Method for isolating and culturing umbilical cord blood stem cells that highly express GDF-3, and uses of GDF-3

Abstract

The present invention relates to a method for isolating and culturing umbilical cord blood stem cells that highly express GDF-3. The umbilical cord blood stem cells isolated and cultured according to the present invention show the characteristic of highly expressing GDF-3. Normally, as the passage proceeds, the proliferation of stem cells decreases, making it difficult to secure a sufficient number of cells. However, in the case of stem cells that highly express GDF-3, the proliferation rate of cells is maintained higher than that of the control group even after passage proceeds, so that a large number of healthy stem cells can be secured. In addition, it has been confirmed that the concentration of extracellular matrix components secreted by the cells is high due to the high proliferation rate of stem cells, and thus a raw material with excellent efficacy / effects can be produced when producing cell and culture medium compositions using umbilical cord blood stem cells that highly express GDF-3.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating and culturing umbilical cord blood stem cells that highly express GDF-3, and various uses of GDF-3. [Background technology]

[0002] There are two types of animal cell culture: primary culture and subculture. Primary culture is the process of isolating and culturing cells from tissue fragments. Subculture is a method of culturing cells obtained through primary culture in order to continuously maintain them.

[0003] Normal animal cell lines are mortal and can only divide a limited number of times. When normal cells are cultured, they grow in the state of undifferentiated stem cells or progenitor cells, and then differentiation begins and proliferation stops. In contrast, cells derived from tumor tissue undergo a mixture of growth and differentiation, allowing them to grow continuously.

[0004] Subculture is a method of culturing cells by periodically transferring them to a new culture dish every 2-7 days to allow for cell proliferation. Cells stop growing due to a lack of nutrients in the limited space within the culture dish and the secretion of metabolic products, so subculture prevents this and provides new space for cells to grow. The most basic subculture method involves cells growing in a monolayer, then removing the cells from the culture dish and culturing them in a new dish. Another method is to use cell disks or microcarriers to perform three-dimensional culture in specialized medium.

[0005] Cells derived from animal tissues are cultured successively through primary, secondary, and tertiary cultures. However, many cells stop growing and die after a certain number of cultures. As the number of cell divisions increases, many cells undergo a phenomenon called cell senescence, and the properties of the cells change over time, so they can only be cultured a certain number of times.

[0006] Stem cells are undifferentiated cells with the ability to self-renew and differentiate into various tissue cells. They can be divided into embryonic stem cells, adult stem cells, and dedifferentiated stem cells. Embryonic stem cells are isolated from the cell mass inside the blastocyst and have the pluripotency to differentiate into all cells and tissues of the human body. They are used for therapeutic purposes, but their transplantation poses ethical concerns, including the high risk of tumor formation. Dedifferentiated stem cells are cells that have the same differentiation potential as embryonic stem cells by introducing genes that cause dedifferentiation into adult somatic cells. While these cells overcome the ethical concerns of embryonic stem cells, their low dedifferentiation efficiency and the use of viruses for gene transfer pose risks for their use in therapeutic applications. Adult stem cells are undifferentiated cells present in body tissues and serve to replace dead or damaged cells. Unlike embryonic stem cells, they differentiate stably, have a low risk of tumorigenesis, and do not destroy fertilized eggs, eliminating ethical concerns.

[0007] Adult stem cells can be collected from fat, bone marrow, umbilical cord blood, etc. Fat and bone marrow stem cells are collected from donors using the immersion method, and the function and efficacy of the stem cells vary depending on the donor's age and health condition. Umbilical cord blood stem cells utilize discarded umbilical cord blood without using the immersion method, and because they use cord blood from similar gestational ages (40 weeks), there is little difference in stem cell function and efficacy between donors.

[0008] Numerous cell therapies utilizing the regenerative and anti-inflammatory functions of adult stem cells are currently under development, and the mechanisms of these functions are due to substances produced by adult stem cells, including proteins and dielectrics such as collagen, TGF-β1, and miRNA. Stem cell therapy agents face challenges in terms of cell engraftment and the development of persistent tumors, and to address these issues, active research is being conducted into stem cell culture media and extracellular vesicles.

[0009] Mesenchymal stem cells are undifferentiated adult stem cells that exist among the differentiated cells of tissues and organs and have the ability to self-renew. They can be easily proliferated outside the body and differentiate into various tissue cells such as fat cells, bone cells, chondrocytes, and muscle cells, and are used for tissue regeneration using their differentiation function.

[0010] Mesenchymal stem cells are known to play an important role in skin regeneration by secreting various growth factors and cytokines, such as epidermal growth factor and fibroblast growth factor, and promoting collagen production from fibroblasts.

[0011] In addition, mesenchymal stem cells isolated from the human body can be improved to be suitable for treatment, and various growth factors or proteins secreted from the stem cells can be used to treat various diseases, such as immunomodulation and anti-inflammation.

[0012] Various growth factors or proteins secreted from stem cells can be used industrially in medicines, cosmetics, etc. Technology for increasing the concentration of growth factors in stem cell culture media or stem cell-derived compositions is currently insufficient, and new methods are needed to produce culture media or compositions containing high concentrations of growth factors for promoting extracellular matrix synthesis.

[0013] The extracellular matrix (ECM) is a cellular structure formed by various substances secreted by cells. It fills the intercellular spaces and connects cells and tissues. The ECM not only provides structural support and intercellular connections, but also facilitates intercellular communication, including signal transduction, and plays an important role in embryonic development and cell differentiation. The ECM is essentially composed of water, proteins, and polysaccharides. Each tissue forms and maintains an ECM with a shape and topology appropriate for its own tissue from the beginning of its development. The ECM binds to cells via extracellular matrix receptors (ECMs), such as integrins, discoidin domain receptors, and syndecans. These connections connect the cytoskeleton and the ECM. In addition, the extracellular matrix has a highly dynamic structure and is constantly remodeled, and its physical and biochemical properties give organs in living organisms unique physical properties, such as tensile strength and elasticity. The extracellular matrix is ​​composed of two large molecules: one is proteoglycan (PG), a polysaccharide complexed with protein, and the other is fibrous proteins such as collagen and keratin, which are insoluble in water.

[0014] Proteoglycans are the most abundant substances between cells and tissues, existing in the form of a hydrated gel and playing various roles with their properties such as buffering, hydration, binding, and resistance.

[0015] Among the many types of fibrous proteins, collagen is the most abundant protein, accounting for approximately 30% of the total protein in multicellular animals. Collagen is responsible for the structure of the extracellular matrix and is involved in elasticity, cell adhesion, cell migration, and tissue development. It is linked to another fibrous protein, elastin. Elastin fibers are responsible for the elasticity of tissues, which require repeated stretching. However, the stretchability of elastin fibers is very limited because they are tightly connected to collagen fibers. Another fibrous protein is fibronectin (FN), which organizes the extracellular matrix and connects cells to the extracellular matrix.

[0016] Meanwhile, the stem cell marker GDF-3 (Growth Differentiation Factor-3) is a member of the TGF-β superfamily and activates Nodal signaling, stimulating the transcription of proteins involved in skin regeneration, such as collagen, fibronectin, and TGF-BI. The correlation between GDF-3 and stem cells has been primarily studied in embryonic stem cells (ESCs), and GDF-3 is known as a stemness marker for ESCs. However, the function of GDF-3 in other adult stem cells remains unclear. Summary of the Invention [Problem to be solved by the invention]

[0017] The present invention provides a method for producing umbilical cord blood stem cells that highly express the GDF-3 gene, and various uses of GDF-3.

[0018] Specifically, the present invention provides a highly effective method for isolating stem cells from umbilical cord blood, characterized by culturing mononuclear cells isolated from umbilical cord blood in a fibronectin-coated culture vessel using a newly designed stem cell culture badge to secure rapidly proliferating umbilical cord blood stem cells. The present invention also provides a method for improving the sustainability of cell proliferation and secretion of extracellular matrix components by inducing increased GDF-3 expression in stem cells. Based on this, the present invention also provides a culture medium composition containing extracellular matrix components by inducing cell proliferation and secretion of extracellular matrix components using umbilical cord blood stem cells with high GDF-3 expression. [Means for solving the problem]

[0019] A first aspect of the present invention provides a method for producing GDF-3, which comprises culturing cord blood stem cells to express GDF-3.

[0020] A second aspect of the present invention provides a method for producing GDF-3-expressing cord blood stem cells, which involves isolating and culturing cord blood stem cells with a fast growth rate (cell doubling time) in order to secure cord blood stem cells with high GDF-3 expression.

[0021] In a third aspect, the present invention provides cord blood stem cells with high GDF-3 expression, which are obtained by seeding cord blood mononuclear cells isolated from cord blood onto a plate containing TGF-β superfamily growth factor and culturing the mononuclear cells that adhere to the culture plate and proliferate relatively rapidly, and then culturing the mononuclear cells.

[0022] A fourth aspect of the present invention provides a composition for increasing fibroblast growth, which comprises GDF-3.

[0023] A fifth aspect of the present invention provides a composition for skin regeneration, which contains GDF-3.

[0024] The present invention will be described in detail below. The inventors conducted various experiments, considering that securing a large number of stem cells would facilitate the establishment of a master cell bank, and that this could be used to secure large quantities of cells and culture media containing high concentrations of active ingredients. As a result, they discovered that: (i) high expression of GDF-3 promotes the synthesis of active substances involved in cell proliferation and regeneration; (ii) there is a correlation between the expression level of GDF-3 in isolated and cultured cord blood stem cells and the proliferation rate of isolated and cultured cord blood stem cells; (iii) high levels of GDF-3 gene expression are observed in cord blood stem cells; (iv) when human epithelial cells (HDF) are treated with GDF-3, the cells exhibit a long and short shape, which is advantageous for proliferation, and the number of cells increases continuously (Example 5 and Figure 7); and (v) when human epithelial cells (HDF) are treated with GDF-3, gene expression of skin constituents such as collagen and fibronectin, which are involved in skin regeneration, also increases (Example 6 and Figure 8). The present invention is based on these findings.

[0025] Umbilical cord blood stem cells isolated and cultured according to the present invention exhibit the characteristic of highly expressing GDF-3. While stem cell proliferation typically declines with increasing passaging, making it difficult to secure a sufficient number of cells, stem cells highly expressing GDF-3 maintain a high level of proliferation ability compared to control groups, even with increasing passaging, making it possible to secure a large number of vigorous stem cells. Furthermore, it has been confirmed that the high proliferation rate of stem cells also leads to a high concentration of extracellular matrix components secreted by the cells, enabling the production of a highly effective raw material for the production of culture medium compositions using umbilical cord blood stem cells highly expressing GDF-3.

[0026] A method for isolating and culturing stem cells with high GDF-3 expression according to one aspect of the present invention utilizes a newly designed stem cell culture badge, which is supplemented with various growth factors including the TGF-β superfamily.

[0027] The cord blood stem cells isolated and cultured according to the present invention can be obtained with a cell doubling time of less than 24 hours.

[0028] As confirmed by the present invention, GDF-3 increases the proliferation of fibroblasts (FIG. 8). A non-limiting example of a fibroblast whose proliferation is increased by GDF-3 is human dermal fibroblast.

[0029] As confirmed by the present invention, GDF-3 is thereby able to increase the expression of GDF-3, collagen and / or fibronectin genes in fibroblasts (FIG. 8).

[0030] Meanwhile, based on the discovery that there is a correlation between the GDF-3 expression level of isolated and cultured umbilical cord blood stem cells and the proliferation rate of isolated and cultured umbilical cord blood stem cells, a method for producing umbilical cord blood stem cells with high GDF-3 expression according to one embodiment of the present invention is characterized by isolating and culturing umbilical cord blood stem cells with a fast growth rate in order to secure umbilical cord blood stem cells with high GDF-3 expression.

[0031] The higher the proliferation rate of the isolated and cultured cord blood stem cells, the higher the expression level of GDF-3 in the isolated and cultured cord blood stem cells.

[0032] Therefore, the present invention allows the establishment of a cell bank for cord blood stem cells with high GDF-3 expression. For example, cord blood mononuclear cells isolated from the umbilical cord blood tract are seeded on a plate containing a TGF-β superfamily growth factor and cultured, and mononuclear cells that adhere to the plate and proliferate relatively quickly are selectively isolated and cultured to establish a cell bank for cord blood stem cells with high GDF-3 expression.

[0033] Furthermore, a method for producing cord blood stem cells with high GDF-3 expression according to one embodiment of the present invention is characterized by isolating and culturing fast-growing cord blood stem cells in a batch culture (Example 1).

[0034] In one embodiment, a method for producing umbilical cord blood stem cells with high GDF-3 expression includes a first step of seeding and culturing umbilical cord blood mononuclear cells isolated from the umbilical cord blood tract onto a batch containing TGF-β superfamily growth factors, and a second step of selectively isolating and culturing mononuclear cells that adhere to the culture dish and proliferate relatively quickly.

[0035] In the second step, cells that have not adhered to the culture dish are removed, and only the adherent mononuclear cells can be cultured.

[0036] The GDF-3-expressing cord blood stem cells produced by the present invention can differentiate into adipocytes, chondrocytes, and / or osteocytes, and therefore, cord blood stem cells highly expressing GDF-3 can be used as a cell therapy agent.

[0037] The types, combinations, and contents of growth factors produced during stem cell culture vary depending on the stem cell origin and the culture method. Therefore, the GDF-3-expressing cord blood stem cells produced according to the present invention can be cultured to produce a GDF-3-containing culture medium. In particular, the present invention allows the isolation and culture of cord blood-derived stem cells that highly express GDF-3 to produce a GDF-3-containing culture medium.

[0038] Surprisingly, we found that the GDF-3-expressing cord blood stem cells produced by the present invention gradually increased in GDF-3 expression level with increasing passage (Figure 5). Therefore, to secure cord blood stem cells with high GDF-3 expression, GDF-3-expressing cord blood stem cells at passages 5 to 7 or higher can be used (Figure 3).

[0039] According to one aspect of the present invention, a method for producing GDF-3-expressing cord blood stem cells is characterized by isolating and culturing cord blood stem cells in a batch containing TGF-β superfamily growth factors, thereby obtaining cord blood stem cells with high GDF-3 expression and excellent growth potential during subculture.

[0040] Therefore, one embodiment of the present invention provides a method for producing GDF-3-expressing cord blood stem cells by isolating and culturing cord blood stem cells from a batch containing various growth factors, including the TGF-β superfamily, and then subculturing them to obtain large quantities of cord blood stem cells (Figure 3).

[0041] By subculturing GDF-3-expressing cord blood stem cells according to one embodiment of the present invention, it is possible to obtain a large number of cord blood stem cells with excellent ability to secrete extracellular matrix components (FIG. 4).

[0042] As mentioned above, the stem cell marker GDF-3 is a member of the TGF-β superfamily. TGF-β is a pleiotropic factor and has the properties of a multifunctional growth factor.

[0043] Transforming growth factor-β (TGF-β) signaling plays a crucial role in various biological processes, performing various functions such as cell growth inhibition, cell death, differentiation, and epithelial-mesenchymal transition (EMT). The TGF-β signaling system is tightly regulated and plays a crucial role in development and organogenesis, as well as maintaining cellular homeostasis. Therefore, disruption of TGF-β signaling can lead to life-threatening diseases such as cancer, fibrosis, and congenital malformations.

[0044] TGF-β is known to exhibit tumor suppressive activity in the early stages of the carcinogenesis process, but to promote cancer growth in the later stages. TGF-β1 is expressed in large amounts in many cancer tissues, and cancer patients with high TGF-β1 expression are known to be more likely to be malignant and have a poor prognosis.

[0045] TGF-β secreted from cells binds to a heterogeneous complex of two types of receptors, type I and type II, to initiate signal transduction. When TGF-β binds to the type II receptor, the type I receptor recognizes it and binds to the type II receptor. The type II receptor then phosphorylates the GS site of the type I receptor, activating the type I receptor kinase. The phosphorylated TGF-β type 1 receptor phosphorylates the C-terminal serine residues of TGF-β signaling mediators, Smad2 and Smad3, thereby inducing their activation. Activated Smad2 and Smad3 then form a complex with Smad4, translocate to the nucleus, and are involved in the expression of target genes.

[0046] The tumor suppressive role of TGF-β has been demonstrated in various studies. Conversely, TGF-β promotes tumor growth, invasion, and metastasis during cancer progression. The detailed molecular mechanisms underlying these opposing roles of TGF-β in cancer progression have yet to be clearly elucidated.

[0047] TGF-β is known to play a key role in determining immune homeostasis and tolerance by inhibiting the function and expansion of many components of the immune system. TGF-β regulates immune tolerance and inflammatory responses. TGF-β signaling directly suppresses the cytotoxic program of CD8+ T cells.

[0048] In the tumor microenvironment, excessive TGF-β1 produced by cancer cells induces excessive changes in the cancer cell signaling system. These changes adversely affect the interaction between cancer cells and tumors. When cross-talk between cells is not normal, permissive stroma is created, creating an environment in which tumor cells can easily spread. TGF-β secreted by cancer cells induces cancer cells with characteristics similar to EMT and cancer stem cells (CSCs).

[0049] Cells that undergo EMT have increased migratory ability and are more susceptible to metastasis; these cells also take on a spindle shape and undergo changes to their cytoskeleton that allow them to more easily migrate to other organs and metastasize.

[0050] In epithelial cells, the EMT process is characterized by the loss of E-cadherin, a protein required for epithelial cell adhesion, and the increased expression of mesenchymal markers such as N-cadherin, vitamin D, and fibronectin. TGF-β is a bona fide mediator of EMT, activating various transcription factors, including SNAI1 / 2, Twist, and ZEB1 / 2. Tumor cells undergoing EMT acquire cancer stem cell (CSC) properties.

[0051] TGF-β, a pleiotropic and multifunctional growth factor, plays opposing roles in cancer progression, and the detailed molecular mechanisms underlying these role reversals have yet to be clearly elucidated. Since GDF-3, a member of the TGF-β superfamily, is also likely to cause similar side effects, it is desirable to mass-produce GDF-3 ex vivo using umbilical cord blood stem cells isolated and cultured according to the present invention, and then administer it in vivo by adjusting the desired administration time, administration site, and / or administration amount.

[0052] As used herein, the term "badge" refers to a composition containing essential components required for cell growth and proliferation in vitro, and includes all stem cell culture badges commonly used in the art, such as, but not limited to, commercially produced or artificially synthesized badges, such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM. As used herein, a batch generally contains a carbon source, a nitrogen source, and trace element components, and may further contain amino acids, antibiotics, and the like.

[0053] Furthermore, as confirmed in the present invention, GDF-3 can increase the expression of the gene encoding at least one protein selected from the group consisting of GDF-3, collagen, and fibronectin in fibroblasts, and / or promote the synthesis of active substances involved in promoting cell proliferation and / or regeneration, and therefore can be used as an active ingredient in compositions for promoting fibroblast growth (FIGS. 6 to 8).A non-limiting example of fibroblasts is human dermal fibroblast (HDF). Therefore, in the present invention, GDF-3 can be used as an active ingredient in a composition for skin regeneration.

[0054] The compositions for skin regeneration include cosmetic compositions for skin regeneration or wrinkle improvement, transdermal pharmaceutical compositions, and pharmaceutical compositions for skin regeneration or wrinkle treatment. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable salts of the active ingredients.

[0055] "Skin regeneration" refers to the recovery of skin tissue from damage caused by external and internal factors, such as ultraviolet rays, external pollutants, wounds, and trauma, and internal factors such as stress.

[0056] "Skin wrinkles" refer to the fine lines that appear when the skin ages, and can be caused by genetic factors, a decrease in collagen and elastin in the dermis, or external factors.

[0057] "Skin wrinkle reduction" refers to suppressing or inhibiting the formation of wrinkles on the skin or alleviating existing wrinkles. For purposes of the present invention, the skin regeneration and wrinkle reduction include enhancing skin elasticity.

[0058] "Wrinkle treatment" means at least partially and temporarily stopping the formation of wrinkles in the skin.

[0059] Skin elasticity is achieved by elastic fibers composed of elastin present in the dermis. These elastic fibers have a very low elastic modulus, like rubber, and are easily deformed by small forces, returning to their original shape when the force is removed. Elastic fibers are composed of microfibrils embedded in an amorphous matrix called elastin. Elastin is a protein composed of very unique amino acids, desmosine and isodesmosine, which are derived from lysine and are found only in elastic fibers. Desmosine and isodesmosine form cross-links within the long peptide chain, and this structure gives elastin its rubber-like properties.

[0060] "Enhancement of skin elasticity" refers to the maintenance or increase of skin elasticity when there is sufficient elastin and collagen, which are elastic fibers made of elastin present together with collagen fibers.

[0061] When the active ingredient is used in a cosmetic composition, it can be prepared in the form of a general oil-forming formulation or a solubilizing formulation, for example, a lotion such as a softening lotion or a nutritious lotion, an emulsion such as a facial lotion or a body lotion, a cream such as a nutritious cream, a moisturizing cream, or an eye cream, an essence, a spray, a gel, a pack, a sunscreen, a makeup base, a liquid type, a powder, a makeup remover such as a cleansing cream, a cleansing lotion, or a cleansing oil, or a cleanser such as a cleansing foam, a soap, or a body wash.

[0062] In addition to the active ingredient, the cosmetic composition may contain adjuvants commonly used in the field of cosmetology, such as fatty substances, organic solvents, solubilizers, thickeners, and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, moisturizing agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredient commonly used in cosmetics.

[0063] When the active ingredient is used in a topical skin preparation, it may further contain adjuvants commonly used in dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, moisturizing topics, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredient commonly used in topical skin preparations, in amounts commonly used in dermatology.

[0064] Furthermore, when the active ingredient is provided in a skin topical formulation, it may be in the form of, but not limited to, an ointment, patch, gel, cream, or spray.

[0065] The pharmaceutically acceptable salts of the active ingredient are acid addition salts formed with organic or inorganic acids, such as formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, and methanesulfonic acid, and inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and boric acid, preferably in the form of hydrochloride or acetate, more preferably in the form of hydrochloride.

[0066] The acid addition salts described above can be prepared by a) directly mixing the active ingredient and the acid, b) dissolving one of them in a solvent or aqueous solvent and mixing them, or c) placing the active ingredient in an acid in a solvent or aqueous solvent and mixing them using a general salt preparation method.

[0067] Further possible salt forms include kava salts, carbapentin salts, pregabalin salts, nicotinate salts, adipate salts, hemimalonate salts, cysteine ​​salts, acetylcysteine ​​salts, methionine salts, arginine salts, lysine salts, ornithine salts, aspartate salts, and the like.

[0068] Furthermore, when the active ingredient of the present invention is used in a pharmaceutical product, it may further contain one or more active ingredients exhibiting the same or similar functions. For example, known skin regeneration or wrinkle-reducing ingredients may be included. The inclusion of additional skin regeneration or wrinkle-reducing ingredients further enhances the skin regeneration or wrinkle-reducing effects of the culture solution of the present invention. When adding such ingredients, consideration should be given to skin safety when used in combination, ease of formulation, and stability of the active ingredient. The additional ingredient(s) may be included in an amount of 0.0001% to 10% by weight of the total composition weight, and this content range may be adjusted depending on factors such as skin safety and ease of formulation of the active ingredient.

[0069] In addition, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0070] Pharmaceutically acceptable carriers include a number of ingredients such as buffer solutions, sterile water for injection, normal saline or phosphate buffered saline, sucrose, histidine, salts, polysorbates, and the like.

[0071] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., transdermally), and can be administered in the form of a general pharmaceutical formulation, for example, in various oral and parenteral dosage forms when administered clinically. When formulated, the composition is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant.

[0072] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the pharmaceutical composition of the present invention with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc.

[0073] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, liquid contents, emulsions, syrups, etc., which contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents.

[0074] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cocoa butter, laurine, and glycerogelatin. [Effects of the Invention]

[0075] In this invention, adult stem cells that highly express GDF-3 were isolated from the umbilical cord blood tract and the role of GDF-3 in umbilical cord blood stem cells was analyzed. As a result, it was revealed that when umbilical cord blood stem cells highly express GDF-3, their cell proliferation ability and extracellular matrix secretion ability are increased, and it was confirmed that such GDF-3 affects cell proliferation and extracellular matrix secretion ability not only in stem cells but also in general somatic cells.

[0076] These characteristics allow for increased subculture of adult stem cells, resulting in the acquisition of more stem cells and the production of large amounts of stem cells themselves and the active substances secreted by them, which can be useful in the development of various compositions utilizing GDF-3-high-expressing umbilical cord blood stem cells and their secreted substances.

[0077] Therefore, the umbilical cord blood stem cells highly expressing GDF-3 produced by the present invention can be used as one of the main raw materials in the field of next-generation biopharmaceuticals.

[0078] Furthermore, if GDF-3 increases the ability to secrete extracellular matrix, the efficacy of cell culture media containing this will be enhanced, which can be useful in developing compositions that can be used to manufacture cosmetics and pharmaceuticals using culture media. [Brief explanation of the drawings]

[0079] [Figure 1] 1 is a graph showing an analysis of whether the cord blood stem cells isolated and cultured in Example 1 express specific surface proteins. [Figure 2] After isolating umbilical cord blood stem cells using a general DMEM badge (CTR) and a new stem cell culture badge (PMS), we are comparing the cell shape and proliferation rate according to passage. [Figure 3] This is a graph comparing the degree of cell proliferation at late passages (after P7) between a general DMEM badge (CTR) and a new stem cell culture badge (PMS). [Figure 4] This graph compares the amount of fibronectin secreted, an extracellular matrix substance secreted by cells, when culturing umbilical cord blood stem cells using a general DMEM badge (CTR) and a new stem cell culture badge (PMS). [Figure 5] This is a graph analyzing the GDF-3 gene expression characteristics expressed in cells from the early stage (P3) to the late stage (P7) of subculture when umbilical cord blood stem cells are cultured in a general DMEM badge (CTR) and a new stem cell culture badge (PMS). [Figure 6] When general skin cells (dermal fibroblasts) were cultured, high expression of the GDF-3 gene was confirmed after treatment with GDF-3 protein. [Figure 7] This is a graph showing the effect of GDF-3 on the growth of human epithelial cells (HDF). It was confirmed that in the case of skin cells with high expression of GDF-3, cell proliferation proceeds rapidly and continuously. [Figure 8] It has been confirmed that normal skin cells in which high GDF-3 expression has been induced show higher gene expression of collagen and fibronectin, which are components of the extracellular matrix, compared to skin cells in the control group. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention encompasses aspects described in the following paragraphs. [Section 1] A method for producing GDF-3, comprising culturing umbilical cord blood stem cells to express GDF-3. [Section 2] Item 2. A method for producing GDF-3 according to Item 1, characterized in that the cord blood stem cells are isolated and cultured from fast-growing cord blood stem cells with a cell proliferation rate of 24 hours or less. [Section 3] Item 1. A method for producing GDF-3 according to Item 1, characterized in that cord blood stem cells with a desired growth rate are isolated and cultured in a batch containing DMEM / F12 to which FGF-2, EGF, TGF-β superfamily growth factors, ascorbic acid, and heparin have been further added. [Section 4] Item 1. The method for producing GDF-3 according to Item 1, wherein the GDF-3 increases the growth of fibroblasts. [Section 5] Item 5. The method for producing GDF-3 according to Item 4, wherein the fibroblasts whose growth is increased by GDF-3 are human epithelial cells (Human Dermal Fibroblasts, HDFs). [Section 6] Item 2. The method for producing GDF-3 according to Item 1, wherein the GDF-3 increases the expression of a gene encoding at least one protein selected from the group consisting of GDF-3, collagen, and fibronectin in fibroblasts. [Section 7] Item 2. The method for producing GDF-3 according to Item 1, wherein the GDF-3 promotes the synthesis of active substances involved in promoting cell proliferation and / or regeneration. [Section 8] A method for producing GDF-3-expressing cord blood stem cells, characterized by isolating and culturing cord blood stem cells with a high growth rate in order to secure cord blood stem cells with high GDF-3 expression. [Section 9] Item 9. A method for producing GDF-3-expressing cord blood stem cells according to Item 8, characterized in that the higher the proliferation rate of the isolated and cultured cord blood stem cells, the higher the GDF-3 expression level of the isolated and cultured cord blood stem cells. [Section 10] Item 9. The method for producing GDF-3-expressing cord blood stem cells according to Item 8, which is used to establish a cell bank for the cord blood stem cells with high GDF-3 expression. [Section 11] A first step of seeding and culturing cord blood mononuclear cells isolated from the cord blood tract onto a badge containing a TGF-β superfamily growth factor; Item 9. A method for producing GDF-3-expressing umbilical cord blood stem cells, as described in Item 8, characterized in that it includes a second step of selectively isolating and culturing mononuclear cells that adhere to a culture dish and proliferate relatively quickly. [Section 12] Item 9. A method for producing GDF-3-expressing cord blood stem cells according to Item 8, wherein the GDF-3-expressing cord blood stem cells are cultured to produce a GDF-3-containing culture medium. [Section 13] Item 9. The method for producing GDF-3-expressing cord blood stem cells according to Item 8, wherein the expression level of GDF-3 in the GDF-3-expressing cord blood stem cells increases with the progression of subculture. [Section 14] Item 9. The method for producing GDF-3-expressing cord blood stem cells described in Item 8, characterized in that the GDF-3-expressing cord blood stem cells are at passage 5 or more to ensure high GDF-3-expressing cord blood stem cells. [Section 15] Item 9. A method for producing GDF-3-expressing cord blood stem cells according to Item 8, characterized in that GDF-3-expressing cord blood stem cells are subcultured to secure a large amount of cord blood stem cells. [Section 16] Item 9. A method for producing GDF-3-expressing cord blood stem cells according to Item 8, characterized in that GDF-3-expressing cord blood stem cells are subcultured to obtain a large number of cord blood stem cells with excellent secretion ability of extracellular matrix components. [Section 17] GDF-3-expressing cord blood stem cells are characterized by the following: cord blood mononuclear cells isolated from umbilical cord blood are seeded and cultured on a plate containing TGF-β superfamily growth factors; and mononuclear cells that adhere to the culture plate and proliferate relatively quickly are selectively isolated and cultured. [Section 18] A composition for increasing fibroblast growth, comprising GDF-3. [Section 19] Item 19. The composition for increasing fibroblast growth according to Item 18, wherein the fibroblasts are human epithelial cells (HDF). [Section 20] Item 19. A composition for increasing fibroblast growth according to Item 18, characterized in that GDF-3 increases the expression of a gene encoding at least one protein selected from the group consisting of GDF-3, collagen, and fibronectin in fibroblasts. [Section 21] Item 19. A composition for increasing fibroblast growth according to Item 18, wherein GDF-3 promotes the synthesis of active substances involved in promoting cell proliferation and / or regeneration. [Section 22] A composition for skin regeneration, characterized by containing GDF-3. [Section 23] Item 23. The composition for skin regeneration according to Item 22, which is a cosmetic composition or a pharmaceutical composition. The present invention will be described in more detail below with reference to examples, which are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of protection of the present invention.

[0081] Example 1. Cord blood stem cell isolation 1.1. Cord blood mononuclear cell isolation Umbilical cord blood collected from a newborn is placed in a Hetasep TM After removing red blood cells by mixing with Ficoll, the cord blood mononuclear cells were isolated by centrifugation and washed with PBS. The cord blood mononuclear cells were seeded onto a culture dish coated with fibronectin (50 μg / ml) for 24 hours at 4°C. The culture medium used here was a novel one developed by adding FGF-2, EGF, TGF-β superfamily growth factors, ascorbic acid, and heparin to DMEM / F12.

[0082] 1.2. Cord blood stem cell isolation After culturing cord blood mononuclear cells for 48 hours, cells that had not adhered to the culture dish were removed, and only the adherent mononuclear cells were cultured. Fast-growing mononuclear cells were selectively isolated and cultured, and then analyzed for stem cell markers. The culture badges were replaced every 2-3 days, and cord blood stem cells with a cell doubling time of less than 24 hours were isolated.

[0083] 1.3. Analysis of cord blood stem cell-specific surface marker expression After isolating umbilical cord blood stem cells (CBSCs) in 1.2, flow cytometry experiments were performed to confirm whether the isolated cells were umbilical cord blood stem cells. For cell surface antigen phenotyping, cells from passages 3–4 were harvested, stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies, and analyzed by FACS (CytoFLEX, Beckman Coulter, CA).

[0084] To analyze the characteristics of the cord blood stem cells isolated in 1.2, CD44 (mesenchymal stem cell marker), CD73 (mesenchymal stem cell marker), and CD105 (mesenchymal stem cell marker) were used as positive marker antigens, and CD11b (hematopoietic stem cell marker), CD19 (immune cell marker), CD34 (hematopoietic stem cell marker), CD45 (non-hematopoietic stem cell marker), and HLA-DR (immune rejection-related marker) were used as negative marker antigens. The percentage of cord blood stem cells positive for each CD antigen is shown in Table 1 and a graph in Figure 1.

[0085] [Table 1]

[0086] Comparative Example 1 Stem cells were isolated and cultured from the umbilical cord blood in the same manner as in Example 1, except that DMEM was used as a batch control in 1.1 Isolation of umbilical cord blood mononuclear cells and 1.2 Isolation of umbilical cord blood stem cells.

[0087] Example 2. Cord blood stem cell culture and proliferation analysis The cord blood stem cells isolated in Example 1 and Comparative Example 1 were cultured at 37°C in 5% CO2 medium in a new stem cell culture medium containing DMEM / F12 containing 10% fetal bovine serum and fibroblast growth factor 2 (FGF2). The cell proliferation rates were compared (Figures 2 and 3).

[0088] As compared in FIG. 2, the cord blood stem cells (PMS) isolated and cultured in Example 1 have a faster proliferation rate than the cord blood stem cells (CTR) isolated and cultured in Comparative Example 1.

[0089] Furthermore, as shown in Figure 3, the cord blood stem cells (CTR) isolated and cultured in Comparative Example 1 stopped growing after the seventh passage, whereas the cord blood stem cells (PMS) isolated and cultured in Example 1 continued to grow for more than eight passages. Therefore, the present invention makes it possible to obtain a larger amount of stem cells.

[0090] Example 3. Comparison of the secretion ability of extracellular matrix (ECM) substances from umbilical cord blood stem cells For the cord blood stem cells isolated in Example 1 and Comparative Example 1, 4 to 10 × 10 cord blood stem cells were cultured at each passage. 3 cells / cm 2 After 24 hours, the cells were washed with PBS, replaced with serum-free media, and cultured for 2 to 7 days to prepare a cord blood stem cell culture medium.

[0091] The fibronectin expression level of the umbilical cord blood stem cell culture medium at each passage was analyzed using an ELISA kit (Figure 4).

[0092] As shown in Figure 4, the fibronectin content in the cord blood stem cell culture medium obtained by subculturing the cord blood stem cells isolated in Example 1, which maintained high GDF-3 expression, was found to be higher than that of the control group obtained by subculturing the cord blood stem cells isolated in Comparative Example 1.

[0093] Compared to the cord blood stem cells isolated in Comparative Example 1, the cord blood stem cells isolated in Example 1 not only showed increased cell proliferation but also increased ECM component content.

[0094] Example 4. Analysis of GDF-3 mRNA expression levels by passage For the cord blood stem cells isolated in Example 1 and Comparative Example 1, the cord blood stem cells at each passage were washed with PBS and suspended in trypsin EDTA. The cells were then placed in a culture plate containing twice the amount of trypsin EDTA used for treatment to inactivate the trypsin EDTA. The suspended cells were centrifuged and the supernatant was removed. The cell pellet was resuspended in PBS and centrifuged, and the supernatant was removed.

[0095] PureLink TM Total RNA was isolated using an RNA Mini Kit (Invitrogen), quantified, and cDNA was synthesized.

[0096] The synthesized cord blood stem cell cDNA was subjected to real-time PCR using the primers listed in Table 2 to confirm the expression level of GDF-3 at each passage. GAPDH, a housekeeping gene, was used as a loading control (Figure 5).

[0097] [Table 2]

[0098] As can be seen from the graph in Figure 5, the rapid proliferation is due to the high expression of GDF-3.

[0099] Furthermore, as shown in Figure 5, it was confirmed that the expression level of GDF-3 was higher in the cord blood stem cells (PMS) isolated in Example 1 compared to the control group of cord blood stem cells (Control) isolated in Comparative Example 1. Specifically, it was confirmed that the expression level of GDF-3 increased gradually as the passage progressed.

[0100] Example 5. Characterization of GDF-3-treated human epithelial cells (HDFs) During human epithelial cell (HDF) culture, mRNA analysis was performed to compare the cell characteristics of the GDF-3-treated and untreated control groups. As shown in Figure 6, the experimental results confirmed that GDF-3 gene expression was elevated in the GDF-3-treated experimental group.

[0101] Example 6. Effect of GDF-3 on HDF growth Human epithelial cells (HDF) were cultured in DMEM batches and treated with GDF-3 at 1-100 ng / ml at 48-hour intervals. As shown in Figure 7, the GDF-3-treated experimental group demonstrated increased HDF growth compared to the untreated group. The GDF-3-treated group exhibited a more elongated and shorter cell shape, favorable for proliferation. Even after long-term culture of over 96 hours, cell proliferation did not stagnate or decline, resulting in a sustained cell growth. This indicates that GDF-3 has an effect on sustained cell growth (Figure 7).

[0102] Example 7. Confirmation of extracellular matrix gene expression levels in HDFs with high GDF-3 expression Real-time PCR confirmed that GDF-3 treatment also increased gene expression of skin constituents such as collagen and fibronectin, which are involved in skin regeneration (Figure 8).

[0103] As shown in Figure 8, we confirmed that when high GDF-3 expression was maintained in HDFs, the gene expression levels of ECM components increased.

Claims

1. Follow these steps: A first step of selectively isolating and culturing umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) having a cell doubling time of 24 hours or less from umbilical cord blood mononuclear cells; The second step involves culturing the UCB-MSCs obtained in the first step to increase GDF-3 mRNA expression and release large amounts of GDF-3. A method for producing GDF-3, comprising:

2. 2. The method for producing GDF-3 according to claim 1, wherein in the first step, the UCB-MSCs are selectively isolated and cultured in DMEM / F12 medium supplemented with FGF-2, EGF, TGF-β superfamily growth factors, ascorbic acid, and heparin.

3. 2. The method for producing GDF-3 according to claim 1, wherein the GDF-3 produced by culturing the UCB-MSCs is used to increase the proliferation of fibroblasts.

4. 4. The method for producing GDF-3 according to claim 3, wherein the fibroblasts whose growth is increased by GDF-3 are human dermal fibroblasts (HDF).

5. The method for producing GDF-3 according to claim 1, characterized in that the GDF-3 produced by culturing the UCB-MSCs is used to increase the expression of a gene encoding at least one protein selected from the group consisting of GDF-3, collagen, and fibronectin in fibroblasts.

6. The method for producing GDF-3 according to claim 1, characterized in that the GDF-3 produced by culturing the UCB-MSCs is used to promote the synthesis of active substances involved in promoting cell proliferation and / or regeneration.

7. A method for producing GDF-3-expressing umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), which have a growth rate (cell doubling time) of 24 hours or less, is selectively isolated from umbilical cord blood mononuclear cells and cultured to obtain the GDF-3-expressing UCB-MSCs, which express GDF-3 at a high level.

8. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, characterized in that the higher the proliferation rate of the GDF-3-expressing UCB-MSCs, the higher the amount of GDF-3 expressed in these cells.

9. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, characterized in that it is used to establish a cell bank for umbilical cord blood stem cells with high GDF-3 expression.

10. Follow these steps: A first step of seeding cord blood mononuclear cells isolated from cord blood onto a culture dish containing a medium supplemented with a TGF-β superfamily growth factor; a second step of selectively isolating and culturing the cord blood mononuclear cells that adhere to a culture dish and grow relatively quickly, with a cell doubling time of 24 hours or less; The method for producing GDF-3-expressing UCB-MSCs according to claim 7, comprising:

11. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, wherein the GDF-3-expressing UCB-MSCs are cultured to produce a GDF-3-containing culture medium.

12. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, wherein the expression level of GDF-3 increases as the passage of the GDF-3-expressing UCB-MSCs progresses.

13. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, wherein the GDF-3-expressing UCB-MSCs are at passage 5 or more to obtain umbilical cord blood stem cells with high GDF-3 expression.

14. 8. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, wherein the GDF-3-expressing UCB-MSCs are propagated by subculture to obtain a large amount of umbilical cord blood stem cells.

15. The method for producing GDF-3-expressing UCB-MSCs according to claim 7, characterized in that the GDF-3-expressing UCB-MSCs are expanded by subculture in order to obtain a large number of umbilical cord blood stem cells with excellent ability to secrete extracellular matrix components.

16. GDF-3-expressing umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are obtained by selectively isolating and culturing umbilical cord blood mononuclear cells (MBCs) isolated from umbilical cord blood, which are seeded onto a culture dish containing a medium supplemented with TGF-β superfamily growth factors, and which adhere to the culture dish and proliferate relatively quickly. These MBCs have a cell doubling time of 24 hours or less, and are then cultured.

17. 2. The method for producing GDF-3 according to claim 1, wherein the GDF-3 produced by culturing the UCB-MSCs is used in a composition for skin regeneration.

18. 18. The method for producing GDF-3 according to claim 17, wherein the GDF-3 produced by culturing the UCB-MSCs is used in a cosmetic composition or a pharmaceutical composition.

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