Method for producing skeletal muscle organoid

Abstract

The present application relates to a method for preparing skeletal muscle organoids, comprising the steps of: inducing paraxial mesoderm stem cells (PMSCs) by directly differentiating human pluripotent stem cells (hPSCs); forming the paraxial mesoderm stem cells in a 3D shape, and then culturing same in a medium; inducing the proliferation of the paraxial mesoderm stem cells; and inducing the maturation of the paraxial mesoderm stem cells.

Description

Method for producing skeletal muscle organoids

[0001] The present invention relates to a method for producing skeletal muscle organoids.

[0002] Skeletal muscles, which account for approximately 40% of adult body weight, control body movement and posture and are composed of individual multinucleated contractile myofibers formed by the fusion of myogenic progenitor cells.

[0003] Meanwhile, sarcopenia is defined as the loss of skeletal muscle mass and strength associated with age, which negatively impacts health. Muscle stem cells (MuSCs), also known as satellite cells, play a crucial role in skeletal muscle regeneration. With aging, the number of MuSCs decreases, and their regenerative capacity in response to exercise and injury is dramatically reduced. Aged satellite cells exhibit impaired self-renewal, activation, proliferation, and differentiation. Furthermore, impaired mitochondrial function is observed in aged satellite cells.

[0004] These age-related sarcopenic features can be induced by chemotherapy. For example, cisplatin, an effective chemotherapy widely used to treat various types of cancer, can cause serious side effects, including nephrotoxicity, neurotoxicity, ototoxicity, and rapid muscle loss. Previous studies have demonstrated that cisplatin treatment can induce mitochondrial dysfunction, overproduction of reactive oxygen species (ROS), and skeletal muscle atrophy. However, most of these studies were limited to mice, such as the C2C12 cell line, in 2D culture systems.

[0005] Therefore, a three-dimensional in vitro culture system using human-derived cells is required for skeletal muscle research, and several studies have been reported on generating 3D skeletal muscle from human pluripotent stem cells (hPSCs). However, there are few reports on muscle fibers containing mature sarcomeres.

[0006] Therefore, there is a need to develop a method for producing hPSC-derived 3D skeletal muscle organoids for studying sarcopenia and cachexia.

[0007] The background technology of this application, Korean Patent Publication No. 10-2022-0153170, relates to a method for producing human pluripotent stem cell-derived cardiac organoids and human pluripotent stem cell-derived cardiac organoids produced thereby.

[0008] The present invention is intended to solve the problems of the above-mentioned conventional technology and provides a method for producing skeletal muscle organoids.

[0009] In addition, a method for producing chemotherapy-induced sarcopenic skeletal muscle organoids is provided by additionally treating a chemotherapy anticancer agent or proinflammatory cytokine in the above production method.

[0010] In addition, a method for screening a treatment agent for sarcopenia using a skeletal muscle organoid manufactured through the above manufacturing method is provided.

[0011] In addition, a skeletal muscle organoid manufactured through the above manufacturing method is provided.

[0012] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0013] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for producing a skeletal muscle organoid, comprising the steps of: directly differentiating human pluripotent stem cells (hPSCs) to induce paraxial mesoderm stem cells (PMSCs); forming the paraxial mesoderm stem cells into a 3D shape and then culturing them in a medium; inducing proliferation of the paraxial mesoderm stem cells; and inducing maturation of the paraxial mesoderm stem cells.

[0014] According to one embodiment of the present invention, the step of inducing the above-described mesenchymal stem cells may be performed using a WNT activator and a BMP (bone morphogenetic protein) inhibitor, but is not limited thereto.

[0015] According to one embodiment of the present invention, the medium may include, but is not limited to, a myogenic specification medium.

[0016] According to one embodiment of the present invention, the progenitor cell growth factor may include, but is not limited to, fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF1), and hepatocyte growth factor (HGF).

[0017] According to one embodiment of the present invention, the step of inducing proliferation may be, but is not limited to, inducing myogenic proliferation of the axial mesenchymal stem cells.

[0018] According to one embodiment of the present invention, the step of inducing the myogenic proliferation may be performed by removing the fibroblast growth factor (FGF), but is not limited thereto.

[0019] According to one embodiment of the present invention, the step of inducing maturation may be, but is not limited to, inducing myogenic maturation of the axial mesenchymal stem cells.

[0020] According to one embodiment of the present invention, the step of inducing the above-mentioned myogenic maturation may be performed by removing the above-mentioned cell growth factor (HGF) and insulin-like growth factor 1 (IGF1) and adding a TGF-β inhibitor, but is not limited thereto.

[0021] In addition, the second aspect of the present invention provides a method for producing a sarcopenic skeletal muscle organoid, which method for producing a skeletal muscle organoid according to the first aspect of the present invention further comprises a step of treating the skeletal muscle organoid with a chemotherapy agent or a proinflammatory cytokine.

[0022] According to one embodiment of the present invention, muscle loss of the skeletal muscle organoid may be caused by treatment with the chemotherapy agent or proinflammatory cytokine, but is not limited thereto.

[0023] According to one embodiment of the present invention, the chemotherapeutic agent may include, but is not limited to, one selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, raltitrexed, and combinations thereof.

[0024] According to one embodiment of the present invention, the proinflammatory cytokine may include, but is not limited to, one selected from the group consisting of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ), Transforming Growth Factor-β (TGF-β), and combinations thereof.

[0025] In addition, the third aspect of the present invention provides a method for screening for a treatment agent for sarcopenia, comprising the steps of treating a skeletal muscle organoid manufactured according to the first aspect of the present invention with a chemotherapeutic agent or a proinflammatory cytokine and a candidate agent for a treatment agent for sarcopenia; and identifying a phenotype associated with sarcopenia in the skeletal muscle organoid.

[0026] Additionally, the fourth aspect of the present invention provides a skeletal muscle organoid prepared by the method according to the first aspect of the present invention.

[0027] According to one embodiment of the present invention, the skeletal muscle organoid is TITIN + muscle fiber, PAX7 + Muscle stem cells, MyHC + skeletal muscle, TUJ1 + Neurons, PDGFRα + May include, but is not limited to, fibroadipogenic precursor cells (FAPs).

[0028] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0029] The method for producing skeletal muscle organoids according to the present invention not only reproduces embryonic muscle formation, but also allows for the production of optimized 3D skeletal muscle organoids containing heterogeneous cell populations such as muscle cells of various stages, mature myofibers, neurons, and fibro-adipogenic progenitors, and can model sarcopenic muscle by performing chemotherapy on the produced skeletal muscle organoids, which can be useful for studying human skeletal muscle development and developing therapeutics for diseases such as sarcopenia and cachexia.

[0030] In addition, the skeletal muscle organoid according to the present invention contains quiescent satellite cells located beneath the basement membrane of muscle fibers, so it can accurately mimic the in vivo muscle structure, and can exhibit characteristics of functionally mature muscle tissue through the formation of neuromuscular junctions and spontaneous contraction.

[0031] In addition, the skeletal muscle organoids according to the present invention can reproduce muscle damage and regeneration processes after TNF-α treatment, so they can be used for research on muscle regeneration mechanisms, and can prevent muscle atrophy caused by chronic inflammation through testosterone treatment, so they can be used as an effective screening platform for developing treatments for sarcopenia.

[0032] In addition, the skeletal muscle organoids according to the present invention can be cultured for a long period of time, can gradually grow until 100 days, and can reach a diameter of approximately 2 mm, and can exhibit structurally stable characteristics through the expression of mature myotube proteins and basement membrane markers.

[0033] In addition, the skeletal muscle organoids according to the present invention contain various cell types such as muscle cells, motor neurons, glial cells, and Schwann cells, and thus can be utilized for research on muscle-nerve interactions and neuromuscular diseases.

[0034] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0035] Figure 1 is a flowchart of a method for producing skeletal muscle organoids according to one embodiment of the present invention.

[0036] Figure 2 is a schematic diagram of a conventional method for producing skeletal muscle organoids (above) and a method for producing skeletal muscle organoids of the present invention (below).

[0037] Figure 3a shows the results of QPCR analysis for the expression of a paraxial mesoderm marker (MSGN1) on days 3 and 6 of culture of skeletal muscle organoids (hSkMO) according to a comparative example (Con) and an example (2D3D) of the present invention, 3b shows the results of QPCR analysis for the expression of a paraxial mesoderm marker (TBX6) on days 3 and 6 of culture of skeletal muscle organoids (hSkMO) according to a comparative example (Con) and an example (2D3D), 3c shows the results of QPCR analysis for the expression of muscle stem cell markers (PAX3, PAX7) on days 20 and 30 of culture of skeletal muscle organoids (hSkMO) according to a comparative example (Con) and an example (2D3D), and 3d shows the results of QPCR analysis for the expression of myogenic differentiation markers (MYOD, MYOG) on days 20 and 30 of culture of skeletal muscle organoids (hSkMO) according to a comparative example (Con) and an example (2D3D). 3e is the result of QPCR analysis for expression, 3e is the result of immunofluorescence analysis for the TITIN+ (skeletal muscle) region on day 50 of culture of skeletal muscle organoids (hSkMO) according to the comparative example (Con) and the exemplary embodiment (2D3D) (scale bar, 200 μm), and 3f is the result of quantification of the TITIN+ (skeletal muscle) region on day 50 of culture of skeletal muscle organoids (hSkMO) according to the comparative example (Con) and the exemplary embodiment (2D3D).

[0038] Figure 4a is an immunofluorescence analysis result of PAX7+ muscle progenitor cells / satellite cells and LAM+ basal layer on day 100 of skeletal muscle organoid (hSkMO) culture according to one embodiment of the present invention, 4b is an observation result of PAX7+ satellite cells on day 100 of hSkMO culture (white arrow indicates PAX7+ cells under the basal layer), 4c is an immunofluorescence analysis result of PAX7+ muscle progenitor / satellite cells and Ki67+ proliferating cells between day 30 and day 100 of hSkMO culture, 4d is a quantification result (n=3) of muscle progenitor / satellite-like PAX7+ cells and proliferating Ki67+ cells between day 30 and day 100 of hSkMO culture, 4e is an immunofluorescence analysis result of PAX7+ muscle progenitor / satellite cells and MYOD+ activated muscle progenitor cells on day 50 and day 100 of hSkMO culture, and 4f is Quantification of PAX7+ muscle progenitor / satellite-like cells and MYOD+ activated muscle progenitor cells between days 50 and 100 of hSkMO culture (n=3), and 4g is the result of immunofluorescence analysis for MyHC, MYOD at days 30 and 100 of hSkMO culture (white arrows indicate activated satellite cells in myofibers, arrowheads indicate myofibrillar bands in myofibers). 4h is the percentage of myofibers with mature sarcomeres at 30 and 100 days of hSkMO culture, 4i is immunofluorescence analysis of skeletal muscle organoid sections showing MyHC+ skeletal muscle and TUJ1+ neurons at day 50 of hSkMO culture (arrows indicate activated satellite cells in myofibers, arrowheads indicate myofibrillar bands in myofibers), and 4j is immunofluorescence analysis of hSkMO sections showing PAX7+ muscle stem cells and PDGFRα+ fibroadipogenic progenitor cells at day 50 of hSkMO culture. [Scale bars, 20 μm (4a, 4c, and 4j), 5 μm (4b and 4g), and 100 μm (4i)].

[0039] Figure 5 is a schematic diagram of a sarcopenic muscle organoid modeling according to one embodiment of the present invention.

[0040] Figure 6a shows the results of MyHC immunofluorescence analysis in normal control, cisplatin-treated hSkMO, and hSkMO treated with hESC-MPC-EV together with cisplatin (scale bar, 5 μm), 6b shows the average fiber width quantified from the immunofluorescence analysis of MyHC (n=5), 6c shows the results of MyHC, MYOG immunofluorescence analysis in normal control, cisplatin-treated hSkMO, and hESC-MPC-EV treated together with cisplatin (scale bar, 20 μm), 6d shows the results of quantification of myofiber cross-sectional area (n=5), and 6e shows the results of percentage of MYOG+ cells per myofiber (n=5).

[0041] Figure 7a shows the results of immunofluorescence analysis of MyHC and cleaved caspase 3 in normal control, cisplatin-treated hSkMO, and hSkMO treated with hESC-MPC-EV together with cisplatin (white arrows indicate apoptotic MyHC; scale bar 20 μm), 7b shows the results of the percentage of cleaved caspase 3+ myofibers per myofiber (n=5), 7c shows the results of immunofluorescence analysis of p21+ senescent cells and MYOG+ muscle cells in normal control, cisplatin-treated hSkMO, and hSkMO treated with hESC-MPC-EV together with cisplatin (white arrow heads indicate p21+ / MYOG+ cells; scale bar, 10 μm), 7d shows the results of quantification of p21+ senescent cells and MYOG+ muscle cells (n=3), and 7e shows the results of immunofluorescence analysis of normal control, cisplatin-treated hSkMO, and Immunofluorescence analysis of MYOG+ muscle cells and γH2AX+ damaged DNA in hSkMO treated with hESC-MPC-EVs together with cisplatin (scale bar, 20 μm), 7f shows the quantification of MYOG+ muscle cells and γH2AX+ damaged DNA (n=3), 7g shows the immunofluorescence analysis of PAX7+ muscle progenitor / satellite cells and MYOD+ activated muscle progenitor cells in the normal control, hSkMO treated with cisplatin, and hSkMO treated with hESC-MPC-EVs together with cisplatin (scale bar, 20 μm), 7h shows the quantification of PAX7+ muscle progenitor / satellite-like cells and MYOD+ activated muscle progenitor cells in the normal control, hSkMO treated with cisplatin, and hSkMO treated with hESC-MPC-EVs together with cisplatin (n=3).

[0042] Figure 8a is a schematic diagram showing the overall strategy for generating hSkMOs from hPSCs and representative images of hSkMOs morphology at each time point (scale bar = 500 μm). Growth diameter represents the average size (mean ± SEM; n = 10).

[0043] Figure 8b is an immunocytochemical image of day-3 paraxial mesenchymal stem cells stained with T / BRA, TBX6, SOX2, and DAPI (scale bar = 200 μm). T / BRA+ is 82.04%, TBX6+ is 78.18%, T / BRA+ / SOX2+ is 15.91%, and TBX6+ / SOX2+ is 11.45% (mean ± SEM; n = 5).

[0044] Figure 8c is a whole mount staining image of day 9 hSkMOs stained with PAX3, TBX6, and DAPI (scale bar = 100 μm, mean ± SEM; n = 5).

[0045] Figure 8d is a frozen section image of day 20 hSkMOs stained with PAX7, Ki67, and DAPI (white scale bar = 200 μm and yellow scale bar = 20 μm, mean ± SEM; n = 5).

[0046] Figure 8e is a cryosection image of day 20 hSkMOs stained with PAX7, MYOD, and DAPI (white scale bar = 200 μm and yellow scale bar = 10 μm, mean ± SEM; n = 5).

[0047] Figure 8f is a cryosection image of day 20 hSkMOs stained with PAX7, MYOG, and DAPI (white scale bar = 200 μm and yellow scale bar = 20 μm, mean ± SEM; n = 5).

[0048] Figure 9a is a schematic diagram showing the functional connections between mature muscle fibers and spinal cord-derived motor neurons.

[0049] Figure 9b shows cryosection images of hSkMOs at 50 and 100 days stained with MyHC, TUJ1, and DAPI (white scale bar = 500 μm and yellow scale bar = 100 μm).

[0050] Figure 9c is a quantification graph of the ratio of TUJ1+ nerve and MyHC+ muscle areas at days 50 and 100 (mean ± SEM; n=14).

[0051] Figure 9d is a quantification graph of the average fiber diameter stained with MyHC (mean ± SEM; n=32).

[0052] Figure 9e is a cryosection image of day 100 hSkMOs stained with MyHC, LAMININ, and DAPI (scale bar = 10 μm).

[0053] Figure 9f is a cryosection image of day 100 hSkMOs stained with PAX7, LAMININ, and DAPI (scale bar = 10 μm).

[0054] Figure 10a is a cryosection image of day 100 hSkMOs stained with ChAT, TUJ1, and DAPI (scale bar = 50 μm).

[0055] Figure 10b is a cryosection image of day 100 hSkMOs stained with GFAP, MAP2, and DAPI (scale bar = 20 μm).

[0056] Figure 10c is a cryosection image of day 100 hSkMOs stained with MyHC, S100β, αBTX, and DAPI (scale bar = 10 μm).

[0057] Figure 10d is a scanning electron microscope image showing skeletal muscle fibers and neuromuscular junctions of 100-day hSkMOs (Mi: mitochondria, M: M-band, Z: Z-line, Ax: axon, SV: synaptic vesicle, SC: synaptic cleft, BL: basement membrane, scale bar = 500 nm).

[0058] Figure 10e shows the results of Fluo4AM calcium imaging visualizing spontaneous contraction and calcium influx in hSkMOs on day 100 (scale bar = 50 μm).

[0059] Figure 10f shows representative traces and amplitude and frequency recordings of spontaneous postsynaptic potential (PSP) responses obtained from muscle fibers (mean ± SEM; n = 8).

[0060] Figure 11a is a schematic diagram showing the acute treatment strategy of TNF-α for hSkMOs on day 100.

[0061] Figure 11b shows the Western blot results of p-NF-κB-p65, p-IκB-α, p-AKT, MYOG, and β-actin expression in the control group, on day 0, day 3, and day 7 after TNF-α treatment (mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n=3).

[0062] Figure 11c is a cryosection image of day 100 hSkMOs stained with PAX7, MYOD, and DAPI (scale bar = 50 μm, mean ± SEM; *p<0.05, **p<0.001; n=3).

[0063] Figure 11d is a frozen section image of day 100 hSkMOs stained with PAX7, Ki67, and DAPI (scale bar = 50 μm, mean ± SEM; *p<0.05, **p<0.001; n=3).

[0064] Figure 11e is a cryosection image of day 100 hSkMOs stained with MYOG and DAPI (scale bar = 100 μm, mean ± SEM; **p<0.001; n=3).

[0065] Figure 11f is a cryosection image of day 100 hSkMOs stained with MyHC, TUJ1, and αBTX (scale bar = 100 μm, mean ± SEM; ****p<0.0001; n=20, **p<0.001, *p<0.01; n=3).

[0066] Figure 12a is a schematic diagram showing the chronic treatment strategy of TNF-α and testosterone for hSkMOs on day 100.

[0067] Figure 12b shows PAX7, AR, and DAPI staining images of hSkMOs on day 100 in the control, TNF-α, and TNF-α + testosterone groups (scale bar = 50 μm, mean ± SEM; **p<0.01, ***p<0.001; n=3).

[0068] Figure 12c is a representative image of hSkMOs in the control, TNF-α, and TNF-α + testosterone groups (scale bar = 500 μm, mean ± SEM; **p<0.01, ****p<0.0001; n=6).

[0069] Figure 13a shows PAX7, MYOD, and DAPI staining images of hSkMOs on day 100 in the control, TNF-α, and TNF-α + testosterone groups (scale bar = 100 μm, mean ± SEM; *p<0.01, **p<0.001; n=3).

[0070] Figure 13b shows PAX7, Ki67, and DAPI staining images of hSkMOs on day 100 in the control, TNF-α, and TNF-α + testosterone groups (scale bar = 100 μm, mean ± SEM; *p<0.01; n=3).

[0071] Figure 13c shows MyHC, MYOG, and DAPI staining images of hSkMOs on day 100 in the control, TNF-α, and TNF-α + testosterone groups (scale bar = 100 μm, mean ± SEM; ***p<0.0001; n=5 and *p<0.01; n=50).

[0072] Figure 13d is an αBTX staining image of hSkMOs on day 100 of the control, TNF-α, and TNF-α + testosterone groups (scale bar = 20 μm, mean ± SEM; *p<0.01, **p<0.001; n=3).

[0073] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0074] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0075] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0076] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0077] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0078] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0079] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0080] Hereinafter, the skeletal muscle organoid production method of the present invention will be described in detail with reference to implementation examples, examples, and drawings. However, the present invention is not limited to these implementation examples, examples, and drawings.

[0081]

[0082] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for producing a skeletal muscle organoid, comprising the steps of: directly differentiating human pluripotent stem cells (hPSCs) to induce paraxial mesoderm stem cells (PMSCs); forming the paraxial mesoderm stem cells into a 3D shape and then culturing them in a medium; inducing proliferation of the paraxial mesoderm stem cells; and inducing maturation of the paraxial mesoderm stem cells.

[0083] The method for producing skeletal muscle organoids according to the present invention not only reproduces embryonic muscle formation, but also allows for the production of optimized 3D skeletal muscle organoids containing heterogeneous cell populations such as muscle cells of various stages, mature myofibers, neurons, and fibro-adipogenic progenitors, and can model sarcopenic muscle by performing chemotherapy on the produced skeletal muscle organoids, which can be useful for studying human skeletal muscle development and developing therapeutics for diseases such as sarcopenia and cachexia.

[0084] Figure 1 is a flowchart of a method for producing skeletal muscle organoids according to one embodiment of the present invention.

[0085] First, human pluripotent stem cells (hPSCs) are directly differentiated to induce paraxial mesoderm stem cells (PMSCs). (S100)

[0086] Conventional skeletal muscle organoid production methods directly form hPSCs into a 3D shape, but this has limitations in differentiation into the paraxial mesoderm lineage. In contrast, the method according to the present invention uses a direct differentiation protocol to induce paraxial mesoderm stem cells (PMSCs) from hPSCs in adherent culture to optimize the skeletal muscle organoid culture protocol, and then forms the PMSCs into a 3D shape and cultures them, thereby accelerating the differentiation protocol into the paraxial mesoderm lineage more than the conventional method.

[0087] In addition, the skeletal muscle organoid according to the present invention contains quiescent satellite cells located beneath the basement membrane of muscle fibers, so it can accurately mimic the in vivo muscle structure, and can exhibit characteristics of functionally mature muscle tissue through the formation of neuromuscular junctions and spontaneous contraction.

[0088] In addition, the skeletal muscle organoids according to the present invention can reproduce muscle damage and regeneration processes after TNF-α treatment, so they can be used for research on muscle regeneration mechanisms, and can prevent muscle atrophy caused by chronic inflammation through testosterone treatment, so they can be used as an effective screening platform for developing treatments for sarcopenia.

[0089] In addition, the skeletal muscle organoids according to the present invention can be cultured for a long period of time, can gradually grow until 100 days, and can reach a diameter of approximately 2 mm, and can exhibit structurally stable characteristics through the expression of mature myotube proteins and basement membrane markers.

[0090] In addition, the skeletal muscle organoids according to the present invention contain various cell types such as muscle cells, motor neurons, glial cells, and Schwann cells, and thus can be utilized for research on muscle-nerve interactions and neuromuscular diseases.

[0091] According to one embodiment of the present invention, the step of inducing the above-described mesenchymal stem cells may be performed using a WNT activator and a BMP (bone morphogenetic protein) inhibitor, but is not limited thereto.

[0092] The WNT activator may include CHIR99021, a GSK3 inhibitor, etc., and the BMP inhibitor may include LDN193189, NOGGIN, DORSOMORPHIN, etc. These activators and inhibitors may be used alone or in combination, and the amount of use and treatment time may be appropriately selected by those skilled in the art.

[0093] During the induction process of the above-mentioned paraxial mesoderm stem cells, the expression of key transcriptional regulators of posterior presomitic mesoderm (pPSM) specification may be upregulated. These transcriptional regulators may include T / Brachyury, TBX6, MSGN1, etc. The generation of the above-mentioned paraxial mesoderm stem cells can be confirmed through various analysis methods commonly used in the art, such as immunocytochemical analysis, gene expression analysis, and protein expression analysis.

[0094] Additionally, to increase the induction efficiency of the aforementioned mesenchymal stem cells, additives such as Rock inhibitors can be additionally used, and culture conditions (temperature, pH, oxygen partial pressure, etc.) can be optimized. These conditions can be appropriately selected and adjusted by those skilled in the art according to the characteristics and yield of the desired mesenchymal stem cells.

[0095] Next, the mesenchymal stem cells are formed into a 3D shape and then cultured in a medium. (S200)

[0096] According to one embodiment of the present invention, the medium may include, but is not limited to, a myogenic specification medium.

[0097] According to one embodiment of the present invention, the progenitor cell growth factor may include, but is not limited to, fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF1), and hepatocyte growth factor (HGF).

[0098] The above-mentioned myogenic specification medium may include growth factors such as hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1), and fibroblast growth factor (FGF). These growth factors are known to promote proliferation and differentiation of muscle cells.

[0099] The above-mentioned source specification medium may also include nutritional supplements such as insulin-transferrin-selenium (ITS), non-essential amino acids (NEAA), and β-mercaptoethanol. Additional supplements such as antibiotics, serum replacement agents, and vitamins may be added as needed.

[0100] Next, it induces proliferation of mesenchymal stem cells (S300).

[0101] According to one embodiment of the present invention, the step of inducing proliferation may be, but is not limited to, inducing myogenic proliferation of the axial mesenchymal stem cells.

[0102] During the above-described process of inducing myogenic proliferation, efficient differentiation of myogenic progenitor markers can occur, and enhanced expression of striated muscle fibers can be observed. This confirms that the method described herein can generate human skeletal muscle organoids more efficiently than conventional methods.

[0103] According to one embodiment of the present invention, the step of inducing the myogenic proliferation may be performed by removing the fibroblast growth factor (FGF), but is not limited thereto.

[0104] FGF is one of the major growth factors that regulates the proliferation of muscle progenitor cells, and its removal can play a crucial role in controlling the differentiation direction of muscle cells. Even after FGF is removed, other growth factors, such as hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF1), can remain in the medium, supporting the survival and differentiation of muscle progenitor cells. Changes in the composition of growth factors can affect the proliferation status and differentiation stage of muscle progenitor cells.

[0105] Next, it induces maturation of mesenchymal stem cells (S400).

[0106] According to one embodiment of the present invention, the step of inducing maturation may be, but is not limited to, inducing myogenic maturation of the axial mesenchymal stem cells.

[0107] Through the above-described myogenic maturation process, not only homogeneous muscle cells but also muscle stem cells of various stages, mature fibers, neurons, and FAPs can be formed, and as a result, skeletal muscle organoids similar to the structure of muscle fibers in vivo can be produced.

[0108] According to one embodiment of the present invention, the step of inducing the above-mentioned myogenic maturation may be performed by removing the above-mentioned cell growth factor (HGF) and insulin-like growth factor 1 (IGF1) and adding a TGF-β inhibitor, but is not limited thereto.

[0109] Deletion of these growth factors and addition of TGF-β inhibitors can play a significant role in promoting terminal differentiation and maturation of muscle cells. TGF-β inhibitors include SB431542, A83-01, and RepSox, which can be used alone or in combination. Inhibition of the TGF-β signaling pathway is known to promote muscle cell differentiation and induce myofiber formation.

[0110]

[0111] In addition, the second aspect of the present invention provides a method for producing a sarcopenic skeletal muscle organoid, which method for producing a skeletal muscle organoid according to the first aspect of the present invention further comprises a step of treating the skeletal muscle organoid with a chemotherapy agent or a proinflammatory cytokine.

[0112] Regarding the method for producing a sarcopenic skeletal muscle organoid according to the second aspect of the present application, detailed descriptions of parts overlapping with the first aspect of the present application are omitted, but even if the descriptions are omitted, the contents described in the first aspect of the present application can be equally applied to the third aspect of the present application.

[0113] According to one embodiment of the present invention, muscle loss of the skeletal muscle organoid may be caused by treatment with the chemotherapy agent or proinflammatory cytokine, but is not limited thereto.

[0114] Using the sarcopenic hSkMO formed through the above chemical anticancer treatment, the phenotypes related to sarcopenia (muscle atrophy, apoptotic muscle fibers, muscle cell aging, muscle cell DNA damage, MuSC depletion) can be confirmed, and this can be utilized for research related to sarcopenia and development of sarcopenia treatment agents.

[0115] In sarcopenic skeletal muscle organoids formed through the above-mentioned proinflammatory cytokine treatment, phenotypes associated with sarcopenia, such as activation of the TNF-α / NF-κB pathway, decreased muscle fiber size, decreased number of neuromuscular junctions, and decreased satellite cell function, can be observed. In particular, chronic proinflammatory cytokine treatment can induce persistent muscle degeneration, mimicking the characteristics of age-related sarcopenia, and can be utilized for evaluating the efficacy of sarcopenia treatments and studying their mechanisms of action.

[0116] According to one embodiment of the present invention, the chemotherapeutic agent may include, but is not limited to, one selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, raltitrexed, and combinations thereof.

[0117] According to one embodiment of the present invention, the proinflammatory cytokine may include, but is not limited to, one selected from the group consisting of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ), Transforming Growth Factor-β (TGF-β), and combinations thereof.

[0118]

[0119] In addition, the third aspect of the present invention provides a method for screening for a treatment agent for sarcopenia, comprising the steps of treating a skeletal muscle organoid manufactured according to the first aspect of the present invention with a chemotherapeutic agent or a proinflammatory cytokine and a candidate agent for a treatment agent for sarcopenia; and identifying a phenotype associated with sarcopenia in the skeletal muscle organoid.

[0120] Regarding the method for screening for a treatment agent for sarcopenia according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted. However, even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the third aspect of the present invention.

[0121] In the step of confirming a phenotype related to sarcopenia in the above skeletal muscle organoid, if a phenotype related to sarcopenia is not confirmed, it can be determined that the candidate for the sarcopenia treatment is effective in treating sarcopenia, but is not limited thereto.

[0122]

[0123] Additionally, the fourth aspect of the present invention provides a skeletal muscle organoid prepared by the method according to the first aspect of the present invention.

[0124] Regarding the skeletal muscle organoid according to the fourth aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the fourth aspect of the present invention.

[0125] According to one embodiment of the present invention, the skeletal muscle organoid is TITIN + muscle fiber, PAX7 + Muscle stem cells, MyHC + skeletal muscle, TUJ1 + Neurons, PDGFRα + May include, but is not limited to, fibroadipogenic precursor cells (FAPs).

[0126] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0127]

[0128] [Example 1-1] Production of skeletal muscle organoids (hSkMO)

[0129] Female H9 (WA09; pass 35), male H1 (WA09; pass 32), and CHA-SCNT-PSC-18 (Korean Stem Cell Registration No. hES12019001), which were previously established by the inventors of the present invention, were used as hPSCs for producing skeletal muscle organoids.

[0130] First, hPSCs were cultured on Matrigel-coated plates (Corning) in mTeSR medium (Stem Cell Technologies) at 37°C and 5% CO2. The cell line was verified to have a normal karyotype and was free of mycoplasma. The hPSC medium was changed daily, and cells were passaged every 5–6 days.

[0131] Next, to induce paraxial mesoderm stem cells (PMSCs), hPSCs were dissociated into single cells using TrypLE (Thermo Fisher Scientific) solution and seeded at 2.3 x 10 per well in 6-well plates (Thermo Fisher Scientific) in mTeSR supplemented with 1 μM Rho kinase (ROCK) inhibitor (Tocris). 5 3.0 x 10 5 Dog cells were inoculated.

[0132] On day 1, cells were plated in PMSC induction medium for up to day 3. PMSC induction medium consisted of DMEM / F12 (Gibco) supplemented with 1% insulin-transferrin-selenium (ITS; Life Technologies), 1% nonessential amino acids (NEAA; Thermo Fisher Scientific), 0.25% penicillin / streptomycin (Gibco), 3 μM CHIR99021 (Stem Cell Technologies), and 0.5 μM LDN193189 (Stemgent).

[0133] PMSCs generated from hPSCs were dissociated using TrypLE solution to generate single-cell suspensions. On day 3 of organoid formation, PMSCs (10,000–15,000 / well, depending on the cell line) were plated in ultra-low binding V-shaped 96-well plates in DMEM / F12 (Gibco) supplemented with 1% insulin-transferrin-selenium (ITS; Life Technologies), 1% nonessential amino acids (NEAA; Thermo Fisher Scientific), 0.25% penicillin / streptomycin (Gibco), 3 μM CHIR99021 (Stem Cell Technologies), 0.5 μM LDN193189 (Stemgent), 20 ng / mL FGF2 (Peprotech), and 1 μM Rho kinase (ROCK) inhibitor (Tocris). The ROCK inhibitor was removed the following day.

[0134] On day 6, organoids were cultured in myogenic specification medium consisting of DMEM / F12, 15% KSR (Thermo Fisher Scientific), 1% NEAA, 1% penicillin / streptomycin, 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific) containing 10 ng / mL HGF (Peprotech), 2 ng / mL IGF1 (Sigma-Aldrich), and 20 ng / mL FGF2.

[0135] On day 9, FGF2 was removed to induce myogenic proliferation.

[0136] On day 30, HGF and IGF1 were removed, and 5 μM SB431542 (Sigma-Aldrich) was added to induce myogenic maturation for up to day 50.

[0137] From day 50, organoids were additionally maintained in basal medium without SB431542.

[0138] [Example 1-2] Modeling of sarcopenic muscle organoids

[0139] We modeled chemotherapy-induced sarcopenia using mature hSkMO, which were cultured for up to 100 days using the skeletal muscle organoids (hSkMO) of Example 1-1. First, cisplatin was added to basal medium without growth factors at 100 μM for 48 hours. The medium was changed daily, and cisplatin was removed 48 hours after chemotherapy, and hESC-MPC-EVs (2×108 particles) were added for 72 hours.

[0140] [Example 2] Production of skeletal muscle organoids (hSkMO)

[0141] Human pluripotent stem cells were dissociated into single cells using TrypLE. After counting the dissociated single cells, they were seeded at 62,500–75,000 / cm in 12-well plates coated with Matrigel (Corning). 2 Plated with a density of .

[0142] On day 1, cells were plated in mTeSR medium (Stem Cell Technologies) supplemented with 1 μM Rock inhibitor (Tocris Bioscience). On day 2, Rock inhibitor was removed, and cells were differentiated until day 3 in paraxial mesoderm induction medium (PIM) supplemented with 3 μM CHIR99021 (Stem Cell Technologies) and 0.5 μM LDN193189 (Stemgent). PIM consisted of DMEM / F12 (Gibco) supplemented with 1% insulin-transferrin-selenium (ITS; Life Technologies), 1% nonessential amino acids (NEAA; Thermo Fisher Scientific), and 0.25% penicillin / streptomycin (P / S; Gibco). The medium was changed daily.

[0143] Differentiated paraxial mesenchymal stem cells were dissociated into a single-cell suspension using TrypLE. On day 3, dissociated paraxial mesenchymal stem cells (5,000–10,000 / well) were plated in ultra-low binding V-bottom 96-well plates (Corning) with PIM medium containing 1 μM Rock inhibitor, 3 μM CHIR99021, and 0.5 μM LDN193189. The initial volume of each well was 100 μl.

[0144] The following day, Rock inhibitor was removed, and cells were maintained in PIM medium supplemented with 3 μM CHIR99021, 0.5 μM LDN193189, and 20 ng / ml bFGF (Peprotech) until day 6. The medium was changed daily.

[0145] On day 6, hSkMOs were cultured in myogenic differentiation medium (MDM) supplemented with 10 ng / mL HGF (Peprotech), 2 ng / mL IGF1 (Sigma-Aldrich), and 20 ng / mL bFGF until day 9. MDM was composed of DMEM / F12 medium containing 15% KSR (Thermo Fisher Scientific), 1% NEAA, 1% P / S, and 0.1 mM β-mercaptoethanol.

[0146] On day 9, hSkMOs were transferred to a 6-well plate equipped with an orbital shaker and cultured in MDM supplemented with 10 ng / mL HGF and 2 ng / mL IGF1. From day 30, they were cultured in MDM supplemented with 5 μM SB431542 (Sigma) until day 50.

[0147] After 50 days, hSkMOs were cultured in muscle maturation medium (MMM). MMM was composed of DMEM / F12 medium supplemented with 1% N2 (Gibco), 2% B27 (vitamin A-free), 1% GlutaMAX, 1% NEAA, 1% P / S, and 0.1% β-mercaptoethanol.

[0148] [Comparative Example] Conventional 3D

[0149] hPSCs were dissociated into single cells using TrypLE (Thermo Fisher Scientific) solution and 1.6 X 10 4 Cells were seeded in ultra-low binding V-shape 96-well plates of mTeSR supplemented with 1 μM Rho kinase (ROCK) inhibitor (Tocris).

[0150] After 24 h, the PMSC induction medium was replaced with DMEM / F12 (Gibco) supplemented with 1% insulin-transferrin-selenium (ITS; Life Technologies), 1% nonessential amino acids (NEAA; Thermo Fisher Scientific), 1% penicillin / streptomycin (Gibco), 3 μM CHIR99021 (Stem Cell Technologies), and 0.5 μM LDN193189 (Stemgent).

[0151] After 3 days, 20 ng / ml FGF2 (Gibco) was added to the PMSC induction medium to induce posterior mesoderm differentiation.

[0152] After 3 days, they were cultured in myogenic specification medium consisting of DMEM / F12, 15% KSR (Thermo Fisher Scientific), 1% NEAA, 1% penicillin / streptomycin, 10 ng / mL HGF (Peprotech), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 2 ng / mL IGF1 (Sigma-Aldrich), and 20 ng / mL FGF2.

[0153] After 24 hours, the organoids were embedded in matrigel and transferred to a 6-well plate, and cultured for a long period of time in an orbital shaker to induce skeletal muscle maturation.

[0154]

[0155] [Experimental Example 1] Analysis of skeletal muscle organoid characteristics according to Example 1-1

[0156] To optimize the skeletal muscle organoid culture protocol, we used a directed differentiation protocol to derive paraxial mesodermal stem cells (PMSCs) from hPSCs in adherent culture. Using WNT activation (CHIR99021) and bone morphogenetic protein (BMP) inhibition (LDN193189) signaling for 3 days, we promoted paraxial mesoderm differentiation. These PMSCs were characterized by the expression of the paraxial mesoderm markers MSGN1 and TBX6 (Figures 3A and 3B). After differentiation into PMSCs in adherent culture, 3D spheroids were generated in low-attachment V-shaped 96-well plates. For further characterization, we performed immunofluorescence analysis of PMSCs and gene expression analysis on day 6. Based on these results, we demonstrated that our 2D3D two-step method is more robust and accelerates the differentiation protocol toward the paraxial mesoderm lineage than conventional 3D methods (Figure 2).

[0157] After 6 days, the organoids were supplemented with some growth factors to induce mesodermal differentiation. Hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF1), and fibroblast growth factor (FGF) were added until day 9 for muscle specification, and hSkMO were cultured in HGF- and IGF1-supplemented medium to expand myogenic cells until day 30. This protocol resulted in efficient differentiation of myogenic progenitor markers, as characterized by gene expression of myogenic progenitor markers (PAX3 and PAX7) and myogenic differentiation markers (MYOD and MYOG) at days 20 and 30 of differentiation (Figs. 3c and 3d). In addition, further enhanced expression of striated muscle fibers was observed by immunofluorescence analysis of the TITIN marker (Figs. 3e and 3f). This confirmed that human skeletal muscle organoids could be generated more efficiently than conventional methods.

[0158] At day 50, many TITIN+ myofibers were observed in hSkMO, suggesting that the organoids were undergoing myogenic maturation (Fig. 3e). To induce the expression of more mature myofibers, hSkMO were cultured for a long period of time, and finally, at day 100, hSkMO with a structure similar to in vivo myofibers could be detected (Fig. 4a). In addition, PAX7+ quiescent muscle stem cells were found beneath the basal lamina of hSkMO, similar to the structure of in vivo myofibers (Fig. 4b). Then, to characterize maturation over time, the muscle stem cell marker PAX7 was stained together with the cell proliferation marker Ki67 or the activated muscle stem cell marker MYOD (Figs. 4c and 4e). For further analysis, proliferating muscle stem cells (PAX7+ / Ki67+) and quiescent muscle stem cells (PAX7+ / Ki67-) were quantified. We found a time-dependent decrease in the PAX7+ / Ki67+ ratio at days 30, 50, and 100. We also quantified quiescent muscle stem cells (PAX7+ / MYOD-), activated muscle stem cells (PAX7+ / MYOD+), and differentiated muscle stem cells (PAX7- / MYOD+), and found a time-dependent decrease in the PAX7+ / MYOD+ ratio and the PAX7MYOD+ ratio at days 50 and 100. To further characterize maturation, we analyzed the expression pattern of the skeletal muscle fiber marker (MyHC) at days 30 and 100 (Fig. 4g). The presence of thicker fibers and sarcomatous bands indicates muscle maturation, and we found an increase in mature myofibers. This confirmed the time-dependent maturation of organoids by long-term culture.

[0159] To investigate the presence of diverse heterogeneous cell populations in hSkMO, we confirmed the expression of TUJ1+ neurons along with MyHC+ skeletal muscle in 50-day hSkMO (Fig. 4i). We found that neuromuscular and skeletal muscle regions were separated in hSkMO. We also confirmed the presence of fibroadipogenic progenitor cells (FAPs), which contribute to muscle regeneration and homeostasis in skeletal muscle tissue. We then detected colocalization of platelet-derived growth factor receptor A (PDGFRα)-positive FAPs with PAX7+ muscle stem cells (Fig. 4j). Based on these results, we confirmed that self-organizing hSkMOs are composed of not only homogeneous muscle cells but also muscle stem cells at various stages, mature fibers, neurons, and FAPs during maturation.

[0160] Through this, it was confirmed that the skeletal muscle organoids manufactured through the method according to the present invention not only reproduce embryonic muscle formation but also are an optimized 3D skeletal muscle organoid model containing heterogeneous cell populations such as muscle cells of various stages, mature myofibers, and neurons and fibroadipogenic precursor cells.

[0161]

[0162] [Experimental Example 2] Modeling of Sarcopenic Muscle Organoids According to Example 1-2 Using Chemotherapy and Treatment Effects through hESC-MPC-EV Treatment

[0163] Considering the mature hSkMO cultured for a long time, we next targeted hSkMO for sarcopenia studies. Age-related sarcopenia can be induced by chemotherapy. Cisplatin (CIS) is an effective chemotherapy widely used to treat various types of cancer. However, cisplatin can cause serious side effects, including nephrotoxicity, neurotoxicity, ototoxicity, and rapid muscle loss. To model sarcopenic muscle organoids using cisplatin treatment, we induced muscle loss in hSkMO and evaluated the therapeutic effect using hESC-MPC-EVs in chemotherapy-induced hSkMO (Figure 5). We then confirmed chemotherapy-induced muscle atrophy using immunofluorescence analysis. Numerous atrophic muscle fibers were detected in the CIS group, but not in the CIS + EV group (Figures 6a and 6c). By quantifying the average fiber diameter, we found that hESC-MPC-EV treatment prevented myofiber atrophy in chemotherapy-induced sarcopenic skeletal muscle organoids (Fig. 6b). Quantification of muscle fiber cross-sectional area (CSA) also demonstrated a significant protective effect of EVs against cisplatin-induced muscle atrophy compared to the control group (Fig. 6d). While the number of MYOG+ myocytes was also reduced in the CIS group, hESC-MPC-EVs were able to prevent cisplatin-induced myocyte loss (Fig. 6e). These results demonstrate the establishment of a model of muscle loss and atrophy using chemotherapy-induced hSkMO and the protective effect of hESC-MPC-EVs against chemotherapy. To investigate other adverse effects of chemotherapy, markers of apoptosis, cellular senescence, and damaged DNA (cleaved caspase 3, p21, γH2AX) were identified by immunofluorescence analysis (Figs. 7a, 7c, and 7e). In the CIS group, numerous apoptotic myofibers, a significant number of p21+ myocytes, and γH2AX+ myocytes were quantified and evaluated.The protective effect of hESC-MPC-EVs was also confirmed against these pathogenic phenotypes (Figures 7b, 7d, and 7f). The proportion of MuSCs was then assessed. In the CIS group, MuSC depletion was detected by immunofluorescence analysis. Interestingly, EV treatment effects were observed only in the quiescent muscle stem cell population (Figures 7g and 7h). Based on these data, we recapitulated the sarcopenia-related phenotypes (muscle atrophy, apoptotic myofibers, myocyte senescence, myocyte DNA damage, and MuSC depletion) using chemotherapy-induced hSkMO. From a therapeutic perspective, we found that hESC-MPC-EVs have the ability to protect hSkMO from chemotherapy-induced sarcopenia.

[0164] Through this, we were able to confirm that the skeletal muscle organoids according to the present invention can model sarcopenic muscles using chemotherapy, and immunofluorescence analysis confirmed the recapitulation of sarcopenia-related phenotypes (muscle atrophy, apoptotic myofibers, myocyte senescence, myocyte DNA damage, MuSC depletion) in chemotherapy-induced hSkMO, and confirmed that hESC-MPC-EVs have the ability to protect hSkMO from chemotherapy-induced sarcopenia.

[0165]

[0166] [Experimental Example 3] Stepwise differentiation for efficient generation of human skeletal muscle organoids according to Example 2

[0167] We previously reported a method for generating human skeletal muscle organoids (hSkMOs) from human pluripotent stem cells (hPSCs), demonstrating the temporal characteristics of skeletal muscle development and the sustainable regenerative capacity of satellite cells upon chemical insult. While a direct induction approach for paraxial mesoderm differentiation and subsequent muscle specification successfully achieved the characteristics of skeletal muscle development, further improvements in the culture system were needed. Therefore, we optimized and modified the previous method to achieve more homogeneous paraxial mesoderm stem cell differentiation. We used a stepwise differentiation protocol, starting with two-dimensional (2D) paraxial mesoderm induction, transitioning to three-dimensional (3D) muscle specification, and ending with a maturation culture system. hSkMOs gradually grew to approximately 2 mm in diameter by day 100 (Fig. 8a).

[0168] First, hPSCs were dissociated into single cells and seeded at 2 × 10 in 12-well plates. 5Cells were seeded at 1 × 10 cells / well. The following day, the cells were exposed to a WNT activator (CHIR99021) and a bone morphogenetic protein (BMP) inhibitor (LDN193189) for 3 days to promote paraxial mesoderm differentiation. On day 3, we observed rapid upregulation of T / Brachyury, T-Box transcription factor 6 (TBX6), and MSGN1, key transcriptional regulators of posterior presomitic mesoderm (pPSM) specification. To characterize the differentiated paraxial mesoderm stem cells, immunocytochemical analysis was performed on day 3 using antibodies against paraxial mesoderm and neural mesoderm precursor markers. As a result, it was observed that most cells were T / Brachyury- and TBX6-positive (+) paraxial mesoderm cells (T / Brachyury+, 82.04%; TBX6+, 78.18%) (Fig. 8b), and SOX2 double-positive neuromesoderm cells showed a relatively low proportion (T / Brachyury+ and SOX2+, 15.91%; TBX6+ and SOX2+, 11.45%) (Fig. 8b).

[0169] Because somites develop from paraxial mesoderm cells through anterior presomitic mesoderm (aPSM) specification, paraxial mesoderm stem cells were dissociated and seeded into V-bottom ultra-low adhesion 96-well plates for transition to a 3D culture system (Fig. 8a). Fibroblast growth factor 2 (FGF2) was supplemented along with continuous CHIR99021 and LDN193189 treatment. On day 6, organoids were exposed to hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) to promote somite specification (Fig. 8a). As a result, upregulation of somite formation markers such as PAX3 and MEOX2 was observed in the organoids. Immunohistochemical analysis confirmed that most cells were PAX3+ (Fig. 8c), and the expression of T / Brachyury, TBX6, and MSGN1 was drastically reduced (Fig. 8c).

[0170] Next, organoids were transferred to 6-well tissue culture plates and cultured on an orbital shaker from day 9 until the day of analysis (Fig. 8a). The use of an orbital shaker significantly improved the culture conditions of the organoids. Organoids were cultured with HGF and IGF for muscle proliferation and differentiation, and FGF2 treatment was omitted (Fig. 8a). To characterize the developing hSkMOs, immunohistochemical analysis was performed on day 20 using antibodies against muscle progenitors. 70.41% of PAX7+ cells co-labeled with Ki67 (Fig. 8d), and approximately 42.30% of PAX7+ muscle progenitors showed double positivity with MYOD, while the remaining 57.70% of cells did not express MYOD (Fig. 8e). Additionally, 5.91% of PAX7+ cells were co-labeled with MYOG, suggesting that proliferation, activation, and differentiation of PAX7+ muscle progenitors in hSkMOs were observed (Fig. 8f).

[0171] These results demonstrate that the in vitro hSkMOs of the present invention successfully reproduce the stepwise developmental characteristics of paraxial mesoderm, prostomes, and somite differentiation.

[0172]

[0173] [Experimental Example 4] Analysis of structural characteristics and functional neuromuscular junction of mature human skeletal muscle organoids according to Example 2

[0174] Human skeletal muscle organoids (hSkMOs) are composed primarily of dermal myocytes and a small number of neuromesoderm neurons. The spatial and structural organization of muscle fibers and their functional connections to spinal cord-derived motor neurons via neuromuscular junctions (NMJs) are key characteristics of skeletal muscle tissue (Fig. 9a). During skeletal muscle development, muscle cells are activated in response to various signals, proliferate, and differentiate into myocytes. These myocytes align to form multinucleated myotubes, an early step in muscle fiber formation (Fig. 9a).

[0175] To investigate the anatomical structure of skeletal muscle in hSkMOs, we assessed the expression of key markers of muscle and nervous system development at days 50 and 100. Immunohistochemical analysis revealed robust expression of myosin heavy chain (MyHC; a mature myotube marker) and TUJ1+ (a neural marker) along with myofibers in both day 50 and day 100 hSkMOs (Fig. 9b, c). Notably, MyHC+ myofibers continued to grow, reaching a thickness twice that at day 100 compared to day 50, indicating that prolonged culture promotes greater myofiber maturation (Fig. 9d).

[0176] Satellite cells, stem cells that play a crucial role in skeletal muscle growth, repair, and regeneration, are located between the basement membrane and the fascicle of muscle fibers. To determine the structural maturity of muscle fibers, immunohistochemical analysis was performed using antibodies against human laminin. MyHC+ muscle fibers were surrounded by a continuous sheath of the basement membrane labeled with human laminin, and satellite cells were found beneath the basement membrane (Fig. 9e, f). These results demonstrated that PAX7+ cells (representing satellite cells) were located beneath the basement membrane in a quiescent state.

[0177] Immunohistochemical analysis of hSkMOs to investigate the neural lineage characteristics revealed that approximately 10.5% of hSkMOs contained neurons (Fig. 9c), and the cells were identified as spinal cord-derived motor neurons expressing choline acetyltransferase (ChAT), an enzyme that synthesizes acetylcholine (ACh) (Fig. 10a). GFAP+ glial cells were also observed in hSkMOs on day 100 (Fig. 10b). In the basement membrane of muscle fibers, abundant α-bungarotoxin (αBTX) immunolabeled ACh receptor clusters connected by TUJ1+ processes were detected, confirming the formation of NMJs, and S100+ Schwann cells were also detected around muscle fibers (Fig. 10c).

[0178] The formation of NMJs in muscle fibers was confirmed by ultrastructural analysis using electron microscopy. Well-developed skeletal muscle was observed, exhibiting organized sarcomeres with distinct bands and mitochondria within dense muscle tissue, and synaptic vesicles at presynaptic axon terminals were detected in specific regions of the basement membrane along the muscle fibers (Fig. 10d).

[0179] hSkMOs exhibited spontaneous contractions during long-term culture, and measurements of contractile activity in organoids confirmed their potential as a powerful model for studying neuromuscular interactions in vitro (Fig. 10e). Calcium imaging revealed calcium influx in morphologically distinct spinal cord-derived motor neurons and muscle fibers (Fig. 10e). Water-immersion optics for electrophysiology allowed the differentiation of hSkMO regions containing neurons or muscle fibers, and the organoids exhibited functional muscle-nerve connections in the form of spontaneous postsynaptic potentials detected by whole-cell patch clamp recordings from muscle fibers (Fig. 10f).

[0180] These results demonstrate that the hSkMOs of the present invention have the characteristics of spatially and structurally well-organized myofibers expressing mature myotubule proteins and basement membrane markers, and form functionally mature neuromuscular junctions.

[0181]

[0182] [Experimental Example 5] Muscle damage and regeneration effects in hSkMOs according to Example 2 through the TNF-α / NF-κB pathway

[0183] Sarcopenia is an age-related condition characterized by a progressive loss of muscle mass, strength, and function. A major contributing factor to sarcopenia is chronic inflammation induced by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α). Previous studies have shown that activation of the TNF-α and nuclear factor-kappa B (NF-κB) pathways promotes age-related changes and is associated with increased muscle breakdown, decreased protein synthesis, and impaired muscle regeneration in sarcopenia due to muscle atrophy.

[0184] To investigate the acute inflammatory response and intrinsic regenerative capacity of 100-day human skeletal muscle organoids (hSkMOs) with mature muscle tissue, the organoids were exposed to TNF-α treatment for 2 days, and muscle damage and regeneration were analyzed on days 0, 3, and 7 post-treatment (Fig. 11a). Western blot analysis revealed that TNF-α / NF-κB pathway-related factors, such as NF-κB p65, IκB-α, and AKT, were highly phosphorylated on day 3 of acute TNF-α treatment and rapidly decreased on day 7 (Fig. 11b). In addition, the expression of myogenin (MYOG), a muscle differentiation-related gene, was significantly increased (Fig. 11b).

[0185] To evaluate changes in satellite cell status, immunohistochemical analysis was performed using antibodies to PAX7, MYOD, MYOG, and Ki67. The results showed that approximately 13.62% of PAX7+ / MYOD+ activated satellite cells rapidly increased on day 0 and decreased over time (Fig. 11c). In addition, 25.67% of PAX7+ / Ki67+ proliferating satellite cells were observed (Fig. 11d), and the number of MYOG+ differentiating muscle cells increased on days 3 and 7 (Fig. 11e).

[0186] To evaluate the effect of TNF-α on muscle atrophy, muscle fiber size was assessed using the myosin heavy chain (MyHC) marker. Muscle fiber size was significantly reduced on days 0 and 3 after treatment. However, muscle fibers recovered to a size similar to that of the control group (Fig. 11f). Analysis of the number of α-bungarotoxin (αBTX) + neuromuscular junctions (NMJs) revealed a transient decrease in the number of NMJs after TNF-α treatment, but this gradually recovered to a level similar to that of the control group over time (Fig. 11f).

[0187]

[0188] [Experimental Example 6] Testosterone's Muscle Wasting Relief Effect

[0189] Based on the acute proinflammatory effects and spontaneous regenerative capacity of mature hSkMOs according to Example 2, we evaluated whether testosterone treatment of hSkMOs prevented muscle atrophy or ameliorated progressive muscle mass loss. To this end, we modeled progressive muscle loss due to aging through chronic TNF-α treatment (Fig. 12a). This approach aimed to mimic the persistent inflammatory effects seen in sarcopenia and chronic muscle degeneration.

[0190] Previous studies have reported that androgen receptors (ARs) are expressed in skeletal muscle cells, including myocytes and satellite cells. In the present study, ARs were primarily expressed in PAX7+ satellite cells, and their expression was significantly increased by testosterone treatment (Fig. 12b). Furthermore, the proportion of PAX7+ satellite cells significantly increased after co-treatment with TNF-α and testosterone compared to TNF-α treatment alone (Fig. 12b).

[0191] A decrease in the size of hSkMOs treated with TNF-α was observed, whereas testosterone-treated hSkMOs showed a relatively similar size to control hSkMOs (Fig. 12c). To investigate whether this prevention of muscle atrophy was due to satellite cell proliferation and differentiation to ensure muscle homeostasis and myogenesis, hSkMOs were immunostained with antibodies against PAX7, MYOD, Ki67, MYOG, and MyHC.

[0192] As a result, the proportion of activated satellite cells (PAX7+ / MYOD+, 7.97%; PAX7+ / Ki67+, 7.03%) was significantly increased in hSkMOs co-treated with TNF-α and testosterone compared to hSkMOs treated with TNF-α alone (Fig. 13a, b). In addition, MYOG+ myocytes (21.23%) were significantly increased in hSkMOs co-treated with TNF-α and testosterone compared to MYOG+ myocytes (5.72%) in hSkMOs treated with TNF-α alone, and the size of MyHC+ myofibers was also increased by testosterone treatment (Fig. 13c).

[0193] Analysis of αBTX+ NMJs revealed that chronic treatment with TNF-α alone resulted in a rapid decrease in NMJs, but co-treatment with testosterone effectively prevented this decrease, maintaining the number of NMJs at a level similar to that of control hSkMOs (Fig. 13d).

[0194] These results suggest that testosterone may prevent muscle atrophy induced by chronic inflammation by accelerating satellite cell activation and differentiation and preserving NMJ integrity.

[0195]

[0196] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0197] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A step of directly differentiating human pluripotent stem cells (hPSCs) to induce paraxial mesoderm stem cells (PMSCs); A step of forming the above-mentioned axial mesenchymal stem cells into a 3D shape and then culturing them in a medium; A step of inducing proliferation of the above-mentioned axial mesenchymal stem cells; and A step of inducing maturation of the above-mentioned axial mesenchymal stem cells; including, Method for producing skeletal muscle organoids.

2. In paragraph 1, The step of inducing the above-mentioned axial mesenchymal stem cells is performed using a WNT activator and a BMP (bone morphogenetic protein) inhibitor. Method for producing skeletal muscle organoids.

3. In paragraph 1, The above medium includes a myogenic specification medium, Method for producing skeletal muscle organoids.

4. In paragraph 3, The above-mentioned progenitor cell growth factor comprises fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF1), and fibroblast growth factor (FGF). Method for producing skeletal muscle organoids.

5. In paragraph 1, The step of inducing the above proliferation is to induce myogenic proliferation of the above-mentioned axial mesenchymal stem cells. Method for producing skeletal muscle organoids.

6. In paragraph 5, The step of inducing the above-mentioned myogenic proliferation is performed by removing the above-mentioned fibroblast growth factor (FGF). Method for producing skeletal muscle organoids.

7. In paragraph 1, The step of inducing the above maturation is to induce the myogenic maturation of the above-mentioned axial mesenchymal stem cells. Method for producing skeletal muscle organoids.

8. In paragraph 7, The step of inducing the above-mentioned myogenic maturation is performed by removing the above-mentioned cell growth factor (HGF) and insulin-like growth factor 1 (IGF1) and adding a TGF-β inhibitor. Method for producing skeletal muscle organoids.

9. In the method for producing a skeletal muscle organoid according to paragraph 1, A method of treating the skeletal muscle organoid with a chemotherapy agent or pro-inflammatory cytokine, comprising: Method for producing sarcopenic skeletal muscle organoids.

10. In paragraph 9, Muscle loss in the skeletal muscle organoid occurs due to treatment with the above chemotherapy agent or proinflammatory cytokine. Method for producing sarcopenic skeletal muscle organoids.

11. In paragraph 9, The above chemotherapeutic agent comprises a drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, raltitrexed, and combinations thereof. Method for producing sarcopenic skeletal muscle organoids.

12. In paragraph 9, The above-mentioned proinflammatory cytokines include those selected from the group consisting of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ), Transforming Growth Factor-β (TGF-β), and combinations thereof. Method for producing sarcopenic skeletal muscle organoids.

13. A step of treating a skeletal muscle organoid manufactured according to paragraph 1 with a chemotherapy agent or pro-inflammatory cytokine and a candidate agent for the treatment of sarcopenia; and A step of confirming a sarcopenia-related phenotype in the above skeletal muscle organoid; including, A method for screening for treatments for sarcopenia.

14. Skeletal muscle organoids manufactured by the method according to paragraph 1.

15. In paragraph 14, The above skeletal muscle organoids are TITIN + muscle fiber, PAX7 + Muscle stem cells, MyHC + skeletal muscle, TUJ1 + Neurons, PDGFRα + Containing fibroadipogenic precursor cells (FAPs), Skeletal muscle organoids.

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