Method for producing senescent neuromuscular organoid, senescent neuromuscular organoid produced thereby, and use thereof

Abstract

Provided are: a method for producing a senescent neuromuscular organoid, the method comprising a step of culturing a neuromuscular organoid in a senescence-inducing medium containing TNF-α, IFN-γ, palmitic acid, and glucose; a senescent neuromuscular organoid produced by the method; and according to another aspect, a composition for producing a senescent neuromuscular organoid, the composition containing TNF-α, IFN-γ, palmitic acid, and glucose.

Description

Method for producing aged neuromuscular organoids, aged neuromuscular organoids produced thereby, and uses thereof

[0001] A method for producing aged neuromuscular organoids, aged neuromuscular organoids produced thereby, and uses thereof are disclosed.

[0002] This invention is the result of the Korean ARPA-H project support of the Korea Health Industry Development Institute with funding from the Ministry of Health and Welfare in 2024 (Project ID: RS-2024-00507183).

[0003] The neuromuscular system is a complex biological system that transmits motor nerve impulses from the central and peripheral nervous systems to peripheral muscle tissue, thereby inducing muscle contraction. The neuromuscular junction (NMJ), the functional connection unit of this neuromuscular system, is formed between the axon terminals of motor neurons and skeletal muscle fibers. Abnormalities in the neuromuscular junction can cause various neuromuscular diseases, such as muscle atrophy, neurotransmission disorders, or loss of motor function. Representative diseases include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Duchenne muscular dystrophy, and Charcot-Marie-Tooth disease (CMT).

[0004] Existing neuromuscular research has relied on two-dimensional cell culture systems or animal models. However, these models have limitations in adequately reproducing human cell-cell interactions and tissue structure, limiting their ability to understand the pathophysiology of human diseases and screen for therapeutics.

[0005] Accordingly, neuromuscular organoids derived from human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) are receiving renewed attention. WO2024 / 232663A1 discloses a technology for forming self-organizing 3D organoids that simultaneously contain both neurons and muscle cells by inducing differentiation of intermediate progenitor cells in the neuromesoderm progenitor (NMP) state. The technology induces the formation of neuromuscular structures through culture conditions including a Sonic Hedgehog (SHH) signaling agonist, a GSK-3 inhibitor, FGF, etc., thereby enabling the reproduction of functional neuromuscular junctions through the maturation and interaction of motor neurons and skeletal muscles.

[0006] These existing technologies have made significant progress in more accurately reproducing human neuromuscular diseases. However, they remain limited in directly mimicking functional decline or pathological changes caused by specific disease states, such as aging.

[0007] Therefore, there is a need for aged neuromuscular organoids and methods for their production.

[0008] One aspect provides a method for producing aged neuromuscular organoids, comprising the step of culturing the neuromuscular organoids in a senescence-inducing medium containing TNF-α, IFN-γ, palmitic acid, and glucose.

[0009] Provided are aged neuromuscular organoids produced by the above method according to different aspects.

[0010] In another aspect, a composition for producing aged neuromuscular organoids containing TNF-α, IFN-γ, palmitic acid, and glucose is provided.

[0011] One aspect provides a method for producing aged neuromuscular organoids, comprising the step of culturing the neuromuscular organoids in a senescence-inducing medium containing TNF-α, IFN-γ, palmitic acid, and glucose.

[0012] In the above method, a "neuromuscular organoid" refers to a three-dimensional artificial tissue capable of simulating a neuromuscular junction. The neuromuscular organoid may comprise nerve cells and muscle cells. The nerve cells may be motor neurons. The muscle cells may be skeletal muscle cells.

[0013] The above-described neuromuscular organoid may be produced by a known method. The neuromuscular organoid may be, for example, derived from pluripotent stem cells (PSCs). The pluripotent stem cells include induced pluripotent stem cells (iPSCs). The induced pluripotent stem cells may be human induced pluripotent stem cells (hiPSCs). The method may include a step of inducing the formation of neuromesoderm progenitors (NMPs) from the pluripotent stem cells, a step of inducing the formation of embryoid bodies (EBs) from the neuromesoderm progenitor cells, and a step of inducing the formation of neuromuscular organoids (NMOs) from the embryoid bodies. The cultured neuromuscular organoids used may not contain cancer cells.

[0014] The method for producing a neuromuscular organoid may be the method described in WO 2024 / 3236633 A1. The method may include the steps of culturing pluripotent stem cells (PSCs) derived from an individual in a medium containing a GSK-3 inhibitor and FGF to differentiate them into neuromesodermal progenitors (NMPs); diluting the neuromesodermal progenitors and dividing them to form embryoid bodies; and culturing the embryoid bodies in a medium containing a sonic hedgehog (SHH) signaling activator to produce a three-dimensional neuromuscular organoid.

[0015] The sonic hedgehog (SHH) signaling activator may be a sonic hedgehog signaling agonist, a sonic hedgehog protein, or a combination thereof. The sonic hedgehog signaling agonist may be purmorphamine, a smoothened agonist (SAG), or a combination thereof.

[0016] The above neural mesoderm progenitor cells may express SOX2 (SRY sex-determining region Y-box 2) or BRA (Brachyury). The dilution may be diluted in a medium containing at least one selected from the group consisting of Y-27632, FGF, IGF, and HGF. The dispensing step may be performed before the neural mesoderm progenitor cells form embryoid bodies, and at least one of dispensing 1,500 to 9,000 neural mesoderm progenitor cells. In the step of producing a three-dimensional neural root organoid, the culturing may be a shaking culture.

[0017] In the above method, the neuromuscular organoid may express at least one selected from the group consisting of neuron-specific class III beta-tubulin (TUJ1), fast myosin heavy chain (MHC), SRY sex-determining region Y-box 2 (SOX2), Sonic Hedgehog (SHH), α-bungarotoxin (α-BTX), Myosin heavy chain 2 (MYH2), and Myosin heavy chain 7 (MYH7). The neuromuscular organoid may have characteristics such as decreased expression of Cdo and Tubb3, increased expression of Olig2 and ChAT (choline acetyltransferase), or a combination thereof.

[0018] In the method for producing the above-described aged neuromuscular organoids, the concentration of glucose in the medium may be a high concentration, for example, a sub-cytotoxic concentration. A "sub-cytotoxic concentration" may be a concentration that induces senescence but does not induce cytotoxicity. The concentration may be a concentration that causes G1-G0 arrest in the cell cycle, a concentration that increases the concentration of intracellular reactive oxygen species (ROS), or a combination thereof. The concentration of the glucose may be 10 mM or more, 15 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 10 to 200 mM, 10 to 100 mM, 10 to 60 mM, 10 to 50 mM, 20 to 200 mM, 20 to 100 mM, 20 to 60 mM, 20 to 50 mM, or 15 to 50 mM.

[0019] The concentration of TNF-α in the medium may be 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, or 5 to 15 ng / mL.

[0020] The concentration of IFN-γ in the medium may be 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 5 to 100 ng / mL, 5 to 50 ng / mL, 5 to 40 ng / mL, 5 to 30 ng / mL, 10 to 100 ng / mL, 10 to 50 ng / mL, 10 to 40 ng / mL, or 10 to 30 ng / mL.

[0021] The concentration of palmitic acid in the medium may be 10 to 250 μM, 10 to 200 μM, 10 to 150 μM, 10 to 130 μM, 20 to 250 μM, 20 to 200 μM, 20 to 150 μM, 20 to 130 μM, 30 to 250 μM, 30 to 200 μM, 30 to 150 μM, 30 to 130 μM, 40 to 250 μM, 40 to 200 μM, 40 to 150 μM, 40 to 130 μM, 50 to 250 μM, 50 to 200 μM, 50 to 150 μM, or 50 to 130 μM.

[0022] In one specific example, the concentrations of TNF-α, IFN-γ, palmitic acid, and glucose may be 5 to 15 ng / mL, 10 to 30 ng / mL, 50 to 150 μM, and 20 to 60 mM, respectively; or 1 to 20 ng / mL, 5 to 50 ng / mL, 30 to 200 μM, and 10 to 200 mM, respectively.

[0023] The above medium may be a basic medium using an aqueous solution, PBS, or N2B27 medium.

[0024] In the above method, the culture may be cultured at 37°C. The culture may include a step of replacing the medium with a new medium at intervals of 1 to 3 days. The culture may be performed for 5 days or more. The culture may be performed for 5 to 15 days, for example, 10 days.

[0025] The method may include, before the culturing step, a step of culturing an embryoid body in a medium to form a neuromuscular organoid, and a step of replacing the medium with the "TNF-α, IFN-γ, palmitic acid, and glucose-containing senescence-inducing medium" and culturing the medium.

[0026] The replacement may be performed after 30 days of embryoid body culture, for example, on days 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The replacement may be performed on days 30 to 60, 30 to 55, 30 to 55, 30 to 50, 35 to 60, 35 to 55, 35 to 55, 35 to 50, 40 to 60, 40 to 55, 40 to 55, or 40 to 50 of embryoid body culture. The above replacement may be 100% replacement with the above "TNF-α, IFN-γ, palmitic acid, and glucose-containing senescence-inducing medium".

[0027] In the above method, before the culturing step, a step of culturing pluripotent stem cells (PSCs) to differentiate them into neuromesodermal progenitors (NMPs); a step of culturing the neuromesodermal progenitor cells to form embryoid bodies; and a step of culturing the embryoid bodies to produce three-dimensional neural root organoids may be included. For example, before the culturing step, a step of culturing pluripotent stem cells (PSCs) in a medium containing a GSK-3 inhibitor and FGF to differentiate them into neuromesodermal progenitors (NMPs); a step of diluting the neuromesodermal progenitor cells and dividing them to form embryoid bodies; And the step of producing a three-dimensional neuromuscular organoid by culturing the embryoid body in a medium containing a sonic hedgehog (SHH) signaling activator may further include;

[0028] The above neural mesoderm progenitor cells may express SOX2 (SRY sex-determining region Y-box 2), BRA (Brachyury), or a combination thereof. The dilution may be diluted in a medium containing at least one selected from the group consisting of Y-27632, FGF, IGF, and HGF. The dispensing step may be performed before the neural mesoderm progenitor cells form embryoid bodies, and at least one of dispensing 1500 to 9000 neural mesoderm progenitor cells. In the step of producing a 3D neural root organoid, the culturing may be a shaking culture.

[0029] In the present invention, the neuromuscular organoid may express at least one 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). The neuromuscular organoid may have characteristics such as decreased expression of Cdo and Tubb3, increased expression of Olig2 and ChAT (choline acetyltransferase), or a combination thereof.

[0030] In the present invention, the senescent neuromuscular organoid may have more senescent characteristics compared to neuromuscular organoids. The senescent characteristics may be increased senescence-associated β-galactosidase, increased centralized nuclei, increased pro-inflammatory cytokines, increased DNA damage, decreased mitochondrial function, or a combination thereof. In the increased senescence-associated β-galactosidase, increased centralized nuclei, increased pro-inflammatory cytokines, and increased DNA damage, the "increase" may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more compared to a control organoid. In the above reduced mitochondrial function, the “reduction” may be greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to a control. The pro-inflammatory cytokine may be TNF-α, IL-6, or a combination thereof. The increased DNA damage may be identified by increased phospho-γH2AX. The aged neuromuscular organoid may have a reduced antioxidant enzyme (e.g., SOD1), a reduced OXPHOS complex gene (e.g., ATP5A, MTC01, UQCRC2, NDUFB8, or SDHB), an increased senescence-associated gene (e.g., CDKN2A or CDKN1A gene), an increased senescence-associated secreted protein gene (e.g., SASP, CCL2, or CXCL1 gene), or a combination thereof compared to a control. The increase or decrease may be an increase or decrease in expression at the mRNA level or protein level.

[0031] The aged neuromuscular organoids may have a reduced overall size, reduced muscle size, or muscle ratio compared to a control.

[0032] The aged neuromuscular organoids may have reduced contractile function compared to the control group. The aged neuromuscular organoids may have reduced synchronized contraction frequency, reduced contraction amplitude, reduced contraction size, or a combination thereof compared to the control group.

[0033] Another aspect provides a composition for producing aged neuromuscular organoids containing TNF-α, IFN-γ, palmitic acid, and glucose. The TNF-α, IFN-γ, palmitic acid, and glucose and their concentrations are as described above.

[0034] The composition may be used in the method. The concentrations of TNF-α, IFN-γ, palmitic acid, and glucose may be 5 to 15 ng / mL, 10 to 30 ng / mL, 50 to 150 μM, and 20 to 60 mM, respectively; or 1 to 20 ng / mL, 5 to 50 ng / mL, 30 to 200 μM, and 10 to 200 mM, respectively.

[0035] The composition may include an excipient, a carrier, or a diluent. The composition may be a liquid composition. The composition may be a medium for cell culture. The medium may be a basic medium such as an aqueous solution, PBS, or N2N27 medium.

[0036] Another aspect provides a senescent neuromuscular organoid produced by the above method. The senescent neuromuscular organoid may be capable of simulating the functional connection structure between senescent neurons and skeletal muscle cells in a three-dimensional environment. The senescent neuromuscular organoid may be used for modeling neuromuscular diseases, studying neuromuscular junction (NMJ) formation and function, drug screening, and toxicity assessment. The senescent neuromuscular organoid may be used for research and drug screening of amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), muscular dystrophy, or a combination thereof. The muscular dystrophy may include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0037] According to a method for producing aged neuromuscular organoids according to an aspect of the present invention, aged neuromuscular organoids can be effectively produced.

[0038] Aged neuromuscular organoids prepared by the above methods according to different aspects can be used for modeling neuromuscular diseases, studying neuromuscular junction (NMJ) formation and function, drug screening, and toxicity assessment.

[0039] Compositions for producing aged neuromuscular organoids according to different aspects can be used in the above method.

[0040] Figure 1 is a schematic diagram showing the process of manufacturing and verifying an aging model using neuromuscular organoids.

[0041] Figure 2 is a drawing showing the results of staining muscle fibers (top), neurites (middle), and extracellular matrix (bottom) using immunofluorescence in cross-sections of neuromuscular organoids 50 and 200 days after organoid formation.

[0042] Figure 3 is a diagram showing the results of qRT-PCR analysis of the expression of pro-inflammatory cytokines in neuromuscular organoids 50 and 200 days after organoid formation.

[0043] Figure 4 is a diagram showing the results of qRT-PCR analysis of the expression levels of antioxidant enzymes (SOD1) and aging-related genes (CDKN2A) in neuromuscular organoids 50 and 200 days after organoid formation.

[0044] Figure 5 is a diagram showing the results of qRT-PCR analysis of the expression of genes for proteins constituting the OXPHOS complex, which performs oxidative phosphorylation in the mitochondria of neuromuscular organoids 50 and 200 days after organoid formation.

[0045] Figure 6 is a diagram showing the results of staining for senescence-associated beta galactosidase (SA-β-gal), a marker of cell senescence, in neuromuscular organoids 50 and 200 days after organoid formation.

[0046] Figure 7 is a diagram showing the results of staining Phospho-γH2AX in neuromuscular organoids 50 and 200 days after organoid formation.

[0047] Figure 8 is a schematic diagram showing the experimental design of simultaneously treating TNF-α, IFN-γ, high glucose, and palmitate to shorten the time to induce muscle loss and maximize efficiency in neuromuscular organoids.

[0048] Figure 9 is a diagram showing the results of qRT-PCR analysis of the expression of senescence marker genes (CDKN2A and CDKN1A) and senescence-associated secreted protein genes (SASP; CCL2 and CXCL1) in neuromuscular organoids between the control group and the senescence-inducing treatment group.

[0049] Figure 10 is a diagram quantitatively comparing the total organoid area, muscle tissue area, and the ratio of muscle tissue to the total area, along with representative images of control and aging-induced treatment group neuromuscular organoids.

[0050] Figure 11a is a graph depicting the change in area of ​​organoids over time to quantitatively analyze the contractile function of control and aging-induced treatment group neuromuscular organoids.

[0051] Figure 11b is a diagram showing the results of deriving the synchronized contraction frequency, average amplitude, and average contraction size of the control group and the aging-induced treatment group neuromuscular organoids based on the measurement results of Figure 11a.

[0052] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0053] Example 1: Production of neuromuscular organoids derived from human induced pluripotent stem cells

[0054] Human induced pluripotent stem cells were cultured in a 5% CO2, 37°C incubator in a Matrigel-coated, tissue culture-treated 6-well plate. The human induced pluripotent stem cells used were purchased from the WC035i-SOD1-D90D cell line (WiCell®). These cells were derived from fibroblasts from amyotrophic lateral sclerosis (ALS). The medium used was N2B27 medium. N2B27 medium is a 1:1 mixture of DMEM / F12 (Gibco) supplemented with 1x N2 (Gibco) and neural basal medium (Gibco) supplemented with 1x B27 (Gibco), 1x Glutamax (Gibco), 0.1 mM beta-mercaptoethanol (Sigma), and 1x Pen-Strep antibiotic (Welgene).

[0055] Pretreatment was performed when the cell density in the above 6-well plate reached approximately 40% confluency. Specifically, the medium in the wells was replaced with N2B27 medium supplemented with 3 μM CHIR99021 and 40 ng / mL bFGF and cultured for 24 hours. Accordingly, the stem cells were differentiated into neural mesoderm progenitors (NMP).

[0056] Next, after 24 hours, Accutase was added to the neural mesoderm precursors in the above wells and incubated at 37℃ for 2 minutes to dissociate cell-cell adhesion. The number of isolated cells was counted and diluted so that 2,000 cells were contained per 100 μL medium. The medium used for dilution was an aggregation medium containing 50 μM Y-27632, 10 ng / mL bFGF, 2 ng / mL IGF-1, and 2 ng / mL HGF in the N2B27 medium. 100 μL of the diluted cells in the aggregation medium was dispensed into each well of an Ultra-Low Attachment round bottom 96-well plate so that approximately 2,000 cells were contained. After centrifugation to collect the dispensed cells at the bottom of the well, they were cultured in a 5% CO2, 37℃ incubator for aggregation. Hereinafter, the incubation start date was set as day 0 of embryoid body formation.

[0057] On day 2 of incubation, 50 μL of medium per well was removed and replaced with 100 μL of N2B27 medium supplemented with 2 ng / mL IGF-1 and 2 ng / mL HGF. On day 4, 80 μL of medium per well was removed and replaced with 100 μL of N2B27 medium supplemented with 1 μM purmorphamine. Purmorphamine was added until day 20, and the medium was replaced every other day. On day 10, organoids from each well of the 96-well plate were divided and transferred to two 60 mm Petri dishes (approximately 48 per dish). Organoid morphology was observed after 4 days of culture. The transferred organoids were suspended and cultured at 75 rpm on an orbital shaker in a 5% CO2, 37°C incubator. On day 20, immunofluorescence was used to confirm the formation of motor neurons and skeletal muscle tissue in each organoid. This confirmed the formation of neuromuscular organoids.

[0058] Figure 1 is a schematic diagram showing the process of manufacturing and verifying an aging model using neuromuscular organoids.

[0059] Example 2. Production of a neuromuscular organoid-based aging model through long-term culture.

[0060] 2-1. Manufacturing of a neuromuscular organoid-based aging model

[0061] For long-term culture, on day 30 of incubation in Example 1, the organoids contained in the 60 mm Petri dishes were transferred to 100 mm Petri dishes and cultured in N2B27 medium. Thereafter, the medium was changed every 3 to 4 days, and it was confirmed that long-term culture of organoids for more than 300 days was possible under these conditions. The neuromuscular organoids of the aging model were harvested on day 200 of incubation.

[0062] 2-2. Histological Characteristics Analysis According to Aging Induction

[0063] The neuromuscular organoids prepared in Example 2-1 were harvested on days 50 and 200 of incubation, respectively. Organoids were fixed overnight in 4% paraformaldehyde (PFA) and washed the following day. For cryosectioning, organoids were stored in Optimal Cutting Temperature compound (OCT), a polymer embedding medium for tissue sections, for one day, then rapidly frozen in liquid nitrogen-cooled isopentane and sectioned at 10 μm thickness in a cryostat. The sectioned tissues were mounted on translucent microscope slides and stored at -80°C until ready for staining.

[0064] For immunofluorescence staining, the sectioned tissues were thawed at room temperature, the remaining OCT was removed with PBS, and re-fixed with 4% PFA for 15 minutes. The tissues were then treated with 0.5% Triton X-100 for 5 minutes and blocked in PBS containing 3% bovine serum albumin and 0.1% Tween-20 for 3 hours at room temperature. The samples were stored in blocking solution containing the primary antibody diluted therein at 4°C until the next day. The following day, the primary antibodies were washed with PBT (0.1% Tween-20) and stored in blocking solution containing the secondary antibodies diluted therein for 1 hour. The secondary antibodies were washed with PBT, the samples were mounted in Mowiol, and images were acquired using CYTATION-C10 (Agilent). The primary antibodies used in this example are listed in Table 1 below.

[0065] Antigen Catalog Number Manufacturer MYH1EMF20DSHBTUBB3Ab18207AbcamLamininL9393SigmaPhospho-γH2AXMA1-2022Invitrogen

[0066] Figure 2 shows the results of immunofluorescence staining of muscle fibers (top), neurites (middle), and extracellular matrix (bottom) in cross-sections of neuromuscular organoids 50 and 200 days after organoid formation. Centralized nuclei, a characteristic of damaged muscle fibers, were observed in muscle fibers of organoids cultured for 200 days (gray arrows), and compared to organoids cultured for 50 days, the neurites extending into muscle fibers and the extracellular matrix proteins surrounding muscle fibers were decreased. Centralized nuclei, a phenomenon in which nuclei located in the periphery of normal mature muscle fibers migrate to the center of the cell, are a common histological feature of damaged muscle fibers. As shown in Figure 2, when muscle cells were observed using MF20 staining, many centralized nuclei, which are observed in damaged muscle cells, were observed only in neuromuscular organoids formed for 200 days. When we stained the neuron-specific marker TUBB3 with MF20 and observed the muscle tissue at the junction where the nerve and muscle tissues meet in the neuromuscular organoids, we found that the neurites extending from the nerve tissue were reduced in the 200-day-old neuromuscular organoids. We also confirmed that laminin, which constitutes the extracellular matrix surrounding muscle fibers, was structurally disrupted in the 200-day-old neuromuscular organoids.

[0067] 2-3. Confirmation of gene expression levels according to aging induction

[0068] On days 50 and 200 of the neuromuscular organoids prepared in step 2-1 above, RNA was isolated using the easy-BLUE Total RNA Extraction Kit (iNtRON) according to the manufacturer's protocol. cDNA synthesis was performed from 500 ng of RNA using PrimeScript RT reagent (TaKaRa). For real-time quantitative reverse transcription-PCR (qRT-PCR), a PCR mixture was prepared using TB Green Premix Ex Taq II (TaKaRa), and the reaction was performed using a Thermal Cycler Dice Real Time System III (TaKaRa). Gene expression values ​​were normalized to GAPDH levels. The primers used in this example are listed in Table 2.

[0069] Gene direction sequence number IL-6 forward 1 reverse 2 TNF-a forward 3 reverse 4 SOD1 forward 5 reverse 6 CDKN2A forward 7 reverse 8 ATP5A forward 9 reverse 10 MTCO1 forward 11 reverse 12 UQCRC2 forward 13 reverse 14 NDUFB8 forward 15 reverse 16 SDHB forward 17 reverse 18 CDKN1A forward 19 reverse 20 CCL2 forward 21 reverse 22 CXCL1 forward 23 reverse 24 GAPDH forward 25 reverse 26 RPLPD forward 27 reverse 28

[0070] Figure 3 is a diagram showing the results of qRT-PCR analysis of pro-inflammatory cytokine expression levels in neuromuscular organoids 50 and 200 days after organoid formation. As shown in Figure 3, it was confirmed that the expression of pro-inflammatory cytokines such as IL-6 and TNF-α increased in aged neuromuscular organoids. Figure 4 is a diagram showing the results of qRT-PCR analysis of the expression levels of antioxidant enzymes (SOD1) and senescence-related genes (CDKN2A) in neuromuscular organoids 50 and 200 days after organoid formation. As shown in Figure 4, the expression levels of antioxidant enzyme genes (SOD1) and CDKN2A, whose expression increases in senescent cells, significantly changed in aged neuromuscular organoids.

[0071] Figure 5 is a diagram showing the results of qRT-PCR analysis of the expression of genes for proteins constituting the OXPHOS complex, which performs oxidative phosphorylation in the mitochondria of neuromuscular organoids 50 and 200 days after organoid formation. As shown in Figure 5, the expression of genes for proteins constituting the OXPHOS complex, which performs oxidative phosphorylation in the mitochondria (ATP5A, MTCO1, UQCRC2, NDUFB8, and SDHB), was confirmed to be reduced in aged neuromuscular organoids.

[0072] 2-4. Identification of cellular aging markers according to aging induction

[0073] The neuromuscular organoids prepared in 2-1 above were harvested on the 30th and 200th days of formation, and cryosections were prepared in the same manner as in 2-1. The expression of pH 6-activated beta-galactosidase, which accumulates in senescent cells, was confirmed in the sectioned tissues through staining using the Senescence β-Galactosidase Staining Kit (Cell Signaling).

[0074] Figure 6 is a diagram showing the results of staining for senescence-associated beta galactosidase (SA-β-gal), a marker of cellular senescence, in neuromuscular organoids 50 and 200 days after organoid formation. As shown in Figure 6, SA-β-gal staining, a marker of cellular senescence, significantly increased in senescence-induced neuromuscular organoids.

[0075] 2-5. Confirmation of DNA damage due to aging

[0076] The neuromuscular organoids manufactured in 2-1 above were harvested on the 50th and 200th days of formation, and the sheared tissues manufactured through cryosectioning were examined for the damaged DNA marker Phospho-γH2AX together with DNA staining (Hoechst) using the same fluorescence immunoassay as in 2-1.

[0077] Figure 7 is a diagram showing the results of Phospho-γH2AX staining in neuromuscular organoids 50 and 200 days after organoid formation. As shown in Figure 7, the proportion of cells in which Phospho-γH2AX staining was observed significantly increased in aging-induced neuromuscular organoids, confirming that genetic damage was induced during the long-term culture process.

[0078] Example 3. Method for manufacturing a neuromuscular organoid-based aging model by creating an aging environment.

[0079] 3-1. Method for producing a neuromuscular organoid-based aging model by inducing an aging environment

[0080] For neuromuscular organoids cultured in N2B27 medium at day 40, the medium was replaced with medium containing the following composition to create a senescence-inducing environment: 10 ng / mL TNF-α, 20 ng / mL IFN-γ, 100 nM palmitic acid, and 42.5 mM glucose. The medium was prepared by adding TNF-α, IFN-γ, and palmitic acid in water and phosphate-buffered saline (PBS) supplemented with glucose and 10% BSA to N2B27 medium. The medium was replaced every two days, and organoids were harvested on day 50 and used for analysis. Controls were cultured with vehicle only at the same time point, for which 21.3 μl / mL of water, 20 μl / mL of 1% ethanol (EtOH), and 10% BSA / PBS were added to the culture medium.

[0081] 3-2. Method for analyzing gene expression according to aging induction

[0082] RNA was isolated from the neuromuscular organoids prepared in step 3-1 above using the easy-BLUE Total RNA Extraction Kit (iNtRON) according to the manufacturer's protocol. cDNA synthesis was performed from 500 ng of RNA using PrimeScript RT reagent (TaKaRa). For real-time quantitative reverse transcription-PCR (qRT-PCR), a PCR mixture was prepared using TB Green Premix Ex Taq II (TaKaRa) and the reaction was performed using a Thermal Cycler Dice Real Time System III (TaKaRa). Gene expression values ​​were normalized to the RPLP0 level. The primers used in this example are listed in Table 2.

[0083] 3-3. Method for analyzing muscle size and function according to aging induction

[0084] Muscle tissue size was determined by analyzing images captured using brightfield optical microscopy using the ImageJ program. Muscle tissue was identified based on its dark contrast and contractile properties, contrasting with neural tissue. The segmented line tool was used to define the muscle area, and its area was calculated. The ratio of muscle tissue area to the total organoid area was calculated and used as a quantitative indicator.

[0085] The muscle function of each organoid was assessed through real-time optical microscopy image analysis. The threshold function in ImageJ was used to define the organoid's perimeter in the acquired images, and the area was measured for each frame to generate a time-dependent area growth curve.

[0086] Synchronized contractions of organoids were defined as peaks in the area change curve, and the average contraction frequency was derived from the number of peaks within a given time period. The difference between the highest point of each peak and the immediately preceding lowest point was defined as the amplitude, and the magnitude of the contraction was calculated by averaging the total amplitude. Finally, the amplitude values ​​were normalized to the muscle area value of each organoid and used as a quantitative indicator of dextral function.

[0087] Figure 8 is a schematic diagram showing the experimental design of simultaneously treating TNF-α, IFN-γ, high glucose, and palmitate to shorten the time to induce muscle loss and maximize efficiency in neuromuscular organoids.

[0088] Figure 9 is a diagram showing the results of qRT-PCR analysis of the expression of senescence marker genes (CDKN2A and CDKN1A) and senescence-associated secreted protein genes (SASP; CCL2 and CXCL1) in neuromuscular organoids between the control group and the senescence-inducing treatment group.

[0089] As shown in Fig. 9, the expression of senescence marker genes (CDKN2A and CDKN1A) and senescence-associated secreted protein genes (SASP; CCL2 and CXCL1) increased in the experimental group organoids compared to the control group organoids.

[0090] Figure 10 is a diagram quantitatively comparing the total organoid area, muscle tissue area, and the ratio of muscle tissue to the total area, along with representative images of control and aging-induced treatment group neuromuscular organoids.

[0091] As shown in Figure 10, compared to the control group organoids, the total organoid area, muscle tissue area, and the ratio of muscle tissue to the total area were reduced in the experimental group organoids.

[0092] Figure 11a is a graph depicting the change in organoid area over time to quantitatively analyze the contractile function of control and aging-induced treatment groups of neuromuscular organoids. Figure 11b is a diagram illustrating the results of deriving the synchronized contraction frequency, average amplitude, and average contraction size of control and aging-induced treatment groups of neuromuscular organoids based on the measurement results of Figure 11a. Through analysis, the synchronized contraction frequency and contraction amplitude were calculated based on each peak.

[0093] As shown in Figures 11a and 11b, the frequency and amplitude of synchronized contractions were reduced in the experimental group organoids compared to the control group organoids.

Claims

1. A method for producing aged neuromuscular organoids, comprising the step of culturing neuromuscular organoids in a senescence-inducing medium containing TNF-α, IFN-γ, palmitic acid, and glucose.

2. A method according to claim 1, wherein the concentrations of TNF-α, IFN-γ, palmitic acid, and glucose are 5 to 15 ng / mL, 10 to 30 ng / mL, 50 to 150 μM, and 20 to 60 mM, respectively; or 1 to 20 ng / mL, 5 to 50 ng / mL, 30 to 200 μM, and 10 to 200 mM, respectively.

3. A method according to claim 1, wherein the culturing comprises a step of replacing the medium at intervals of 1 to 3 days.

4. A method according to claim 1, wherein the culturing is performed for 5 days or more.

5. A method according to claim 4, wherein the culturing is performed for 5 to 15 days.

6. In claim 1, before the culturing step, a step of culturing pluripotent stem cells (PSCs) and differentiating them into neuromesodermal progenitors (NMPs); A step of culturing the above neural mesoderm progenitor cells to form an embryoid body; and A method further comprising a step of culturing the embryoid body to produce a neuromuscular organoid.

7. A method according to claim 1, comprising, before the culturing step, a step of culturing an embryoid body in a medium to form a neuromuscular organoid, and a step of replacing the medium with a TNF-α, IFN-γ, palmitic acid, and glucose-containing senescence-inducing medium and culturing the medium.

8. A method according to claim 7, wherein the replacement is performed 30 to 60 days after embryonic body culture.

9. The method of claim 1, wherein the senescent neuromuscular organoid has the characteristics of increased senescence associated β-galactosidase, increased centralized nuclei, increased pro-inflammatory cytokines, increased DNA damage, decreased mitochondrial function, or a combination thereof.

10. A senescent neuromuscular organoid produced by the method of claim 1.

11. A composition for producing aged neuromuscular organoids containing TNF-α, IFN-γ, palmitic acid, and glucose.

12. A composition for use in the method of claim 1, according to claim 11.

13. A composition according to claim 11, wherein the concentrations of TNF-α, IFN-γ, palmitic acid, and glucose are 5 to 15 ng / mL, 10 to 30 ng / mL, 50 to 150 μM, and 20 to 60 mM, respectively; or 1 to 20 ng / mL, 5 to 50 ng / mL, 30 to 200 μM, and 10 to 200 mM, respectively.

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