KR20140070794A - Compositions for Improving or Treating Muscle Atrophy Diseases Comprising Stem Cells Derived from Umbilical Cord - Google Patents

Abstract

The present invention provides a pharmaceutical composition to improve or treat muscle atrophy disease comprising stem cells derived from umbilical cord as an active ingredient. Upon administering the stem cells derived from umbilical cord of the present invention into muscles, the administered stem cells increase the muscle cells in stem cell administered areas and increase muscle fiber size and myotility, thus the stem cells derived from umbilical cord can be effective to improve or treat muscle atrophy disease. The stem cells derived from the umbilical cord of the present invention are cultivated in a medium including an umbilical cord extract as a replacement, thereby being safely applicable clinically without concerns of safety problems such as cross-species contamination according to use of fetal bovine serum and the like.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for improving or treating a muscular atrophy comprising stem cells derived from umbilical cord,

The present invention relates to a composition for improving or treating a muscle atrophy disease comprising umbilical cord stem cells.

The umbilical cord is composed of two arteries, one vein and a part of the extracellular matrix, Wharton's jelly. The extracellular matrix is composed of fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), platelet-derived growth factor (PDGF) and transforming growth factor beta (Sobolewski K. et al., Placenta 26, 747-752 (2005)), which helps to promote blood transport, prevent vascular compression in the umbilical cord and dry the umbilical cord after delivery, It functions to shrink.

Muscle atrophy is caused by various causes such as absence of mechanical stimulation, movement member, oil field, starvation and cancer. Muscle regression occurs primarily from significant loss of myofibrillar proteins, resulting in decreased muscle and athletic performance. For example, even though motion or other countermeasures (e.g., electrical stimulation) are applied continuously (e.g., 1 to 3% loss of muscle strength per week in astronauts during long-term space flight) As with the conditions, muscle regression inevitably proceeds. Therefore, research on other countermeasures to overcome obstacles to assist astronauts or to improve their performance is needed, either as an alternative to mechanical stimulation or as an addendum to mechanical stimulation.

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.

The present inventors have made extensive efforts to develop a stem cell capable of effectively treating a muscular atrophy disease and safely applicable to a human body, and as a result, it has been found that stem cells obtained from a umbilical cord are very effective in improving or treating a muscular atrophy disease Thereby completing the present invention.

It is therefore an object of the present invention to provide a pharmaceutical composition for improving or treating muscle atrophy disease.

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 comprising (a) a pharmaceutical effective amount of mammalian umbilical cord stem cells; And (b) a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition for improving or treating atrophy.

The present inventors have made extensive efforts to develop a stem cell capable of effectively treating a muscular atrophy disease and safely applicable to a human body, and as a result, it has been found that stem cells obtained from a umbilical cord are very effective in improving or treating a muscular atrophy disease Respectively.

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

The mammalian umbilical stem cell of the present invention is preferably at least one, preferably at least two selected from the group consisting of CD9, CD29, CD73, CD105, CD166 and HLA-ABC which are stem cell marker expression antigens, At least three, more preferably at least four, even more preferably at least five, and most preferably six, surface antigens. According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention express 70-100% of the stem cell surface surface antigens CD9, CD29, CD73, CD105, CD166 and HLA-ABC, 80-100%, and even more preferably 85-95%.

Meanwhile, the mammalian umbilical cord stem cells of the present invention show the immunological characteristics of the negative hematopoietic stem cell surface surface antigens CD31, CD34 and / or CD45. According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention express 0-10% of hematopoietic stem cell surface surface antigens CD31, CD34 and / or CD45, more preferably 0.1-7% And most preferably 0.16-5.6%.

The term "% " used in reference to the expression rate of the surface antigen means the ratio of cells expressing surface antigens in the cells to be analyzed. For example, a 95% expression rate of the CD29 surface antigen means that 95% of the cells express the CD29 surface antigen in the cell sample to be analyzed.

The mammalian umbilical cord stem cells of the present invention preferably comprise at least one, preferably at least two, more preferably at least three, most preferably undifferentiated stem cell marker proteins selected from the group consisting of Oct4, Sox2, KLF4 and Nanog Four types are expressed in cells.

According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention are selected from the group consisting of activin A, activated leukocyte cell adhesion molecule (ALCAM), angiogenin, CV-2 , DEC-1 (Dickkopf-related protein-1), Dkk-3, EDA-A2 (Ectodysplasin A2), EMT-II (Endothelial-monocyte-activating polypeptide- Endothelin, FGF-16, FSL1 (Follistatin-like 1), Galectin-3, GCSF (Granulocyte colony-stimulating factor) , Growth differentiation factor 15 (GDF-15), Glypican 3, GPC5 (Glypican 5), Granulocyte-macrophage colony-stimulating factor (GM-CSF), GRO (growth-related oncogene) IGF-I, IL-2 receptor alpha (IGFBP-1), IGFBP-4, IGFBP-6, IGFBP-rpl, IGFBP- , IL-6, IL-8, IL-12 receptor beta 2, IL-13 receptor alpha 1, IL-15, IL-1 5 receptor alpha, IL-16, IL-23, IL-28A, Inhibin B, Interferon-inducible T-cell alpha chemoattractant, LTBPl (Latent TGF-beta bp1), Leukaemia inhibitory factor, low density lipoprotein receptor-related protein (LRP-6), monocyte chemotactic protein-1, MCP-3, macrophage migration inhibitory factor (MIF) protein 2, MMP-1, MMP-3, MMP-10, Nidogen-1, OPG, TNFRSF11B, 3 (Pentraxin 3), PGRN (Progranulin), sFRP-4 (Secreted frizzled-related protein 4), sgp130 (soluble glycoprotein 130), Siglec-9 (sialic acid binding immunoglobulin-like lectin 9), Smad 4 proteins 4), secreted protein acidic and rich in cysteine (SPARC), tissue factor pathway inhibitor-1 (TFPI), TSP (Thrombospondin), TSP-1, tissue inhibitor of metalloproteinases 1, TIMP- -3, uPA (urokinase type plasminogen activat or a protein selected from the group consisting of urokinase plasminogen activator receptor (uPAR) and vascular endothelial growth factor (VEGF), more preferably activin A, Ectodysplasin A2, GRO, IGFBP- (IGF binding protein 7), IL-6 (Interleukin 6), MIP 2 (Macrophage inflammatory protein 2), TSP (Thrombospondin), TSP-1, tissue inhibitor of metalloproteinases 1 Secretes a protein selected from the group.

According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention can be produced by culturing pluripotent stem cells such as Actin A, angiogenin, decolin, Dkk-1, Dkk-3, EDA-A2, FGF- IGF-I, IL-2 receptor alpha, IL-2, IL-2, IL-2, IL-6, IL-6, 15, IL-15 receptor alpha, IL-16, IL-23, IL-28A, LRP-6, MCP-1 , MCP-3, MIP-1a, MIP2, TNFRSF11B, pentactoxin 3, PGRN, sFRP-4, sgp130, Siglec-9, Smad4, SPARC, TSP, TSP-1, TIMP-1, TIMP- -3. ≪ / RTI >

According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention are cultured in a three-dimensional culture, IGFBP-1, IGFBP-7, IL-6, IL-8, IGFBP-1, MMP-1, MMP-3, MMP-10, Nidogen-1, PGRN, sgp130, Smad 4, SPARC, TSP, TSP-1, TIMP-1, TIMP-2, uPA, uPAR and VEGF.

According to a preferred embodiment of the present invention, the mammalian umbilical cord stem cells of the present invention are cultured in a two-dimensional culture and three-dimensional culture, such as Acivin A, Dkk-1, EDA-A2, GDF-15, GRO, IGFBP- 6, MCP-1, MIP2, Smad4, SPARC, TSP, TSP-1, TIMP-1 and TIMP-2.

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)) and the like can be used. The culture medium used in the culturing process preferably further comprises a umbilical cord extract.

The present inventors have found that, in order to efficiently and economically cultivate stem cells while avoiding the safety problem associated with the use of an animal-derived protein source such as fetal bovine serum, the umbilical cord extract is obtained by minimizing protein denaturation from umbilical cord, As a serum substitute.

Hereinafter, a method for preparing a umbilical cord extract for use in stem cell culture will be described in detail.

(a) Cutting of umbilical cord

According to the present invention, the umbilical cord is first cut. In the present invention, the amputation of the umbilical cord is carried out in order to widen the contact area with the buffer solution, thereby facilitating dissolution of the useful substance present in the umbilical cord tissue.

The umbilical cord may be cut through various methods known in the art.

According to a preferred embodiment of the invention, the umbilical cord is cut to a length of 0.5-3.0 cm, more preferably 0.5-2.5 cm, even more preferably 1-2 cm.

(b) Cutting the umbilical cord into the buffer Impregnation

Then, the cut umbilical cord is inserted into the buffer solution. The buffer used in the present invention may be any buffer having a buffering power and includes, for example, sodium acetate, sodium phosphate, glycine-HCl, Tris-HCl and PBS (phosphate buffered saline). In particular, PBS can be used, and the pH is 2-11, preferably 4-10, more preferably 4-8, even more preferably 6.8-7.6.

The amount of the buffer (impregnated) of the amputated umbilical cord is not particularly limited and is preferably 2 to 5 times, more preferably 2 to 4 times, more preferably 2.5 to 3.2 times the umbilical cord weight .

According to a preferred embodiment of the present invention, the amputated umbilical cord is washed with a suitable solution (for example, a buffer) before being added to the buffer.

(c) stirring the resultant of step (b)

Stir the result of step (b).

According to a preferred embodiment of the present invention, stirring is carried out at 4 캜 to 10 캜, more preferably 4 캜 to 6 캜. Preferably stirring is carried out for 7-24 hours, more preferably 12-24 hours, even more preferably 18-24 hours.

Stirring can be carried out through various methods known in the art, and most preferably, stirring is performed using a magnetic bar.

(d) Centrifugation of the stirring result

The resultant mixture is centrifuged to obtain a supernatant as a final umbilical cord extract. The supernatant contains various useful proteins that are bound to extracellular matrix such as growth factors and cytokines.

According to a preferred embodiment of the present invention, centrifugation is carried out at 3,000-6,000 rpm, more preferably at 4,000-4,500 rpm. The centrifugation is carried out at 4 ° C to 10 ° C, more preferably at 4 ° C to 6 ° C.

According to a preferred embodiment of the present invention, the centrifugation is carried out for 5 min-30 min, more preferably 5 min-20 min, even more preferably 10 min-15 min.

According to a preferred embodiment of the present invention, the umbilical cord stem cells of the present invention cultured in a cell culture solution containing the umbilical cord extract prepared by the above-mentioned method are cultured in a culture medium containing the umbilical cord stem cells / Immunological characteristics (see Figs. 2 to 4). The umbilical cord stem cells cultured in the serum-containing culture medium exhibit a spreading shape as the number of subculture increases, while the umbilical cord stem cells of the present invention show an increase in the number of subculture, Maintain cell morphology. In addition, as the frequency of cell passage increases, the umbilical cord stem cell of the present invention significantly increases the expression of stem cell surface antigen (CD73, CD105 and CD166) as compared with the umbilical cord stem cell cultured in the medium containing serum , And the expression of undifferentiated stem cell marker protein is also longer lasting. Meanwhile, as a result of inducing the umbilical cord stem cells of the present invention to differentiate into osteoblasts, adipocytes and muscle cells, differentiation into osteoblasts, adipocytes and muscle cells was effectively induced and markers specific to each cell were expressed . Thus, the umbilical cord stem cells of the present invention show not only the muscle cells but also the ability to differentiate into osteoblasts and adipocytes.

According to a preferred embodiment of the present invention, the umbilical cord stem cells of the present invention exhibit activity to proliferate or differentiate into muscle cells. In other words, the umbilical cord stem cells of the present invention are proliferated or differentiated into muscle cells at the site where the stem cells are injected, preferably in the muscles, thereby to prevent muscle diseases, preferably muscular atrophy diseases, Lt; RTI ID = 0.0 > and / or < / RTI >

The muscle atrophy disease treated by the pharmaceutical composition of the present invention is a muscle disease derived from damage of muscle tissue due to a genetic, pathological or physical cause, and is preferably a muscle disease caused by muscular dystrophy, myasthenia gravis, muscular dystrophy, Myasthenia gravis, myasthenia gravis, muscle weakness or myasthenia gravis, and most preferably, muscular dystrophy. The present invention also relates to a method for preventing or treating at least one of muscle weakness, muscle stiffness, muscular degeneration, atrophic lateral sclerosis or myasthenia gravis.

As used herein, the term " muscle atrophy " refers to a decrease in muscle tissue or muscle regression, and a muscle atrophy refers to a disease caused by direct or indirect causes of muscle atrophy.

According to a preferred embodiment of the present invention, the umbilical cord stem cells of the present invention promote the proliferation of muscle cells (cf. Examples 4 and 6). As demonstrated in the following examples, when the umbilical cord stem cells of the present invention were administered to the hypodermic animals, the weight of the soleus muscle was increased and the expression of actin and desmin, which are specific markers of muscle development, was increased. Resulting from the proliferation of muscle cells by the umbilical cord stem cells of the present invention.

According to a preferred embodiment of the present invention, the umbilical cord stem cells of the present invention increase muscle contraction force (see Example 5). When the umbilical cord stem cells of the present invention were administered to an animal having a decreased muscle contraction ability due to induction of hippocampus, the extensor function of the soleus muscle was improved to a level similar to that of a normal control.

According to a preferred embodiment of the present invention, the umbilical cord stem cells of the present invention promote the degradation of lactic acid in muscle (see Example 7) and increase the size of muscle fibers (see Example 9).

According to a preferred embodiment of the present invention, the pharmaceutical composition for improving or treating atrophy of the present invention is a composition for intramuscular administration, and when the composition is administered into a muscle, Promotes muscle cell proliferation and lactate degradation, increases muscle fiber size, and increases muscle contraction, thereby improving or treating an amyotrophic disease.

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

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, methyl cellulose, 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 preparations are described 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, . Typical dosages of the pharmaceutical compositions of this invention are 10 < ² > -10 < ¹⁰ > cells per day on an adult basis.

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 improving or treating a muscle atrophy disease comprising mammalian umbilical cord stem cells as an active ingredient.

(Ii) When the umbilical cord stem cells of the present invention are administered intramuscularly, the administered stem cells are effective for the prophylaxis or treatment of muscular atrophy diseases by proliferating the muscle cells of the administered site and increasing the muscle fiber size and muscular contractility .

(Iii) Since the umbilical cord stem cells of the present invention are cultured in a medium containing umbilical cord extract as a serum substitute, they can be safely applied to clinical applications without fear of safety problems such as interspecies contamination due to use of fetal serum.

FIG. 1 is a graph showing the results of comparing the myofiber area of the muscle-induced animal model with the normal control group in order to establish an animal model to induce an atrophy-inducing animal and to confirm the induction of the atrophy.
FIG. 2 is a photograph showing the shape of umbilical cord stem cells cultured in a medium supplemented with Umbilical cord extract (UCE).
FIG. 3 is a graph showing FACS analysis of whether MSCs markers are maintained according to subculture when umbilical cord stem cells are cultured in a medium supplemented with umbilical cord extract. FIG.
FIG. 4 is a result of PCR to determine whether expression of ESCs gene is maintained according to subculture when umbilical cord stem cells are cultured in a medium supplemented with umbilical cord extract.
FIG. 5 shows the result of PCR to determine whether the ability of the stem cells to differentiate when umbilical cord stem cells were cultured in a medium supplemented with umbilical cord extract
FIG. 6 is a view showing a cytokine profile secreted upon two-dimensional culture of umbilical cord stem cells. FIG.
FIG. 7 shows the results of measurement of gene expression of human umbilical cord-mesenchymal stem cell by time. (A) Western blot results for expression of Myo D and Pax 3/7, (B) mRNA expression levels measured by quantitative RT-PCR.
Fig. 8 shows the results of a comparison of normalized muscle mass (muscle weight / weight, saline group ** P < 0.05, *** P < 0.001, ^# P < 0.05 compared to group returned to normality after umbilical mesenchymal stem cell administration) Fig.
FIG. 9 shows the result of measuring the strong axis ability of the soleus muscle at 1 week after injection of umbilical cord mesenchymal stem cells.
And Figure 10 is compared with the actin and desmin expression level measurement is a graph showing the results (compared with the saline group, * P = 0.05, ** P = 0.005, umbilical cord mesenchymal stem cells after administration group returned to normal life in gajamigeun ^# P = 0.05, ^## P = 0.005).
FIG. 11 is a graph showing the lactic acid accumulation level measured from an animal model inducing an isthmus of suspension at 1 week after injection of mesenchymal stem cells through a lactate assay ( ^&& P = 0.05, ^&& P <0.05, saline compared to the group ** P <0.05, *** P < 0.005, as compared to after administration of umbilical cord mesenchymal stem cells group returned to normal activity ^## P <0.05).
FIG. 12 is a histological analysis result of the effect of regenerating myofibers by stem cell transplantation. FIG. Muscle tissue is stained with hematoxylin and eosin (H & E).
13 is a graph showing the measurement results of changes in myofiber size due to stem cell transplantation. Cross-sectional area (CSA) was significantly higher in the human umbilical cord mesenchymal stem cell group than in the saline group (* P <0.05 compared to saline group, ** P <0.005, ^# P < 0.05 compared to the group).
FIG. 14 is a graph showing the results of immunofluorescence staining performed one week after injection of human umbilical cord mesenchymal stem cells. Desmin (red), human cell nucleus (green), nucleus (blue).

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

Example 1: Establishment of an animal model causing an atrophy and verification of induction of atrophy

A special cage was made to create an unloading environment on the ground similar to the gravity environment of the universe. A lifter of 360 degrees was lifted from the bottom of the animal, Were fixed together with the tail. The body slope of the experimental animal was maintained at about 30 degrees from the surface to make it similar to the microcosmic environment of the real universe.

Two weeks after the suspension, the staphylococcus aureus was extracted and fixed in 4% neutral buffered formalin for 24 hours. The paraffin block was prepared for histological observation. Hematoxylin-eosin staining revealed that the size of the muscle fibers surrounded by the endomysium was smaller than that of the control, and the size of the muscle fibers was reduced by about 2.5 times (Fig. 1). In addition, the decrease in desmin and skeletal muscle actin indicates that the muscle skeleton is damaged by the isthmus. As a result of this study, we established a pediculence model similar to the cosmic microwave gravitational environment and verified the induction of muscular atrophy through this animal model.

Example 2: Cell culture and experimental method

2-1. Cell culture

After removing the umbilical artery and vein, Wharton's jelly tissue was cut into 0.5 cm ³ size, placed on a 100 mm dish, and cultured with α-MEM medium for 2 weeks. After culturing, cells were grown from Wurton meat tissue to a dish, and the remaining tissue was removed and cultured until the cells reached 70% saturation. The cells were started with passage 0, the cells were removed with 0.05% trypsin-EDTA, and subcultured at a concentration of ¹ × 10 ³ cells / cm ² . Cells were cultured by adding 0.3 mg / ml umbilical cord extract (UCE) to the medium (FIG. 2).

The method of preparing the umbilical cord extract is as follows:

The umbilical cord was cut to a length of 0.5 to 2.0 cm and washed twice with PBS (pH 7.0). Approximately 15 ml of PBS was added to about 8 g of umbilical cord and homogenized, stirred (at 4 ° C. for 24 hours) Or incubation (37 &lt; 0 &gt; C for 24 hours). The supernatant collected by centrifugation at 4,500 rpm and 4 ° C for 10 minutes was used as an umbilical cord extract, and protein quantification was carried out by Bradford assay.

2-2: FACS analysis

Cells in culture were separated in a flask using 0.5% trypsin-EDTA and washed three times with D-PBS. The cells were suspended in PBS at a concentration of 3 × 10 ⁵ cells / 200 μL, and 10 μL of each antibody was added The reaction was carried out in the dark room for a minute. Cells were then washed twice with PBS, and 500 μL of the same PBS was added followed by flow cytometry (FC 500; Beckman Coulter, USA). Antibodies were detected in PE mouse anti-human CD 105 (BD Biosciences, MD., USA), FITC mouse anti-human CD9 (BD Biosciences, MD., USA), PE mouse anti-human CD29 Human CD79 (BD Biosciences, MD., USA), PE mouse anti-human CD166 (BD Biosciences, MD., USA), PE mouse anti-human CD73 Human CD34 (BD Biosciences, MD., USA), FITC mouse anti-human CD45 (BD Biosciences, MD., USA), FITC mouse anti-human CD34 (BD Biosciences, MD., USA) and FITC mouse anti-human HLA-DR (BD Biosciences, MD.

2-3: RT-PCR analysis

1 ml of lysis buffer was added to the UC-MSC cultured in the medium containing each FBS or UCE, and the total RNA was isolated according to the manufacturer's instructions. CDNA was synthesized from 1 μg of RNA using ELPIS (reverse transcription master premix). Using this cDNA as a template, the gene to be observed was amplified by PCR (Table 1). Gene amplification was performed for a total of 32 cycles. Each cycle consisted of denaturation at 94 ° C for 30 seconds, annealing for 30 seconds, and extension at 72 ° C for 30 seconds. After completion of the reaction, the PCR products were stained with ethidium bromide after electrophoresis and irradiated with ultraviolet light to obtain DNA images (FIG. 4).

lineage gene /
Gene storage number direction The sequence (5'-3 ') Annealing temperature
(° C) PCR product size (bp) Stem castle Oct 4 / NM_002701 Forward GAG TGA GAG GCA ACC TGG AG 60 256 Reverse TAG CCT GGG CTA CCA AAA TG SOX 2 / NM_003106 Forward AAT GCC TTC ATG GTG TGG TC 60 357 Reverse GTT CAT GTG CGC GTA ACT GT Nanog / NM_024865 Forward TTC TTC CAC CAG TCC CAA AG 60 354 Reverse CAT CCC TGG TGG TAG GAA GA KLF4 / NM_004235 Forward CCC AAT TAC CCA TCC TCC CT 60 309 Reverse ACG GTA CTG CCT GGT CAG TT Osteogenesis Collagen type I / NM_000089 Forward ACC TGG TCA AAC TGG TCC TG 60 306 Reverse CGT CCT CTC TCA CCA GGA AG Osteopontin / NM_000582 Forward CAT CAC CTG TGC CAT ACC AG 60 503 Reverse CCA TGT GTG AGG TGA TGT CC ALP / BC021289 Forward TGA CCT CCT CGG AAG ACA CT 60 242 Reverse AGA CTG CGC CTG GTA GTT GT Local tax saturation LPL / NM_000237 Forward AAG AAC CGC TGC AAC AAT CT 60 351 Reverse ACT GCT CCA CCA GTC TGA CC Leptin / NM_000230 Forward CAC ACA CGC AGT CAG TCT CC 60 298 Reverse ACC ACC TCT GTG GAG TAG CC FABP4 / NM_001442 Forward TGG AAA CTT GTC TCC AGT GAA 60 353 Reverse GTG GAA GTG ACG CCT TTC AT Control group GAPDH / NM_002046 Forward GAA GGT GAA GGT CGG ACTCE 60 448 Reverse GGA GGC ATT GCT GAT GAT CT

LPL: lipoprotein lipase

ALP: alkaline phosphatase

FABP4: fatty acid binding protein 4

KLF4: Kruppel-like factor 4

2-4: cell differentiation

Osteoblast differentiation Osteoblast )

The cells were treated with 0.05% trypsin-EDTA, separated from the culture medium, suspended in the medium, inoculated at a concentration of 1 × 10 ⁵ cells / well in a 6-well plate, and after 3 days, STEMPRO bone formation differentiation kit (Invitrogen, USA ) Was used to induce differentiation. Differentiation induction medium was changed to new one every 3-4 days and cultured for 2 weeks at 5% CO ₂ and 37 ° C. In order to confirm the differentiation into osteoblasts, we observed Alizarin Red S staining reagent which specifically stained osteoblasts. In addition, RT-PCR method was used to confirm the expression of specific genes in osteoblasts (FIG. 5, Table 1).

Adipocyte differentiation ( Adipocyte )

Differentiation was induced using a STEMPRO lipid-derived differentiation kit (Invitrogen, USA) when inoculated at a concentration of 1 × 10 ⁵ cells / well in a 6-well plate and became over-confluent after 3 days. Differentiation induction medium was replaced with new medium once every 3-4 days and cultured for 2 weeks at 5% CO ₂ and 37 ° C. To confirm the expression pattern of lipid droplets in adipose tissue, oil - red O staining was performed. In addition, expression of specific genes of adipocytes was confirmed by RT-PCR (FIG. 5, Table 1).

2-5: Cytokine array

The cytokines present in the umbilical cord extract were analyzed according to the manufacturer's instructions of the RayBio human cytokine array V kit (RayBiotech, USA). The array membrane used in this experiment can analyze 507 different growth factors / cytokines. Briefly, biotin was added to the umbilical cord extract and reacted at room temperature for 30 minutes to prepare biotin-labeled umbilical cord extract. The array membrane was blocked for 1 hour at room temperature using a blocking buffer, and then the biotin-labeled umbilical cord extract was reacted at room temperature for 2 hours after buffer removal. Then, the cells were washed three times with washing buffer, diluted 1: 500 with HRP-conjugated streptavidin in blocking buffer, and reacted at room temperature for 2 hours. After washing three times with wash buffer, the detection buffer was treated and observed with a chemiluminescence detection system. The obtained dot blot image was analyzed for the amount of cytokine expression using the Multi Gauge V3.0 program (Fig. 6).

Example  3: Muscle of stem cells Ability to distinguish  Confirm

To confirm the ability of stem cells to differentiate into muscle cells, 10 μM 5-azacytidine (Sigma-Aldrich, USA) was added to the cultured stem cells over time and MyoB1 (Myoblast determination protein 1), Pax3 / 7 (Paired box gene 3/7 protein) protein expression. RT-PCR was performed using desmin, MHC (Myosin heavy chain), and NKX2.5 (Homeobox protein NK-2 homolog E), which are specific markers of muscle development, Respectively. Over time, MyoD1 and Pax3 / 7 increased about 2.1-fold, and the increase of muscle-specific genes at the gene level confirmed that the stem cells could differentiate into muscle cells (FIG. 7).

Example  4: Measurement of the weight change of the soleus muscle by stem cell transplantation

After weighing, weighed and weighed weights were measured for each experimental group. Based on the measured weights, the changes in the weight of the legs were analyzed in relation to the weight.

Five-week-old SD-rat females were used. After 2 weeks of induction period, umbilical cord-derived MSCs, adipose-derived MSCs, bone marrow-derived stem cells 1 × 10 ⁶ cells / 100 μl of bone marrow-derived MSC were suspended in saline and placed in a syringe of 0.25 mm (31 G) × 8 mm (Insulin syringe, BD ULtra-Fine II) And injected twice between the gastronemius muscle and below.

After weighing, weighed and weighed weights were measured for each experimental group, and weighed weight changes were measured against the weight based on the measured weights.

As a result, when the saline solution was administered to the hypodermic animals, the weight of the soleus muscle was reduced to about twice that of the normal group. After 2 weeks of stem cell administration, no significant change in weight was observed. However, the weight of the soleus muscle was increased more than that of the group that did not receive stem cells and received a normal life after stem cell administration (FIG. 8).

Example  5: Stem cell transplantation Muscle contraction force  increase

After removing the spermatic muscle of the test animal, the tendons were fixed to the muscle as much as the surgical suture and temporarily stored in physiological saline solution. The extracted muscle was placed in an aluminum laboratory bath containing oxygenated saline solution and maintained at a temperature of 25 ° C. One of the muscles connected by a suture was connected to a fine screw to determine the optimum length for maximum muscle strength . A dual-mode Laver Arm system (Aurora scientific, Inc.) and a Grass S48 stimulator (Grass Instrument) were installed using DCM (Dynamic Muscle Control, solwood Enterprise, Inc.) software, The stimulus was transmitted to the platinum electrode through the spermatocyte to allow the muscle to contract. The tetanic force due to stimulation was converted to an electrical signal through a transducer (Aurora 300B-LR motor) and then input to a computer and analyzed using DMA (Dynamic Muscle Analysis, Solwood Enterprise, Inc.) software. As a result, it was confirmed that the strong axis ability of the soleus muscle of the group transplanted with stem cells was improved similarly to the control group (Fig. 9).

Example  6: Identification of muscle-specific gene expression by stem cells

 The stem cells were divided into the normal group and the non - stem cell group. The control group was a normal group without stem cells. The expression of actin and desmin, which are specific markers of muscle development, was increased in the experimental group that maintained the hypothalamus for 2 weeks after administration of the stem cells compared to the control group administered with saline solution. Although all of the treated stem cells showed muscle regeneration effect, remarkable muscle regeneration effect was observed in the umbilical cord stem cells. In the group that maintained normal life after stem cell administration, the degree of muscle recovery rapidly appeared, which means that the muscles contracted by gravity were restored to their original function through contraction and relaxation. Even in the group that maintained normal life without stem cell administration, the expression of actin and desmin was lower than that of the group treated with stem cells (Fig. 10).

Example  7: Identification of lactic acid degradation by stem cell transplantation

Stem cell transplantation regenerates atrophied muscle, which is expected to break down accumulated lactic acid and eliminate muscle fatigue due to lactic acid. In order to confirm the effect of lactic acid degradation by stem cell transplantation, the lactic acid accumulation level was checked in an animal model induced by suspension culture, and the volume (SV, sample volume) of the sample used was determined by the amount of lactic acid (La, Lactate amount). The amount of lactic acid accumulated about 2 times as much as that of the control group after the induction of the hypothalamus and increased about 3 times after one week of saline administration. On the other hand, the content of lactic acid was decreased by about 0.5 times in the group treated with umbilical cord stem cells compared with that in the saline group, and about 0.6 times in the group administered with adipose derived stem cells and bone marrow derived stem cells (FIG. 11) .

Example  8: Histological analysis of muscle fiber regeneration by stem cells

Histopathologic analysis of the effect of stem cell stimulation on muscle atrophy was performed. As a result of H & E staining, it was observed that the size of muscle fibers was decreased in the induction group, and the muscle fiber size was increased and the density was also high in the group to which the stem cells were specifically injected. According to the type of stem cells, the size of the muscle fibers is increased after 2 weeks of adipose tissue-derived stem cells, but the endomysium is spread more widely than the normal tissue, and is regenerated by satellite cells The density of the tissue was not found to be high. In the group treated with bone marrow-derived stem cells, the size of muscle fibers was larger than that of the group administered with adipose tissue-derived stem cells. However, muscle regeneration by satellite cells was not observed after 2 weeks of administration. On the other hand, the group treated with umbilical cord stem cells showed a large number of muscles being regenerated by satellite cells from the first week of administration, and muscles showing destruction and regeneration of injured muscles after two weeks. The group that maintained normal life after umbilical cord stem cell administration showed a higher frequency of muscle fiber regeneration than other stem cells (FIG. 12).

Example  9: Measurement of muscle fiber size change by stem cells

In order to confirm the effect of the stem cell treatment of muscular dystrophy, changes in the muscle fiber size after the hip flexion were measured. To quantitatively measure the size of muscle fibers visually identified through tissue staining, the size of 100 muscle fibers was randomly measured and analyzed using the Image J program. When the umbilical cord stem cells were administered, the size of the muscle fibers increased by about 1.7 times as compared with saline, 1.1 times as much as when administered with fat-derived stem cells, and 1.3 times as much as with bone marrow-derived stem cells (FIG. In addition, groups that remained normal after stem cell administration showed similar increases within the error range. These results are in agreement with the results of histological analysis, and increase in muscle fiber size indicates that muscle fibers are regenerating due to the activity of satellite cells. In addition, the physiological that is one of the cross-sectional area of muscle is an important factor in determining the strength of the muscle, between the cross-sectional area and muscle of the muscle to act on the muscular strength exerted force that is common to that there is a correlation, the muscles 1 cm ² demonstrated The muscle strength is 4-6 kg, and muscle strength increases as the muscle cross-sectional area increases. The greater the cross-sectional area of the muscle, the more force can be exerted because of the abundant amount of actin and myosin, which are components of muscle contraction, so that a cross bridge action for muscle contraction can be sufficiently formed.

Example  10: Immunofluorescent staining  Identification of stem cells through

Immunofluorescent staining was performed using samples prepared for histological analysis. FITC - labeled human nuclear antibody was used to confirm whether the actually administered stem cells acted. Immunofluorescent staining was carried out using desmin antibody to confirm the degree of atrophy or regeneration of muscle fibers. After 2 weeks, the muscle fiber was reduced to about 1/4 of the size, and the structure was very irregular. It was confirmed that the muscular atrophy was caused by the hypothalamus, and in the group treated with stem cells, not only the damaged muscle fibers but also the regenerated muscle fibers were restored to their original shape. As a result of immunofluorescence staining, fluorescence was observed under the basal lamina in which the satellite cells were located only in the group to which the stem cells were administered. This result suggests that the stem cells induce muscle regeneration by supporting the satellite cells of the experimental animals (Fig. 14 ).

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 (18)

(a) a pharmaceutically effective amount of mammalian umbilical cord stem cells; And (b) a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 1, wherein the mammalian umbilical cord stem cells are mesenchymal stem cells.
The mammalian umbilical cord stem cell according to claim 1, wherein said mammalian umbilical cord stem cells are (i) positive for at least one surface antigen selected from the group consisting of CD9, CD29, CD73, CD105, CD166 and human leukocyte antigen Immunological characteristics; And (ii) have negative immunological properties against at least one surface antigen selected from the group consisting of surface antigens CD31, CD34 and CD45, and (iii) selected from the group consisting of Oct4, Sox2, KLF4 and Nanog Wherein the stem cell is a stem cell that expresses at least one undifferentiated stem cell marker protein.
The method of claim 1, wherein the mammalian umbilical cord stem cells are selected from the group consisting of activin A, activated leukocyte cell adhesion molecule (ALCAM), angiogenin, CV-2 (Crossveinless-2), decolin (DEC-1), Dkk-3, EDTA-A2 (Ectodysplasin A2), EMT-II (Endothelial-monocyte-activating polypeptide-II), FGF-7 (Epidermal growth factor- ), KGF (Keratinocyte growth factor), Endothelin, FGF-16, FSL1 (Follistatin-like 1), Galectin-3, GCSF (Granulocyte colony-stimulating factor) Growth differentiation factor 15), GPC3 (Glypican 3), GPC5 (Glypican 5), GM-CSF (Granulocyte-macrophage colony-stimulating factor), GRO (growth-related oncogene), GRO- IGFBP-1, IGFBP-1, IGFBP-3, IGFBP-4, IGFBP-6, IGFBP-rp1, IGFBP-7, IGF-I, Interleukin 2 receptor alpha, IL- IL-16 receptor IL-8, IL-12 receptor beta 2, IL-13 receptor alpha 1, IL-15, IL-28A, Inhibin B, Interferon-inducible T-cell alpha chemoattractant, LTBP1, Leukaemia inhibitory factor (LIF), LRP-6 (low density lipoprotein receptor -related protein 6), MCP-1 (monocyte chemotactic protein-1), MCP-3, MCP (macrophage migration inhibitory factor), MIP-1a, MIP2 (macrophage inflammatory protein 2) ), MMP-3, MMP-10, Nidogen-1, OPG, TNFRSF11B, Pentraxin3, 4 (secreted frizzled-related protein 4), sgp130 (soluble glycoprotein 130), Siglec-9 (sialic acid binding immunoglobulin-like lectin 9), Smad 4 (Sma and Mad related proteins 4), SPARC TIMP-2, TIMP-3, uPA (urokinase type plasminogen activator), uPAR-1, TSP-1, tissue inhibitor of metalloproteinases 1, (urokinase plasminogen ac IGFBP-7 (IGF-binding protein 7), and more preferably a protein selected from the group consisting of TGF-κB, tivator receptor and vascular endothelial growth factor (VEGF), more preferably activin A, Ectodysplasin A2, GRO, , IL-6 (Interleukin 6), MIP 2 (Macrophage inflammatory protein 2), TSP (Thrombospondin), TSP-1, tissue inhibitor of metalloproteinases 1 and TIMP- Wherein the stem cell is a stem cell.
5. The method according to claim 4, wherein said mammalian umbilical cord stem cells are selected from the group consisting of actin A, angiogenin, decoline, Dkk-1, Dkk-3, EDA-A2, FGF-7, KGF, endothelin, IGF-I, IL-2 receptor alpha, IL-6, IL-6, IGFBP-4, IGFBP-6, IGFBP-rpl, IGFBP-7, FSL1, GCSF, GDF-15, GPC3, GPC5, GRO, IGFBP- IL-15 receptor alpha, IL-16, IL-23, IL-28A, LRP-6, MCP-1, MCP-3, MIP-1a, MIP2, TNFRSF11B, pentactaxin 3, PGRN, sFRP-4, sgp130, Siglec-9, Smad4, SPARC, TSP, TSP-1, TIMP-1, TIMP-2 and TIMP- &Lt; / RTI &gt; wherein the stem cell is a stem cell that secretes a protein selected from the group consisting of:
4. The method according to claim 4, wherein said mammalian umbilical cord stem cells are selected from the group consisting of actin A, ALCAM, CV-2, Dkk-1, Dkk-3, EDA-A2, EMAP-II, endothelin, FGF-16 IGFBP-7, IL-6, IL-8, Inhibin B, IGFBP-1, IGFBP- TAC, LTBP1, LIF, MCP-1, MCP-3, MIF, MIP-1a, MIP2, MMP-1, MMP-3, MMP-10, , TSP-1, TIMP-1, TIMP-2, uPA, uPAR and VEGF.
The method according to claim 4, wherein the umbilical cord stem cells of the mammal are selected from the group consisting of actin A, Dkk-1, EDA-A2, GDF-15, GRO, IGFBP-7, IL- 1, MIP2, Smad4, SPARC, TSP, TSP-1, TIMP-1 and TIMP-2.
The pharmaceutical composition of claim 1, wherein the stem cells are derived from Wharton's jelly tissue of the mammalian umbilical cord.
The pharmaceutical composition according to claim 1, wherein the atrophic disease is at least one selected from the group consisting of muscular atrophy, myasthenia gravis, muscular dystrophy, muscular weakness, muscle weakness, muscle stiffness, muscular degeneration atrophy, amyotrophic lateral sclerosis or myasthenia gravis.
2. The pharmaceutical composition according to claim 1, wherein the composition is for parenteral administration.
11. The pharmaceutical composition according to claim 10, wherein the parenteral administration is intramuscular administration or intravascular administration.
The pharmaceutical composition according to claim 1, wherein the stem cell promotes proliferation of muscle cells.
The pharmaceutical composition according to claim 1, wherein the stem cells increase muscle contraction.
The pharmaceutical composition according to claim 1, wherein the stem cell promotes lactic acid degradation in muscle.
The pharmaceutical composition according to claim 1, wherein the stem cells increase the size of muscle fibers.
The pharmaceutical composition according to claim 1, wherein the stem cells are cultured in a cell culture solution containing a umbilical cord extract.
17. The pharmaceutical composition according to claim 16, wherein the umbilical cord extract is prepared by a method comprising the steps of:
(a) cutting the umbilical cord;
(b) inserting the amputated umbilical cord into a buffer;
(c) stirring the result of step (b); And
(d) centrifuging the result of step (c) to obtain a supernatant as a umbilical cord extract.
2. The pharmaceutical composition according to claim 1, wherein the mammal is a human, a pig, a horse, a cow, a mouse, a rat, a hamster, a rabbit, a goat or a sheep.

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