments of the present invention, the bipolarizing neuronal mesoderm precursor may, but is not limited to, express SOX2 (SRY sex-determining region Y-box 2) or BRA (BRACHYURY) in step (S0).

[0024] In still other embodiments of the present invention, in the step (S1), the culture medium composition for diluting the bipotent neural mesoderm precursor may include, but is not limited to, any one or more selected from the group consisting of Y-27632, FGF (Fibroblast Growth Factor), IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

[0025] In still other embodiments of the present invention, the step (S1) is characterized by any one or more selected from the group consisting of, but not limited to: (a) being performed before the bipotent neural mesoderm precursor forms a germ body; and (b) dispensing 1500 to 9000 bipotent neural mesoderm precursors.

[0026] In still other embodiments of the present invention, in the step (S2), the culturing is also an orbital shaking culture, but is not limited thereto.

[0027] The present invention provides a three-dimensional neuromuscular organoid produced by the above production method.

[0028] In one embodiment of the present invention, the organoid can express any one or more selected from the group consisting of, but not limited to, TUJ1 (neuron-specific class III beta-tubulin), fast MHC (fast myosin heavy chain), SOX2 (SRY sex-determining region Y-box 2), SHH (Sonic Hedgehog), α-BTX (α-bungarotoxin), MYH2 (Myosin Heavy Chain 2), and MYH7 (Myosin Heavy Chain 7).

[0029] In yet another embodiment of the present invention, the neuromuscular organoid is characterized by one or more selected from the group consisting of: (a) Decreased expression of Cdo and Tubb3; and (b) Expression of Olig2 and ChAT (choline acetyltransferase) increases.

[0030] In yet another embodiment of the present invention, the neuromuscular organoid may feature one or more selected from the group consisting of: (a) Promote differentiation in motor neurons; (b) Increase in the size of muscle fibers; and (c) The muscle fibers are mature.

[0031] In yet another embodiment of the present invention, the neuromuscular organoid may be characterized by synchronous contraction, but is not limited thereto.

[0032] The present invention provides a neuromuscular disease model characterized by including the aforementioned neuromuscular organoids.

[0033] In one embodiment of the present invention, the neuromuscular disease is a degenerative neuromuscular disease, but is not limited to it.

[0034] In other embodiments of the present invention, the degenerative neuromuscular disease may be selected from, but is not limited to, the group consisting of spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, and mytonic dystrophy.

[0035] The present invention provides a method for screening neuromuscular disease treatment agents, comprising the following steps: (1) A step of treating neuromuscular organoids derived from individuals with neuromuscular diseases produced by the above manufacturing method with a candidate substance for the treatment of neuromuscular diseases; and (2) A screening method comprising the step of comparing the morphological characteristics of neuromuscular organoids treated with the candidate substance.

[0036] In one embodiment of the present invention, the screening method may further include, but is not limited to, the step of determining that the candidate substance is a therapeutic agent for neuromuscular diseases if the morphological characteristics of the neuromuscular organoid treated with the candidate substance exhibit the morphological characteristics of normal neuromuscular tissue.

[0037] Furthermore, the present invention provides a culture medium composition containing a sonic hedgehog (SHH) signaling activator as an active ingredient for use in three-dimensional culture of pluripotent stem cells (PSCs) to neuromuscular organoids.

[0038] Furthermore, the present invention provides a composition containing a sonic hedgehog (SHH) signaling activator as an active ingredient for the production of neuromuscular organoids from pluripotent stem cells (PSCs).

[0039] Furthermore, the present invention provides a screening application for the three-dimensional neuromuscular organoid of the present invention for the treatment of neuromuscular diseases. [Effects of the Invention]

[0040] The neuromuscular organoids of the present invention, when cultured in a culture medium containing a composition with a sonic hedgehog signaling agonist as an active ingredient, undergo accelerated differentiation of nerve cells constituting the neuromuscular organoids into motor nerve cells, and also promote the development and maturation of muscle fibers. Since the interaction between motor nerve cells and muscle tissue via the neuromuscular junction is crucial to the pathogenesis and treatment of degenerative neuromuscular diseases, the neuromuscular organoids of the present invention can be used as a model of neuromuscular disease (specifically, a model of degenerative neuromuscular disease). Therefore, it is expected to be applicable to mechanistic research and drug screening of nerve and muscle diseases. [Brief explanation of the drawing]

[0041] [Figure 1] This is a diagram illustrating the neuromuscular organoid manufacturing protocol. [Figure 2] This diagram illustrates the changes in neuromuscular organoids caused by treatment with a sonic hedgehog signaling agonist. [Figure 3A] This diagram illustrates a typical image of germ formation induction by a pretreatment method, showing failed aggregation (left), successful germ formation (center), and the ratio of successful germ formation using the pretreatment method (right). [Figure 3B] This diagram illustrates a typical image of neuromuscular morphogenesis induction by elongation, showing the ratio of failed morphogenesis (left), successful neuromuscular morphogenesis by elongation (center), and successful morphogenesis by pretreatment method (right). [Figure 4] This diagram shows the successful formation of neuromuscular organoids by confirming their morphogenesis over time. [Figure 5]This diagram shows that human pluripotent stem cells are differentiated into bipolar neuronal mesoderm precursors (NMPs) through a pretreatment method, and that this was confirmed by the expression of NMPs markers SOX2 and BRA (BRACHYURY). The diagram also shows that the induction of NMPs through the pretreatment method can be confirmed by the simultaneous expression of SOX2 and BRA via immunofluorescence starting 24 hours after pretreatment. [Figure 6] This diagram shows immunofluorescence results for neural stem cells (SOX2) and neuronal cells (neuron-specific class III beta-tubulin, TUJ1) markers in neuromuscular organoids at various time points. It confirms that on day 4 of organoid formation, localized expression of SOX2 in the area where nerve tissue is formed indicates the formation of the neuroectoderm (left), and from day 7 of organoid formation onwards, differentiation from neural stem cells into neuronal cells is confirmed through TUJ1 expression (center). [Figure 7A] This diagram shows the immunofluorescence results of neuronal and skeletal muscle markers in successful neuromuscular organoids (day 20). The left figure shows neuromuscular organoids expressing the neuronal marker (TUJ1) and the embryonic myosin heavy chain isoform marker (eMHC), while the right figure shows a higher magnification image of the region indicated by the dotted rectangle in the left figure. [Figure 7B] The image shows the immunofluorescence results of neuronal cell markers and skeletal muscle markers in successful neuromuscular organoids (day 40), demonstrating that neuromuscular organoids were successfully formed by the manufacturing method of the present invention. Specifically, the middle figure shows neuromuscular organoids expressing the neuronal cell marker (TUJ1) and the mature skeletal muscle marker (fast myosin heavy chain, Fast MHC). The right figure shows a higher magnification image of the skeletal muscle portion of the neuromuscular organoid, illustrating that exons extended from the nerve tissue into the skeletal muscle tissue to form a neuromuscular junction. [Figure 7C]This diagram shows the immunofluorescence results of neuronal and skeletal muscle markers in successful neuromuscular organoids (day 60). The left figure shows neuromuscular organoids expressing the neuronal marker (TUJ1) and the mature skeletal muscle marker (Fast MHC), while the right figure shows a higher magnification image of the region indicated by the dotted rectangle in the left figure. [Figure 8] This figure shows that sonic hedgehog signaling (SHH signaling) was activated in neuromuscular organoids via treatment with purmorphamine, as a result of qRT-PCR. (Expression values ​​were normalized to L32 levels and further normalized to control organoid values. Error bars indicate standard deviation. Data were analyzed by Tukey's multiple comparison test following one-way ANOVA (****P<0.0001).) [Figure 9] This figure shows that motor neuron differentiation was induced through treatment with purmorphamine, based on the expression levels of motor neuron markers. It also displays the results of qRT-PCR analysis of the motor neuron progenitor cell marker (OLIG2), motor neuron cell marker (ChAT), and general neuron marker (TUBB2). (Expression values ​​were normalized to L32 levels and further normalized to control organoid values. Error bars indicate standard deviation. Data were analyzed using Tukey's multiple comparison test following one-way ANOVA (***P<0.001, ****P<0.0001).) [Figure 10] This figure shows the frequency of neuromuscular junction formation induced by purmorphamine treatment, confirmed via α-bungarotoxin (α-BTX). [Figure 11] This diagram shows the morphological differences of organoids after adding purmorphamine to the culture medium from day 4 of neuromuscular organoid formation, illustrating that the size of organoids generally increases with longer treatment periods, particularly in muscle tissue areas. [Figure 12A]This figure quantifies the size of neuromuscular organoids treated with a sonic hedgehog signaling agent (purmorphamine, SAG). It shows that neuromuscular organoid size increases when treated with the sonic hedgehog signaling agent compared to the control group. (Neuromuscular organoid size was measured by the area of ​​a bright-field image 20 days after purmorphamine treatment. Error bars indicate the standard deviation. Data were analyzed using Tukey's multiple comparison test following one-way ANOVA (****P<0.0001).) [Figure 12B] This figure quantifies the size of neuromuscular organoids treated with a sonic hedgehog signaling agent (purmorphamine, SAG). It shows that neuromuscular organoid size increases when treated with the sonic hedgehog signaling agent compared to the control group. (Neuromuscular organoid size was measured by the area of ​​a bright-field image 20 days after purmorphamine treatment. Error bars indicate the standard deviation. Data were analyzed using Tukey's multiple comparison test following one-way ANOVA (****P<0.0001).) [Figure 13] This figure shows that treatment with purmorphamine increases the expression of mature muscle fiber markers as a result of qRT-PCR. (Expression values ​​were normalized to L32 levels and further normalized to control organoid values; error bars indicate standard deviation; data were analyzed by Tukey's multiple comparison test following one-way ANOVA (*P<0.05, ***P<0.001, ****P<0.0001).) [Figure 14] This figure shows that Fast MHC, which is specifically expressed in mature muscle fibers, is expressed earlier in the group compared to the control group. [Figure 15] This diagram shows that, due to the promotion of muscle tissue maturation on day 14 of neuromuscular organoid formation, synchronous muscle tissue contraction induced by glutamate treatment is observed earlier compared to the control group. [Figure 16]This figure shows that, compared to the spontaneous contractions observed with conventional neuromuscular organoids, the neuromuscular organoid according to the present invention exhibits synchronized contractions of the entire skeletal muscle tissue that are remarkably similar to the contractions of actual muscles in the human body. [Figure 17] This diagram demonstrates that synchronized contraction of the entire skeletal muscle tissue, which has not been observed in conventional neuromuscular organoids, is observed in the neuromuscular organoid according to the present invention. The diagram shows the results of defining the region of interest in a video of the neuromuscular organoid on day 50, and quantifying the area displacement over time. Specifically, the diagram shows the results of comparing the contraction patterns of three different types of neuromuscular organoids. [Figure 18] This diagram shows a comparison of germinal body size based on the number of starting cells. When the number of starting cells exceeds 4,500 (top 3 cells), the inside of the germinal body is excessively large, and empty space is formed inside. When the number of starting cells is excessively low (500 cells, bottom right), the NMPs do not form a germinal body. The diagram shows that starting cell counts between 3,000 (bottom left) and 1,500 (bottom center) resulted in the formation of well-aggregated germinal bodies. [Figure 19A] Immunofluorescence results demonstrating the cellular typology diversity of neuromuscular organoids are shown. Figure 19A shows acetylcholine receptor clusters labeled with α-bungarotoxin (αBTX) using fast MHC (left) and neuronal filaments + synaptophysin (NF+SV, right). [Figure 19B] The immunofluorescence results showing the cellular typology diversity of neuromuscular organoids are shown. Figure 19B shows the satellite cell marker (PAX7) labeled with LAMININ. [Figure 19C] The image shows immunofluorescence results illustrating the cellular typology diversity of neuromuscular organoids. Figure 19C shows motor neuron markers (ChAT) representing a subset of neurons (TUJ1), with the right panel showing a higher magnification image of the region in the left panel, indicated by the dotted square. [Figure 20A]This figure shows the results of synchronous contraction of skeletal muscle formed from neuromuscular organoids. Synchronized contraction of skeletal muscle formed from neuromuscular organoids on day 50 was tracked via live video, and the contraction rate is shown as the average (Figure 20A). Changes in the synchronous contraction rate were also recorded as the organoids were sequentially treated with glutamate and curare, and the error bars are plots showing the standard deviation (n=5). [Figure 20B] This figure shows the results of synchronous contraction of skeletal muscle formed from neuromuscular organoids. Synchronized contraction of skeletal muscle formed from neuromuscular organoids on day 50 was tracked via live video, and the contraction rate is shown individually (Figure 20B). The organoids were sequentially treated with glutamate and curare, and the changes in the synchronous contraction rate were also recorded. The error bars are a diagram showing the standard deviation (n=5). [Figure 21A] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of myosin heavy chain isoforms. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21B] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of myosin heavy chain isoforms. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21C]This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of myosin heavy chain isoforms. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21D] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of neural markers. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21E] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of neural markers. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21F] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of neuromuscular junction-related genes. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 21G] This figure shows the results of a comparison between synchronously contractile and non-contractile neuromuscular organoids. It also shows the results of qRT-PCR analysis of neuromuscular junction-related genes. (Expression values ​​were normalized to L32 levels and further normalized to non-contractile organoid values. Error bars indicate standard deviation, and statistical significance was calculated using Student's t-test (pairs, two-sided) (*P<0.05, **P<0.01, ***P<0.001).) [Figure 22] This diagram shows a schematic representation of how neuromuscular organoids can be used in customized medical treatment. [Figure 23A] This diagram shows typical morphologies of neuromuscular organoids generated from hiPSCs derived from ALS patients. Specifically, it shows typical morphologies of neuromuscular organoids generated from wild-type cells. [Figure 23B] This shows typical morphologies of neuromuscular organoids generated from hiPSCs derived from ALS patients. Specifically, it is a diagram showing typical morphologies of neuromuscular organoids generated from hiPSCs derived from ALS patients. [Modes for carrying out the invention]

[0042] The inventors of this invention have completed the invention by developing neuromuscular organoids through three-dimensional culture of human pluripotent stem cells.

[0043] The present invention provides a culture medium composition for neuromuscular organoids for three-dimensional culture of pluripotent stem cells (PSCs) into neuromuscular organoids, comprising a sonic hedgehog (SHH) signaling activator as an active ingredient.

[0044] In this invention, "for cultivation" means "can be used in any cultivation process," and may include, for example, "for use in cultivation" and "useful for cultivation." Therefore, "for cultivation," "for use in cultivation," and "useful for cultivation" are interchangeable but not limited to each other.

[0045] In the present invention, Sonic hedgehog (SHH) is expressed from the notochord, which is temporarily present beneath the spinal cord before vertebral formation during development. Neural stem cells in the spinal cord located ventrally near the notochord can, but are not limited to, differentiate into floor plate and motor neurons through a concentration gradient of Sonic hedgehog.

[0046] In this invention, "Sonic hedgehog (SHH)" refers to a protein that is one of three or more proteins in the mammalian signaling pathway family called hedgehog, the others being DHH (desert hedgehog) and IHH (Indian hedgehog), but not limited to these. SHH interacts with two or more transmembrane proteins through interactions with transmembrane molecules (Patched, PTC) and SMO (Smoothened). SHH typically binds to PTC and then allows activation of SMO as a signaling transducer. In the absence of SHH, PTC typically represses SMO, which can, but not limited to, reactivate transcription repressors to prevent transcription of specific genes. Furthermore, if SHH is present and binds to PTC, PTC cannot interfere with the function of SMO. If SMO is not suppressed, a specific protein can enter the nucleus and act as a transcription factor that activates a specific gene, but it is not limited to this.

[0047] In the present invention, sonic hedgehog signaling (SHH signaling) may be involved in the differentiation of neural stem cells into motor neurons, such as lower motor neurons (LMNs). Here, lower motor neurons are those that form the neuromuscular junction between skeletal muscle and are formed on the ventral side of the spine, but are not limited to these.

[0048] In the present invention, "Sonic hedgehog (SHH) signal propagation activator" includes, but is not limited to, any molecule or compound that binds to PTC or a smoothed activator, etc., and activates the SHH signal propagation pathway.

[0049] In the present invention, "signal" refers to internal and external factors that regulate changes in cellular structure and function, and may include, but is not limited to, chemical or physical factors.

[0050] In the present invention, the terms "activator" or "activator" may also refer to, but are not limited to, any molecule, such as a small molecule, peptide, protein, or compound, for activating the molecule that causes the specified differentiation of cells in the present invention.

[0051] In the present invention, "active ingredient" means an ingredient that exhibits the desired activity on its own, or an ingredient that can exhibit the desired activity together with a carrier or the like that is inactive on its own.

[0052] In the present invention, "organoid" refers to a three-dimensional cell aggregate formed through autoregeneration and autoorganization from pluripotent stem cells (PSCs), adult stem cells (ASCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), etc., and may include organoids or cell clusters formed from suspension cell cultures. The organoid may also be named a miniature organ-like body, organ-like body, or organ-like body. Specifically, the organoid contains one or more cell types from the diverse types of cells that constitute an organ or tissue, and can reproduce the morphology and function of a tissue or organ. Furthermore, by re-aggregating and recombining cells using a three-dimensional culture method to make them similar to the biological environment, it is possible to overcome the limitations of 2D cell lines cultured using a 2D culture method and reproduce the physiological activity and function of living organisms in a similar manner, making it applicable to, but not limited to, disease modeling and drug screening.

[0053] In the present invention, "stem cells" may mean undifferentiated cells that have the ability to self-renew and differentiate and proliferate. Stem cells may include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability. Pluripotent stem cells may mean cells that have the ability to differentiate into all tissues and cells that make up the living body. Multipotent stem cells mean cells that have the ability to differentiate into multiple types of tissues and cells, though not all types. Unipotent stem cells mean cells that have the ability to differentiate into a specific tissue or cell. Examples of pluripotent stem cells include germinal stem cells (ES cells), undifferentiated gonadal cells (EG cells), and reverse-differentiated stem cells (induced pluripotent stem cells, iPS cells). Examples of multipotent stem cells include, but are not limited to, mesenchymal stem cells (derived from adipose tissue, bone marrow, umbilical cord blood, etc.), hematopoietic stem cells (derived from bone marrow or peripheral blood, etc.), neural stem cells, germline stem cells, and other adult stem cells.

[0054] In the present invention, "pluripotent stem cell (PSC)" may be used interchangeably with, but is not limited to, "universal stem cell."

[0055] In the present invention, "pluripotency" or "pluripotent" may refer to, but is not limited to, stem cells that have the potential to differentiate into any one of the three germ layers: endoderm (e.g., the inner layer of the gastrointestinal tract, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, genitourinary tract), or ectoderm (e.g., epidermal tissue and nervous system).

[0056] In the present invention, "induced pluripotent stem cells (iPSCs)" are cells induced by artificially performing a dedifferentiation process (reprogramming) on ​​already differentiated adult cells, and may possess pluripotency. The induced pluripotent stem cells may differentiate into various organ cells such as brain and heart cells, but are not limited to these. Furthermore, in the present invention, "induced pluripotent stem cells" may also be induced pluripotent stem cells derived from fibroblasts, peripheral blood mononuclear cells, skin keratinocytes, nerve cells, or umbilical cord blood, but are not limited to these. According to one embodiment of the present invention, the induced pluripotent stem cells may also be induced pluripotent stem cells derived from fibroblasts, but are not limited to these.

[0057] In the present invention, "somatic cell" is a term opposite to germ cell and refers to a cell derived from a living adult. According to one embodiment of the present invention, the somatic cell is a fibroblast, peripheral blood mononuclear cell, skin keratinocyte, nerve cell, or umbilical cord blood. In the present invention, the somatic cell may be derived from, but is not limited to, a fibroblast, peripheral blood mononuclear cell, skin keratinocyte, nerve cell, or umbilical cord blood.

[0058] In this invention, "neuromuscular organoid" refers to a tissue formed by the co-development of nerve tissue, skeletal muscle tissue, and neuromuscular junctions from the same progenitor cells, but is not limited to these. Furthermore, in this invention, "neuromuscular junction" refers to a synaptic connection between the terminal of a motor nerve and muscle (e.g., skeletal, smooth, or cardiac), but is not limited to these.

[0059] In the present invention, "neuromuscular organoids" are different from, but not limited to, conventional neuromuscular organoids that lack a notochord expressing sonic hedgehog (SHH) in the embryo and have insufficient sonic hedgehog signaling (SHH signaling), which is essential for differentiation into motor neurons.

[0060] In the present invention, "neuromuscular organoids" can be used, but are not limited to, for studying the mechanisms of neurological and muscular diseases and for drug screening models by reproducing nerve tissue, muscle tissue, and the junctions between the two tissues in a test tube from human-derived cells.

[0061] In the present invention, "neuromuscular organoids" are motor nerve cells that have been successfully differentiated and can be used as models for research on neuromuscular diseases (e.g., degenerative neuromuscular diseases) and for the development of therapeutic agents, but are not limited to that use.

[0062] In the present invention, the "neuromuscular organoid" is one that undergoes synchronous contraction not observed in conventional neuromuscular organoids when applying common technical knowledge, and this synchronous contraction is remarkably similar to the contraction of skeletal muscle tissue in the human body, but is not limited to that.

[0063] In the present invention, "synchronous contraction" is interchangeable with, but not limited to, synchronized contraction and synchronous contraction.

[0064] In the present invention, synchronous contraction, unlike skeletal muscle spasms, also refers to, but is not limited to, a larger contraction of the entire skeletal muscle of the neuromuscular organoid. In the present invention, "synchronous contraction" is caused by the proliferation and maturation of skeletal muscle cells, but is not limited to that.

[0065] In the present invention, "neuromuscular disease" refers to a wide range of diseases involving damage or dysfunction of peripheral nerves and muscles. Specifically, the damaged sites may be, but are not limited to, cell bodies, lateral columns, Schwann cells, neuromuscular junctions, muscles, or combinations thereof.

[0066] In the present invention, "neuromuscular disease" also refers to degenerative neuromuscular diseases. Specifically, in the present invention, degenerative neuromuscular diseases include, but are not limited to, spinal muscular atrophy (SMA), Lou Gehrig's disease (Amyotrophic Lateral Sclerosis, ALS), Duchenne muscular dystrophy, and mytonic dystrophy.

[0067] In the present invention, "cultivation" can mean, but is not limited to, all actions performed to grow cells under appropriately artificially controlled environmental conditions.

[0068] According to one embodiment of the present invention, the culture may include, but is not limited to, a two-dimensional culture or a three-dimensional culture.

[0069] In the present invention, the sonic hedgehog signaling activator is one or more selected from the group consisting of sonic hedgehog signaling agonists and sonic hedgehog proteins, but is not limited thereto.

[0070] In the present invention, the sonic hedgehog signaling agent is one or more selected from the group consisting of purmorphamine and smoothed agonist (SAG), but is not limited thereto.

[0071] In the present invention, "purmorphamine" refers to a purine derivative, for example, CAS number 483367-10-8, which activates the Hedgehog pathway, including targeting Smoothened, and is also the compound shown in [Chemical Formula 1] below, but is not limited thereto.

[0072] [ka]

[0073] In a specific embodiment of the present invention, purmorphamine is Stemolecule TM This may also include purmorphamine (Stemgent, Inc. Cambridge, Massachusetts, United States), and other substances that are directly manufactured or commercially distributed may be obtained and used, but are not limited to these.

[0074] The purmorphamine of the present invention is a sonic hedgehog signaling agent that promotes the differentiation of neural stem cells into motor neurons, and the purmorphamine of the present invention may, but is not limited to, activate sonic hedgehog signaling (SHH signaling) during the formation of neuromuscular organoids.

[0075] The "smoothed agonist (SAG)" of the present invention is a chlorobenzothiophene-containing compound that acts as an activator of the G protein-binding receptor Smoothed (SMO), for example, one with CAS number 364590-63-6, or other compounds that can be obtained and used by direct manufacture or commercially available, including, but not limited to, the compound shown in [Chemical Formula 2] below.

[0076] [ka]

[0077] In the present invention, the culture medium composition may further contain, but is not limited to, one or more selected from the group consisting of GSK-3 (Glycogen synthase kinase 3) inhibitor, FGF (Fibroblast Growth Factor), Y-27632, IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

[0078] In the present invention, "GSK-3 (Glycogen synthase kinase 3) inhibitor" is an inhibitor of serine / threonine protein kinase that mediates the addition of phosphate molecules to serine and threonine amino acid residues, but is not limited to that. Furthermore, in the present invention, "GSK-3 (Glycogen synthase kinase 3) inhibitor" may include, for example, CHIR99021, but is not limited to that.

[0079] In the present invention, "CHIR99021" is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, and is a substance that targets GSK1 / 2, an upstream molecule of GSK1 / 2 involved in the GSK signaling process, as a GSK (glycogen synthase kinase) inhibitor, but is not limited to this. CHIR99021 is used at concentrations of 2 to 4 μM or 2.5 to 3.5 μM, and more specifically, CHIR99021 may be used at a concentration of 3 μM, but is not limited to this.

[0080] In the present invention, "FGF (Fibroblast Growth Factor)" is a secreted molecule that plays a role in stem cell proliferation, differentiation, and post-injury recovery, but is not limited to that. Furthermore, in the present invention, "FGF (Fibroblast Growth Factor)" may include, for example, bFGF (Basic Fibroblast Growth Factor), but is not limited to that.

[0081] In the present invention, "bFGF (basic fibroblast growth factor)" is a basic fibroblast growth factor that promotes the proliferation or induces differentiation of various cells and exhibits activity by binding to basic fibroblast growth factor receptors on the cell surface, but is not limited to that.

[0082] In this invention, bFGF (basic fibroblast growth factor) is a factor involved in angiogenesis, wound healing, embryonic development, and various endocrine signaling pathways, and can, but is not limited to, playing an important role in the proliferation and differentiation processes of various cells and tissues. bFGF can be used at concentrations of 7-12 ng / mL or 8-11 ng / mL. More specifically, bFGF can, but is not limited to, a concentration of 10 ng / mL.

[0083] In the present invention, Y-27632 is used at a concentration of 40-60 μM or 45-55 μM, and more specifically, at a concentration of 50 μM, but is not limited thereto.

[0084] In this invention, "IGF (insulin-like growth factor)" is a factor mainly secreted in the liver in response to stimulation by growth hormone (GH). Most cells in the human body (especially cells of muscle, bone, liver, kidney, nerve, skin, and lungs) are affected by IGF1 and may, but are not limited to, functions that regulate cell growth (especially nerve cells) and development, as well as cellular DNA synthesis, in addition to insulin-like effects. Furthermore, in this invention, "IGF (insulin-like growth factor)" is also IGF-1 (insulin-like growth factor-1), but is not limited to that. IGF-1 may be used at concentrations of 1-3 ng / mL or 1.5-2.5 ng / mL, and more specifically, at a concentration of 2 ng / mL, but is not limited to that.

[0085] In the present invention, HGF (hepatocyte growth factor) is a lateral secretory cell growth, motility, and morphogenetic factor, secreted by mesenchymal cells and acting mainly on epithelial and endothelial cells, but also acting on hematopoietic progenitor cells and T cells, although it is not limited to these. HGF is used at a concentration of 1-3 ng / mL or 1.5-2.5 ng / mL, and more specifically, it may be used at a concentration of 2 ng / mL, but it is not limited to this.

[0086] In this invention, "culture medium" means a medium that enables the proliferation, survival, and differentiation of neuromuscular organoids in vitro. This includes, but is not limited to, any common culture medium suitable for the culture and differentiation of neuromuscular organoids used in this field. Furthermore, the type of culture medium and culture conditions may be appropriately selected depending on the type of cell, but is not limited to that.

[0087] In the present invention, one or more substances selected from the group consisting of GSK-3 (Glycogen synthase kinase 3) inhibitors, FGF (Fibroblast Growth Factor), Y-27632, IGF (insulin-like growth factor), and HGF (hepatocyte growth factor) may be added to the basic culture medium to produce an effect, but are not limited to this.

[0088] In this invention, the basic culture medium may be any basic culture medium commonly used in the industry, but is not limited thereto.

[0089] In one embodiment of the present invention, a stabilized feeder-free maintenance medium for human pluripotent stem cells (hPSCs) may be used as the basic medium, but is not limited thereto. Specifically, it may be mTeSR1 Plus medium or N2B27 medium, but is not limited thereto.

[0090] In the present invention, mTeSR1 Plus medium (also known as catalog number 100-1130 or 100-0276, but not limited thereto) is also known as mTeSR1 Plus medium (Stem Cell Technologies), which maintains cell quality characteristics and enhances cell proliferation rates through stabilized key medium components including FGF2 and enhanced pH buffering capabilities.

[0091] In the present invention, N2B27 medium is a mixture of DMEM / F12 supplemented with 1xN2 and neurobasal medium supplemented with 1xB27, 1xGlutamax, 0.1 mM β-mercaptoethanol (Simga), and 1xPen-Strep antibiotic, but is not limited to this.

[0092] In the present invention, the N2B27 medium is a mixture of DMEM / F12 supplemented with 1xN2 and neurobasal medium supplemented with 1x B27, 1x Glutamax, 0.1 mM β-mercaptoethanol (Simga), and 1x Pen-Strep antibiotic in a ratio of 1:10-10:1, 2:10-10:2, 3:10-10:3, 4:10-10:4, 6:10-10:6, 8:10-10:8, 9:10-10:9, or 1:1.

[0093] In this invention, 1xN2 (which may also be catalog number 17502048 or 17502001, but is not limited thereto) may also be 1xN2 (Gibco), but is not limited thereto.

[0094] In the present invention, DMEM / F12 (also known as catalog number 11320033 or 11320082, but not limited thereto) is also known as DMEM / F12 (Gibco), and DMEM / F12 is a basic culture medium used to support the growth of mammalian cells. Cells successfully cultured in DMEM / F12 may include, but are not limited to, MDCK, glial cells, fibroblasts, human endothelial cells, and mouse fibroblasts.

[0095] In this invention, 1xB27 (which may also be catalog number 17504044 or 17504001, but is not limited thereto) may also be 1xB27 (Gibco), but is not limited thereto.

[0096] In this invention, 1xGlutamax (which may also be catalog number 35050061 or 35050079, but is not limited thereto) may also be 1xGlutamax (Gibco), but is not limited thereto.

[0097] In the present invention, the 1xPen-Strep antibiotic (which may also be, but is not limited to, catalog number LS202-01 or LS202-02) may also be, but is not limited to, the 1xPen-Strep antibiotic (Welgene).

[0098] In the present invention, Neurobasal medium (also catalog number 21103049, but not limited thereto) is also Neurobasal medium (Gibco), and is a basic medium designed for the long-term retention and maturation of pure prenatal and germinal neuronal populations without the need for an astrocyte trophoblast when used with Gibco B-27 supplement, but not limited thereto.

[0099] In one embodiment of the present invention, the "culture medium composition" is a culture medium composition, but is not limited to that.

[0100] In one embodiment of the present invention, the culture medium composition may be processed individually using a combination of one or more components, but is not limited thereto. For example, the culture medium composition of the present invention may be divided into three or more stages and independently named as a first to third composition.

[0101] In one embodiment of the present invention, the first composition is a culture medium composition for differentiating individual-derived pluripotent stem cells into NMPs, and may contain CHIR99021 and bFGF, but is not limited thereto. In the present invention, the step of differentiating individual-derived pluripotent stem cells into NMPs using the first composition can be called the "pretreatment" step, and therefore, the first composition of the present invention can be used interchangeably with the "pretreatment composition," but is not limited thereto.

[0102] Furthermore, the second composition is also a culture medium composition for further diluting the separated NMP to prevent intercellular bonding, and may, but is not limited to, Y-27632, bFGF, IGF-1, and HGF. The second composition of the present invention may, but is not limited to, be used before the NMP pretreated with the first composition forms embryos.

[0103] Finally, the third composition is also a culture medium composition for culturing germ cells in neuromuscular organoids, and contains an SHH signaling agent, further comprising one or more of the five components (CHIR99021, bFGF, Y-27632, IGF-1, and HGF), and the further components may be IGF-1 and HGF, but are not limited to these.

[0104] In one embodiment of the present invention, the culture medium composition includes the five components (CHIR99021, bFGF, Y-27632, IGF-1, and HGF), each component of which may, but is not limited to, be included in and processed independently in one or more steps.

[0105] In the present invention, neuromuscular organoids may include, but are not limited to, one or more selected from the group consisting of nerve tissue, skeletal muscle tissue, and neuromuscular junctions.

[0106] In one embodiment of the present invention, it was confirmed that when neuromuscular organoids were produced using the culture medium composition of the present invention, neuromuscular organoids containing nerve tissue, skeletal muscle tissue, and neuromuscular junctions were produced. According to common knowledge in the art, it is obvious that there is considerable difficulty in producing neuromuscular organoids containing all of these neuromuscular components. Nevertheless, the inventors of the present invention have demonstrated that when using the culture medium composition of the present invention, they were able to produce neuromuscular organoids that contained not just some but all of the components of nerve tissue, skeletal muscle tissue, and neuromuscular junctions, thus producing neuromuscular organoids similar to human neuromuscle. This result also suggests the excellent effect of the present invention.

[0107] In the present invention, pluripotent stem cells (PSCs) are also, but not limited to, human induced pluripotent stem cells (hiPSCs).

[0108] In one embodiment of the present invention, the pluripotent stem cells are, but are not limited to, human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0109] The present invention provides a method for producing neuromuscular organoids, comprising the following steps: (S0) The stage in which human pluripotent stem cells (hPSCs) derived from an individual are cultured in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate into bipolar neuromesodermal progenitors (NMPs); (S1) The step of diluting the bidifferentiating neuronal mesoderm precursor and dispensing it to form an embryoid body; and (S2) A step of culturing the embryo in a culture medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0110] The present invention provides a method for producing neuromuscular organoids by treatment with a sonic hedgehog (SHH) signaling activator (specifically, purmorphamine, but not limited to it), which activates sonic hedgehog signaling and successfully induces differentiation into motor neurons without inhibiting the formation of neuromuscular organoids, but is not limited to this.

[0111] In one embodiment of the present invention, each of the stages (S0), (S1), and (S2) is 1-60 days, 1-55 days, 1-50 days, 1-45 days, 1-40 days, 1-35 days, 1-30 days, 1-28 days, 1-26 days, 1-24 days, 1-22 days, 1-20 days, 1-18 days, 1-16 days, 1-14 days, 1-12 days, 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-60 days, 2-55 days, 2-50 days, 2-45 days, 2-40 days, 2-35 days, 2- 30th, 2nd-28th, 2nd-26th, 2nd-24th, 2nd-22nd, 2nd-20th, 2nd-18th, 2nd-16th, 2nd-14th, 2nd-12th, 2nd-10th, 2nd-9th, 2nd-8th, 2nd-7th, 2nd-6th, 2nd-5th, 2nd-4th, 2nd-3rd, 3rd-60th, 3rd-55th, 3rd-50th, 3rd-45th, 3rd-40th, 3rd-35th, 3rd-30th, 3rd-28th, 3rd-26th, 3rd-24th, 3rd-22nd, 3rd-20th, 3rd-18th, 3rd-16th, 3rd-14th, 3rd-12th, 3rd-10th, 3rd-9th, 3rd-8th, 3rd-7th, 3rd-6th , 3-5 days, 3-4 days, 5-60 days, 5-55 days, 5-50 days, 5-45 days, 5-40 days, 5-35 days, 5-30 days, 5-28 days, 5-26 days, 5-24 days, 5-22 days, 5-20 days, 5-18 days, 5-16 days, 5-14 days, 5-12 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 10-60 days, 10-55 days, 10-50 days, 10-45 days, 10-40 days, 10-35 days, 10-30 days, 10-28 days, 10-26 days, 10-24 days, 10-22 days, 10-20 days, 10- It takes place on the 18th, 10-16th, 10-14th, 10-12th, 15-60th, 15-55th, 15-50th, 15-45th, 15-40th, 15-35th, 15-30th, 15-28th, 15-26th, 15-24th, 15-22nd, 15-20th, 15-18th, 15-16th, 20-60th, 20-55th, 20-50th, 20-45th, 20-40th, 20-35th, 20-30th, 20-28th, 20-26th, 20-24th, and 20-22nd, but is not limited to these dates.

[0112] In one embodiment of the present invention, each of the stages (S0), (S1), and (S2) may be calculated starting from day 0, where the embryo is formed, but is not limited thereto.

[0113] In one embodiment of the present invention, the (S0) stage may begin on -10 to 0 days, -9 to 0 days, -8 to 0 days, -7 to 0 days, -6 to 0 days, -5 to 0 days, -4 to 0 days, -3 to 0 days, -2 to 0 days, -1 to 0 days, -5 days, -4 days, -3 days, -2 days, or -1 day, but is not limited thereto.

[0114] In one embodiment of the present invention, stage (S1) may begin on day 0, but is not limited thereto.

[0115] In one embodiment of the present invention, the stage of (S2) is: 1-15 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-15 days, 2-14 days, 2-13 days, 2-12 days, 2-11 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, It can, but is not limited to, start on the 2nd-3rd, 3rd-15th, 3rd-14th, 3rd-13th, 3rd-12th, 3rd-11th, 3rd-10th, 3rd-9th, 3rd-8th, 3rd-7th, 3rd-6th, 3rd-5th, 3rd-4th, 4th-15th, 4th-14th, 4th-13th, 4th-12th, 4th-11th, 4th-10th, 4th-9th, 4th-8th, 4th-7th, 4th-6th, 4th-5th, or 4th.

[0116] Unlike the method of separating pluripotent stem cells into individual cells (single cell state) and then differentiating them, the method of producing neuromuscular organoids according to the present invention allows differentiation of pluripotent stem cells in a colony state, but is not limited to this method.

[0117] The present invention relates to a method for producing neuromuscular organoids, comprising the step of culturing in a culture medium containing a sonic hedgehog signaling activator to produce a three-dimensional neuromuscular organoid, wherein the neuromuscular organoid may be characterized by one or more selected from the group consisting of the following, but is not limited thereto: (a) Increased expression of motor neuron markers, (b) Promoting the proliferation of skeletal muscle cells, (c) Promoting the maturation of skeletal muscle cells; and (d) Synchronous contraction.

[0118] The present invention's method for producing neuromuscular organoids may include, but is not limited to, a process of inducing the differentiation of pluripotent stem cells into neuromesodermal progenitors (NMPs), which are bidifferentiating progenitor cells that differentiate into nerve cells and muscle cells constituting the posterior spinal cord.

[0119] While the present invention's pluripotent stem cells can induce excellent embryonic formation and differentiation by promoting differentiation while in a colony-like state, it is not limited to this.

[0120] In the present invention, "neuromesodermal progenitors (NMPs)" are those that contribute to the paraxial mesoderm adjacent to the spinal cord and are capable of differentiating into multiple cell types, specifically, those capable of differentiating into both nerve cells and skeletal muscle cells, but are not limited to these.

[0121] Unlike pluripotent stem cells, the NMPs of the present invention have limited differentiation potential, and their differentiation into other cell types, excluding nerve cells and cells derived from the somatic mesoderm, is limited, but not limited.

[0122] In the present invention, "individual" is not limited to any vertebrate, but specifically applies to humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, and cats, and in one embodiment of the present invention, it may also be a human, but is not limited to that.

[0123] In one embodiment of the present invention, the individual may be a normal individual or an individual with a neuromuscular disease, but is not limited to either. Furthermore, in this invention, it has been confirmed that when pluripotent stem cells derived from a normal individual are used, normal neuromuscular organoids can be produced, and when pluripotent stem cells derived from an individual with a neuromuscular disease are used, neuromuscular organoids from an individual with a neuromuscular disease can be produced.

[0124] In the present invention, "dilution" is intended to prevent intercellular bonding and to adjust the cell concentration in the culture medium so that a certain level of cells are present, but is not limited to these purposes.

[0125] In specific embodiments of the present invention, dilution is for adjusting the cell concentration in the culture medium so that it contains 2000 cells, but is not limited thereto.

[0126] In the present invention, "differentiation" means, but is not limited to, the process by which a non-specialized cell acquires the characteristics of a specialized cell, such as a specific type of neuron, muscle cell, brain cell, heart cell, or liver cell. Furthermore, "differentiation" is also, but is not limited to, a process regulated by the interaction of cellular genes with extracellular physical and chemical conditions through signaling pathways typically involving proteins contained on the cell surface.

[0127] In the present invention, non-specialized cells include, but are not limited to, pluripotent stem cells, bipolar mesoderm precursors, and germ cells.

[0128] In the present invention, "dispensing" can refer to dispensing the dichotomous neuronal mesoderm precursor of the present invention in order to form an embryo, but is not limited to that.

[0129] In the present invention, at the (S0) stage, human pluripotent stem cells are in a state where they have formed colonies, but are not limited to that.

[0130] In the present invention, the "colony-formed state" does not include, but is not limited to, the step of treating pluripotent stem cells with a separation preparation such as Accutase to separate them into individual cells.

[0131] In the present invention, the "colony-formed state" can induce the formation or differentiation of germ cells that are superior to those formed when cultured as individual cells and differentiated into bipolar neuromesodermal progenitors (NMPs), but is not limited to this state.

[0132] In the present invention, step (S0) can be used interchangeably with the "pretreatment" step, but is not limited thereto.

[0133] In the present invention, at the (S0) stage, the bipolarizing neuronal mesoderm precursor may express SOX2 (SRY sex-determining region Y-box 2) or BRA (BRACHYURY), but is not limited to these.

[0134] In the present invention, at the (S0) stage, the differentiated bipolar mesoderm precursor may express SOX2 (SRY sex-determining region Y-box 2) or BRA (BRACHYURY), but is not limited to these.

[0135] In step (S1) of the present invention, the culture medium composition for diluting the bidifferentiating neuronal mesoderm precursor may, but is not limited to, include one or more selected from the group consisting of Y-27632, FGF (Fibroblast Growth Factor), IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

[0136] In step (S1) of the present invention, the culture medium composition for diluting the bidifferentiating neuronal mesoderm precursor may, but is not limited to, include one or more selected from the group consisting of Y-27632, FGF (Fibroblast Growth Factor), IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

[0137] In the present invention, step (S1) is characterized by one or more selected from the group consisting of the following: (a) The bifurcation ability is performed before the mesoderm precursor forms the germ; and (b) Dispense 1500 to 9000 bipolarizing neuronal mesoderm precursors.

[0138] In the present invention, step (S1) may be carried out for 0 to 6 days, but is not limited thereto.

[0139] In the present invention, day 0 can mean, but is not limited to, the period immediately following the completion of stage (S0) or a period consecutive to stage (S0). Furthermore, in the present invention, day 0 can mean, but is not limited to, the period immediately following the completion of the pretreatment stage or a period consecutive to the pretreatment stage.

[0140] In the present invention, step (S1) may be performed before or during the formation of the bipolar mesoderm precursor germinal body.

[0141] In one embodiment of the present invention, the bidifferentiating neuronal mesoderm precursors dispensed in the (S1) stage are 500-10000, 500-9500, 500-9000, 500-8500, 500-8000, 500-7500, 500-7000, 500-6500, 500-6000, 500-5500, 500-5000, and 5 00~4500 pieces, 500~4000 pieces, 500~3500 pieces, 500~3000 pieces, 500~2500 pieces, 500~2000 pieces, 1000~10000 pieces, 1000~9 500 pieces, 1000~9000 pieces, 1000~8500 pieces, 1000~8000 pieces, 1000~7500 pieces, 1000~7000 pieces, 1000~6500 pieces, 1000~6 000 pieces, 1000~5500 pieces, 1000~5000 pieces, 1000~4500 pieces, 1000~4000 pieces, 1000~3500 pieces, 1000~3000 pieces, 1000~ 2500 pieces, 1000~2000 pieces, 1500~10000 pieces, 1500~9500 pieces, 1500~9000 pieces, 1500~8500 pieces, 1500~8000 pieces, 1500 pieces ~7500, 1500~7000, 1500~6500, 1500~6000, 1500~5500, 1500~5000, 1500~4500, 1500~4000, 1500~3500, 1500~3000, 1500~2500, 1500~2000, or 2000, but not limited to these. In one embodiment of the present invention, it was confirmed that neuromuscular organoids with excellent morphological characteristics were produced by dispensing 2000 units.

[0142] In one embodiment of the present invention, stage (S1) was carried out for four days, but is not limited to that.

[0143] In the present invention, in step (S2), the culture is a shaking culture, but is not limited to that.

[0144] In the present invention, the (S2) stage is calculated from the completion time of the (S0) stage as follows: 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3- These dates may also start on the 5th, 3-4th, 4-10th, 4-9th, 4-8th, 4-7th, 4-6th, 4-5th, 5-10th, 5-9th, 5-8th, 5-7th, 5-6th, 6-10th, 6-9th, 6-8th, 6-7th, 7-10th, 7-9th, 7-8th, 8-10th, 8-9th, and 9-10th, but are not limited to these dates.

[0145] In the present invention, the (S2) stage is calculated from the completion time of the (S0) stage as follows: 1-30 days, 1-28 days, 1-26 days, 1-24 days, 1-22 days, 1-20 days, 1-18 days, 1-16 days, 1-14 days, 1-12 days, 1-10 days, 1-8 days, 1-6 days, 1-4 days, 1-2 days, 2-30 days, 2-28 days, 2-26 days, 2-24 days, 2-22 days, 2-2 These events may also end on, but are not limited to, days 0, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-30, 4-28, 4-26, 4-24, 4-22, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, and 4-6.

[0146] In one embodiment of the present invention, step (S2) was carried out for 4-6 days, 10 days, 14 days, or up to 20 days based on the culture day, and was carried out for a period of at least approximately 1-2 days or more, but is not limited to these.

[0147] In the present invention, shaking culture may include, but is not limited to, rotational shaking culture.

[0148] This invention provides a three-dimensional neuromuscular organoid manufactured by the manufacturing method of the present invention.

[0149] In the present invention, the organoid can express one or more selected from the group consisting of TUJ1 (neuron-specific class III beta-tubulin), fast MHC (fast myosin heavy chain), SOX2 (SRY sex-determining region Y-box 2), SHH (Sonic Hedgehog), α-BTX (α-bungarotoxin), MYH2 (Myosin Heavy Chain 2), and MYH7 (Myosin Heavy Chain 7), but is not limited to these.

[0150] In the present invention, a neuromuscular organoid may be characterized by one or more selected from the group consisting of the following, but is not limited thereto: (a) Decreased expression of Cdo and Tubb3; and (b) Increased expression of Olig2 and ChAT (choline acetyltransferase).

[0151] In the present invention, a neuromuscular organoid may be characterized by one or more selected from the group consisting of the following, but is not limited thereto: (a) Promote differentiation into motor neurons, (b) The size of the muscle fibers increases; and (c) Muscle fibers mature.

[0152] In the present invention, a neuromuscular organoid may be characterized by one or more selected from the group consisting of the following, but is not limited thereto: (a) Differentiation into motor neurons is promoted, (b) The size of the muscle fibers increases; and (c) Muscle fibers mature.

[0153] In the present invention, the phrase "neuromuscular organoids promote differentiation into motor neurons" may mean, but is not limited to, that the neuromuscular organoids of the present invention significantly promote differentiation into motor neurons compared to conventional neuromuscular organoids, resulting in an increase in the number of cells differentiated into motor neurons.

[0154] In this invention, neuromuscular organoids are used for motor neuron differentiation, but are not limited to that purpose. According to common practice in the industry, when conventional techniques are applied, only 6% of neuromuscular organoids differentiate into motor neurons. However, the inventors of this invention have confirmed that when treated with purmorphamine, neuromuscular organoids achieve a remarkably superior level of motor neuron differentiation efficiency.

[0155] In the present invention, an increase in the size of muscle fibers in neuromuscular organoids means that the skeletal muscle tissue becomes larger, specifically that the nerve tissue becomes smaller and the skeletal muscle tissue becomes larger, and more specifically that the growth rate of skeletal muscle tissue is relatively higher than that of nerve tissue, but is not limited to these interpretations.

[0156] In the present invention, an increase in the size of muscle fibers in neuromuscular organoids means that, by treating with an SHH signaling agent, the nerve tissue becomes smaller and the skeletal muscle tissue becomes larger, and that the increase in the overall size of the organoid is due to the growth of skeletal muscle tissue rather than nerve tissue, but is not limited to this.

[0157] In the present invention, the maturation of muscle fibers in neuromuscular organoids is confirmed by an increase in the expression of mature muscle fiber markers, and also signifies the early expression of mature muscle fiber markers. When muscle fibers mature, it can be confirmed that they exhibit synchronous contraction, but the invention is not limited to this.

[0158] In the present invention, neuromuscular organoids are characterized by synchronous contraction, but are not limited thereto.

[0159] The present invention provides a neuromuscular disease model characterized by including the neuromuscular organoid of the present invention.

[0160] The aforementioned neuromuscular disease model overcomes the shortcomings of conventional nerve tissue, skeletal muscle tissue, and neuromuscular junction models (specifically animal models). Because it has a relatively simple manufacturing process and allows for large-scale drug screening, it can, but is not limited to, be used as a platform for the development of preventive and / or therapeutic agents for neuromuscular diseases.

[0161] In this invention, neuromuscular diseases are, but are not limited to, degenerative neuromuscular diseases.

[0162] In the present invention, degenerative neuromuscular diseases may be selected from, but are not limited to, the group consisting of spinal muscular atrophy (SMA), Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS), Duchenne muscular dystrophy, and mytonic dystrophy.

[0163] In the present invention, degeneration or death of motor neurons can lead to weakening of the neuromuscular junction and atrophy of muscle tissue, and conversely, muscular dystrophy can induce weakening of the neuromuscular junction and death of motor neurons, but is not limited to these.

[0164] The present invention provides a method for screening neuromuscular disease treatment agents, comprising the following steps: (1) The step of treating neuromuscular organoids derived from individuals with neuromuscular disease produced by the method of the present invention with a candidate substance for the treatment of neuromuscular disease; and (2) A screening method comprising the step of comparing the morphological characteristics of neuromuscular organoids treated with the candidate substance.

[0165] In the present invention, the "method for screening neuromuscular disease therapeutic agents" can be used interchangeably with, but is not limited to, the "method for screening neuromuscular disease therapeutic agents," the "method for use in screening neuromuscular disease therapeutic agents," and the "method useful for screening neuromuscular disease therapeutic agents."

[0166] In the present invention, the "candidate therapeutic substance" may be, but is not limited to, individual nucleic acids, proteins, other extracts or natural products, or compounds that are presumed to have the potential to prevent or treat neuromuscular disease-related disorders by conventional selection methods, or that are randomly selected.

[0167] In the present invention, "treatment" may mean, but is not limited to, any action that results in improvement or significant modification of neuromuscular disease symptoms through the administration of a candidate therapeutic substance according to the present invention.

[0168] The "neuromuscular organoids derived from individuals with neuromuscular diseases" of the present invention also have morphological characteristics in which the muscle tissue (specifically, skeletal muscle tissue, but not limited to it) is formed smaller than that of normal neuromuscle, but are not limited to it.

[0169] In order to confirm whether the abnormal morphological features of the neuromuscular organoids derived from individuals with neuromuscular diseases in this invention are simply the result of batch-to-batch variability and mutant genotypes, it would be necessary to repeat and further characterize the neuromuscular organoids derived from individuals with neuromuscular diseases, but this is not the only way to do so.

[0170] The size of muscle tissue formed from neuromuscular organoids derived from individuals with neuromuscular diseases according to the present invention is 0.1-0.9 times, 0.1-0.8 times, 0.1-0.7 times, 0.1-0.6 times, 0.1-0.5 times, 0.1-0.4 times, 0.1-0.3 times, 0.1-0.2 times, 0.2-0.9 times, 0.2-0.8 times, 0.2-0.7 times, 0.2-0.6 times, 0.2-0.5 times, 0.2-0.4 times, 0.2-0.3 times, 0.3-0.9 times, 0 The multipliers are also, but not limited to, 0.3-0.8x, 0.3-0.7x, 0.3-0.6x, 0.3-0.5x, 0.3-0.4x, 0.4-0.9x, 0.4-0.8x, 0.4-0.7x, 0.4-0.6x, 0.4-0.5x, 0.5-0.9x, 0.5-0.8x, 0.5-0.7x, 0.5-0.6x, 0.6-0.9x, 0.6-0.8x, 0.6-0.7x, 0.7-0.9x, 0.7-0.8x, and 0.8-0.9x.

[0171] The screening method of the present invention may further include, but is not limited to, the step of determining that the candidate substance is a therapeutic agent for neuromuscular diseases if the morphological characteristics of a neuromuscular organoid treated with the candidate substance exhibit the morphological characteristics of normal neuromuscular tissue.

[0172] In the present invention, the morphological characteristics of normal neuromuscular tissue include, but are not limited to, the formation of larger muscle tissue compared to neuromuscular organoids derived from individuals with neuromuscular diseases.

[0173] Furthermore, the present invention provides a culture medium composition containing a sonic hedgehog (SHH) signaling activator as an active ingredient for use in three-dimensional culture of pluripotent stem cells (PSCs) to neuromuscular organoids.

[0174] Furthermore, the present invention provides a composition containing a sonic hedgehog (SHH) signaling activator as an active ingredient for the production of neuromuscular organoids from pluripotent stem cells (PSCs).

[0175] Furthermore, the present invention provides a culture medium composition containing a sonic hedgehog (SHH) signaling activator as an active ingredient for the production of neuromuscular organoids from pluripotent stem cells (PSCs).

[0176] Furthermore, the present invention provides a screening application for the three-dimensional neuromuscular organoid of the present invention for the treatment of neuromuscular diseases.

[0177] Furthermore, the present invention provides a three-dimensional neuromuscular organoid manufactured by a method for manufacturing neuromuscular organoids, comprising the following steps, for use in screening for neuromuscular disease treatment agents: (S0) The stage in which human pluripotent stem cells (hPSCs) derived from an individual are cultured in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate into bipolar neuromesodermal progenitors (NMPs); (S1) The step of diluting the bidifferentiating neuronal mesoderm precursor and dispensing it to form an embryoid body; and (S2) A step of culturing the embryo in a culture medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0178] Furthermore, the present invention provides a neuromuscular disease model containing a three-dimensional neuromuscular organoid produced by a method for producing neuromuscular organoids that includes the following steps for screening neuromuscular disease therapeutic agents: (S0) The stage in which human pluripotent stem cells (hPSCs) derived from an individual are cultured in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate into bipolar neuromesodermal progenitors (NMPs); (S1) The step of diluting the bidifferentiating neuronal mesoderm precursor and dispensing it to form an embryoid body; and (S2) A step of culturing the embryo in a culture medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0179] In the present invention, the "therapeutic agent" can be used interchangeably with, but is not limited to, a pharmaceutical composition.

[0180] The renal muscle organoids of the present invention may, but are not limited to, be included in a pharmaceutical composition at a concentration of 1 pg to 30 g w / v%.

[0181] The present invention may provide a pharmaceutical composition containing renal muscle organoids as an active ingredient.

[0182] In the present invention, “administration” means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention can be through a variety of oral or parenteral routes, as long as it can reach the target tissue.

[0183] In the present invention, "prevention" means all actions that delay the progression of renal muscle disease by administering the composition according to the present invention; "treatment" means all actions that improve or significantly alter the symptoms of renal muscle disease by administering the pharmaceutical composition according to the present invention; and "improvement" means all actions that reduce parameters related to renal muscle disease, such as the severity of symptoms, by administering the composition according to the present invention.

[0184] In the present invention, the pharmaceutical composition may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

[0185] In the present invention, "carrier" is also called a "vehicle" and refers to a compound that facilitates the addition of proteins or peptides into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic substances into the cells or tissues of living organisms.

[0186] In this invention, "diluent" is defined as a compound that not only stabilizes the biologically active form of the target protein or peptide but is also diluted with water that dissolves the protein or peptide. Salts dissolved in buffer solutions are used as diluents in this field. The buffer solution commonly used is phosphate-buffered saline, because it mimics the salt state of human body fluids. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents do not often alter the biological activity of the compound. Compounds containing azelaic acid used herein may be administered to human patients as themselves, or in combination with other components, such as in conjugation therapy, or as a pharmaceutical composition mixed with appropriate carriers or excipients.

[0187] Furthermore, the pharmaceutical compositions according to the present invention can be prepared by conventional methods in the form of topical preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injectable solutions. Examples of carriers, excipients, and diluents that may be contained in the compositions include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil. When preparing the compositions, they are typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients are used, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Possible suppository bases include witepsol, macrogol, tween 61, cocoa butter, lauric butter, and glycerogenous gelatin.

[0188] The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, transdermal administration, etc.

[0189] The appropriate dosage of the pharmaceutical composition of the present invention can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0190] The pharmaceutical compositions of the present invention may be manufactured in unit dose form or encapsulated in large containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant or stabilizer.

[0191] The terminology used in this invention has been selected as widely used and general terms as possible, taking into account the function of the invention; however, this may vary depending on the intentions of the articulators, precedents, the emergence of new technologies, etc. In certain cases, the applicant may have arbitrarily selected terms, in which case their meanings will be described in detail in the description of the invention. Therefore, the terminology used in this invention is not merely a set of names, but must be defined based on the meaning of the term and the overall content of the invention.

[0192] Terms such as "first," "second," etc., may be used to describe a variety of components, but such components should not be limited by such terms. The terms are used solely for the purpose of distinguishing one component from another. For example, without exceeding the scope of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component. The term "and / or" includes combinations of multiple items described or any of the multiple items described.

[0193] Throughout the specification of this invention, when a part of it "includes" a component, it means that, unless otherwise specifically stated, it does not exclude other components, but rather may include other components. Terms such as "about," "substantially," etc., used throughout the specification of this invention, are used to mean either the numerical value or an approximate value of the manufacturing and material tolerances inherent to the meaning referred to, when such values ​​are presented, and are used to ensure accuracy for understanding the invention or to prevent unscrupulous infringers from unfairly using disclosures that refer to absolute numerical values.

[0194] Throughout the specification of the present invention, the term “these combinations” as used in the Marcoush expression means one or more mixtures or combinations selected from the group of components described in the Marcoush expression, and means including one or more selected from the group of components.

[0195] The above-described information may, but is not limited to, the same application of the neuromuscular organoid culture medium composition, neuromuscular organoid manufacturing method, three-dimensional neuromuscular organoid, neuromuscular disease model, neuromuscular disease treatment agent screening method, uses of the neuromuscular organoid culture medium composition, uses of the composition, neuromuscular disease treatment agent screening applications, and neuromuscular disease prevention or treatment agent screening applications.

[0196] The following are preferred embodiments for understanding the present invention. However, these embodiments are provided to further facilitate understanding the present invention and do not limit the scope of the present invention.

[0197] Experimental Examples: Materials and Methods Experimental Example 1. Human pluripotent stem cell lines (hiPSCs) and their maintenance and management Human iPSC lines and isogenic control materials possessing SOD1 mutations derived from ALS patients were obtained from WiCell (WC035i-SOD1-D90A; WC035i-SOD1-D90D), and their use in this embodiment was approved by the Sungkyunkwan University Clinical Trials Review Board (SKKU2023-10-041). Cells were maintained in a 5% CO2, 37°C incubator in mTeSR1 Plus medium (Stem Cell Technologies) in Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning). 0.5 mg of Matrigel dissolved in DMEM / F12 was used for each 6-well cell culture plate (Corning), and the wells were coated at least 2 hours before use. Cells were subcultured every 3-4 days using Versene solution (Gibco). Initially, cells were rinsed with Versene and fresh Versene was added to the cells. Incubation time was optimized for each cell line (4-5 minutes). On the first day after subculturing, 10 μM Y-27632 (Caymen Chemical) was added to the culture medium. For cryopreservation of hiPSCs, the cells were resuspended in mFreSR (Stem Cell Technologies) and stored in a liquid nitrogen tank. Mycoplasma contamination of the cell lines was checked periodically.

[0198] Experimental Example 2. Induction of neural mesodermal progenitor cells from hiPSCs. Human iPSCs were cultured under maintenance conditions for at least two passages, and allowed to stand until approximately 40% confluence was reached. The retention medium was washed and removed, and replaced with N2B27 medium supplemented with pretreatment medium: 3 μM CHIR99021 (Caymen Chemical) and 40 ng / mL bFGF (Gibco). The following day, fresh pretreatment medium was replenished, and the cells were retained in the pretreatment medium for 24–36 hours until dissociation for germinal formation. N2B27 medium is a 1:1 mixture of DMEM / F12 (Gibco) supplemented with 1 x N2 (Gibco) and basal neuronal medium (Gibco) supplemented with 1 x B27 (Gibco), 1 x Glutamax (Gibco), 0.1 mM β-mercaptoethanol (Simga), and 1 x Pen-Strep antibiotic (Welgene).

[0199] Experimental Example 3. Generation of neuromuscular organoids derived from hiPSCs Cell morphology was monitored until the colony margins showed signs of pluripotency. Once the cells were prepared, they were gently isolated into single cells by culturing in Accutase at 37°C for 2 minutes. The single cells were counted and resuspended in N2B27 medium supplemented with 50 μM Y-27632, 10 ng / mL bFGF, 2 ng / mL IGF-1 (R&D Systems), and 2 ng / mL HGF (R&D Systems), resulting in a cell density of 20,000 cells / mL. 100 μL of medium containing 2,000 cells was seeded into each well of an Ultra-Low Attachment 96-well plate (Corning) via a multichannel pipette. The plate was centrifuged at 350 x g for 2 minutes and placed in a 5% CO2, 37°C incubator for aggregation. On day 2, half of the existing medium (50 μL) was removed, and each well was replenished with 100 μL of new N2B27 medium supplemented with 2 ng / mL IGF-1 and 2 ng / mL HGF. Pipetting was performed in a constant and slow manner to remove the medium from the wells without disturbing the organoids. On day 4, half of the existing medium (75 μL) was removed, and each well was replenished with 100 μL of new N2B27 medium without added growth factors. Purmorphamine (1 μM) or SAG (1 μM) was added to the N2B27 medium to activate the SHH signaling pathway. From this point onward, new N2B27 medium was replenished every other day. On day 10, the organoids were collected in a 96-well plate, transferred to two 60 mm Petri dishes containing N2B27 medium, and placed in an orbital shaker rotating at 70 rpm inside a 5% CO2, 37°C incubator. On day 30, the organoids from each 60 mm petri dish were transferred to a 100 mm dish containing N2B27 medium. Replenishing the medium in the petri dishes was done by tilting the dish and collecting the organoids to one side. After collecting the organoids to the bottom without any floating organoids, the used media was carefully aspirated until sufficient media covered the organoids, and then replenished with new media. The organoids were cultured in the same petri dish for the entire period unless an excessive number of cells separated from the organoids were visible under a microscope.To prevent damage to the organoids during transport, the pipette ends were trimmed with sterile scissors. The organoids were retained indefinitely without any signs of cell death on the surface, with fresh culture medium being replenished regularly.

[0200] Experimental Example 4. Bright-field microscopy and live imaging Brightfield images were acquired via a Nikon Eclipse Ti2 inverted microscope. Organoids were imaged directly in the wells of an Ultra-Low Attachment 96-well plate or in a 35mm Petri dish of N2B27 medium. For live imaging, the AVI acquisition option in Leica software was utilized (25 frames per second, 1–5 minute duration).

[0201] Experimental Example 5. Contraction Analysis To quantify spasmodic muscle contractions, live video was captured in the contracted region using 20x zoom. The acquired video was used with ImageJ software to define the organoid boundaries using thresholds, and a binary stack was generated. A region of interest was defined (10x50 pixels), and the region displacement was normalized to the initial region and plotted over time.

[0202] To quantify synchronized contractions, videos of the entire organoid were acquired, and the number of contractions was manually calculated. 50 μM glutamate (Sigma-Aldrich) was added to the culture medium to activate motor neurons. 10 μM Curare (Sigma-Aldrich) was added to the culture medium to inhibit acetylcholine receptors.

[0203] Experimental Example 6. Frozen Sections The organoids were fixed overnight in 4% PFA at 4°C. The following day, the PFA solution was washed off, and the organoids were transferred to a 30% sucrose solution in PBS and stored overnight at 4°C. After the organoids had settled to the bottom of the tube, they were incubated at room temperature for 30 minutes in a 1:1 mixture of 30% sucrose solution and OCT. Subsequently, the organoids were washed three times with fresh OCT and placed in Tissue-Tek Cryomold. The samples were frozen in isopentane cooled with liquid nitrogen and cut into 10 μm thick slices through a cryogenic storage device. The thin slices were placed on translucent microscope slides and stored at -80°C until ready for histochemical analysis.

[0204] Experimental Example 7. Immunofluorescence For whole-mount staining, organoids were fixed overnight in 4% PFA at 4°C. The samples were permeabilized with 0.5% Triton X-100 for 5 minutes and blocked at room temperature for 3 hours in PBS containing 3% bovine serum albumin and 0.1% Tween-20. The samples were then cultured overnight at 4°C in primary antibody diluted in the blocking solution. The following day, the primary antibody was washed with PBT (0.1% Tween-20) and cultured at room temperature for 1 hour in secondary antibody diluted in the blocking solution. The secondary antibody was washed with PBT, and the samples were placed on 80% glycerol in PBS. Confocal images were acquired via ZEISS LSM710 and CYTATION-C10 (Agilent). The primary antibodies used in this embodiment are listed in Table 1 below.

[0205] [Table 1]

[0206] For frozen sections, sample slides were washed with PBS to remove OCT and fixed in 4% PFA at room temperature for 15 minutes. The same steps as described above were followed from clearmiosis to immunolabeling. Samples were mounted on Mowiol and imaged through ZEISS LSM710 and CYTATION-C10.

[0207] Experimental Example 8. Real-time quantitative reverse transcription PCR RNA was isolated using the easy-BLUE Total RNA Extraction Kit (iNtRON) according to the manufacturer's protocol. cDNA synthesis was performed from 0.5 mg of RNA using PrimeScript RT reagent (TaKaRa) according to the manufacturer's instructions. For real-time quantitative reverse transcription PCR (qRT-PCR), the PCR mixture was prepared using TB Green Premix Ex Taq II (TaKaRa), and the reaction was carried out using Thermal Cycler Dice Real Time System III (TaKaRa). Gene expression levels were normalized against GAPDH levels. The primer sequences used in this embodiment are listed in Table 2.

[0208] [Table 2]

[0209] Experimental Example 9. Quantification and Statistical Analysis To measure organoid size, binary images were automatically generated from bright-field images using ImageJ software, and the total area of ​​each organoid was quantified through particle analysis. To quantify fast MHC expression regions, the stained regions were calculated and separated into DNA-stained regions using the Image Statistics instrument in Gen5 software (Agilent Technologies). To quantify the number of AChR clusters, an object mask was generated using the cell analyzer in Gen5 software, and the number of objects was calculated in the overall field of view. The size of individual AChR clusters was recorded for each individual region from the calculated index of the cell analyzer.

[0210] All data were reported as mean ± standard error of the mean. Statistical significance was calculated using the Student's t-test, and a p-value less than 0.05 was considered statistically significant. For comparisons between multiple groups, the data were analyzed using one-way ANOVA, followed by Tukey's multiple comparison tests using Prism 9.1.0, GraphPad.

[0211] Embodiment. Experimental results. Example 1. Production of neuromuscular organoids Example 1-1. Pretreatment steps for differentiating hiPSCs into NMPs Human induced pluripotent stem cells were cultured in Matrigel-coated tissue culture-treated 6-well plates. To induce differentiation of the stem cells into neural mesoderm precursors (NMPs), pretreatment was carried out at approximately 40% confluency. For this pretreatment, 3 μM CHIR99021 and 40 ng / mL bFGF were added to N2B27 medium.

[0212] Examples 1-2. Embryoformation stage where cells are seeded at an appropriate density. After 24 hours of pretreatment, intercellular junctions were separated by treating with Accutase at 37°C for 2 minutes to induce embryoid body formation. The number of separated cells was calculated using a hemocytometer, and the cell concentration was diluted in aggregation medium to contain 2000 cells per 100 μL. For the aggregation medium, 50 μM Y-27632, 10 ng / mL bFGF, 2 ng / mL IGF-1, and 2 ng / mL HGF were added to N2B27 medium. The cells diluted in the aggregation medium were dispensed in 100 μL portions into each well of an Ultra-Low Attachment round bottom 96-well plate, so that approximately 2000 cells were placed in each well. After collecting the dispensed cells at the bottom of the wells using a centrifuge, they were cultured in a 37°C, 5% CO2 incubator.

[0213] Examples 1-3. Formation stages of neuromuscular organoids cultured in a medium supplemented with purmorphamine. On the second day of embryogenesis (day 2), 50 μL of culture medium was removed from each well, and 100 μL of N2B27 medium supplemented with 2 ng / mL IGF-1 and 2 ng / mL HGF was dispensed into each well.

[0214] On day 4 of embryonic development, 80 μL of culture medium was removed from each well, and 100 μL of N2B27 medium supplemented with 1 μM purmorphamine was dispensed into each well. From then until day 20, the cells were cultured in N2B27 medium supplemented with 1 μM purmorphamine. The culture medium was changed every two days. During this period, as the embryonic bodies lengthened, the precursor cells of nerve tissue and muscle tissue became geographically separated.

[0215] On day 10 of embryogenesis, the organoids from each 96-well plate were transferred to two 60mm Petri dishes. The transferred organoids were cultured at 37°C in a 5% CO2 incubator with orbital shake at a rate of 75 rpm.

[0216] The characteristics of the neuromuscular organoid of the present invention manufactured by Example 1 (Figure 1) were confirmed as shown in Figure 2. The specific characteristics were confirmed as follows.

[0217] Example 2. Confirmation of morphological characteristics of neuromuscular organoids Example 2-1. Confirmation of germ cell formation and neuromuscular organoid morphological characteristics using a pretreatment method. Specifically, we compared embryo formation and neuromuscular morphogenesis induction using different pretreatment methods (Figures 3A and 3B). Figure 3A shows representative images of successful embryos (center) and unsuccessful embryos (left), and the ratio of successful embryo formation using the pretreatment method (right). Figure 3B shows representative images of successful neuromuscular morphogenesis by elongation (center) and unsuccessful morphogenesis (left), and the ratio of successful morphogenesis using the pretreatment method (right).

[0218] Conventionally, pretreatment was carried out in conjunction with cell passage, but in the present invention, it was carried out through medium exchange during the cell culture process. Furthermore, conventionally, stem cells were separated into individual cells (single cell state) via Accutase treatment before pretreatment, but in the present invention, pretreatment was carried out while the stem cells were in a colony state, and it was confirmed that this method is more suitable for inducing successful embryonic body formation and differentiation (Figures 3A and 3B).

[0219] In other words, it was found that the method of pre-treating stem cells in a colony-like state without separating them into individual cells according to the present invention can increase the ratio of successful embryo formation to excellent neuromuscular morphogenesis.

[0220] Example 2-2. Confirmation of excellent morphological characteristics of neuromuscular organoids over time. We confirmed the successful formation of neuromuscular organoids through the temporal morphogenesis of neuromuscular organoids formed according to the protocol of the present invention (Figure 4). Specifically, successful neuromuscular organoids formed both nerve and skeletal muscle tissue in a single organoid. The two tissues separated from each other, forming a "peanut"-like morphology, with each tissue tending to form on opposite sides (Figure 4).

[0221] The two tissues could be distinguished by the relatively dark color of the skeletal muscle tissue. The distinctive hue of the skeletal muscle became even more pronounced as the organism matured. On the lateral side of the nerve tissue, the neurodermis appeared as several small rose-like or continuous neurodermis along the overall surface of the nerve tissue portion. For reference, there were also cases where the two tissues were not completely separated from each other. In these cases, the skeletal muscle portion was found as a small projection, which was confirmed to be surrounded by nerve tissue.

[0222] Example 3. Confirmation of biomarkers in neuromuscular organoids Example 3-1. Confirmation of NMP marker expression by pretreatment method Through pretreatment, we confirmed that human pluripotent stem cells differentiated into bipolar mesoderm precursors (NMPs) by the expression of the NMP markers SOX2 (SRY sex-determining region Y-box 2) and BRA (BRACHYURY) (Figure 5).

[0223] As a result, the induction of NMPS was also observed to be associated with the co-expression of SOX2 and BRA (BRACHYURY). Furthermore, before pretreatment, hiPSCs expressed only SOX2, one of the pluripotency factors, along with OCT4 and NANOG (Figure 5, left). Cells expressing both SOX2 and BRA simultaneously were shown approximately 24 hours after pretreatment (Figure 5, center). One day after aggregation, germ cells produced from pretreated hiPSCs also co-expressed SOX2 and BRA, suggesting that most cells were successfully differentiated into NMPs (Figure 5, right).

[0224] Example 3-2. Confirmation of markers involved in neuroectoderm formation in neuromuscular organoids. Since the sonic hedgehog (SHH) signaling pathway is a major factor in the ventralization of spinal neurons, we added purmorphamine, an SHH signaling agent, to organoids to induce motor neuron development in neuromuscular organoids. During embryonic development, SHH was expressed in the notochord when neural stem cells emerged and formed the neural plate. On day 4, one of the organoid tails of the neuromuscular organoid began to express the neural stem cell marker (SOX2), and from day 7, the expression of the neural marker (TUJ1) was confirmed at the site where the neural stem cell marker (SOX2) was expressed (Figure 6).

[0225] On day 4 of organoid formation, localized expression of SOX2 in the area where nerve tissue is formed confirmed the formation of the neuroectoderm (left side of Figure 6). From day 7 of organoid formation, differentiation from neural stem cells into nerve cells was confirmed through TUJ1 expression (center of Figure 6). Based on this, it was decided to treat organoids with a sonic hedgehog signaling agent from day 4 of organoid formation. Since neural stem cells were shown in neuromuscular organoids on day 4, purmorphamine was administered at various intervals from day 4 (days 4-6, 4-10, and 4-20).

[0226] Example 3-3. Confirmation of markers involved in neuromuscular junction formation in neuromuscular organoids. The successful formation of neuromuscular organoids using the putotocor of the present invention was confirmed by staining for the neuronal cell marker TUJ1 and the mature skeletal muscle marker Fast MHC (Figure 7B). The right panel (yellow frame) shows a magnified view of the skeletal muscle portion of the neuromuscular organoid, illustrating that axons extended from the nerve tissue into the skeletal muscle tissue to form a neuromuscular junction.

[0227] Furthermore, the successful formation of nerve and skeletal muscle tissue was confirmed through immunolabeling. Day 20 neuromuscular organoids formed extensive neurites visualized by neuron-specific class III beta-tubulin (TUJ1) (Figure 7A). Germ myosin heavy chains (eMHCs), which label developing muscle fibers, were found in the same organoids surrounded by TUJ1 labeling. Closer observation of the skeletal muscle tissue revealed root canals with extended neurites in the surrounding nerve tissue. The skeletal muscle portion of day 40 neuromuscular organoids showed fast myosin heavy chains (MYH2), indicating mature muscle fiber formation (Figure 7B). Higher magnification of the skeletal portion revealed even more extensive neurites intertwined with muscle fibers. Individual muscle fiber sarcomeres were observed in day 60 neuromuscular organoids (Figure 7C).

[0228] Example 4. Confirmation of SHH signaling activation in neuromuscular organoids by purmorphamine. Neuromuscular organoids were collected on day 20, and the effect of purmorphamine on motor neuron induction was quantitatively evaluated via qRT-PCR.

[0229] As a result, we confirmed that SHH expression was significantly induced by purmorphamine in proportion to the duration of treatment (Figure 8). This indicates that SHH signaling was activated in organoids through purmorphamine treatment.

[0230] Specifically, we confirmed that treatment with prumorphamine significantly increased SHH expression, while the expression of CDO, a co-receptor of SHH, decreased inversely proportional to SHH expression due to negative feedback.

[0231] Example 5. Confirmation of differentiation induction of motor neurons in neuromuscular organoids by purmorphamine. We confirmed whether motor neuron differentiation was induced by treatment with purmorphamine by measuring the expression levels of motor neuron markers (Figure 9). The expression of both the motor neuron progenitor cell marker OLIG2 and the motor neuron marker ChAT was significantly increased in the organoid group treated with purmorphamine for the longest period (days 4-20).

[0232] This study demonstrated that maintaining SHH signaling from 10 to 20 days after organoid formation is important for motor neuron formation in neuromuscular organoids, and that activation of SHH signaling via purmorphamine promotes motor neuron development in neuromuscular organoids.

[0233] In addition, the increase in the expression of motor neuron markers was accompanied by a decrease in the expression of the general neuron marker (TUBB3) in the organoids treated with pulmorphamine. This suggests that the effect of pulmorphamine on the differentiation into motor neurons is actually greater than the results in Figure 9, as the longer the treatment with pulmorphamine, the more the expression of general nerve tissue markers decreases.

[0234] That is, it was confirmed that the expression of motor neuron markers increased in the neuromuscular organoids of the present invention (Figure 9), and it was confirmed that the treatment with pulmorphamine decreased nerve tissue in the organoids while promoting the growth of skeletal muscle tissue.

[0235] Example 6. Confirmation of the effect of increasing the formation of neuromuscular junctions in neuromuscular organoids by pulmorphamine As a result of confirming the frequency of formation of neuromuscular junctions by the treatment with pulmorphamine through α-bungarotoxin (α-BTX), a tendency to increase was confirmed (Figure 10).

[0236] This suggests that neuromuscular junctions were formed in the neuromuscular organoids of the present invention.

[0237] Example 7. Confirmation of morphological characteristics in neuromuscular organoids by pulmorphamine Example 7-1. Confirmation of increase in muscle tissue volume due to promorphamine treatment period. After adding pulmorphamine to the culture medium on the 4th day of organoid formation and comparing the differences in the morphology of the organoids according to the treatment period, it was confirmed that generally, the size of the organoids increased with the length of the treatment period of pulmorphamine centered on the muscle tissue site (Figure 11). Also, after adding pulmorphamine to the culture medium on the 4th day of organoid formation and comparing the differences in the morphology of the organoids according to the treatment period with that on the 50th day, it was confirmed that generally, the longer the treatment period of pulmorphamine, the smaller the nerve tissue and the larger the skeletal muscle tissue.

[0238] When considering that the neural tissue part and the skeletal muscle part of the neuromuscular organoid are distinguished by color under a bright-field microscope as shown in Fig. 11, the increase in size by purmorphamine suggests that it is mostly due to the growth of skeletal muscle tissue rather than neural tissue.

[0239] Example 7-2. Confirmation of overall size increase by promorphamine One of the first prominent effects of purmorphamine on the neuromuscular organism was an increase in the size of the organism. The organoid became larger in proportion to the purmorphamine treatment period (Fig. 12A).

[0240] Such an effect on the size of the organoid was also observed when the neuromuscular organoid was further treated with the smoothened agonist (SAG), another type of sonic hedgehog signaling agent. Specifically, as a result of quantifying and comparing the size of the organoid by SAG treatment, it was confirmed that the size of the organoid became larger when treated with SAG compared to the control group (Fig. 12B).

[0241] That is, the present invention suggests that it increases the overall size of the organoid, thereby promoting the proliferation of skeletal muscle cells in the neuromuscular organoid (Figs. 11 and 12).

[0242] Example 8. Confirmation of the effect of promoting muscle tissue maturation in neuromuscular organoids by purmorphamine Example 8-1. Confirmation of increased expression of mature muscle fiber markers. It was confirmed through qRT-PCR that the expression of the mature myofiber marker increased by the treatment of purmorphamine (Fig. 13). Such a result was confirmed from the qRT-PCR analysis of the 20-day-old neuromuscular organoids treated with purmorphamine for various periods, because the expression of the myosin heavy chain isoform was significantly increased by purmorphamine (Fig. 13).

[0243] Specifically, we confirmed that the expression of MYH2 (Myosin heavy chain 2) and MYH7 (Myosin heavy chain 7), markers for fast-twitch oxidative (IIA) and slow-twitch (I) muscle fibers, respectively, was induced by purmorphamine treatment, but the expression of MYH4, a marker for fast-twitch glycolytic (IIB) muscle fibers, was not affected.

[0244] Example 8-2. Confirmation of early expression of mature muscle fiber markers. We confirmed that the expression timing of Fast MHC, which is specifically expressed in mature muscle fibers, was advanced compared to the control group (Figure 14). This suggests that the method of the present invention promotes the maturation of skeletal muscle cells in neuromuscular organoids.

[0245] Example 9. Confirmation of the excellent muscle tissue contraction effect of purmorphamine on neuromuscular organoids. Example 9-1. Confirmation of premature muscle tissue contraction On day 14 of organoid formation, we confirmed that the timing of observed muscle tissue contraction was advanced compared to the control group, due to the accelerated maturation of muscle tissue (Figure 15).

[0246] Furthermore, we confirmed that the timing of synchronous contractions induced by glutamate treatment was significantly accelerated in organoids treated with purmorphamine. This provides further evidence of purmorphamine's involvement in the maturation of skeletal muscle tissue.

[0247] Specifically, organoids treated with purmorphamine for a short period (4-6 days) and the control group did not respond to glutamate, but organoids treated with purmorphamine for a longer period (4-10 days; 4-14 days) showed synchronous contraction in response to glutamate treatment on day 14 (Figure 15). Overall, these data suggest that purmorphamine promotes skeletal muscle growth and maturation in neuromuscular organoids.

[0248] Example 9-2. Confirmation of the synchronized contraction effect of the entire skeletal muscle tissue. In the neuromuscular organoid of the present invention, synchronous contraction of the entire skeletal muscle tissue was observed, which is more similar to the contraction of actual human muscles compared to the spontaneous contraction observed in existing neuromuscular organoids (Figure 16). In other words, it was confirmed that the promotion of skeletal muscle cell proliferation resulted in synchronous contraction similar to the contraction of actual human skeletal muscle tissue, which has not been observed in existing neuromuscular organoids.

[0249] Specifically, previous studies described spontaneous contractions of skeletal muscle tissue formed in neuromuscular organoids at 40–50 days as further evidence of successful skeletal muscle formation. Such small, rapid, spasmodic contractions were also observed in the neuromuscular organoids formed in the present invention. To quantify such contractions, the skeletal muscle portion of the neuromuscular organoids was imaged in real time at high density. Regional displacement within the region of interest was plotted over time in the acquired images. The contractile motion of the organoids tracked over time showed inter-organoid variability (Figure 17).

[0250] Unlike the skeletal muscle spasms described in previous studies, we also observed much larger synchronous contractions of the entire skeletal muscle in the neuromuscular organoids.

[0251] Specifically, after magnifying the muscle tissue of neuromuscular organoids and recording videos, the changes in the area of ​​muscle tissue within the ROI (region of interest) over time were tracked, and muscle tissue contraction was visualized. In "Organoid 3," the area indicated by the arrow shows the overall contraction of the entire muscle tissue.

[0252] In other words, the proliferation and maturation of skeletal muscle cells in neuromuscular organoids suggest that synchronized contractions similar to those observed in actual human skeletal muscle tissue were observed, which have not been observed in existing neuromuscular organoids.

[0253] Example 10. Optimization of the neuromuscular organoid protocol Example 10-1. Confirmation of the optimal cell seeding density after pretreatment. A problem with conventional techniques is that it is difficult to determine the appropriate seeding density of cells after pretreatment. 2 When cells were seeded in ) , excessive fusion of cells often occurred after 3 days in the pre-treated medium. Reducing the initial seeding density produced cells of sub-best quality due to the fact that hiPSCs prefer colony formation for survival (Rivera, T., et al., Human-Induced Pluripotent Stem Cell Culture Methods Under cGMP Conditions. Current Protocols in Stem Cell Biology, 2020. 54(1): p. e117.). These difficulties resulted in inconsistent outcomes in the formation of healthy embryos and successful organoids.

[0254] Therefore, to overcome these problems, the inventors tested other procedures for pretreatment of hiPSCs. Another paper (Olmsted, ZT and JL Paluh, Co-development of central and peripheral neurons with trunk mesendoderm in human elongating multi-lineage organized gastruloids. Nature Communications, 2021. Hereinafter referred to as Olmsted and Paluh) described a protocol for generating elongated multilineage organization (EMLO) that forms tissue derived from any of the three germ layers using the same pretreatment medium containing CHIR99021 and bFGF. When the cellular confluency was approximately 60%, the culture medium was briefly replaced with the pretreatment medium to pretreat the hiPSCs. Such a procedure for pretreatment of hiPSCs was combined with the rest of the neuromuscular organoid protocol of Martins et al. A remarkable effect was observed in that more consistent results were obtained because the control of cellular confluency was further smoothed.

[0255] Example 10-2. Confirmation of the optimal cell number for single embryo production. The number of starting cells for producing a single embryoid body of a determined size was optimized. The embryoid bodies of the prior art (Martins et al.) were made of 4,500 - 9,000 cells depending on the cell line used. When such a number of cells was used for a single embryoid body, the size of the embryoid body on day 1 was much larger than the embryoid body size (diameter ~75 μm) described by Martins et al. (9,000 cells: diameter ~500 μm; 4,500 cells: diameter ~250 μm, Figure 18). The large embryoid bodies could not successfully form neuromuscular organoids and remained round, suggesting that the cell size / number per embryoid body is yet another mediating variable that needs to be optimized for successful organoid formation.

[0256] By comparing various numbers of cells for making a single embryoid body, the optimal number of 2,000 cells (diameter ~150 μm) for neuromuscular organoid formation was calculated (center of Figure 3A).

[0257] Example 11. Confirmation of morphological characteristics of the skeletal muscle part related to the reproduction of neuromuscular organoid formation Acetylcholine receptor clusters were shown by α - bungarotoxin (αBTX) labeling (Figure 19A). The extensive presence of satellite cells within the skeletal muscle region was discovered by PAX7 expression (Figure 19B). On the other hand, only a small subset of motor neurons was identified by choline acetyltransferase (ChAT) expression near the boundary between the nerve and skeletal muscle tissues (Figure 19C). Such characteristics suggest the successful reproduction of neuromuscular organoid formation through protocol optimization.

[0258] Example 12. Confirmation of the cause of the generation of synchronous contractions formed in neuromuscular organoids This type of contraction (hereinafter referred to as "synchronous contraction" to distinguish it from spasmodic contractions) occurred infrequently, approximately once every two minutes (Figures 20A and 20B). To determine whether the synchronous contractions observed in neuromuscular organoids were regulated by motor neurons that activate skeletal muscle fibers through neuromuscular junctions, changes in contraction velocity were monitored after treatment with glutamate and curare. After treatment with glutamate, an excitatory neurotransmitter, the average synchronous contraction velocity increased slightly, though not statistically significant. More importantly, when the organoids were treated with curare, a toxin that inhibits acetylcholine receptor function in muscle fibers, synchronous contractions were completely cessated.

[0259] This suggests that synchronous contraction is a result of muscle fibers being activated through acetylcholine receptors at the neuromuscular junction.

[0260] Example 13. Confirmation of markers involved in synchronous contraction of neuromuscular organoids. Neuromuscular organoids exhibiting synchronous contraction were collected separately from the remaining organoids to illuminate the difference between synchronous contraction and the remaining non-contraction neuromuscular organoids. qRT-PCR analysis confirmed that organoids with synchronous contraction expressed significantly higher levels of myosin heavy chain in all isoforms tested (Figures 21A to 21C). However, there was no significant difference in the expression levels of neuronal markers between the two groups (Figures 21D and 21E). Furthermore, the expression of neuromuscular junction-related genes was significantly increased in organoids with synchronous contraction compared to the remaining organoids (Figures 21F and 21G). These data suggest that synchronous contraction in neuromuscular organoids is due to skeletal muscle development and maturation within the organoid.

[0261] Example 14. Analysis of morphological features of neuromuscular organoids applicable to customized medicine for amyotrophic lateral sclerosis (ALS). The most intriguing application of neuromuscular organoids is in personalized medicine. Neuromuscular organoids generated from patient-derived hiPSC lines can model disease causes and select appropriate treatment options in a patient-specific manner (Figure 22). For this purpose, we obtained SOD1 mutant hiPSC lines derived from amyotrophic lateral sclerosis (ALS) patients and homogeneous control hiPSC lines with modified SOD1 genes and used them to generate neuromuscular organoids for a proof-of-concept. Organoids generated from the SOD1 mutant hiPSC line exhibited abnormal morphology in relatively small skeletal muscle portions and indistinguishable morphological features not seen in organoids from the control hiPSC line (Figures 23A and 23B).

[0262] The above-described description of the present invention is merely illustrative, and a person with ordinary skill in the art to which the present invention belongs will understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, the above-described embodiments should be understood in all respects as illustrative and not limiting. [Industrial applicability]

[0263] The neuromuscular organoids of the present invention, when cultured in a culture medium containing a composition with a sonic hedgehog signaling agonist as an active ingredient, not only is the process of differentiating the neurons constituting the neuromuscular organoids into motor neurons promoted, but the development and maturation of muscle fibers are also promoted. The interaction between motor neurons and muscle tissue through the neuromuscular junction is central to the pathogenesis and treatment of degenerative neuromuscular diseases, and the neuromuscular organoids of the present invention can be used as a model of neuromuscular disease (specifically, a model of degenerative neuromuscular disease). Therefore, it is expected to be applicable to mechanistic research of nerve and muscle diseases and drug screening, and thus has industrial potential.

Claims

1. A culture medium composition for neuromuscular organoids, for three-dimensional culture of pluripotent stem cells (PSCs) into neuromuscular organoids, containing Sonic hedgehog (SHH) signaling activator as an active ingredient.

2. The neuromuscular organoid culture medium composition according to claim 1, wherein the sonic hedgehog signaling activator is one or more selected from the group consisting of sonic hedgehog signaling agonists and sonic hedgehog proteins.

3. The neuromuscular organoid culture medium composition according to claim 2, wherein the sonic hedgehog signaling agent is one or more selected from the group consisting of purmorphamine and smoothed agonist (SAG).

4. The culture medium composition for neuromuscular organoid culture according to claim 1, further comprising one or more selected from the group consisting of a GSK-3 (Glycogen synthase kinase 3) inhibitor, FGF (Fibroblast Growth Factor), Y-27632, IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

5. The neuromuscular organoid culture medium composition according to claim 1, wherein the neuromuscular organoid comprises one or more selected from the group consisting of nerve tissue, skeletal muscle tissue, and neuromuscular junctions.

6. The culture medium composition for neuromuscular organoids according to claim 1, wherein the pluripotent stem cell (PSC) is a human induced pluripotent stem cell (hiPSC).

7. A method for manufacturing neuromuscular organoids, including the following steps: (S0) A step in which human pluripotent stem cells (hPSCs) derived from an individual are cultured in a medium containing a GSK-3 (Glycogen synthase kinase 3) inhibitor and FGF (Fibroblast Growth Factor) to differentiate into neuromesodermal progenitors (NMPs) capable of bidifferentiation, (S1) The step of diluting the bidifferentiating nerve mesoderm precursor and dispensing it to form an embryoid body, (S2) A step of culturing the embryo in a culture medium containing a Sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

8. The manufacturing method according to claim 7, wherein in the (S0) stage, the human pluripotent stem cells are in a state of having formed colonies.

9. The method for producing a bipolarizing neuronal mesoderm in the (S0) stage, wherein the bipolarizing neuronal mesoderm precursor expresses SOX2 (SRY sex-determining region Y-box 2) or BRA (BRACHYURY), according to claim 7.

10. The method for producing the product according to claim 7, wherein the culture medium composition for diluting the bidifferentiating neuronal mesoderm precursor in step (S1) comprises one or more selected from the group consisting of Y-27632, FGF (Fibroblast Growth Factor), IGF (insulin-like growth factor), and HGF (hepatocyte growth factor).

11. The manufacturing method according to claim 7, characterized in that step (S1) is one or more selected from the following group: (a) The bifurcation ability is carried out by the neural mesoderm precursor before the formation of the germ cell; and (b) Dispense 1,500 to 9,000 bidifferentiating neuronal mesoderm precursors.

12. The manufacturing method according to claim 7, wherein in step (S2), the culture is orbital shaking culture.

13. A three-dimensional neuromuscular organoid manufactured by the manufacturing method described in any one of claims 7 to 12.

14. The neuromuscular organoid according to claim 13, wherein the organoid expresses one or more selected from the group consisting of TUJ1 (neuron-specific class III beta-tubulin), fast MHC (fast myosin heavy chain), SOX2 (SRY sex-determining region Y-box 2), SHH (Sonic Hedgehog), α-BTX (α-bungarotoxin), MYH2 (Myosin Heavy Chain 2), and MYH7 (Myosin Heavy Chain 7).

15. The neuromuscular organoid described in claim 13 is characterized by one or more selected from the following group: (a) Decreased expression of Cdo and Tubb3; (b) Expression of Olig2 and ChAT (choline acetyltransferase) increases.

16. The neuromuscular organoid according to claim 13 is characterized by one or more selected from the following group: (a) Promote differentiation into motor neurons, (b) The size of the muscle fibers increases; (c) Muscle fibers mature.

17. The neuromuscular organoid according to claim 13, characterized in that the neuromuscular organoid contracts synchronously.

18. A neuromuscular disease model characterized by including the neuromuscular organoid described in claim 13.

19. The neuromuscular disease model according to claim 18, wherein the neuromuscular disease is a degenerative neuromuscular disease.

20. The neuromuscular disease model according to claim 19, wherein the degenerative neuromuscular disease is selected from the group consisting of spinal muscular atrophy (SMA), Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS), Duchenne muscular dystrophy, and mytonic dystrophy.

21. A screening method for neuromuscular disease treatment agents, including the following stages: (1) The step of treating neuromuscular organoids derived from individuals with neuromuscular disease produced by the method of claim 7 with a candidate substance for the treatment of neuromuscular disease, (2) A screening method comprising the step of comparing the morphological characteristics of neuromuscular organoids treated with the candidate substance.

22. The aforementioned screening method is (3) The screening method according to claim 21, further comprising the step of determining that the candidate substance is a therapeutic agent for neuromuscular diseases if the morphological characteristics of a neuromuscular organoid treated with the candidate substance are those of normal neuromuscular tissue.

23. A culture medium composition containing Sonic hedgehog (SHH), a signaling activator, as an active ingredient, for three-dimensional culture applications from pluripotent stem cells (PSCs) to neuromuscular organoids.

24. Applications for the production of neuromuscular organoids from pluripotent stem cells (PSCs) using a composition containing Sonic hedgehog (SHH) signaling activator as an active ingredient.

25. The use of the three-dimensional neuromuscular organoid described in claim 13 for screening agents for neuromuscular disease treatment.

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