KR102330083B1 - Pharmaceutical Compositions for Improving or Treating Muscle Atrophy Comprising Conditioned Medium of Stem Cells Derived from Umbilical Cord - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising a culture solution of mammalian umbilical cord-derived stem cells as an active ingredient. When the umbilical cord-derived stem cell culture solution of the present invention is administered intramuscularly, the administered stem cells are effective for the prevention or treatment of muscular atrophy by proliferating muscle cells at the administered site, increasing muscle mass, and rapidly recovering muscle fatigue. do. Since the present invention uses a stem cell culture medium, it can overcome the technical and practical limitations of the treatment using stem cells, and has the advantage of being relatively easy to secure, so it can be very effectively used for the prevention and treatment of muscular atrophy.

Description

A pharmaceutical composition for the prevention or treatment of muscular atrophy comprising a mammalian umbilical cord-derived stem cell culture medium as an active ingredient {Pharmaceutical Compositions for Improving or Treating Muscle Atrophy Comprising Conditioned Medium of Stem Cells Derived from Umbilical Cord}

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

The muscle mass of the human body is normally maintained by balancing protein synthesis and degradation, but when protein degradation increases or synthesis decreases, muscle atrophy occurs. In addition to malnutrition and decreased activity, muscle atrophy is commonly observed in chronic wasting diseases such as cancer, diabetes, uremia, chronic lung disease, heart failure, and AIDS, but it is also known to appear in acute diseases such as sepsis, surgery, trauma, and burns. Even if exercise or other treatment methods (electrical stimulation, chemical drug treatment) are continuously received, it cannot be a fundamental solution, and it is not a fundamental treatment method because it is difficult to recover completely.

As stem cells have been proposed as a solution for overcoming incurable diseases, they have attracted attention because of their strong regeneration potential. Although embryonic stem cells and adult stem cells differ in their differentiation ability, they can be expected to have the effect of regenerating damaged parts because they differentiate into cells constituting various tissues. According to recent reports, adult stem cells are obtained from various human sources, such as umbilical cord blood, bone marrow, fat, and amniotic fluid, and these mesenchymal stem cells (MSCs) suggest their potential as a treatment for various disease models. have. In addition, in order to treat musculoskeletal disorders, treatment studies using stem cells have been preceded, and it has been reported that stem cells have the potential for muscle regeneration. The present inventors also preceded the study of muscle atrophy treatment using umbilical cord-derived mesenchymal stem cells (UC-MSCs), and reported results of improving muscle atrophy after directly injecting the umbilical cord-derived stem cells into the local area where the lesion occurred. (ref 1).

Recently, reports of paracrine action, one of the disease treatment mechanisms of adult stem cells, are increasing. That is, when the mesenchymal stem cells are exposed to the lesion site, they secrete various therapeutic proteins in response to the micro-environment. Secreted therapeutic proteins include growth factors, cytokines, extracellular matrix, and antioxidant proteins, which are analyzed and reported through various proteomics techniques. Therefore, when mesenchymal stem cells, which perform paracrine action, are applied to various disease models, there are increasing reports that these secreted proteins improve body functions and exhibit therapeutic effects on diseases. However, despite the potential of stem cells, development and utilization as a therapeutic agent are limited due to practical technical problems. For example, in order to use cells as a therapeutic agent, more than a certain number of cells are required. is needed

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field 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.2,2015 2. Zhang C, et al., J Biol Chem. 18;288(3):1489-99,2013 3. Romero-Suarez S, et al., Aging (AlbanyNY). 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 intensive research efforts to develop a material that can effectively treat muscular atrophy and can be safely applied to the human body, and as a result, by identifying that the culture medium of umbilical cord-derived stem cells is very effective for preventing or treating muscular atrophy, the present invention has been completed

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle atrophy.

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

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising a mammalian umbilical cord-derived stem cell culture medium prepared by the following method as an active ingredient:

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

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

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

The present inventors have made intensive research efforts to develop a substance that can effectively treat muscular atrophy and can be safely applied to the human body, and as a result, it was found that the culture solution of umbilical cord-derived stem cells is very effective in preventing or treating muscular atrophy.

According to the present invention, the umbilical cord-derived stem cell culture medium of the present invention increases the proliferation of muscle cells, increases the muscle mass, and promotes the decomposition of lactic acid in the muscle. In addition, it increases the expression of the muscle protein desmin in an animal model of muscular atrophy, increases the activity of Akt and FoxO in the AKT-FoxO pathway related to muscle atrophy, and thus the expression of the muscle atrophy-related genes MuRF-1 and Atrogin-1 By reducing , it exhibits amyotrophic protective activity.

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

The umbilical cord-derived stem cells used in the present invention can be obtained from various mammals. more preferably human, pig, horse, cow, mouse, rat, hamster, rabbit, goat or sheep, even more preferably human, pig or mouse, most preferably human It can be obtained from the umbilical cord.

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

The umbilical cord-derived stem cells used in the present invention can be obtained in the manner described in Examples below. For example, after removing the arteries and veins in the umbilical cord, the Wharton's soft tissue is cut and the cut Wharton's soft tissue is directly cultured to grow cells (outgrowth). Then, by subculturing the grown cells, stem cells are obtained. The medium used in the culturing process may be any medium commonly used for culturing animal cells, for example, Eagle's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), α- 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)), Way-mouth'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 the MCDB series (Ham) , RG et al., In Vitro 14:11 (1978)) and the like can be used.

The culture medium of the umbilical cord-derived stem cells used in the present invention is obtained by culturing the stem cells in a medium containing serum, and when the cells become 80-90% density, the medium is exchanged with a serum-free medium and then cultured for 42-52 hours. Then, the culture solution is centrifuged to obtain a supernatant. More preferably, the culture medium obtained after exchanging the medium with a serum-free medium and then culturing for 48 hours is used.

As demonstrated in the examples below, in the absence of serum, the survival rate of umbilical cord-derived stem cells was maintained as in normal conditions until 48 hours, but after 48 hours, the cell viability decreased, and the adhesion of the cells was also rapidly decreased. As a result of measuring the concentration of the same amount of stem cell culture medium obtained for each, it was confirmed that 48 hours was the time to secure the most useful substances, and it was decided to obtain a culture medium after 48 hours of culture.

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

According to one embodiment of the present invention, the umbilical cord-derived stem cell culture medium can be concentrated by cutting-off 3 kDa or by freeze-drying, and the culture medium promotes the proliferation and migration of muscle cells regardless of the concentration method.

As demonstrated in the Examples below, as a result of checking whether the culture solution of the powder formulation stored at 4° C. after freeze-drying was changed according to the storage period, protein denaturation according to the storage period did not occur. Therefore, the culture solution of the present invention can be stored for a long time after freeze-drying.

According to one embodiment of the present invention, the umbilical cord-derived stem cell culture medium is thrombospondin (Thrombospondin, TSP), thrombospondin-1, EDA-A2 (Ectodysplasin-A2), IGFBP-rp1 (Insulin-like growth factor binding) protein-related protein 1)/IGFBP-7, TIMP-2 (tissue inhibitor of metalloproteinase-2), IL-6, GRO, MIP2, SPARC, MCP-1, Dkk-1, Activin A, TIMP -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” refers to a decrease in muscle tissue or degeneration of a muscle.

According to a preferred embodiment of the present invention, the pharmaceutical composition for preventing or treating muscular atrophy of the present invention is a composition for intramuscular administration, and when the composition is administered intramuscularly, the administered umbilical cord-derived stem cells are intramuscular. It promotes cell proliferation and lactic acid degradation, increases the size of muscle fibers, and increases muscle contractility, thereby exhibiting preventive or therapeutic efficacy against muscular atrophy.

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

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

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered PBS (PBS) saline) or medium, but is not limited thereto.

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

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, more preferably intramuscularly or intravascularly.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. can be

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

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

(i) The present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising a culture medium of mammalian umbilical cord-derived stem cells as an active ingredient.

(ii) When the umbilical cord-derived stem cell culture solution of the present invention is administered intramuscularly, the administered stem cells proliferate muscle cells in the administered site, increase muscle mass, and rapidly recover muscle fatigue to prevent muscular atrophy or effective for treatment.

(iii) Since the present invention uses a stem cell culture medium, it can overcome the technical and practical limitations of the treatment using stem cells and has the advantage of being relatively easy to secure, so it can be very effectively used for the prevention and treatment of muscular atrophy.

1 is a photograph showing the production of a lower extremity suspension model (maintaining 30° with the ground) for inducing muscle atrophy.
Figure 2 is a schematic diagram for the design of the muscle atrophy animal model.
3 is a result of measuring cell viability under serum-free conditions.
4 is a result of measuring the concentration of the same amount of stem cell culture medium for each time in the absence of serum.
5 is a result of measuring the effect on cell proliferation according to the concentration method of the useful stem cell material.
6 is a result of measuring the effect (Wound healing assay) on cell migration according to the method of enriching the useful stem cell material.
7 is a result of measuring the effect (Migration assay) on cell migration according to the concentration method of the useful stem cell material.
8 is a result of confirming the denaturation of the protein according to the elapsed time after storage at 4° C. of the freeze-dried stem cell useful material.
9 is a result of measuring the expression of the muscle protein desmin after treatment with dexamethasone for each concentration.
Figure 10 is a result of comparing the change in the expression of muscle protein desmin by treating dexamethasone and a stem cell useful substance.
11 is a schematic diagram showing a signal transduction system related to muscle atrophy.
12 is a result of confirming the muscle atrophy signaling related markers after treatment for each concentration of dexamethasone.
13 is a result of measuring the protective effect on dexamethasone-induced muscular atrophy signal transduction using a stem cell useful substance.
14 is a comparison result of standardized soleus muscle weight of experimental animals according to the number of transplants.
15 is a comparison result of standardized soleus muscle weights of experimental animals.
16 is a result of comparing the concentration of lactic acid accumulation in experimental animals.
17 is a result of comparing the expression level of desmin in experimental animals.
18 is a result of comparing the expression level of skeletal muscle actin in experimental animals.
19 is a result confirming the effect of the stem cell useful substance on the muscle atrophy mechanism through western blot.
20 is a cytokine array analysis result of human umbilical cord-derived mesenchymal stem cell useful material (culture solution).
21 is a result of analyzing the expression of the exosome marker.
22 is a result of analyzing the expression levels of markers related to muscle atrophy signaling (PI3K/Akt-FoxO pathway) according to the presence or absence of exosomes.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

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

In order to secure umbilical cord-derived stem cell useful substances (culture solution), the umbilical cord-derived stem cells obtained through a series of processes from the umbilical cord through a prior patent were treated with 10% fetal bovine serum (FBS, Hyclone Laboratories Inc., Logan, UT, USA). ^{A 175 cm 2} culture flask (SPL Life Sciences Co., Ltd.) with 25 mL of MEM alpha modified medium (α-MEM, Hyclone) containing ., Pocheon, Korea) 37 ℃, 5% CO ₂ conditions were cultured. After dispensing the umbilical cord tissue-derived stem cells, they were cultured for 3 days, and when the cells reached a density of 80-90%, they were replaced with serum-free MEM alpha improved medium (serum-free condition). After 48 hours of incubation, the culture solution was recovered in a 50 mL conical tube (SPL), and centrifuged at 1500 rpm for 5 minutes to remove the cell debris from which the adhesion fell. After that, the supernatant was transferred, and concentrated in two ways to store and maintain a stable formulation and used for the experiment. The first method was freeze-dried (FDU-8612, Operon, Gimpo, Korea), stored in a vacuum state, and stored at 4°C until experiment, and the second method was 15 mL of 3K Centricon (Milipore, Bedford, MA, USA). ) and centrifuged at 4,000 x g for 30 minutes, and only obtained 3K or higher and stored at 4°C until testing. The thus-prepared stem cell culture medium was allowed to be treated by concentration according to the experimental conditions for which the efficacy was to be reported.

2. Measurement of cell viability

In order to determine the optimal harvesting time without toxicity to the growth of cells in the absence of serum, useful stem cell materials (cultures) were recovered for each time period and cell viability was measured. It was dispensed into a 96-well plate (SPL), treated with reagents, and _{cultured at 37° C. and 5% CO 2} conditions for 24 hours and 48 hours, respectively, and then WST-1 assay was performed. The amount of the medium contained in each well and Cyto X water soluble tetrazolium salt (WST, LPS solution, Daejeon, Korea) were diluted in a ratio of 1:10 and treated, followed by reaction at 37°C for 1 hour. After the reaction was completed, the absorbance value was measured at 450 nm using an ELISA microplate reader (Epoch, Bio-Tek., Winooski, CA, USA), and cell viability was evaluated using this value.

3. L6 muscle cells culture and muscle atrophy Induction model construction

To create a muscle atrophy-inducing cell model, a rat skeletal muscle cell line, L6 cell line (CRL1458, ATCC, Manassas, VA, USA) was used. This was based on a medium containing 10% fetal bovine serum and 1% P/S in DMEM-high glucose (Hyclone). After confirming that the L6 muscle cells were attached to the bottom of the flask and proliferated, the new medium was replaced every 2 days. When the cell density reached about 90%, the cells were suspended using trypsin-EDTA (Hyclone), and then only the cells were recovered and subcultured by centrifugation at 1,500 rpm for 5 minutes. In addition, to induce muscle atrophy, 1 μM of dexamethasone (Dexamethasone; Sigma Aldrich, St. Louis, MO, USA) was treated and the expression of the muscle atrophy-related transcription factors (MuRF-1 and Atrogin-1) was confirmed accordingly. was built. In addition, it was attempted to prove its efficacy by adding a stem cell useful substance (culture solution) to L6 muscle cells together with dexamethasone.

4. Observation of cell migration

1 x 10 ⁵ L6 myocytes were planted in a 6-well plate (SPL) and a wound was made using a 200 ml tip. The cells were cultured under each culture condition, and cell morphology and movement were observed with a microscope over time.

5. Establishment of muscle atrophy-induced animal model and measurement of weight change in soleus muscle

A 5-week-old female Sprague-Dawley (SD) rat was used as an experimental animal, and the experiment was conducted after the adaptation period. Muscular atrophy was induced using the lower extremity suspension method (Groups 2 and 3), and the tail was fixed on the top of the cage using dressing tape, and the body angle was maintained at 30° with the floor to keep the hind legs suspended. (Fig. 1). In addition, according to the experimental conditions, it was divided into three groups, the 1-week untreated control group (Group 1), the 1-week group muscle atrophy induction group (Group 2), the 1-week group muscle atrophy induction group, the stem cell culture medium transplanted and the hind limb rich experimental group ( Group 3) (Fig. 2). After inducing muscle atrophy for 2 weeks, 100 μl of 0.9% saline was injected intramuscularly into the soleus muscle of the control group, and 2 mg/100 μl of a useful stem cell substance (culture solution) was injected into the soleus muscle of the test group. . The lower extremity state was maintained continuously for 1 week after injection. One week after administration of the test substance, the soleus muscle was removed, and the weight was measured, standardized in proportion to the body weight, and then compared.

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

RNA was extracted from the extracted soleus root using an easy-spin total RNA extraction kit (iNtRON Biotechnology Inc., Seoul, Korea). After adding 1 ml of lysis buffer to the soleus muscle, it was homogenized with a homogenizer. 200 μl of chloroform was added and mixed. Centrifuge at 4°C at 13,000 rpm for 10 minutes, transfer only the supernatant to a new tube, add 400 μl of binding buffer, and shake well 2-3 times. The mixed solution was put into the column and centrifuged at 13,000 rpm for 30 seconds to remove the solution that came down from the column, and 700 μl of washing buffer A was added to the column, and then centrifuged again at 13,000 rpm for 30 seconds to remove the solution that came down. After adding 700 μl of wash buffer B to the column, centrifugation was performed at 13,000 rpm for 30 seconds to remove the solution, and again centrifuged at 13,000 rpm for 2 minutes to dry the column membrane. A new tube is mounted under the column, 50 μl of elution buffer is added to the column, and then centrifuged at 13,000 rpm for 1 minute to extract RNA from the tube. After quantification of the extracted RNA, 500 ng of RNA was denatured at 70°C for 5 minutes, 1/5 volume of 5x RT master mix was added, and then reacted at 37°C for 1 hour to synthesize cDNA. Dilute the synthesized cDNA in distilled water, mix desmin and skeletal muscle actin primers and SYBR Green Master Mix, measure the amount of amplified RNA using Real-Time PCR equipment, A graph was prepared for comparison.

6. Confirmation of lactic acid accumulation concentration following transplantation of stem cell useful substances (culture solution)

500 μl of lactic acid assay buffer (Eton Bioscience, San Diego, CA, USA) was added to the extracted soleus muscle, homogenized with a homogenizer, and quantified using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein amount was diluted to 1 mg/ml and the protein was removed at -80°C. To prepare a standard curve to be compared with the sample, add 990 μl of lactic acid assay buffer to 10 μl of lactic acid standard solution, dilute it to 1 nmol/μl, and dispense 0, 2, 4, 6, 8, and 10 μl into 96-well plates. Samples were also dispensed by 10 μl and filled with lactic acid assay buffer so that the total volume was 50 μl. 46 μl of lactic acid assay buffer, 2 μl of probe, and 2 μl of enzyme mix were mixed according to the composition, and 50 μl of the reaction mix was dispensed into each well and mixed well. After blocking the light and reacting at room temperature for 30 minutes, O.D. The absorbance was measured at 570 nm, and the concentration was calculated and compared by substituting the standard curve.

7. Confirmation of expression of muscle atrophy mechanism markers following transplantation of stem cell useful substances (culture)

L6 muscle cells cultured in 6-well plates were treated with 1 μ dexamethasone and conditioned medium 1, 5, 10, and 100 μ, followed by incubation at 37° C. and 5% CO ₂ conditions 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 fresh added protease inhibitor cocktail) was dispensed into each well and reacted. did it And after taking the supernatant through centrifugation at 4 ℃, 13,000 rpm for 15 minutes, the extracted protein was measured for absorbance in an ELISA microplate reader 595 nm to determine the protein concentration of each sample. Each sample was heated at 100° C. for 3 minutes with SDS-PAGE sample buffer (Bio-Rad, Seoul, Korea), electrophoresed on SDS-pliacrylamide gel (Bio-Rad), and then subjected to SDS-PAGE. To transfer the separated protein to the membrane, a nitrocellulose transfer membrane (Protran®BA83, Whatman, Dassel, Germany) was used. After blocking with 3% bovine serum albumin (BSA, Bio Basic Inc., Ontario, Canada) for 1 hour, primary antibodies desmin (1:1000 dilution), p-Akt (1:1000 dilution), Akt (1:2000 dilution) p-Foxo1/3α(1:1000 dilution), FoxO1(1:1000 dilution), FoxO3α(1:1000 dilution), MuRF-1(1:1000 dilution), MAFbx(1:1000 dilution) ), β-actin (1:5000 dilution) was added and left at 4°C overnight. After washing 3 times for 15 minutes each with TBST (Tris-buffered saline and Tween 20) to prevent non-specific binding other than antigen-antibody binding, a secondary antibody goat anti-mouse (Santa Cruz, sc) labeled with horseradish peroxidase (HRP) -2005), goat anti-rabbit (Santa Cruz, sc-2004) were diluted to each concentration and reacted for 1 hour. After washing three times for 10 minutes in TBST, a color reaction was induced using an ECL (Enhanced chemiluminescence) kit (Bio-Rad) and confirmed using LAS-4000 (Fuji photo film Co., Tokyo, Japan).

Additionally, 500 μl of lysis buffer was added to the soleus muscle extracted from the animal model, homogenized with a homogenizer, and quantified using a Bradford assay, and the above experiment was performed in the same manner.

8. Stem Cells From useful substances (culture solution) exosomes extraction and exosomes marker including expression confirmation muscle atrophy Confirmation of effect on signaling mechanism

To investigate the function of exosomes secreted by umbilical cord-derived stem cells, and to confirm their potential as biomarkers, exosomes were isolated from the secured umbilical cord-derived stem cell useful substances (culture solution). The newly obtained umbilical cord-derived useful material (culture solution) was centrifuged at 4℃, 3000 x g for 15 minutes to remove residual impurities and the supernatant was taken, and then ExoQuick-TC Exosome precipitation solution (System Biosciences; Mountain View, California, USA) in a ratio of 5:1 (culture solution: precipitation mixture), and mix by turning the tube upside down. Then, store at 4°C for more than 12 hours without rotation conditions. After 12 hours, the mixed solution is centrifuged at 4° C., 1500 x g for 30 minutes, and exosome-containing beige or white pellets can be identified. After removing the supernatant, the remaining supernatant is further removed by centrifugation at 4°C and 1500 x g for 30 minutes. Confirmation of the expression of exosomes was confirmed through the expression of CD9 and CD81 antibodies (Santa Cruz; 1:1000 dilution) as exosome markers using a Western blot method.

Additionally, by using the exosomes obtained in this way and treating them together with L6 cells, we tried to check the effect on the muscle atrophy signaling mechanism. As already mentioned in method 8, the expression of markers related to amyotrophic signal transduction was investigated, and the function of exosomes was confirmed.

Experiment result

1. Establishment of conditions for securing umbilical cord-derived stem cell culture medium

By establishing conditions for securing/treating the culture medium of umbilical cord-derived stem cells, it was attempted to maximize the efficiency.

Decide when to harvest the culture medium

Through culturing of umbilical cord-derived stem cells under serum-free conditions, the cell viability was compared to determine the culture medium harvest time (FIG. 3).

It was confirmed that the viability of the umbilical cord-derived stem cells in the serum-free condition was maintained as in the normal condition until 48 hours, and the cell shape and adhesion were also maintained. However, after 48 hours, the cell viability decreased, and the cell adhesion was also rapidly decreased, confirming the cell death. As a result of measuring the concentration of the same amount of stem cell culture medium obtained for each hour, it was determined as the time when the greatest amount could be secured at 48 hours (FIG. 4).

In the analysis results of various growth factors and cytokines secreted by stem cells, which were included in the prior patent, the largest amount was confirmed at 48 hours, and it was determined that this time point was an important time point affecting cell growth. Through this, it was decided to collect the useful stem cell material (culture solution) 48 hours after culturing in a serum-free medium.

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

In order to implement a stable formulation of the collected stem cell useful material (culture solution), it was concentrated to secure a large amount and at the same time to check how it affects the growth or migration of other cells (FIG. 5).

When compared with the control group in which the L6 muscle cells were cultured in the serum-free medium, the serum-free culture condition was treated with the stem cell useful substances (culture solution) concentrated in different ways. The effect on cell proliferation was confirmed by time.

It was confirmed that the 3 kDa cut-off method of concentration or freeze-drying concentration was effective for cell proliferation after 24 hours when compared to serum-free medium conditions. However, there was no significant difference according to the concentration method, and it was confirmed that a similar effect was shown on cell proliferation. Cell morphology was observed through a wound healing assay to see how effective stem cell useful substances (culture medium) for cell proliferation actually have an effect on cell migration (FIG. 6). As a result of culturing wounded L6 muscle cells under each culture condition and observing cell morphology and migration by time, compared to 48 hours for cell migration under normal conditions, the growth of cells in serum-free medium was It was confirmed that the movement ability was significantly reduced. On the other hand, it was confirmed that the condition of the serum-free medium, but when the concentrated stem cell useful material (culture solution) was mixed and treated together, it was confirmed that it also affects the movement of cells. Regardless of the concentration method of the stem cell useful material (culture solution), the result of promoting cell migration was confirmed compared to the serum-free medium condition. Additionally, the number of migrated cells was compared using a migration assay based on 48 hours of observation of the cell migration ( FIG. 7 ).

Compared to the serum-free medium condition, it was confirmed that the number of migrated cells was increased when a stem cell useful substance (culture solution) was added. Also, there was no significant difference according to the concentration method of the stem cell useful substances (culture solution), and significant results were confirmed in both groups. Based on these results, it was confirmed that it promotes the proliferation and migration of cells regardless of the concentration method of the stem cell useful material (culture medium). However, as an alternative method to overcome the limitations of stem cells, it is necessary to secure a large amount of the task we need to solve for the optimization using stem cell useful substances (culture medium) and development of therapeutic agents, and to prevent storage and deterioration. devising a way to do it. It is important that a method that can satisfy all of these points secures a large amount of the sample through the concentration process and secures it in a stable state that can be stored for a long time.

Stem cell useful material (culture solution) was dried by cooling the cells containing moisture in a vacuum, freezing once, and sublimating on ice. The stem cell useful material (culture solution) in the powder formulation thus made was stored in an airtight container and stored at 4° C. until the experiment. In order to check whether protein denaturation occurs according to the elapsed time of storage, it was confirmed through CBB (coomassie brilliant blue) staining (FIG. 8).

The protein was extracted by dissolving the stem cell useful material (culture solution) in the form of a powder that was freeze-dried and stored at 4°C, which was separated by membrane electrophoresis to detect the protein. The pattern of the protein band was confirmed through protein staining, and as a result of checking whether there was a change according to the elapsed time, it was confirmed that there was no significant difference in the pattern of the protein band of the stem cell useful substances stored for one month and two years. Through these results, it was confirmed that protein denaturation according to the storage period did not occur after the concentration method, and it was confirmed that long-term storage after securing the sample through this could be confirmed.

2. Proof of muscle atrophy protective effect using stem cell useful material (culture solution)

2-1. amyotrophic cell model

Building a muscle atrophic cell model

In order to confirm the efficacy of the stem cell useful substance (culture medium), a muscle atrophy model was tested using a cell model (skeletal muscle cell line, L6 muscle cell) of the same species (rat) before proceeding with a subsequent study (animal experiment). built. In order to induce muscle atrophy, dexamethasone was treated for 24 hours at each concentration, and the results were confirmed using Western blot (FIG. 9). In L6 muscle cells, it was confirmed that the expression of the muscle-specific protein desmin decreased as the concentration of dexamethasone increased depending on the concentration of dexamethasone. In many reports using dexamethasone, it was shown that the expression of desmin decreased from the concentration of 1 μM used, and it was confirmed as a statistically significant result. Based on this result, the concentration of dexamethasone to induce muscle atrophy was set to 1 μM, and subsequent experiments were performed at the same concentration.

Confirmation of protective effect using stem cell useful substances (culture solution)

In order to induce muscle atrophy by using dexamethasone in L6 muscle cells and to check the effect of the stem cell useful substance (culture solution; CM) to be confirmed at the same time, the cells were treated with dexamethasone at various concentrations along with the stem cell useful substance (culture solution) for 24 hours and confirming the results using Western blot (FIG. 10).

In L6 muscle cells, it was confirmed that the expression of the myoprotein desmin induced by treatment with dexamethasone gradually increased as the concentration increased in a CM concentration-dependent manner, and it was confirmed that the result was statistically significant. Based on these results, it was confirmed that the stem cell useful substances have a protective effect against the induction of muscle atrophy by dexamethasone.

Effect of muscle atrophy mechanism by stem cell useful substances (culture solution)

In the present invention, the expression of each signaling marker was confirmed by targeting the PI3k/Akt-FoxO pathway, which is well known as a muscle atrophy mechanism ( FIG. 11 ). When muscle atrophy was induced by treatment with dexamethasone in L6 muscle cells, the muscle atrophy-inducing signaling mechanism (PI3K/Akt-FoxO pathway) was confirmed through Western blot results (FIG. 12).

When muscle atrophy was induced by treatment with dexamethasone in L6 muscle cells, it was confirmed that it was induced through the PI3K/Akt-FoxO pathway, which is well known as the mechanism of muscle atrophy. Dexamethasone concentration-dependently decreased Akt activity, leading to a decrease in FoxO transcription factor phosphorylation and consequent decrease in FoxO activity. It was confirmed that this transcription factor increased the expression of the gene MuRF-1 related to muscle atrophy.

Wang et al. (ref) reported that when a mouse muscle cell line was stimulated with dexamethasone, proteolysis was increased by about 20%, and in addition to ubiquitin-proteasome activity as a muscle atrophy mechanism, the PI3k/Akt-FoxO pathway was mediated. Therefore, it is reported that it suppresses the activity of FoxO, a transcription factor, and is related to the regulation of the activity of genes involved in muscle atrophy, such as Atrogin-1 and MuRF-1.

In order to induce muscle atrophy by using dexamethasone in L6 muscle cells and at the same time confirm the effect of the stem cell useful substance (culture solution) on the muscle atrophy signal transduction (PI3K/Akt-FoxO pathway), various Stem cell useful substances (culture solution; CM) of the concentration were treated for 24 hours, and the results were confirmed using Western blot (FIG. 13).

The muscle atrophy signaling mechanism induced by dexamethasone was confirmed, and by treating the cells with a stem cell useful substance (culture solution) at various concentrations together with dexamethasone, the activity of Akt reduced by dexamethasone was reduced by the concentration of the stem cell useful substance (culture solution). It was confirmed that the increase was dependent on . In addition, it was confirmed that the expression of the muscle atrophy-related genes MuRF-1 and Atrogin-1 was decreased by the increase in FoxO activity. These results were significant from the concentration of 0.1 mg or more of the useful substance (culture solution). Through these results, the muscular atrophy induced by dexamethasone passes through the AKT-FoxO pathway, and it was possible to confirm the muscle atrophy protective effect using the stem cell useful substance. It is thought to be an important data for optimizing treatment technology by identifying

2-2. amyotrophic animal model

Comparison of Effects of Stem Cell Culture Transplantation after Induction of Muscle Atrophy

① Weight/weight of soleus muscle of the experimental animal

Inducing muscle atrophy with lower limb suspension for 2 weeks, injecting a useful stem cell substance (culture solution), and standardizing the weight of the soleus muscle by dividing the weight of the soleus muscle by the body weight of the experimental animal to check the weight of the soleus muscle relative to the weight of each experimental animal 1 week later ( muscle mass) (FIG. 14). As a result of checking the standardized soleus muscle weight of the experimental animals according to the number of times of administration of the stem cell useful substance (culture solution), the group administered with the stem cell useful substance (culture solution) compared to the saline group induced muscle atrophy with lower extremity suspension An increase in this muscle mass was confirmed.

In order to optimize the transplantation method, the effect according to the number of administrations was comparatively analyzed, and the group in which the stem cell useful substance (culture solution) was administered after induction of muscle atrophy was more effective than the group in which the group was administered repeatedly at repeated cycles. It was confirmed that it was statistically significant. Based on these results, the method of repeated administration was selected for the number of administrations.

The two groups, except for the untreated group, induced muscle atrophy with lower extremity suspension for 2 weeks, and then injected stem cell culture. The weight of the soleus muscle was standardized (muscle mass) by dividing the weight by the body weight (FIG. 15). As a result of checking the change in the weight of the soleus muscle on the left and right sides of each animal, the weight of the soleus muscle was recovered in the group administered with the stem cell useful substance (culture solution) without any significant difference, which was also confirmed to be statistically significant.

② Comparison of lactic acid accumulation following transplantation of stem cell useful substances (culture solution) in experimental animals

Lactic acid is generated when the blood is not supplied to muscle cells smoothly, creating a hypoxic state and generating ATP through anaerobic metabolism.

In order to measure the lactate concentration in the soleus muscle due to hypovolemia caused by the suspension of the lower extremities, a lactate assay was performed (FIG. 16). As the stem cell useful material (culture solution) was injected into the experimental animals that induced muscle atrophy with the lower extremity suspension, the mRNA expression levels of desmin and skeletal muscle actin, which are muscle regeneration markers, were confirmed by real-time PCR to compare the differences (Fig. 17). , 18). As a result of comparing the expression of muscle-specific genes in the 1 week group, which showed statistical significance, the expression of desmin in the saline group, which induced muscle atrophy with lower extremity suspension, decreased compared to the control group that maintained a normal life for 3 weeks. and it was confirmed that desmin was significantly increased in the stem cell useful substance (culture) injection group. In addition, mRNA expression of skeletal muscle actin was also decreased in the saline group, which induced muscle atrophy compared to the control group, and it was confirmed that the expression of the stem cell useful substance (culture solution) injection group was significantly increased. Based on these results, it is thought that the muscle regeneration is rapidly restored after the induction of the lower extremity suspension and the injection of useful substances (culture solution) for stem cells.

Proof of protective effect on muscle atrophy treatment effect mechanism

As identified in the cell model, it was attempted to confirm the protective effect of using a stem cell useful substance (culture solution) by targeting the therapeutic mechanism of muscle atrophy in the animal model.

Proteins were extracted from the soleus muscle of an animal model of muscle atrophy and the markers of the muscle atrophy signaling mechanism (PI3K/Akt-FoxO pathway) were analyzed. As a result, the following results were obtained (FIG. 19). Similarly, in the animal model in which muscle atrophy was induced, the activity of Akt and FoxO was decreased, and it was confirmed that the transcription factors MuRF-1 and MAFbx expressed when muscle atrophy was induced by the FoxO gene transferred into the nucleus were increased. On the other hand, the activity of Akt, which was decreased by the injection of the stem cell useful substance (culture), increased, and thus the activity of FoxO was increased to decrease the expression of the muscle atrophy genes MuRF-1 and MAFbx, which was also statistically significant. did. Through these results, the protective effect on the Akt-FoxO pathway among the muscle atrophy mechanisms using stem cell useful substances (culture medium) was demonstrated, and this potential will be used as an important data for preventing or treating muscle atrophy.

3. Protein Cytokine Analysis

umbilical cord origin mesenchymal Protein investigation of stem cell useful substances (culture solution)

In order to examine the cytokine secretion pattern of the umbilical cord-derived mesenchymal stem cells secreted and proliferated according to the present invention, a human cytokine antibody array (RayBiotech Inc., Norcross, USA) was used.

First, the cell number of umbilical cord-derived stem cells was obtained, and the culture medium was collected 48 hours after replacement with a serum-free medium. 20, TSP (Thrombospondin), TSP-1, EDA-A2, IGFBP-rp1 (IGFBP-7), TIMP-2, IL-6, GRO, MIP 2 in the culture medium of umbilical cord-derived mesenchymal stem cells , SPARC, MCP-1, Dkk-1, Activin A (Activin A), TIMP-1, VEGF, GDF-15, Smad 4, HGF, Angiogenin, Angiopoietin-1 (Angiopoietin- 1), it was confirmed that cytokines such as IGFBP-6, FGF-7 (KGF), and MMP-1 were secreted (Fig. 20a: hUC-MSCs-array analysis picture of culture medium, Fig. 19b, c: antibody array order).

As a result of examining the function of each of these identified cytokines, IL-6 is an essential factor in regulating muscle satellite cells, and is a very important factor in promoting muscle satellite cell proliferation to recover from diseases caused by not using muscles. is (ref 2). MIP-2 plays an important role in osseous muscle function, which is a potential therapeutic target for treating diseases such as aging and sarcopenia by regulating calcium homeostasis (ref 3). MCP-1 is an important factor in the regeneration of tissues damaged by injuries or wounds (ref 4). Activin A is a factor that regulates 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 during 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).

It is expected that the various factors secreted from the umbilical cord-derived mesenchymal stem cell useful material (culture medium) in this study will play a key role in inhibiting muscle atrophy and regenerating muscle.

4. Investigate the function of secretions of human-derived umbilical cord stem cells, and evaluate their potential as biomarkers

Confirmation of exosome marker expression

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

muscle atrophy Confirmation of effect on signaling mechanism

In order to induce and confirm muscle atrophy using dexamethasone in L6 muscle cells, stem cell useful substances (culture medium), exosomes obtained from the culture medium, and exosome-free culture medium conditions were treated together, and then muscle atrophy signal transduction (PI3K/Akt) -FoxO pathway) expression of related markers was confirmed (FIG. 21).

Analyzing the results corresponding to the previously demonstrated results, the exosomes obtained from the umbilical cord-derived stem cell culture medium were similar to the condition treated with the culture medium compared to the condition in which muscle atrophy occurred (dexamethasone treated condition, Dexa-treated). When treated together, the decreased Akt activity was increased, and thus FoxO activity was increased to decrease the expression of the muscle atrophy genes MuRF-1 and MAFbx, which was also confirmed to be statistically significant. However, it is important to consider through this result, when the culture medium condition without exosomes was treated, it was not possible to confirm the change in the expression of these markers. Based on these results, in regulating the muscle atrophy mechanism (Akt-FoxO pathway), the exosomes in the umbilical cord-derived stem cell culture medium have a function in cell signal transduction and can be predicted as a new biomarker. In addition, if further research on RNA and protein in exosomes is conducted to prove their function, it can be considered as a breakthrough as a development for the treatment of muscular atrophy disease.

Summary and Conclusion

A muscle atrophy model was established using muscle cells and animal models, and the expression of muscle-related proteins according to muscle atrophy stimulation was compared. In addition, the expression change of the muscle-related protein was compared by treating the stem cell useful material (culture medium) together. Importantly, the therapeutic/protective effect of the stem cell useful substance (culture medium) was confirmed from the mechanism of muscle atrophy. Based on the results obtained through this study, the technical and practical limitations of the treatment using stem cells were overcome, and at the same time, it showed the possibility of a therapeutic effect on muscle atrophy even with a relatively easy to obtain stem cell useful material (culture solution). This is a groundbreaking study that deviated from the conventional treatment methods using only stem cells. In addition, it can be said that it is a research that takes a step forward for the development of biomarkers for the treatment of muscular atrophy disease through functional investigation using microvesicles such as exosomes by the secretory action of stem cells.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

As a pharmaceutical composition for the prevention or treatment of muscular atrophy comprising a mammalian umbilical cord-derived stem cell culture medium prepared by the following method as an active ingredient,
The culture medium does not contain stem cells, comprising exosomes secreted from umbilical cord-derived stem cells, a pharmaceutical composition for preventing or treating muscular atrophy:
(a) isolating the stem cells from the mammalian umbilical cord;
(b) culturing the stem cells in a serum-free medium that does not contain an umbilical cord extract; and
(c) collecting the culture solution 48 hours after incubation and centrifuging to obtain a supernatant.
According to claim 1, wherein the culture medium is thrombospondin (Thrombospondin, TSP), thrombospondin-1, EDA-A2 (Ectodysplasin-A2), IGFBP-rp1 (Insulin-like growth factor binding protein-related protein 1) / IGFBP-7, TIMP-2 (tissue inhibitor of metalloproteinase-2), IL-6, GRO, MIP2, SPARC, MCP-1, Dkk-1, Activin A, TIMP-1, VEGF, GDF- 15, Smad 4, HGF, angiogenin (Angiogenin), angiopoietin-1 (Angiopoietin-1), IGFBP-6, FGF-7 / KGF and MMP-1, characterized in that it contains the prevention of muscular atrophy or A therapeutic pharmaceutical composition.
The pharmaceutical composition for the prevention or treatment of muscular atrophy according to claim 1, wherein the culture medium promotes the proliferation of muscle cells.
The pharmaceutical composition for preventing or treating muscular atrophy according to claim 1, wherein the culture medium increases the expression of desmin.
The pharmaceutical composition for the prevention or treatment of muscular atrophy according to claim 1, wherein the culture medium increases the activity of Akt and FoxO.
The pharmaceutical composition for preventing or treating muscular atrophy according to claim 1, wherein the culture medium reduces the expression of MuRF-1 (Muscle RING-finger protein-1) and Atrogin-1/MAFbx.
The pharmaceutical composition for the prevention or treatment of muscular atrophy according to claim 1, wherein the culture medium promotes the decomposition of lactic acid in muscles.
The pharmaceutical composition for the prevention or treatment of muscular atrophy according to claim 1, wherein the culture medium increases muscle mass.

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