KR20160137864A - Pharmaceutical Compositions for Improving or Treating Muscle Atrophy Comprising Conditioned Medium of Stem Cells Derived from Umbilical Cord - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle atrophy comprising a culture medium of stem cells derived from mammal umbilical cords. When administering the culture medium of stem cells derived from umbilical cords into the muscle, the administered stem cells proliferate the muscular cells at the administration site, increase the amount of muscle, restore muscular fatigue rapidly, and thus are effective for preventing or treating muscle atrophy. Since the pharmaceutical composition according to the present invention uses a culture medium of stem cells, it can overcome technological and practical limitations of therapies using stem cells and has an advantage of easy availability, and thus is significantly effective for preventing and treating muscle atrophy.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating muscular dystrophy comprising stem cell culture derived from mammalian umbilical cord as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating muscular dystrophy comprising a mammalian umbilical cord stem cell culture liquid as an active ingredient.

The amount of muscle in the human body normally maintains a balanced balance of protein synthesis and degradation. However, when the protein decomposition increases or the synthesis decreases, muscular atrophy occurs. In addition to nutritional deficiencies and activity deterioration, it is known that it is also found in chronic wasting diseases such as cancer, diabetes, uraemia, chronic lung disease, heart failure and AIDS, but also in acute diseases such as sepsis, surgery, trauma and burns. Although exercise or other therapies (electrical stimulation, chemical medication) are continuous, they are not a fundamental solution, and they are difficult to complete and difficult to cure.

Stem cells have been noticed as a solution to the overcoming of incurable diseases because of the strong regeneration potential of stem cells. Actually, embryonic stem cells and adult stem cells have differentiation ability but differentiate into cells constituting various tissues. Therefore, it is expected to regenerate damaged parts. Recent reports suggest that adult stem cells are obtained from a variety of human sources, such as umbilical cord blood, bone marrow, fat, amniotic fluid, etc., and these mesenchymal stem cells (MSCs) have. In addition, in order to treat musculoskeletal diseases, treatment studies using stem cells have been preceded, and stem cells have been reported to have potential for muscle regeneration. The inventors of the present invention also conducted studies on the treatment of muscle atrophy using umbilical cord-derived mesenchymal stem cells (UC-MSCs), and reported that the umbilical cord-derived stem cells were directly injected into the localized lesion, (ref 1).

Recently, there have been increasing reports of paracrine action, one of the mechanisms of adult stem cell disease treatment. In other words, when mesenchymal stem cells are exposed to the lesion, they secrete various therapeutic proteins in response to the micro-environment. The secreted therapeutic proteins include growth factors, cytokines, extracellular matrix, and antioxidant proteins, which are being analyzed and reported through various proteomics techniques. Therefore, when mesenchymal stem cells performing peripheral secretion function are applied to various disease models, it is increasingly reported that these secretory proteins enhance body function and show therapeutic efficacy against diseases. However, in spite of the potential of stem cells, development and application as therapeutic agents are limited due to realistic technical problems. For example, in order to use a cell as a therapeutic agent, a certain number or more of cells are required. For this purpose, the cells must be maintained and cultured in a constant state, and the efficiency and economical efficiency caused by a series of processes related to transplantation for treatment .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

1. Kim MJ, et al., J Stem Cell Res Ther., 5 (266): p. 2. Zhang C, et al., J Biol Chem. 18; 288 (3): 1489-99, 2013 3. Romero-Suarez S, et al., Aging (Albany, NY). 2 (8): 504-13,2010 4. Shireman PK, et al., J Leukoc Biol. 81 (3): 775-85, 2007 5. Souza TA, et al., Mol Endocrinol. 22 (12): 2689-702, 2008 6. Messina S, et al., FASEBJ. 21 (13): 3737-46, 2007 7. Hayashi S, et al., Histochem Cell Biol. 122 (5): 427-34, 2004 8. Floss T, et al., Genes Dev. 15: 11 (16): 2040-51, 1997 9. Wang W, et al., Am J Pathol. 174 (2): 541-9, 2009

The present inventors have made extensive efforts to develop a substance that can effectively treat muscular dystrophy and can be safely applied to a human body. As a result, it has been found that a culture solution of umbilical cord stem cells is very effective for preventing or treating muscular dystrophy. It was completed.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of muscle atrophy.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating muscular dystrophy comprising, as an active ingredient, a mammalian umbilical cord stem cell culture prepared by the following method:

(a) isolating stem cells from the mammalian umbilical cord;

(b) culturing the stem cells in a serum-free medium; And

(c) collecting the culture solution at 48 hours after culturing and centrifuging to obtain a supernatant.

The present inventors have made intensive researches to develop a substance that can effectively treat muscular dystrophy and can be safely applied to human body. As a result, it has been found that a culture solution of umbilical cord stem cells is very effective for preventing or treating muscular dystrophy.

According to the present invention, the umbilical cord stem cell culture solution of the present invention increases muscle cell proliferation, increases muscle mass, and promotes lactic acid degradation in muscles. In addition, in the model of muscle atrophy, the expression of muscle protein, desmin, increases the activity of Akt and FoxO in the AKT-FoxO pathway associated with atrophy, and thus the expression of MuRF-1 and Atrogin-1 Lt; RTI ID = 0.0 > asymmetric < / RTI >

According to one embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention are mesenchymal stem cells.

The umbilical cord stem cells used in the present invention can be obtained from various mammals. More preferably a human, a pig, a mouse, a human, a pig, a horse, a cow, a mouse, a rat, a hamster, a rabbit, a goat or a sheep, Can be obtained from the umbilical cord.

The umbilical cord stem cells used in the present invention can be obtained from Wharton's jelly tissue of various mammalian umbilical cord.

The umbilical cord stem cells used in the present invention can be obtained in the manner described in the following examples. For example, after removing the umbilical artery and vein, the Warton muscle tissue is cut and the cut Warton mesh tissue is directly cultured to outgrow the cells. Then, the grown cells are subcultured to obtain stem cells. The medium used in the culturing may be any medium conventionally used for culturing animal cells. For example, Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), MEM (Stanner, CP et al, Nat New Biol 230:... 52 (1971)), Iscove's MEM (Iscove, N. et al, J. Exp Med 147:... 923 (1978)), 199 medium ( Morgan et al, Proc Soc Exp Bio Med, 73:...... 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al, J. Amer Med Assoc 199:.... 519 (1967)) , F12 (Ham, Proc Natl Acad Sci USA 53:.... 288 (1965)), F10 (Ham, RG Exp Cell Res 29:.. 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al, Virology 8:. 396 (1959)), a mixture of DMEM and F12 (Barnes, D. et al, Anal Biochem 102:... 255 (1980)), Waymouth's MB752 / 1 (Waymouth, . C. J. Natl Cancer Inst 22: . 1003 (1959)), McCoy's 5A (McCoy, TA, et al, Proc Soc Exp Biol Med 100:...... 115 (1959)) and MCDB series (Ham , RG et al., In Vitro 14: 11 (1978)), etc. .

The culture medium of the umbilical cord stem cells used in the present invention can be obtained by culturing the stem cells in a medium containing serum and changing the medium to serum-free medium when the cells become 80-90% density, then culturing for 42-52 hours Next, the culture was centrifuged to obtain a supernatant. More preferably, the medium is replaced with a serum-free medium, followed by culture for 48 hours, and then the obtained culture medium is used.

As evidenced in the following examples, the survival rate of umbilical cord stem cells in serum-free conditions was maintained at the normal condition until 48 hours, but after 48 hours, the cell survival rate was lowered and the cell adhesion was also decreased rapidly As a result of measuring the same amount of the stem cell culture solution obtained at the same time, it was confirmed that 48 hours was the time to obtain the most useful substance, and it was determined that the culture solution was obtained after culturing for 48 hours.

The umbilical cord stem cell culture of the present invention can be concentrated using various methods of concentration known in the art.

According to one embodiment of the present invention, the umbilical cord stem cell culture can be concentrated by 3 kDa cut-off and concentrated by lyophilization. Regardless of the concentration method, the culture solution promotes the proliferation and migration of muscle cells.

As evidenced in the following examples, the culture of the powder formulations stored at 4 캜 after lyophilization was checked whether there was any change in storage period. As a result, no protein denaturation occurred during the storage period. Therefore, the culture solution of the present invention can be stored for a long period after lyophilization.

According to an embodiment of the present invention, the umbilical cord stem cell culture solution is selected from the group consisting of Thrombospondin (TSP), Trombospondin-1, Ectodysplasin-A2, IGFBP-rpl protein-related protein 1) / IGFBP-7, tissue inhibitor of metalloproteinase-2 (TIMP-2), IL-6, GRO, MIP2, SPARC, MCP-1, Dkk-1, 1, VEGF, GDF-15, Smad 4, HGF, Angiogenin, Angiopoietin-1, IGFBP-6, FGF-7 / KGF and MMP-1.

As used herein, the term " muscle atrophy " means reduction of muscle tissue or muscle regression.

According to a preferred embodiment of the present invention, the pharmaceutical composition for preventing or treating muscular atrophy according to the present invention is a composition for intramuscular administration, wherein the umbilical cord stem cells administered when the composition is administered into muscle are muscular Promotes cell proliferation and lactic acid degradation, increases muscle fiber size, and increases muscle contraction to prevent or treat amyotrophic syndromes.

The composition of the present invention comprises (a) a pharmaceutical effective amount of a mammalian umbilical cord stem cell culture; And (b) a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the therapeutic efficacy or activity of the above-described mammalian umbilical cord stem cell culture.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, or a medium, but are not limited thereto.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably a parenteral administration method, more preferably intramuscular administration or intravascular administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a pharmaceutical composition for the prophylaxis or treatment of muscular dystrophy comprising a culture medium of mammalian umbilical cord stem cells as an active ingredient.

(Ii) When the umbilical cord stem cell culture solution of the present invention is administered intramuscularly, the administered stem cells proliferate the muscle cells of the administered site, increase the amount of muscle, and quickly recover muscular fatigue, It is effective for treatment.

(Iii) Since the present invention utilizes a stem cell culture solution, it can overcome technological and practical limitations of stem cell therapy and is relatively easy to secure. Thus, it can be effectively used for prevention and treatment of muscular dystrophy.

Fig. 1 is a photograph showing a model of a hip-hanging model for maintaining atrophy induced (maintaining 30 ° to the ground).
Figure 2 is a schematic diagram of an animal model of an atrophy.
Figure 3 shows the results of cell viability measurement under serum-free conditions.
FIG. 4 shows the results of measurement of the same amount of stem cell culture liquid in the absence of serum.
FIG. 5 shows the results of measuring the effect on the cell proliferation according to the concentration method of stem cell useful material.
FIG. 6 shows the result of measuring the effect of the Wound healing assay on cell migration according to the method of concentrating stem cell useful material.
FIG. 7 shows the result of measuring the migration effect of the stem cell useful substance on the cell migration according to the concentration method.
FIG. 8 shows the results of confirming denaturation of protein with the elapsed time after storage at 4 ° C of the material useful for freeze-dried stem cells.
FIG. 9 shows the results of measuring the expression of muscle protein desmin after treatment with dexamethasone by concentration.
FIG. 10 shows the results of comparing changes in muscle protein desmin expression by treating dexamethasone and a stem cell useful substance.
11 is a schematic diagram showing a signal transmission system related to an atrophy.
FIG. 12 shows the result of confirming the markers related to the atrophy signal transmission after treatment with the concentration of dexamethasone.
FIG. 13 is a result of measuring the protective effect on dexamethasone-induced atrophy signaling using a stem cell useful substance.
FIG. 14 shows the results of comparing the standardized weights of the soleus muscles according to the number of transplants.
Fig. 15 shows the results of comparing the standardized weights of the soleus muscles of experimental animals.
Fig. 16 shows the results obtained by comparing lactic acid accumulation concentrations of experimental animals.
17 shows the results of comparing the degree of expression of desmin in experimental animals.
18 shows the results of comparing the degree of expression of skeletal muscle actin in experimental animals.
FIG. 19 shows the effect of the stem cell useful material on the muscle atrophy mechanism through Western blotting.
Fig. 20 is a cytokine array analysis result of a human umbilical cord-derived mesenchymal stem cell useful substance (culture solution).
21 shows the results of analysis of exosomal marker expression.
FIG. 22 shows the results of analysis of the degree of expression of the markers related to the presence of the extracellular signal transduction (PI3K / Akt-FoxO pathway) with or without exosomes.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

1. Cultivation of umbilical cord-derived stem cells and securing useful substances (culture medium)

In order to secure the umbilical cord stem cell useful material, the umbilical cord stem cells obtained from the umbilical cord through a previous patent were cultured in a 10% fetal bovine serum (FBS, Hyclone Laboratories Inc., Logan, UT, USA) And 25 mL of MEM alpha-modified medium (α-MEM, Hyclone) containing 100 mg / mL streptomycin and 100 IU / mL penicillin (1% P / S, Hyclone) were placed in a 175 cm ² culture flask ., Pocheon, Korea) and cultured at 37 ° C and 5% CO ₂ . Umbilical cord tissue-derived stem cells were seeded and cultured for 3 days. When the cells reached a density of 80-90%, serum-free condition was replaced with serum-free MEM alpha-supplemented medium. Forty-eight hours after culture, the culture was collected in a 50 mL conical tube (SPL) and centrifuged at 1500 rpm for 5 minutes to remove debris from adherent cells. Thereafter, the supernatant was transferred, and then concentrated and used for the experiment in order to store and maintain the stable formulation. The first method was stored at 4 ° C until it was stored in a vacuum and stored at 4 ° C in a freeze-dried (FDU-8612, Operon, Gimpo, Korea) ), Centrifuged at 4,000 xg for 30 min, and then stored at 4 ° C until the experiment was performed. The stem cell cultures thus prepared were prepared according to the experimental conditions to be tested for efficacy.

2. Measurement of cell survival rate

Cell viability was measured by collecting stem cell useful material (culture medium) at each time point in order to determine the optimum collection timing without toxicity in the growth of cells under serum-free conditions. The cells were cultured in 96-well plates (SPL), treated with the reagents, and cultured at 37 ° C and 5% CO ₂ for 24 hours and 48 hours, respectively, and WST-1 assay was performed. The amount of the medium contained in each well and Cyto X water solubilized tetrazolium salt (WST, LPS solution, Daejeon, Korea) were diluted at a ratio of 1:10, and the mixture was reacted at 37 ° C for 1 hour. After completion of the reaction, the absorbance was measured at 450 nm using an ELISA microplate reader (Epoch, Bio-Tek., Winooski, CA, USA) and the cell viability was evaluated using the absorbance.

3. L6 Muscle cell  Culture and Muscle atrophy  Guided model construction

The rat skeletal muscle cell line, L6 cell line (CRL 1458, ATCC, Manassas, VA, USA) was used to generate an atrophy induction cell model. This was based on medium supplemented with 10% fetal bovine serum and 1% P / S in DMEM-high glucose (Hyclone). After confirming that L6 muscle cells were attached to the bottom of the flask, the new medium was replaced every two days. When the density of the cells reached about 90%, the cells were suspended using trypsin-EDTA (Hyclone), followed by centrifugation at 1,500 rpm for 5 minutes to recover only the cells. (MuRF-1 and Atrogin-1) were expressed by treatment with 1 μM of dexamethasone (Sigma Aldrich, St. Louis, Mo., USA) . In addition, stem cell useful material (culture solution) was added to L6 muscle cells together with dexamethasone to prove its efficacy.

4. Cell migration observation

1 x 10 ⁵ L6 muscle cells were planted in 6-well plates (SPL) and wounded using a 200 ml tip. The cells were cultured under the respective culture conditions and the morphology and migration of the cells were observed with a microscope.

5. Establishment of an animal model to induce atrophy and measure the weight change of the soleus muscle

Five-week-old female Sprague-Dawley (SD) rats were used as experimental animals and the experiment was performed after the adaptation period. Muscle atrophy was induced using a suspension method (Groups 2 and 3), and the tail was fixed to the top of the cage using a dressing tape to keep the body angle at 30 ° to the floor so that the hind legs were kept floating (Fig. 1). The groups were divided into three groups according to the experimental conditions. Group 1, group 1, group 2, group 1, group 1, group 1, group 1, Group 3) (Fig. 2). After induction of 2-week muscle atrophy, 100 μl of 0.9% saline was injected intramuscularly into the soleus muscle of the control group, and 2 mg / 100 μl of stem cell useful substance (culture solution) was injected into the soleus muscle . After the injection, the suspension was maintained for 1 week. One week after administration of the test substance, the soleus muscle was extracted, weighed, and standardized in proportion to the body weight.

5. Identification of muscle-specific gene expression following transplantation of stem cell useful material (culture medium)

The extracted soleus muscle was extracted with iZ-spin total RNA extraction kit (iNtRON Biotechnology Inc., Seoul, Korea). 1 ml of dissolution buffer was added to the goat muscle and homogenized with a homogenizer. 200 μl of chloroform was added and mixed. Centrifuge at 13,000 rpm for 10 minutes at 4 ° C, transfer the supernatant to a new tube, add 400 μl of binding buffer, and shake it 2-3 times. The mixed solution was added to the column, centrifuged at 13,000 rpm for 30 seconds to remove the solution from the column, 700 μl of the washing buffer A was added to the column, and centrifuged at 13,000 rpm for 30 seconds to remove the solution. Wash Buffer B 700 μl into the column, centrifuge at 13,000 rpm for 30 seconds, remove the solution, and centrifuge again at 13,000 rpm for 2 minutes to dry the membrane of the column. Place a new tube under the column, add 50 μl of elution buffer to the column, and centrifuge at 13,000 rpm for 1 minute to extract the RNA into the tube. After quantification, 500 ng of RNA was denatured at 70 ° C for 5 minutes, and a 5x RT master mix was added thereto. The RNA was reacted at 37 ° C for 1 hour to synthesize cDNA. The synthesized cDNA was diluted in distilled water, and the desmin and skeletal muscle actin primers were mixed with the SYBR Green master mix, and the amount of RNA amplified by real-time PCR was measured. And the graph was compared.

6. Identification of accumulation of lactic acid by transplantation of stem cell useful material (culture solution)

500 μl of lactic acid assay buffer (Eton Bioscience, San Diego, Calif., USA) was added to the extracted soleus muscle, homogenized with a homogenizer, and quantified using a Bradford assay (Bio-Rad Laboratories, Hercules, Calif., USA) The amount of protein was diluted to 1 mg / ml and proteins were removed at -80 캜. In order to prepare a standard curve to be compared with the sample, 990 μl of lactate assay buffer was added to 10 μl of lactate standard solution, and diluted to 1 nmol / μl, and 0, 2, 4, 6, 8 and 10 μl were added to 96- The samples were also divided into 10 μl portions and filled with a lactic acid assay buffer to give a total volume of 50 μl. 46 μl of lactate assay buffer, 2 μl of probe and 2 μl of enzyme mix were mixed, and 50 μl of each reaction mixture was dispensed and mixed well. Light was blocked and allowed to react at room temperature for 30 minutes. Absorbance was measured at 570 nm and substituted for the standard curve to calculate and compare the concentration.

7. Expression of markers of muscle atrophy following transplantation of stem cell useful material (culture solution)

1 μM dexamethasone and conditioned media 1, 5, 10, and 100 μL were cultured in L6 muscle cells cultured in 6-well plates and cultured at 37 ° C and 5% CO ₂ for 24 hours. For protein extraction, 100 μL of RIPA lysis buffer (50 mM Tris / HCl pH 8, 150 mM NaCl, 0.5% Natriumdesoxycholat, 1% Triton X-100, 0.1% SDS and freshly added protease inhibitor cocktail) . The supernatant was centrifuged at 13,000 rpm for 15 minutes at 4 ° C, and the protein concentration of each sample was determined by measuring absorbance at 595 nm using an ELISA microplate reader. Each sample was heated at 100 ° C. for 3 minutes with SDS-PAGE sample buffer (Bio-Rad, Seoul, Korea), electrophoresed on SDS-polyacrylamide gel (Bio-Rad) A nitrocellulose transfer membrane (Protran®BA83, Whatman, Dassel, Germany) was used to transfer the separated proteins to the membrane. After 1 hour blocking with 3% bovine serum albumin (BSA, Bio Basic Inc., Ontario, Canada), primary antibody desmin (1: 1000 dilution), p-Akt (1: 1000 dilution), FoxO1 (1: 1000 dilution), FoxO3 (1: 1000 dilution), MuRF-1 (1: 1000 dilution), MAFbx ), β-actin (1: 5000 dilution) and allowed to stand overnight at 4 ° C. (Horseradish peroxidase) -labeled secondary antibody goat anti-mouse (Santa Cruz, sc) was added to each well, followed by washing three times for 15 minutes to prevent nonspecific binding in addition to antigen-antibody binding with TBST (Tris-buffered saline and Tween 20) -2005) and goat anti-rabbit (Santa Cruz, sc-2004) were diluted to the respective concentrations and reacted for 1 hour. After washing three times for 10 minutes each with TBST, the color reaction was induced using an ECL (Bio-Rad) kit and confirmed using LAS-4000 (Fuji Photo Film Co., Tokyo, Japan).

In addition, 500 μl of the dissolution buffer was added to the gut extracted from the animal model, homogenized with a homogenizer, quantitated using a Bradford assay, and the above experiment was carried out in the same manner.

8. Stem cells From the useful substance (culture medium) Exosome  Extraction and Exosome Marker  Including confirmation of expression Muscle atrophy  Identification of effects on signal transduction mechanism

To examine the function of exosomes secreted by umbilical cord stem cells, exosomes were isolated from the obtained umbilical cord stem cell useful material (culture solution) in order to check the possibility as a biomarker. The newly obtained umbilical cord-derived stem cell useful material (culture solution) was centrifuged at 3000 x g for 15 minutes to remove residual impurities, and the supernatant was taken. ExoQuick-TC Exosome precipitation solution (System Biosciences; Mountain View, California, USA) at a ratio of 5: 1 (culture solution: precipitation mixture), and the tube is turned upside down and mixed. After that, store at 4 ℃ for 12 hours without rotating condition. After 12 hours, the mixture was centrifuged at 4O < 0 > C at 1500 x g for 30 minutes to identify beige or white pellets containing exosomes. After removing the supernatant, further centrifuge at 4 ° C and 1500 x g for 30 minutes to remove the supernatant. Expression confirmation of exosomes was confirmed by expression of CD9, CD81 antibody (Santa Cruz; 1: 1000 dilution) as an exosomal marker using the Western blot method.

In addition, we tried to confirm the effect of the exosomes obtained on L6 cells in the treatment of muscle atrophy signal. As already mentioned in Method 8, the expression of the markers related to the atrophy signal transduction was examined and the function of the exosome was confirmed.

Experiment result

1. Establishment of conditions for ensuring umbilical cord stem cell culture fluid

And establishing conditions for securing / treating the culture medium of umbilical cord stem cells, thereby maximizing the efficiency.

Determination of culture time

The stem cell-derived stem cells were cultured under serum-free conditions, and cell viability was compared to determine the culturing time (FIG. 3).

The survival rate of umbilical cord stem cells under serum - free conditions was maintained as normal for 48 hours, and cell morphology and adhesion were maintained. However, after 48 hours, the survival rate of the cells decreased, and the adhesion of the cells dropped sharply, confirming the death of the cells. As a result of measuring the same amount of stem cell culture solution obtained over time, it was determined that the most amount of the stem cell culture solution was obtained at 48 hours (FIG. 4).

In the results of various growth factors and cytokines assayed by stem cells contained in the prior patent, the highest amount was observed at 48 hours, and it was considered that this point is an important time point affecting cell growth. Through this, the stem cell useful material (culture solution) was determined to be collected at 48 hours after culturing in serum-free medium.

Comparison of potency according to concentration method of stem cell useful substance (culture solution)

In order to obtain a stable formulation of the stem cell useful material (culture solution), it was intended to obtain a large amount by concentration and to influence the growth or migration of other cells (FIG. 5).

When L6 muscle cells were cultured in serum-free medium and compared with the control group, which was confirmed as a serum-free medium, the concentration of the stem cell useful material (culture medium) The effect on cell proliferation was confirmed by time.

3 kDa cut-off concentration, or lyophilization and concentration were confirmed to be effective for cell proliferation after 24 hours when compared to the conditions of serum-free medium. However, there was no significant difference according to the concentration method, and it was confirmed that the effect was similar to the cell proliferation. Cellular morphology was observed through a wand healing assay to see how the stem cell useful material (culture medium) effective for cell proliferation actually affected cell migration (Fig. 6). L6 muscle cells with wound were cultured under the respective culture conditions and the morphology and migration of the cells were observed over time. As a result, it was found that the cells migrated under normal conditions for 48 hours, It was confirmed that the moving ability was remarkably decreased. On the contrary, it was confirmed that the culture condition of serum - free media influences the cell migration when mixed with the enriched stem cell useful material (culture solution). The results showed that cell migration was promoted as compared with that of medium without serum regardless of the concentration method of stem cell useful material (culture medium). In addition, the number of migrated cells was compared using a migration assay based on 48 hours of observation of cell migration (FIG. 7).

Compared with the serum - free media conditions, the number of cells migrated when the stem cell useful material (culture medium) was added was increased. In addition, there was no significant difference according to the concentration method of stem cell useful material (culture solution), and both groups showed significant results. Based on these results, it was confirmed that cell proliferation and migration were promoted irrespective of the method of concentrating stem cell useful substance (culture solution). However, as an alternative method to overcome the limitations of stem cells, it is necessary to secure a large amount of work to be solved for the optimization of the stem cell useful material (culture solution) and to develop the therapeutic agent, It is to devise a method that can be done. It is important to secure a large amount of the sample through the concentration process and to stabilize it so that it can be stored for a long period of time.

Stem cell useful material (culture solution) was prepared by rapidly cooling water-containing cells in a vacuum and once freezing, sublimating the ice and drying. The stem cell useful material (culture solution) of the powder formulation thus prepared was stored in an airtight container and stored at 4 ° C until the experiment. In order to confirm protein denaturation according to the storage elapsed time, CBB (coomassie brilliant blue) staining was performed (Fig. 8).

Protein was extracted by lyophilizing the stem cell useful material (culture solution) of the powder form stored at 4 ° C, and the protein was separated by membrane electrophoresis. Protein bands were identified by protein staining. The results showed that there was no significant difference in the protein bands of stem cell useful materials stored for one month and two years. From these results, it was confirmed that the protein denaturation did not occur during the storage period after the concentration method. As a result, it was confirmed that the sample could be easily stored for a long period after securing the sample.

2. Proof of protection of muscle atrophy using stem cell useful substance (culture solution)

2-1. Model of muscle atrophy

Establishment of muscle atrophy cell model

In order to confirm the efficacy of the stem cell useful material (culture medium), a muscle model of the rat (skeletal muscle cell line, L6 muscle cell) was used before the subsequent study (animal experiment) Respectively. To induce atrophy, dexamethasone was treated by concentration for 24 hours, and the results were confirmed using Western blot (Fig. 9). The concentration of dexamethasone in L6 muscle cells increased with increasing concentrations, indicating that the expression of muscle - specific protein desmin decreased. A number of reports using dexamethasone showed that desmin expression decreased from 1 μM concentration and was statistically significant. Based on these results, the concentration of dexamethasone to induce atrophy was set at 1 μM, and the subsequent experiments were conducted at the same concentration.

Confirmation of protection effect using stem cell useful substance (culture solution)

In order to confirm the effect of the stem cell useful substance (culture medium; CM) to induce atrophy by using dexamethasone in L6 muscle cells, the stem cell useful substance (culture solution) was treated with dexamethasone at various concentrations for 24 hours , And the results were confirmed using Western blot (Fig. 10).

It was confirmed that the expression of desmin in the L6 muscle cells was gradually increased as the concentration of the muscle protein-induced desmin was induced by the CM concentration-dependent increase in the dexamethasone treatment, and this was statistically significant. Based on these results, it was confirmed that stem cell useful material has protective effect against dexamethasone induced atrophy.

Effects of muscle atrophy induced by stem cell useful material (culture medium)

In the present invention, expression of signal transduction markers was confirmed by targeting the PI3k / Akt-FoxO pathway, which is well known as an atrophy mechanism (Fig. 11). When L6 muscle cells were treated with dexamethasone to induce atrophy, the mechanism of the atrophy-induced signal transduction pathway (PI3K / Akt-FoxO pathway) was confirmed by western blotting (Fig. 12).

When L6 muscle cells were treated with dexamethasone to induce atrophy, they were induced through the PI3K / Akt-FoxO pathway, which is known to be a mechanism of atrophy. Deactivation of Akt is dependent on the concentration of dexamethasone, leading to a decrease in phosphorylation of FoxO transcription factor and consequently degradation of FoxO activity. This transcription factor has been shown to increase the expression of MuRF-1, a gene associated with atrophy.

Wang et al. (Ref) increased protein degradation by about 20% when stimulated with dexamethasone in muscle cell lines of rats. In addition to the activity of ubiquitin-proteasome by the mechanism of atrophy, PI3k / Akt- , Which is involved in the regulation of the activity of genes involved in atrophy, such as Atrogin-1 and MuRF-1, and inhibits the activity of FoxO, a transcription factor.

 In order to examine the effect of the stem cell useful substance (culture medium) to induce the atrophy by using dexamethasone in the L6 muscle cells and to confirm the effect of the stem cell useful substance (culture solution) on the atrophy signal transmission (PI3K / Akt-FoxO pathway) (Culture medium; CM) was treated for 24 hours, and the results were confirmed using Western blot (Fig. 13).

The mechanism of dexamethasone-induced atrophy signal transduction was confirmed, and the activity of Akt reduced by dexamethasone was determined by treating the stem cell useful substance (culture solution) with various concentrations of dexamethasone in the cells. The activity of the stem cell useful substance (culture solution) Dependent manner. In addition, we confirmed that the expression of MuRF-1 and Atrogin-1 in the atrophy-related genes was decreased by the increase of FoxO activity. These results were significant from a concentration of 0.1 mg or more of the useful substance (culture medium). These results indicate that dexamethasone-induced atrophy is mediated by the AKT-FoxO pathway, and that the effect of the stem cell-mediated anti-atrophy protection is confirmed. , It will be important data for optimization of treatment technology.

2-2. Model of muscle atrophy

Comparison of effects of stem cell culture fluid after induction of atrophy

① Experimental animal weed muscle weight / weight

In order to determine the weight of the soleus muscle relative to the weight of each experimental animal, the weight of the soleus muscle of the experimental animal was divided into body weights to standardize the weight of the soleus muscle muscle mass) (Fig. 14). As a result of examining the standardized weed weight of the experimental animals according to the number of times of administration of the stem cell useful substance (culture solution), compared to the saline group in which the muscle atrophy was induced, This increase in muscle mass was confirmed.

In order to optimize the transplantation method, we compared the effect of the administration of the drug on the number of times of administration, and repeatedly repeated administration of the stem cell useful material (culture solution) after induction of atrophy was more effective. Statistically significant. Through these results, repeated dosing was selected.

Two groups, except for the control group, were induced to induce muscle atrophy for 2 weeks, and the stem cell culture fluid was injected. After 1 week, the weight of the soleus muscle of each experimental animal was examined to determine the weight of the soleus muscle The weight was divided into body weights and muscle masses (Figure 15). The weight change of the left and right saddle muscles of each animal was examined. As a result, the weight of the saddle muscle was recovered and the statistical significance was confirmed in the group to which stem cell useful material (culture medium) was administered.

② Comparison of accumulation of lactic acid by transplantation of stem cell material (culture medium) in experimental animals

It is known that lactic acid occurs when ATP is produced through anaerobic metabolism by making hypoxic state because blood is not smoothly supplied to muscle cells, and lactic acid accumulation causes degeneration of muscle contraction protein and also affects muscle strength.

In order to measure the concentration of lactic acid in the soleus due to hypotonic phenomena caused by the horseshoe crab, a lactic acid assay was carried out (Fig. 16). The mRNA expression levels of desmin and skeletal muscle actin, which are muscle regeneration markers, were examined by real-time PCR after injecting a stem cell useful substance (culture solution) into an experimental animal inducing muscle atrophy with the hippocampus (Fig. 17 , 18). As a result of comparing the expression of muscle-specific genes in the 1 week group with statistical significance, the expression of desmin in the saline group inducing muscle atrophy was reduced compared to the control group in which the normal life was maintained for 3 weeks , And that the desmin was significantly increased in the group injected with stem cells. In addition, mRNA expression of skeletal muscle actin was decreased in the saline group inducing muscle atrophy as compared with the control group, and the expression of the stem cell active material (culture medium) injection group was significantly increased. Based on these results, it is considered that the muscle regeneration is rapidly restored after injecting stem cell useful material (culture solution) after induction of suspension.

Protective effect of mechanism of treatment effect of atrophy

The aim of this study was to investigate the protective effect of stem cell useful material (culture solution) on the mechanism of muscle atrophy in animal model as identified in cell model.

(PI3K / Akt-FoxO pathway) markers were analyzed by extracting proteins from the muscle of the muscle atrophy model. The results were as follows (Fig. 19). Similarly, the activity of Akt and FoxO was decreased in the animal model induced by the atrophy, and the increase of the transcription factors MuRF-1 and MAFbx expressed upon the induction of atrophy by the FoxO gene transferred into the nucleus was confirmed. On the other hand, the activity of Akt decreased by the injection of the stem cell useful substance (culture medium) was increased, and the activity of FoxO was increased, thereby decreasing the expression of MuRF-1 and MAFbx, which are muscle atrophy genes, Respectively. These results demonstrate the protective effect of Akt-FoxO pathway during the atrophy of the stem cell useful material (culture solution) and will be important data for preventing or treating atrophy using this potential.

3. Protein cytokine analysis

Umbilical origin Intermediate lobe  Protein investigation of stem cell useful substance (culture solution)

In order to examine the secretion patterns of the umbilical cord-derived mesenchymal stem cells secreted and proliferated according to the present invention, human cytokine antibody arrays (RayBiotech Inc., Norcross, USA) were examined.

First, the number of cells of the umbilical cord stem cells was secured and the medium was replaced with serum-free medium. After 48 hours, the culture medium was collected, and the collected medium was quantitated by protein quantification and arrayed. 20, TSP (Thrombospondin), TSP-1, EDA-A2, IGFBP-rpl (IGFBP-7), TIMP-2, IL-6, GRO and MIP 2 1, VEGF, GDF-15, Smad 4, HGF, Angiogenin, Angiopoietin-1, 1), IGFBP-6, FGF-7 (KGF), and MMP-1 (Fig. 20a: array analysis of hUC-MSCs-culture medium, Fig. 19b, order).

IL-6 is an essential factor in regulating muscle cells. It is important to regenerate muscular satellite cells and to promote muscle cell proliferation. (Ref 2). MIP-2 plays a crucial role in the function of bone muscle, a potentially therapeutic target for regulating calcium homeostasis to treat diseases such as aging or myopenia (ref 3). MCP-1 is an important factor in the regeneration of injured tissue due to injury or wound (ref 4). Activin A is a regulator of muscle mass (ref 5), and VEGF is essential for bone muscle regeneration and promotes muscle function (ref 6). HGF plays a role in regulating growth and differentiation in muscle regeneration (ref 7), and FGF-7 plays an essential role in cell proliferation, differentiation and morphogenesis by stimulating or activating muscle satellite cells (ref 8) . MMP-1 also promotes muscle cell migration and differentiation (ref 9).

We expect that the various factors secreted from umbilical cord-derived mesenchymal stem cell cultures of this study will play a key role in muscle atrophy inhibition and muscle regeneration.

4. Investigate the function of the secretion of human umbilical cord stem cells to assess the potential as a biomarker

Confirm expression of exosomal markers

After the precipitation reaction, exosome extraction was confirmed through expression of CD9 and CD81 markers to confirm the obtained exosomes (FIG. 20).

Muscle atrophy  Identification of effects on signal transduction mechanism

L6 muscle cells were treated with dexamethasone to induce atrophy and simultaneous atrophy signaling (PI3K / Akt) after treatment of stem cell useful material (culture medium), exosome obtained from culture medium, and culture condition without exosome -FoxO pathway) related markers (Fig. 21).

Analysis of the results of the above results demonstrated that the exosomes obtained from the umbilical cord stem cell cultures were similar to the conditions in which the culture medium was treated in comparison with the condition in which the atrophy occurred (dexamethasone treatment, Dexa-treated) When treated together, the activity of reduced Akt was increased, and the activity of FoxO was increased to decrease the expression of MuRF-1 and MAFbx, which are the atrophy genes, and this proved statistically significant. However, it is important to note from this result that changes in the expression of these markers could not be confirmed when cultured conditions without exosomes were processed. Based on these results, in controlling the Akt-FoxO pathway, exosomes in the umbilical cord stem cell culture fluid have functions in cell signaling and can be predicted as new biomarkers. In addition, research on RNA and protein in exosomes can be further developed and proved to be a breakthrough in the development of treatments for atrophic diseases.

Summary and Conclusions

Muscle cells and animal models were used to establish an atrophy model and to compare the expression of muscle-associated proteins with atrophic stimulation. In addition, the expression of muscle - related proteins was compared by treating stem cell useful material (culture medium). Importantly, the treatment / protection effect of stem cell useful material (culture medium) was confirmed from the mechanism of atrophy. Based on the results obtained from this study, we have overcome the technical and practical limitations of stem cell treatment and showed the possibility of treatment of muscle atrophy as a relatively stable stem cell useful substance (culture medium). This is an epoch-making study that deviates from the current treatment method of using stem cells alone. In addition, stem cell-derived secretory action of exocose-like micro vesicles, such as the function of the research to develop a biomarker for the treatment of muscle atrophy can be a step ahead.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A pharmaceutical composition for preventing or treating muscular dystrophy comprising the mammalian umbilical cord stem cell culture solution prepared by the following method as an active ingredient:
(a) isolating stem cells from the mammalian umbilical cord;
(b) culturing the stem cells in a serum-free medium; And
(c) collecting the culture solution at 48 hours after culturing and centrifuging to obtain a supernatant.
The method according to claim 1, wherein the culture medium is selected from the group consisting of Thrombospondin (TSP), Trombospondin-1, EDTA-A2, Insulin-like growth factor binding protein-related protein 1 (IGFBP- IGFBP-7, tissue inhibitor of metalloproteinase-2 (TIMP-2), IL-6, GRO, MIP2, SPARC, MCP-1, Dkk-1, Activin A, TIMP- 15, Smad 4, HGF, Angiogenin, Angiopoietin-1, IGFBP-6, FGF-7 / KGF and MMP-1.
2. The pharmaceutical composition according to claim 1, wherein the culture solution promotes muscle cell proliferation.
2. The pharmaceutical composition according to claim 1, wherein the culture medium increases the expression of desmin.
2. The pharmaceutical composition according to claim 1, wherein the culture liquid increases the activity of Akt and FoxO.
The pharmaceutical composition according to claim 1, wherein the culture medium reduces expression of MuRF-1 (Muscle RING-finger protein-1) and Atrogin-1 / MAFbx.
The pharmaceutical composition according to claim 1, wherein the culture liquid promotes lactic acid degradation in muscle.
The pharmaceutical composition according to claim 1, wherein the culture medium increases the amount of muscle.

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