Co-differentiated cell therapy

Abstract

The present invention relates to a codifferentiated cell therapy agent, and more specifically, the cell therapy agent of the present invention is manufactured through the codifferentiation of immature myotubes that have been differentiated in vitro, thereby allowing the proliferation and early terminal differentiation processes of the transplanted cells at the transplant site after transplantation to be omitted. It provides a solution to the problems of satellite stem cells, which have quantitative limitations, and skeletal muscle progenitor cells, which have the disadvantage of low success rates for proliferation and early terminal differentiation of the transplanted cells at the transplant site after the transplantation process, and can increase the contractility of the skeletal muscle (physiological activity of the skeletal muscle) by enhancing the ability to supply calcium for skeletal muscle contraction at the transplant site.

Description

Technology Field

[0001] The present invention relates to a codifferentiated cell therapy agent. Background Technology

[0003] Skeletal muscle cell transplantation is being attempted in the affected area to address skeletal muscle atrophy caused by genetic or acquired skeletal muscle diseases, skeletal muscle atrophy occurring during the long-term treatment of other diseases (especially cancer treatment), loss of skeletal muscle due to fire or physical accidents, skeletal muscle atrophy resulting from abnormalities or loss of nerves innervating the skeletal muscles, skeletal muscle atrophy due to insufficient nutrient intake such as malnutrition or fasting, skeletal muscle atrophy due to low frequency of use such as maintaining a sedentary posture for long periods due to occupation or lifestyle, skeletal muscle atrophy caused by zero gravity conditions such as in astronauts, or sarcopenia occurring during the natural aging process.

[0004] In this process, 'satellite stem cells' isolated from tissue or 'myoblasts' obtained through cell culture are used as transplant cells; however, satellite cells are difficult to obtain in large quantities due to their inherently small volume, and myoblasts have the disadvantage of a low success rate for cell proliferation and early terminal differentiation at the transplant site after transplantation. Prior art literature

[0006] 1. Briggs, D.; Morgan, JE Recent progress in satellite cell / myoblast engraftment—Relevance for therapy. FEBS J. 2013, 280, 4281-4293.2. Schiaffino, S.; Dyar, K.A.; Ciciliot, S.; Blaauw, B.; Sandri, M. Mechanisms regulating skeletal muscle growth and atrophy. FEBS J. 2013, 280, 4294-4314.3. Yin, L.; Li, N.; Jia, W.; Wang, N.; Liang, M.; Yang, X.; Du, G. Skeletal muscle atrophy: From mechanisms to treatments. Pharmacol. Res. 2021, 172, 105807.4. Sacco, A.; Doyonnas, R.; Kraft, P.; Vitorovic, S.; Blau, HM Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008, 456, 502-506.5. Montarras, D.; Morgan, J.; Collins, C.; Relaix, F.; Zaffran, S.; Cumano, A.; Partridge, T.; Buckingham, M. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005, 309, 2064-2067. The problem to be solved

[0007] The objective of the present invention is to provide a cell therapy agent with a high transplant success rate for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy, a method for manufacturing the same, and uses thereof. means of solving the problem

[0009] To achieve the above objective, the present invention [compares] skeletal muscle progenitor cells (myoblasts) differentiated in vitro for 5 days with control skeletal muscle cells (myotubes).

[0010] The expression of MyoD, MyHC II, RyR1, SERCA1, and STIM1 proteins increases, and

[0011] As the thickness of the cell increases,

[0012] It provides mature co-differentiated myotubes with increased calcium supply from inside or outside the cell for skeletal muscle contraction.

[0013] The present invention also provides a method for producing a mature co-differentiated skeletal muscle cell (D2-to-D3 mature co-differentiation myotube), comprising the step of using an immature differentiated skeletal muscle cell (D2 immature myotube) obtained on the second day after the initiation of terminal differentiation of a myoblast as a transfer cell, and an immature differentiated skeletal muscle cell (D3 immature myotube) obtained on the third day as a host cell, and transferring the immature differentiated skeletal muscle cell (D2 immature myotube) from the second day to the immature differentiated skeletal muscle cell (D3 immature myotube) from the third day to co-differentiate together.

[0014] The present invention also provides a pharmaceutical composition for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy, comprising the above-mentioned fully co-differentiated skeletal muscle cells (D2-to-D3 mature co-differentiation myotubes) as an active ingredient.

[0015] The present invention also provides a method for treating a muscle disease associated with skeletal muscle atrophy, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to an individual requiring treatment. Effects of the invention

[0017] Since the cell therapy agent of the present invention is manufactured through the co-differentiation of incompletely differentiated skeletal muscle cells differentiated in vitro, the proliferation and early terminal differentiation processes of the transplanted cells at the transplant site after transplantation can be omitted. Furthermore, it provides a solution to the problems of satellite stem cells, which have quantitative limitations, and skeletal muscle progenitor cells, which have low success rates for proliferation and early terminal differentiation at the transplant site after transplantation, and can increase the contractility of skeletal muscle (physiological activity of skeletal muscle) by enhancing the ability to supply calcium used for skeletal muscle contraction at the transplant site. Brief explanation of the drawing

[0019] Figure 1 shows a schematic of the codifferentiation strategy during terminal differentiation of skeletal muscle progenitor cells into myotubes. Figure 2 shows the results of comparing the degree of intracellular calcium movement for skeletal muscle contraction in co-differentiated myotubes (A: control group, Mb-to-D2, D2-to-D2, D3-to-D2; B: control group, Mb-to-D3, D2-to-D3, D3-to-D3). Figure 3 shows the results of a comparison of cell widths and expression levels of major proteins involved in skeletal muscle contraction and relaxation of fully differentiated codifferentiated skeletal muscle cells (D2-to-D3 co-differentiated myotubes) on day 5 of differentiation: (A) The result of measuring the width of D2-to-D3 codifferentiated differentiated skeletal muscle cells on day 5 of differentiation. (B) The results of comparing the expression levels of MyoD, myogenin (terminal differentiation-associated protein) and MyHC II (contraction-mediated protein) in D2-to-D3 codifferentiated skeletal muscle cells on day 5. (C) The results of comparing the expression levels of RyR1, DHPR, SERCA1a, and CASQ1, which are key proteins that mediate the contraction and relaxation of skeletal muscle. (D) Results of immunolithography analysis performed to compare the expression levels of triad formation-mediated proteins. Figure 4 shows the results of comparing external calcium influx (SOCE) mediating external calcium procurement during skeletal muscle contraction in D2-to-D3 co-differentiated myotubes on day 5 of differentiation, comparison of cytoplasmic calcium concentration, amount of calcium stored in the myocytoplasmic reticulum, and amount of expression of SOCE-mediated proteins. (A) This is the result of measuring the external calcium influx (SOCE) resulting from depleting the amount of calcium stored in the myocytoplasmic reticulum (SR) by treatment with tapsi-gajin (TG). (B) Comparison of the amount of calcium in the cytoplasm and (C) Comparison of the amount of calcium stored in the myocytoplasmic reticulum (or the amount of calcium that can be released from the myocytoplasmic reticulum into the cytoplasm, i.e., the amount of calcium available for skeletal muscle contraction) by tapsigazine (TG) treatment. (D) Results of comparing the expression levels of SOCE-mediated proteins using immunoassay (TRPC: trip cation channel, STIM: steam). Specific details for implementing the invention

[0020] The configuration of the present invention will be described in detail below.

[0021] The present invention relates to a control group of myoblasts differentiated in vitro for 5 days compared to myotubes.

[0022] The expression of MyoD, MyHC II, RyR1, SERCA1, and STIM1 proteins increases, and

[0023] As the thickness of the cell increases,

[0024] This relates to D2-to-D3 mature co-differentiation myotubes with increased calcium procurement from inside or outside the cell for skeletal muscle contraction.

[0025] In addition, the present invention provides a method for producing a mature co-differentiated skeletal muscle cell (D2-to-D3 mature co-differentiation myotube), comprising the step of using an immature differentiated skeletal muscle cell (D2 immature myotube) obtained on the second day after the initiation of terminal differentiation of a myoblast as a transfer cell, and an immature differentiated skeletal muscle cell (D3 immature myotube) obtained on the third day as a host cell, and transferring the immature differentiated skeletal muscle cell (D2 immature myotube) from the second day to the immature differentiated skeletal muscle cell (D3 immature myotube) from the third day to co-differentiate together.

[0026] In this specification, the term "terminal differentiation of myoblasts" refers to the process in which mononuclear myoblasts form multinuclear myotubes through fusion. Myoblasts, which correspond to precursor cells of skeletal muscle, undergo Pax7 when self-renewal. + It can be distinguished using markers, and in the case of proliferation, Pax7 + / MyoD + It can be classified as such. In addition, cells in the differentiation stage that form skeletal muscle cells (myotubes) are Pax7 - MyoD + MyoG +It can be distinguished using markers. Cells in the early stages of differentiation forming the aforementioned myotubes show increased expression of myogenic transcription factors such as Myosin D (MyoD), while myogenin increases during the mid-stage. In the late stage, when differentiation is nearly complete, the expression of the myosin heavy chain, a protein directly involved in skeletal muscle contraction, increases.

[0027] In order to overcome the disadvantages of satellite stem cells and skeletal muscle progenitor cells associated with cell transplantation in the treatment of muscle diseases related to skeletal muscle atrophy, the inventors used immature myotubes with partial terminal differentiation as transferred cells and also used immature myotubes as host cells to co-differentiate them, and as a result, obtained mature co-differentiated myotubes (D2-to-D3 mature co-differentiation myotubes) in which the expression of MyoD, MyHC II, RyR1, SERCA1, and STIM1 proteins increased, cell thickness increased, and calcium procurement from inside or outside the cell for skeletal muscle contraction increased compared to control myotubes in which myoblasts were differentiated in vitro for 5 days.

[0028] Specifically, the complete co-differentiated skeletal muscle cell of the present invention can be obtained by using an immature differentiated skeletal muscle cell (D2 immature myotube) obtained on the 2nd day after the initiation of terminal differentiation of a myoblast as a transfer cell, and an immature differentiated skeletal muscle cell (D3 immature myotube) obtained on the 3rd day as a host cell, and transferring the immature differentiated skeletal muscle cell (D2 immature myotube) from the 2nd day to the immature differentiated skeletal muscle cell (D3 immature myotube) from the 3rd day to co-differentiate together.

[0029] The above myoblasts may be mouse myoblasts, house mouse myoblasts, frog myoblasts, rabbit myoblasts, guinea pig myoblasts, pig myoblasts, monkey myoblasts, or human myoblasts, but are not limited thereto.

[0030] The above codifferentiation may involve additionally culturing day 2 immature differentiated skeletal muscle cells (D2 immature myotube) and day 3 immature differentiated skeletal muscle cells (D3 immature myotube) together in vitro for 2 days, but is not limited thereto.

[0031] In addition, differentiation and codifferentiation of myoblasts can be performed in a differentiation medium.

[0032] As used in this specification, the term "medium" refers to a test tube containing elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals ( in vitroIt refers to a mixture for the growth and proliferation of cells. In particular, the medium of the present invention is a medium for the differentiation and codifferentiation of skeletal muscle progenitor cells, and includes cell growth and proliferation. The medium may be prepared by adding supplementary components for differentiation or codifferentiation to various basic media. The "basic medium" is a mixture containing essential sugars, amino acids, water, etc., necessary for cell survival, and may be prepared by artificial synthesis or a commercially manufactured medium may be used. Examples of commercially manufactured media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium.

[0033] Specifically, the differentiation medium of the present invention uses 5% heat-inactivated horse serum and low-glucose DMEM instead of 20% FBS and F-10 Nutrient Mixture, and does not use bFGF.

[0034] The above differentiation or codifferentiation can be performed in a 10% CO2 incubator.

[0035] In the transplantation of skeletal muscle cells into areas of skeletal muscle atrophy caused by genetic or acquired skeletal muscle diseases, skeletal muscle atrophy occurring during the long-term treatment of other diseases, or sarcopenia occurring during the natural normal aging process, the fully codifferentiated skeletal muscle cells are manufactured through the codifferentiation of incompletely differentiated skeletal muscle cells differentiated in vitro; thus, the proliferation and initial terminal differentiation processes of the transplanted cells at the transplantation site after transplantation can be omitted. This provides a solution to the problems associated with satellite stem cells, which have quantitative limitations, or skeletal muscle progenitor cells, which have low success rates for proliferation and initial terminal differentiation at the transplantation site after transplantation, and can increase the contractility of the skeletal muscle (physiological activity of the skeletal muscle) by enhancing the ability to supply calcium for skeletal muscle contraction at the transplantation site.

[0036] Therefore, the completed codifferentiated skeletal muscle cells of the present invention can be used as a cell therapy agent for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy.

[0037] Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy, comprising the above-mentioned fully co-differentiated skeletal muscle cells (D2-to-D3 mature co-differentiation myotubes) as an active ingredient.

[0038] The muscle disease associated with the aforementioned skeletal muscle atrophy is a muscle disease resulting from the atrophy or damage of muscle tissue caused by genetic, pathological, or physical causes.

[0039] Primary skeletal muscle atrophy can occur directly due to various hereditary muscle disorders, including congenital and hereditary myopathy. Hereditary myopathy is associated with progressive atrophy, inflammation, impaired muscle fiber metabolism, and muscle spasms or spasticity. Hereditary myopathy can be further subdivided into muscular atrophy, congenital myopathy, mitochondrial myopathy, and metabolic myopathy. The more commonly observed hereditary myopathy are mitochondrial and metabolic myopathy. Congenital myopathy includes Nemalin myopathy, and muscular atrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, and myotonic muscular dystrophy (Type 1 and Type 2).

[0040] Conversely, acquired causes, such as systemic diseases or physical conditions, can lead to secondary skeletal muscle atrophy. Pathological conditions that induce skeletal muscle atrophy include age-related sarcopenia, cachexia due to cancer, chronic obstructive pulmonary disease (COPD), diabetes and obesity, chronic kidney disease, heart failure, neurodegenerative diseases, sepsis, burns, and trauma. Physiological responses such as fasting or malnutrition can also result in skeletal muscle atrophy. Skeletal muscle atrophy can occur in immobilized patients, such as those with leg fractures, disuse, immobilization, or bed rest, but it can also occur in the general population who maintain a weightless state, lead a sedentary lifestyle, or have occupations involving prolonged sitting. Ultimately, the signaling pathway for skeletal muscle atrophy is initiated by a lack of muscle contraction and stimulation due to various causes, leading to protein loss and apoptosis. As these diseases progress, muscle atrophy occurs when the rate of protein degradation exceeds the rate of protein synthesis.

[0041] Sarcopenia refers to the gradual decline in skeletal muscle mass and strength in adults as they age. Skeletal muscle atrophy and fatigue due to aging are long-term processes; the causes are multifactorial and complex, involving both external and internal factors. In aged skeletal muscle, metabolism slows, biosynthesis decreases, and mitochondrial size shrinks, leading to a rapid loss of muscle mass and efficiency parameters. As average muscle volume decreases during the aging process, instability, an increased risk of falls, and bone fractures occur, consequently increasing the need for medical and social services.

[0042] Specifically, the muscle diseases associated with skeletal muscle atrophy according to the present invention may include, but are not limited to, muscular dystrophy, myasthenia gravis, muscular dystrophy, myasthenia gravis, muscle weakness, muscle degenerative atrophy, amyotrophic lateral sclerosis or myasthenia gravis, myasthenia gravis caused by genetic mutation, malignant hyperthermia, Duchenne muscular dystrophy, or Becker muscular dystrophy.

[0043] In addition, examples of muscular atrophy include sarcopenia, disuse atrophy, muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, or muscular dystrophy, but are not limited thereto.

[0044] In this specification, the term "prevention" refers to any act of suppressing or delaying the onset of skeletal muscle atrophy by administering a composition according to the present invention, and "treatment" refers to any act of improving or beneficially changing the symptoms of an individual suspected of or suffering from skeletal muscle atrophy by administering the pharmaceutical composition.

[0045] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of fully codifferentiated skeletal muscle cells for the treatment of muscle diseases associated with skeletal muscle atrophy. A therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician, and includes an amount that induces the alleviation of symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the content (number) of fully codifferentiated skeletal muscle cells included in the pharmaceutical composition of the present invention will vary according to the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health status, gender, diet, time of administration, route of administration, secretion rate of the composition, duration of treatment, and concurrently used drugs, but, for example, 1 x 10⁶ 4　 cells / kg ~ 1X10 8 Completely differentiated skeletal muscle cells of cell / kg may be included, but are not limited thereto.

[0046] In the present invention, the composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the composition may be characterized in that it is intended for mammals, preferably humans.

[0047] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, flavoring agent, etc. For injectable preparations, it may include a mixture of a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, etc. For topical administration, it may include a base, excipient, lubricant, preservative, etc. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0048] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0049] The routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0050] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathoracic, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques.

[0051] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0052] The pharmaceutical composition of the present invention may be used alone or in combination with conventional treatments for muscle diseases associated with skeletal muscle atrophy, or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers, or exercise therapy or rehabilitation therapy.

[0053] The present invention also provides a method for treating a muscle disease associated with skeletal muscle atrophy, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to an individual requiring treatment.

[0054] In this specification, the term "individual requiring treatment" refers to a patient who has developed or is suspected of having a muscle disease associated with skeletal muscle atrophy.

[0055] In this specification, the term “individual” means a human and an animal such as a monkey, dog, goat, pig, mouse, rat, guinea pig, rabbit, or primate having a disease whose symptoms may be improved by administering the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention may be applied not only to humans (for treatment, inhibition, or prevention) but also to other animals that are commercially useful.

[0056] In the treatment method of the present invention, when the pharmaceutical composition of the present invention is administered once or several times a day, the cell therapeutic agent (fully codifferentiated differentiated skeletal muscle cells) included in the composition is 1 x 10 4 cell / kg to 1X10 8 It is desirable to include an amount of cell / kg.

[0057] In the treatment method of the present invention, the pharmaceutical composition of the present invention may be administered in a conventional manner through the rectum, intravenous, arterial, abdominal cavity, intramuscular, sternal, transdermal, local, ocular, or intradermal routes.

[0058] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0059] <Example 1> Preparation of Codifferentiated Differentiated Skeletal Muscle Cells

[0060] In order to overcome the blind spots of satellite stem cells and skeletal muscle progenitor cells, the inventors conducted an experiment to determine whether (1) the success rate of transplantation can be increased, (2) whether additional differentiation occurs well upon transplantation to generate mature myotubes, and (3) whether the mature myotubes exhibit high physiological activity for skeletal muscle contraction, when codifferentiated using immature myotubes with partially undergone terminal differentiation as transferred cells and host cells (i.e., a state in which immature myotubes are included or maintained because the tissue itself cannot resolve skeletal muscle atrophy or sarcopenia).

[0061] (Method for isolating skeletal muscle satellite cells and differentiating them into myotubes)

[0062] Skeletal muscle satellite cells were isolated from the skeletal muscle of mouse fetuses, and myoblasts were obtained by primary culture.

[0063] Cells were treated with cell culture medium (F10 Nutrient Mixture composition: 20% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine, 20 nM basic fibroblast growth factor) and cultured at 37°C in a 5% CO2 incubator. Depending on the application, 10-cm, 96-well, or 6-well culture dishes were used, and all dishes were coated with Matrigel. Differentiation into differentiated skeletal muscle cells (myotubes) was induced when cells proliferated to occupy approximately 65% ​​of the culture dish (differentiation medium composition: 5% heat-inactivated horse serum and low-glucose DMEM were used instead of 20% FBS and F-10 Nutrient Mixture in the cell culture medium, bFGF was not added, and a 10% CO2 incubator was used).

[0064] (Co-differentiation method of skeletal muscle satellite cells)

[0065] Myoblasts obtained from primary culture were differentiated in a differentiation medium for 2 or 3 days to prepare immature differentiated skeletal muscle cells (D2 or D3 immature myotubes). Microdifferentiated cells used as a control were differentiated in a differentiation medium for 5 days as usual to obtain mature differentiated skeletal muscle cells.

[0066] Three types of cells to be used as transfer cells (skeletal muscle progenitor cells, D2 incompletely differentiated skeletal muscle cells, and D3 incompletely differentiated skeletal muscle cells) were harvested on differentiation days 0, 2, or 3, respectively, and the three harvested cells were each transferred to a host cell culture dish containing D2 incompletely differentiated skeletal muscle cells or D3 incompletely differentiated skeletal muscle cells to be used as host cells. Subsequently, the transfer cells and the host cells were co-differentiated with respect to the host cells until day 5 of differentiation to obtain mature co-differentiated myotubes.

[0067] The codifferentiated skeletal muscle cells obtained in this way were named Mb-to-D2, D2-to-D2, D3-to-D2, Mb-to-D3, D2-to-D3, and D3-to-D3 codifferentiated skeletal muscle cells, respectively.

[0068] (Method for measuring the width of codifferentiated skeletal muscle cells)

[0069] Images of codifferentiated skeletal muscle cells were obtained by magnifying a randomly selected portion of a specific size (1,000 μm width and 600 μm length, PregRes Capture Pro v2.8.8, JENOPTIK Optical Systems, Inc.) using a backlight microscope (Nikon Eclipse TS100, Nikon Instruments, Inc.) equipped with a camera (monochrome camera, ProgRes MF, JENOPTIK Optical Systems, Inc.), and the length of the thickest part of the codifferentiated skeletal muscle cells was measured using the ImageJ program.

[0070] (Method for obtaining lysate of codifferentiated differentiated skeletal muscle cells)

[0071] Codifferentiated differentiated skeletal muscle cells were harvested and obtained, and lysed (solubilized) at 4°C for 24 hours with the addition of a lysate (1% Triton X-100, 10 mM Tris-HCl, 1 mM Na3VO4, 10% glycerol, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail, pH 7.4) to obtain lysed samples of codifferentiated differentiated skeletal muscle cells.

[0072] (Immunoblotting)

[0073] Lysate samples of codifferentiated differentiated skeletal muscle cells were separated on a 10% or 12% SDS-PAGE gel, and the proteins separated on the gel were transferred to a PVDF (poly-vinylidenefluoride) membrane (100 V, 2 hours). After treatment with 5% non-fat milk for 1 hour, the corresponding primary antibody (1:1000) was applied, followed by treatment with the corresponding secondary antibody (horseradish peroxidase-conjugated secondary antibody) for 45 minutes, followed by a colorimetric reaction (SuperSignal ultrachemiluminescent Visualization and analysis were performed using a substrate. The antibody information used is listed in Table 1. The antibodies were used after diluting them to 1:1,000. However, the secondary antibody was used after diluting it to 1:50,000.

[0074] Antibody Information antibody name Antibody manufacturing company name Order Information RyR1 antibody Thermo Fisher Scientific (Waltham, MA, USA) MA3-925 DHPR antibody MA3-920 SERCA1a antibody MA3-912 CASQ1 antibody MA3-913 JP1 antibody 40-5100 TRPC1 antibody Alomone Labs (Jerusalem, Israel) ACC-010 TRPC3 antibody ACC-016 TRPC4 antibody ACC-018 TRPC6 antibody ACC-017 Orai1 antibody Santa Cruz Biotechnology (Dallas, TX, USA) sc-377281 α-actin antibody sc-58671 MyoD antibody sc-377460 myogenin antibodies sc-398002 JP2 antibody sc-134875 MyHC II antibody Abcam (Cambridge, MA, USA) ab37484 STIM1 antibody Cell Signaling Technology (Danvers, MA, USA) 4916 STIM2 antibody Proteintech (Rosemont, IL, USA) 21192-1-AP Goat secondary antibody Jackson Immuno Research Laboratories (West Grove, PA, USA) 205-035-108 Mouse secondary antibody 715-035-151 Rabbit secondary antibodies 711035-152

[0076] (Measurement of calcium response of codifferentiated differentiated skeletal muscle cells to KCl, cytoplasmic calcium concentration, and amount of calcium stored in the myocytoplasmic reticulum using single-cell calcium imaging techniques)

[0077] Calcium fluorescent dye (Ca) that emits fluorescence of a different wavelength than before binding with calcium when bound to calcium. 2+The dyes fura-2 (5 μM, for measuring cytoplasmic calcium levels) or fluo-4 (5 μM, for other single-cell calcium imaging experiments) were injected into codifferentiated skeletal muscle cells while maintaining 37°C for 45 minutes (incubation). During this time, the codifferentiated skeletal muscle cells were treated with imaging solution (125 mM NaCl, 5 mM KCl, 2 mM KH2PO4, 2 mM CaCl2, 25 mM HEPES, 6 mM glucose, 1.2 mM MgSO4, 0.05% BSA (fraction V), pH 7.4). Fluorescence microscopy was used to measure intracellular calcium transport (Nikon x40 oil-immersion objective, NA 1.30, ECLIPSE Ti, Nikon). Fluorescence changes of the calcium fluorescent stain were transmitted to a computer using a 75-watt xenon lamp (FSM150Xe, Bentham Instruments, Ltd) and a 12-bit CCD camera (DVC-340M-OO-CL, Digital Video Camera Company) connected to a fluorescence microscope, and analyzed using the associated program (InCyt Im1 image acquisition and analysis software, v5.29, Intracellular Imaging Inc). Calcium transport from the myocytoplasmic reticulum (SR) to the cytoplasm induced by KCl treatment, calcium concentration within the cytoplasm, and the amount of calcium stored in the myocytoplasmic reticulum (in other words, the amount of calcium that can be transported from the myocytoplasmic reticulum to the cytoplasm) induced by Thapsigagin (TG) treatment were measured, and the values ​​for the peak heights of the graphs were statistically analyzed (showing the same trend as the statistical analysis of the graph area). The absolute concentration of calcium in the cytoplasm was measured using the Calcium Calibration Buffer Kit #1 (Thermo Fisher Scientific).

[0078] (Measurement of external calcium influx (SOCE) using single differentiated skeletal muscle cell calcium imaging technique)

[0079] Calcium fluorescent dye (Ca) that emits fluorescence of a different wavelength than before binding with calcium when bound to calcium. 2+ Fluo-4 (5 μM), a dye, was injected into codifferentiated differentiated skeletal muscle cells while maintaining the temperature at 37°C for 45 minutes (incubation). At this time, the differentiated skeletal muscle cells were treated with imaging solution (125 mM NaCl, 5 mM KCl, 2 mM KH2PO4, 2 mM CaCl2, 25 mM HEPES, 6 mM glucose, 1.2 mM MgSO4, 0.05% BSA (fraction V), pH 7.4). A fluorescence microscope (Nikon x40 oil-immersion objective, NA 1.30, ECLIPSE Ti, Nikon) was used to measure calcium movement within the codifferentiated differentiated skeletal muscle cells. Fluorescence changes of the calcium fluorescent stain were transmitted to a computer using a 75-watt xenon lamp (FSM150Xe, Bentham Instruments, Ltd) and a 12-bit CCD camera (DVC-340M-OO-CL, Digital Video Camera Company) connected to a fluorescence microscope, and analyzed using the associated program (InCyt Im1 image acquisition and analysis software, v5.29, Intracellular Imaging Inc). Calcium (store-operated Ca) that flows from outside the cell into the cell when calcium in the calcium storehouse (i.e., the myocytoplasmic reticulum (SR)) is depleted... 2+ For the experiment to measure the amount of entry (SOCE), calcium-free (Ca 2+After treating differentiated skeletal muscle cells with a β-free imaging solution for 5 minutes, calcium depletion within the myocytoplasmic reticulum was induced by treatment with TAPSGAZINE, and the amount of calcium entering the extracellular space was measured by applying 2 mM calcium extracellularly. TG was dissolved in Me2SO₄ (< 0.05%) and manually treated into the cells, and it was confirmed that there was no change in cellular calcium transport caused by Me2SO₄ (< 0.05%) itself. The results for the SOCE and TG response measurements were statistically analyzed by the area under the calcium transport graph.

[0080] (Data Analysis: Statistical Analysis)

[0081] All data were aggregated from data obtained from multiple experiments and expressed as ± SE. Except for the comparison of cytoplasmic calcium concentration, values ​​obtained from the control group were set to 1, and the results were expressed as normalized ratios representing relative changes. The number of replicates and cell counts used in the data analysis are indicated in the plots or tables. Significant differences were unpaired t It was performed using the t-test or one-way ANOVA-Tukey's post hoc test (GraphPad InStat, v2.04), and the significant difference P Values ​​<0.05 were indicated (* or #). The graph was created using Origin 2019b. .

[0083] <Experimental Example 1> Preparation of Differentiated Skeletal Muscle Cells under Different Codifferentiation Conditions

[0084] Figure 1 shows a schematic of the codifferentiation strategy during terminal differentiation of skeletal muscle progenitor cells into myotubes.

[0085] Days 0 through 5 represent days 0 through 5 of terminal differentiation initiation, and differentiated myotubes refer to multinucleated myotubes that are fully differentiated on day 5 of differentiation. D2 or D3 host cells are D2 or D3 immature myotubes, and codifferentiation condition 1 or 2 refers to cases where D2 or D3 immature myotubes are used as host cells. The number of cells used for each codifferentiation condition and the cell selection method are shown in Tables 2 and 3.

[0086] Table 2 shows the number of myoblasts initially used to obtain myoblasts and immature myotubes under six different conditions (Mb-to-D2, D2-to-D2, D3-to-D2, Mb-to-D3, D2-to-D3, D3-to-D3). All data were aggregated from multiple experiments (see Table 2 below) and expressed as ±SE. *Significant differences are defined as relative to Mb-to-D2 (codifferentiation condition 1) or relative to Mb-to-D2 (codifferentiation condition 2), P It was indicated for cases <0.05. There was no significant difference.

[0087] Table 3 shows the number of spots observed in cell culture dishes that were randomly selected to count the number of myoblasts in Table 2, with each spot (1,000 μm wide and 600 μm long) randomly selected from different cell culture wells.

[0088] Number of myoblasts on day 0 of differentiation under different codifferentiation conditions Codifferentiation conditions Number of skeletal muscle progenitor cells on differentiation day 0 Mobile cell D2 or D3 host cells Codifferentiation condition 1 (D2 host cell) control group None 137.53 ± 19.96 Mb-to-D2 111.80 ± 22.93 112.30 ± 19.94 D2-to-D2 99.28 ± 17.90 102.73 ± 18.41 D3-to-D2 114.26 ± 20.11 108.26 ± 20.90 Codifferentiation condition 2 (D3 host cell) control group None 144.49 ± 17.12 Mb-to-D3 117.59 ± 19.33 101.90 ± 18.75 D2-to-D3 92.64 ± 15.96 93.87 ± 15.56 D3-to-D3 103.08 ± 18.21 104.33 ± 17.76

[0089] In Table 2, the number of spots randomly selected and observed in the cell culture dish to count the number of myoblasts. Codifferentiation conditions Number of designated points Codifferentiation condition 1 control group 120 points from 10 wells Mb-to-D2 192 points from 16 wells D2-to-D2 240 branches from 20 wells D3-to-D2 192 points from 16 wells Codifferentiation condition 2 control group 120 points from 10 wells Mb-to-D3 240 branches from 20 wells D2-to-D3 288 branches from 24 wells D3-to-D3 240 branches from 20 wells

[0091] As described above, Mb-to-D2, D2-to-D2, D3-to-D2, Mb-to-D3, D2-to-D3, and D3-to-D3 codifferentiated differentiated skeletal muscle cells were obtained through six different codifferentiation conditions.

[0093] <Experimental Example 2> Comparison of the extent of intracellular calcium movement for skeletal muscle contraction in codifferentiated skeletal muscle cells

[0094] The relative amount of calcium released from the sarcoplasmic reticulum (SR) into the cytoplasm for skeletal muscle contraction was measured by treating codifferentiated skeletal muscle cells with KCl, a cell membrane depolarizing agent (an excitation-contraction coupling inducer for skeletal muscle contraction), and the results are shown in Figure 2. The statistical results are represented as a bar graph on the right side of Figure 2. * Significant differences were indicated compared to the control group ( P <0.05). The number of experiments used for analysis and statistics and the obtained values ​​are shown in Table 4. In Table 4, the data were aggregated from the number of codifferentiated differentiated skeletal muscle cells indicated in parentheses and expressed as ± SE. The values ​​obtained from the control group were set to 1, and the results were expressed as a normalized ratio representing the relative change. * The significant difference is compared to the control group. P Indicated when <0.05

[0095] As shown in Figure 2, it was confirmed that intracellular calcium supply for skeletal muscle contraction in D2-to-D3 codifferentiated skeletal muscle cells for KCl under different codifferentiation conditions was significantly increased compared to control cells. This suggests that when D2 incompletely differentiated skeletal muscle cells are used as transplant cells and D3 incompletely differentiated skeletal muscle cells are used as host cells, the contractile activity of skeletal muscle can be enhanced through cell transplantation.

[0096] Intracellular calcium movement occurring in codifferentiated differentiated skeletal muscle cells in response to KCl on day 5 of differentiation Codifferentiation condition 1 control group Mb-to-D2 D2-to-D2 D3-to-D2 Day 5 of the eruption 1.00 ± 0.13 (121 differentiated skeletal muscle cells from 17 wells) 0.86 ± 0.11 (122 differentiated skeletal muscle cells from 17 wells) 0.99 ± 0.12 (147 differentiated skeletal muscle cells from 17 wells) 0.88 ± 0.07 (136 differentiated skeletal muscle cells from 16 wells) Codifferentiation condition 2 control group Mb-to-D3 D2-to-D3 D3-to-D3 Day 5 of the eruption 1.00 ± 0.14 (109 differentiated skeletal muscle cells from 14 wells) 0.94 ± 0.34 (74 differentiated skeletal muscle cells from 14 wells) 3.15 ± 0.72 * (84 differentiated skeletal muscle cells from 12 wells) 1.13 ± 0.23 (75 differentiated skeletal muscle cells from 12 wells)

[0098] <Experimental Example 3> Comparison of cell width of D2-to-D3 co-differentiated myotubes on day 5 after differentiation initiation and comparison of expression levels of major proteins involved in skeletal muscle contraction and relaxation

[0099] On day 5 after the initiation of differentiation, the cell width of D2-to-D3 co-differentiated myotubes and the expression levels of major proteins involved in skeletal muscle contraction and relaxation were compared.

[0100] Figure 3A and Table 5 show the results of measuring the width of D2-to-D3 codifferentiated differentiated skeletal muscle cells on day 5 of differentiation. The control group value was set to 1, and the relative change was expressed as a normalized ratio. The number of experiments used for analysis and statistics and the obtained values ​​are indicated in parentheses in Table 5. *Significant differences compared to the control group are indicated. P <0.05).

[0101] D2-to-D3 codifferentiated differentiated skeletal muscle cells became thicker compared to control cells, indicating that terminal differentiation was better achieved, which is supported by a quantitative increase in MyoD, a factor involved in terminal differentiation, and MyHC II, one of the skeletal muscle contraction proteins.

[0102] Width of codifferentiated differentiated skeletal muscle cells on day 5 of differentiation Codifferentiation condition 1 Codifferentiation condition 2 control group Mb-to-D2 D2-to-D2 D3-to-D2 control group Mb-to-D3 D2-to-D3 D3-to-D3 Day 5 of the eruption 1.00 ± 0.03 0.87 ± 0.03 * 0.93 ± 0.03 0.97 ± 0.04 1.00 ± 0.03 0.86 ± 0.03 * 1.35 ± 0.03 * 1.14 ± 0.03 *

[0104] Next, on day 5, immunolithography was performed to compare the expression levels of MyoD and myogenin (terminal differentiation-associated protein) and MyHC II (contraction-mediated protein), RyR1, DHPR, SERCA1a and CASQ1, which are key proteins mediating the contraction and relaxation of skeletal muscle, and triad formation-mediated proteins in D2-to-D3 codifferentiated skeletal muscle cells.

[0105] The expression level of α-actin, a structural phase protein of all cells, was confirmed (no change). CASQ1: Calciquestrin 1, JP: Transtophyllin. The control value was set to 1, and the relative change was expressed as a normalized ratio; the number of experiments used for analysis and statistics and the obtained values ​​are indicated in parentheses in Table 6. Information on the antibodies used is shown in Table 1. α-actin was used as a control to indicate that the total protein amount of the samples placed in the gel did not differ. *Significant differences compared to the control group are indicated ( P < 0.05).

[0106] As shown in Figures 3B to 3D and Table 6, the increased expression of RyR1 and SERCA1, which are key proteins for the contraction and relaxation of skeletal muscle, is consistent with the increased response to KCl (Table 4), an excitation-contraction coupling inducing substance. In other words, compared to the control group, D2-to-D3 codifferentiated skeletal muscle cells show high levels of both the outward completion of terminal differentiation and physiological functional completion.

[0107] Comparison of the intensities of immunological bands for differentiation factor proteins, excitation-contraction-mediating proteins, and triad formation-mediating proteins in lysate samples of codifferentiated differentiated skeletal muscle cells obtained on day 5 of differentiation control group D2-to-D3 Differentiation Factors and MyHC II MyoD 1.00 ± 0.00 1.69 ± 0.20 * Myogenin 1.00 ± 0.00 1.02 ± 0.07 MyHC II 1.00 ± 0.00 1.65 ± 0.23 * Differentiation factors and α-actin on MyHC II 1.00 ± 0.00 1.00 ± 0.02 Excitation-contraction-mediating proteins RyR1 1.00 ± 0.00 1.80 ± 0.37 * DHPR 1.00 ± 0.00 0.99 ± 0.08 SERCA1a 1.00 ± 0.00 1.94 ± 0.37 * CASQ1 1.00 ± 0.00 0.98 ± 0.02 α-actin on excitation-contraction-related proteins 1.00 ± 0.00 1.02 ± 0.04 Triad formation-mediating proteins JP1 1.00 ± 0.00 1.06 ± 0.15 JP2 1.00 ± 0.00 0.96 ± 0.04 α-actin on triad formation-mediating proteins 1.00 ± 0.00 1.00 ± 0.04

[0109] <Experimental Example 4> Comparison of extrinsic calcium influx (SOCE) mediating extrinsic calcium procurement during skeletal muscle contraction, cytoplasmic calcium concentration, amount of calcium stored in the myocytoplasmic reticulum, and expression level of SOCE-mediated proteins in D2-to-D3 co-differentiated myotubes on day 5 after differentiation initiation

[0110] After depleting the amount of calcium stored in the myocytoplasmic reticulum (SR) by treatment with tapxigazine (TG), the resulting external calcium influx (SOCE) was measured and the amount of calcium in the cytoplasm was compared. Additionally, the amount of calcium stored in the myocytoplasmic reticulum (or the amount of calcium that can come out of the myocytoplasmic reticulum into the cytoplasm, i.e., the amount of calcium that can be used for skeletal muscle contraction) was compared. The control group value was set to 1, and the relative change was expressed as a normalized ratio. The number of experiments used for analysis and statistics and the obtained values ​​are shown in parentheses in Table 7. * Indicates a significant difference compared to the control group ( P <0.05). The data were aggregated from the number of codifferentiated differentiated skeletal muscle cells indicated in parentheses and expressed as ± SE. The values ​​obtained from the control group were set to 1, and the results were expressed as a normalized ratio representing the relative change. * The significant difference is compared to the control group, P Indicated when <0.05

[0111] In addition, immunolithography was performed to compare the expression levels of SOCE-mediated proteins, and the expression level of α-actin, a structural phase protein of all cells, was confirmed. The control group was set to 1, and the relative change was expressed as a normalized ratio; the number of experiments used for analysis and statistics and the obtained values ​​are indicated in parentheses in Table 8. The data were aggregated from three different independent experiments and expressed as ± SE, and the values ​​obtained from the control group were set to 1, and the results were expressed as normalized ratios representing the relative change. α-actin was used as a control to indicate that the total protein amount of the samples placed in the gel was not different. *Significant differences are relative to the control group, P Indicated when <0.05

[0112] As shown in Figures 4A to 4D, the amount of calcium flowing from the outside into the cell of D2-to-D3 codifferentiated skeletal muscle cells was significantly increased compared to control cells, which means that this is due to an increase in STIM1, a SOCE-mediated protein. Furthermore, the increased SOCE means that it contributed to an increase in cytoplasmic calcium and calcium within the myocytoplasmic reticulum.

[0113] Overall, this means that the amount of calcium used for skeletal muscle contraction increases, and as a result, skeletal muscle contraction can occur more effectively.

[0114]

[0115]

[0116] As described above, regarding the transplantation of skeletal muscle cells into sites of skeletal muscle atrophy / wasting caused by genetic or acquired skeletal muscle diseases, skeletal muscle atrophy occurring during the long-term treatment of other diseases, or sarcopenia occurring during the natural normal aging process, the present invention provides a solution to the problems of satellite stem cells, which have quantitative limitations, or myoblasts, which have the disadvantage of low success rates for proliferation and initial terminal differentiation of transplanted cells at the transplantation site after transplantation.

[0117] When D2 immature myotubes, which are incompletely differentiated on day 2 with some degree of terminal differentiation, are used as transplant cells, the cells transplanted to the transplant site proliferate and can skip the initial terminal differentiation process. Therefore, by using D2 immature myotubes, which are incompletely differentiated on day 2 and cultured outside the human body and partially differentiated (in vitro culture), as transplant cells, the low success rate of skeletal muscle cell transplantation, which is inevitably low due to the proliferation of transplant cells and failure of the initial terminal differentiation process, can be overcome.

[0118] In addition to the success rate of skeletal muscle cell transplantation by using day 2 immature myotubes, if 'mature co-differentiated myotubes' produced by co-differentiating using day 3 immature myotubes as host cells are used as transplant cells, the contractility of the skeletal muscle (physiological activity of the skeletal muscle) can also be increased due to the enhancement of the ability to supply calcium for skeletal muscle contraction at the transplant site.

Claims

Claim 1 D2-to-D3 mature co-differentiation myotubes, in which the expression of MyoD, MyHC II, RyR1, SERCA1, and STIM1 proteins is increased, cell thickness is increased, and calcium procurement from inside or outside the cell for skeletal muscle contraction is increased, compared to control myotubes, in which myoblasts were differentiated in vitro for 5 days. Claim 2 In claim 1, the myoblast is a fully codifferentiated skeletal muscle cell selected from the group consisting of mouse myoblasts, rat myoblasts, frog myoblasts, rabbit myoblasts, guinea pig myoblasts, pig myoblasts, monkey myoblasts, and human myoblasts. Claim 3 In claim 1, the completed co-differentiated skeletal muscle cell is obtained by using an immature differentiated skeletal muscle cell (D2 immature myotube) obtained on the 2nd day after the initiation of terminal differentiation of a myoblast as a transfer cell, and an immature differentiated skeletal muscle cell (D3 immature myotube) obtained on the 3rd day as a host cell, and transferring the immature differentiated skeletal muscle cell (D2 immature myotube) from the 2nd day to the immature differentiated skeletal muscle cell (D3 immature myotube) from the 3rd day to co-differentiate together. Claim 4 A pharmaceutical composition for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy, comprising the completed co-differentiated skeletal muscle cells (D2-to-D3 mature co-differentiation myotubes) of claim 1 as an active ingredient. Claim 5 A pharmaceutical composition for the prevention or treatment of a muscle disease associated with skeletal muscle atrophy, wherein the muscle disease associated with skeletal muscle atrophy is selected from the group consisting of muscular atrophy, myasthenia gravis, muscular dystrophy, myasthenia gravis, hypotonia, muscle weakness, myasthenia gravis, amyotrophic lateral sclerosis or myasthenia gravis, myasthenia gravis caused by genetic mutation, malignant hyperthermia, Duchenne muscular atrophy, and Becker muscular atrophy. Claim 6 A pharmaceutical composition for the prevention or treatment of muscle diseases associated with skeletal muscle atrophy, wherein the muscle atrophy is selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

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