Method for extending telomere length in stem cells

Abstract

The present invention relates to a method for extending the length of telomeres in stem cells. The method according to the present invention exhibits excellent effects of extending the length of telomeres in stem cells and enhancing the synthesis ability of vascular endothelial growth factor (VEGF) and the synthesis ability of collagen. By securing high-quality stem cells with elongated telomeres in a short period, the method is expected to be effectively applied to the development and production of highly efficient cell therapies as well as to a composition for the prevention or alleviation of skin aging.

Description

Methods for extending the length of telomeres in stem cells

[0001] The present invention relates to a method for extending the length of telomeres of stem cells.

[0002] Within the nucleus of a cell are chromosomes, which are composed of DNA and proteins that contain genetic information. Telomeres exist at both ends of the chromosomes. Telomeres protect the ends of chromosomes during cell division and are known to play a crucial role in determining the lifespan of a cell. However, as cells continue to divide, telomeres shorten and, when they are no longer able to protect DNA, the cell stops dividing, ages, and dies. This is why telomeres are called the "cellular aging clock." In 1982, Dr. Elizabeth Blackburn and others discovered that when telomeres shorten, chromosomes cannot replicate and cell division stops, thereby discovering a link between telomeres, aging, and lifespan, and were awarded the 2009 Nobel Prize in Physiology or Medicine.

[0003] The purposes of culturing human or animal cells are diverse, ranging from understanding life phenomena to producing physiologically active substances, testing the efficacy and safety of drugs, foods, and cosmetics, producing therapeutic proteins, and producing cell therapies. Stem cells, in particular, are undifferentiated cells capable of differentiating into various tissue types, and are currently being utilized in regenerative medicine and cosmetics development.

[0004] Meanwhile, vascular endothelial growth factor (VEGF) is a factor that plays an important role in the creation of new blood vessels by promoting the proliferation of cells inside blood vessels. It is known that the new blood vessels created in this way supply oxygen and nutrients and remove waste products generated during metabolic processes, and therefore it is used in the development of cosmetics for skin regeneration and anti-aging. In addition, collagen is the main component of connective tissue and is an essential element that makes up muscles, bones, skin, hair, etc. in our body. It is known that collagen synthesis in the body decreases significantly as we age, and therefore it is used in the development of anti-aging cosmetics for elasticity and moisturizing.

[0005] Meanwhile, Korean Patent No. 2048067 discloses a 'method for increasing the stem cell capacity of human mesenchymal stem cells' using Endothelin-1, and Korean Patent No. 2209234 discloses a 'method for extending telomeres of cells' through physical stimulation, but the 'method for extending the length of telomeres of stem cells' of the present invention is not described.

[0006] The present invention was derived from the above-mentioned needs, and the inventors prepared CM60 consisting of 2 ng / ㎖ of epidermal growth factor (EGF), 2 ng / ㎖ of basic fibroblast growth factor (bFGF), 40 mM of L-ascorbic acid, and 400 mM of N-acetyl-cysteine, and confirmed that the telomere length, VEGF (vascular endothelial growth factor) synthesis ability, and collagen synthesis ability of stem cells cultured three-dimensionally while treating with the CM60 significantly increased compared to the control group (two-dimensional culture treated with CM60), thereby completing the present invention.

[0007] To solve the above problem, the present invention provides a method for extending the length of telomeres of stem cells, which comprises a step of three-dimensionally stirring and culturing stem cells in a medium containing CM60.

[0008] In addition, the present invention provides stem cells cultured by the above method with extended telomere length and increased VEGF synthesis ability and collagen synthesis ability.

[0009] In addition, the present invention provides a cosmetic composition for preventing or improving skin aging containing a culture solution of the stem cells as an effective ingredient.

[0010] In addition, the present invention provides a medium composition for culturing stem cells with extended telomere lengths, containing CM60 as an active ingredient.

[0011] The method according to the present invention is excellent in increasing the length of telomeres of stem cells, the ability to synthesize VEGF (vascular endothelial growth factor), and the ability to synthesize collagen. Therefore, it is expected that it can be usefully used for the development and production of cell therapeutic agents with improved efficiency and as a composition for preventing or improving skin aging by securing high-quality stem cells with extended telomere lengths in a short period of time.

[0012] Figure 1 shows the results of confirming the proliferation of stem cells after 3, 5, or 7 days of two-dimensional culture of human stem cells in a medium containing CM60 or a medium not containing CM60.

[0013] Figure 2 shows the results of measuring the length of telomeres after two-dimensional culture of human stem cells in a medium not containing CM60, two-dimensional culture in a medium containing CM60, three-dimensional culture in a medium not containing CM60, or three-dimensional culture in a medium containing CM60.

[0014] Figure 3 shows the results of measuring the content of VEGF (vascular endothelial growth factor) after two-dimensional culture of human stem cells in a medium containing CM60 or three-dimensional culture in a medium containing CM60.

[0015] Figure 4 shows the results of measuring the content of collagen after two-dimensional culture of human stem cells in a medium containing CM60 or three-dimensional culture in a medium containing CM60.

[0016] In order to achieve the purpose of the present invention, the present invention provides a method for extending the length of telomeres of stem cells, which comprises a step of three-dimensionally stirring and culturing stem cells in a medium containing CM60.

[0017] In the method for extending the length of telomeres of stem cells of the present invention, the stem cells may be human adipose-derived stem cells, but are not limited thereto.

[0018] In the method for extending the length of telomeres of stem cells of the present invention, the CM60 may preferably be composed of 1 to 100 ng / ml of epidermal growth factor (EGF), 1 to 100 ng / ml of basic fibroblast growth factor (bFGF), 10 to 200 mM of L-ascorbic acid and 100 to 900 mM of N-acetyl-cysteine, more preferably 1 to 10 ng / ml of epidermal growth factor, 1 to 10 ng / ml of basic fibroblast growth factor, 10 to 70 mM of L-ascorbic acid and 200 to 600 mM of N-acetyl-cysteine, and still more preferably 1 to 3 ng / ml of It may be composed of epidermal growth factor, 1 to 3 ng / ml of basic fibroblast growth factor, 30 to 50 mM of L-ascorbic acid and 300 to 500 mM of N-acetyl-cysteine, and most preferably, but not limited to, 2 ng / ml of epidermal growth factor, 2 ng / ml of basic fibroblast growth factor, 40 mM of L-ascorbic acid and 400 mM of N-acetyl-cysteine.

[0019] In addition, in the method for extending the length of telomeres of stem cells of the present invention, the CM60 is characterized by improving the proliferation ability of stem cells.

[0020] In the method for extending the length of telomeres of stem cells of the present invention, the three-dimensional stirred culture may be cultured by attaching stem cells to a microcarrier, but is not limited thereto. The microcarrier may be made of at least one selected from the group consisting of gelatin, collagen, hyaluronic acid, chondroitin sulfate, alginate, chitosan, aminopropylsilane, polydopamine, poly-L-lysine, RGD peptide, and graphene, but is not limited thereto.

[0021] The method of extending the length of telomeres of stem cells of the present invention may preferably be, but is not limited to, three-dimensionally stirring culture of human adipose-derived stem cells in DMEM / F12 medium containing CM60 consisting of 1 to 10 ng / ml of epidermal growth factor, 1 to 10 ng / ml of basic fibroblast growth factor, 10 to 70 mM of L-ascorbic acid, and 200 to 600 mM of N-acetyl-cysteine.

[0022] The present invention also provides stem cells cultured by the above method, in which the length of telomeres is extended and VEGF synthesis ability and collagen synthesis ability are increased.

[0023] Stem cells with extended telomere lengths according to the present invention are characterized by increased vascular endothelial growth factor (VEGF) and collagen synthesis capabilities.

[0024] The present invention also provides a cosmetic composition for preventing or improving skin aging, containing a culture solution of the stem cells as an effective ingredient.

[0025] The cosmetic composition of the present invention is characterized by an excellent effect in preventing or improving skin aging, as it contains as an active ingredient a culture solution of stem cells with increased vascular endothelial growth factor (VEGF) and collagen synthesis ability.

[0026] In a cosmetic composition according to one embodiment of the present invention, the cosmetic composition for preventing or improving skin aging may have any one formulation selected from the group consisting of a cream, a softening lotion, a nourishing lotion, a pack, an essence, a hair tonic, a shampoo, a rinse, a hair conditioner, a hair treatment, a gel, a skin lotion, a skin softener, a skin toner, an astringent, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisture cream, a hand cream, a foundation, a nourishing essence, a sunscreen, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, and a body cleanser, but is not limited thereto. A cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for formulating the formulation, and the types and amounts of these ingredients may be easily selected by a person skilled in the art.

[0027] When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0028] When the cosmetic composition of the present invention is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane-butane or dimethyl ether may be additionally included.

[0029] When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0030] When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0031] When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0032] The cosmetic composition of the present invention may additionally contain excipients including fungicides, hydrotropism inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc.

[0033] The present invention also provides a medium composition for culturing stem cells with extended telomere lengths, containing CM60 as an active ingredient.

[0034] In the badge composition according to the present invention, the CM60 is as described above.

[0035] In the medium composition according to one embodiment of the present invention, the CM60 may be added to various commercially available media known for stem cell culture, but is not limited thereto.

[0036] In addition, when a medium composition containing the above CM60 as an active ingredient is used for stem cell culture, it is characterized by extending the length of telomeres of stem cells and increasing the ability to synthesize vascular endothelial growth factor (VEGF) and collagen.

[0037]

[0038] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention and the scope of the present invention is not limited thereto.

[0039]

[0040] Materials and Methods

[0041] 1. 3D culture of stem cells

[0042] In the present invention, human stem cells isolated from the abdominal adipose tissue of a healthy natural person approved by the Institutional Review Board (IRB) were used. Stem cells were cultured in DMEM / F12 (Dulbecco's Modified Eagle Medium Nutrient Mixture / F-12) medium supplemented with 1% antibiotic-antimycotic solution (cat no. 15-240-062, Gibco) and 2-10% fetal bovine serum (FBS) at 37°C and 5% CO2, with the medium replaced every two days and subcultured once every four days, and the primary flasks were cultured. Afterwards, the primary cultured stem cells were attached to microcarriers (microcarriers, cat no. M9418, Sigma-Aldrich) with a diameter of 100-350 μm and cultured at 37°C and 5% CO2. Three-dimensional circulation culture was performed under conditions of 15–50 rpm. The three-dimensional culture was cultured for 1–2 weeks, with half of the culture medium replaced with fresh medium every two days.

[0043]

[0044] 2. Statistical analysis

[0045] Statistical analysis of the data obtained from each experiment was performed using the Sigma plot (Sigma plot, Systat Software Inc.) and Prism 5.0 (GraphPad Software Inc.) programs. Statistical analysis of the control and experimental groups was performed using one-way ANOVA, and post hoc tests were performed using Dunnett's test. All results are expressed as the mean ± standard error, and significance was determined based on a p<0.05 (**).

[0046]

[0047] Manufacturing Example 1. Manufacturing of CM60 for 3D culture of stem cells

[0048] Three-dimensional culture of stem cells was performed by adding epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), L-ascorbic acid, and N-acetyl-cysteine ​​to DMEM / F12 medium supplemented with 1% antibiotic-antimycotic solution and 2–10% fetal bovine serum (FBS). Each component added to the medium was added so that the final concentrations were as shown in Table 1 below, and this composition was designated as CM60.

[0049] CM60 composition ingredient name final concentration epidermal growth factor (EGF) 2 ng / ㎖ basic fibroblast growth factor (bFGF) 2 ng / ㎖ L-ascorbic acid 40 mM N-acetyl-cysteine ​​400 mM

[0050]

[0051] Example 1. Measurement of proliferation capacity

[0052] 1×10 human stem cells per test group 4 After inoculating the dogs and culturing them for 7 days in DMEM / F12 medium containing CM60 or DMEM / F12 medium not containing CM60 using a two-dimensional plate culture method, the proliferation capacity of the stem cells was investigated by direct counting using a hemocytometer.

[0053] As a result, when cultured in two dimensions without CM60, the number of proliferating cells after 3 days, 5 days, or 7 days of culture was 2.74×10 4 Dog, 6.84×10 4 Dog or 8.99×104 On the other hand, when cultured in two dimensions with CM60, the number of proliferating cells after 3 days, 5 days, or 7 days of culture was 4.41×10 4 Dog, 14.07×10 4 dog or 37.80×10 4 I checked the individual.

[0054] Through the above results, it was confirmed that the number of stem cells significantly increased by more than 420% (p<0.05) by adding CM60, thereby showing that CM60 significantly increases the proliferation capacity of stem cells (Fig. 1).

[0055]

[0056] Example 2. Measurement of telomere length

[0057] In order to secure the quantity of stem cells suitable for the treatment of patients, stem cells have been conventionally proliferated through subculture using a two-dimensional plate culture method. However, it is known that when stem cells are proliferated using this method, the length of telomeres becomes shorter as the subculture progresses.

[0058] Human adipose-derived stem cells were cultured in two dimensions or three dimensions in DMEM / F12 medium containing CM60, with a total cell number of 1 × 10 8 After culturing each cell until it reached 100 μg / ml, the telomere length was calculated according to the manual using the Absolute Human telomereLength Quantification qPCR Assay Kit (#8918, Science Cell Research Lab).

[0059] As a result, the telomere length when cultured two-dimensionally including CM60 was 8.80±0.64 Kb, which increased by about 1.3 Kb (17.0%) during the culture period, and the telomere length when cultured three-dimensionally including CM60 was 12.85±0.94 Kb, which increased by about 5.33 Kb (70.1%) during the culture period. It was confirmed that the telomere length when cultured three-dimensionally including CM60 significantly (p<0.05) increased by about 46% or more compared to when cultured two-dimensionally including CM60 (Fig. 2).

[0060]

[0061] Example 3. Measurement of vascular endothelial growth factor (VEGF) synthesis ability

[0062] Human stem cells were cultured at 1.5 × 10 in DMEM / F12 medium containing CM60. 5 After inoculation with a dog and culturing for 7 days in two dimensions or three dimensions, the medium was recovered and the VEGF content was measured using the Human VEGF Quantikine ELISA Kit (#DVE00, R&D systems) according to the manual.

[0063] As a result, it was confirmed that the VEGF content when cultured two-dimensionally including CM60 was 4,738±71 pg / ㎖, whereas the VEGF content when cultured three-dimensionally including CM60 was 7,476±72 pg / ㎖, thereby confirming that the VEGF content when cultured three-dimensionally increased significantly (p<0.05) by about 58% or more compared to when cultured two-dimensionally (Fig. 3).

[0064]

[0065] Example 4. Measurement of collagen synthesis capacity

[0066] Human stem cells were cultured at 1.5 × 10 in DMEM / F12 medium containing CM60. 5After inoculation with a dog and culturing for 7 days in two-dimensional or three-dimensional culture, the medium was recovered and tested with the Soluble Collagen assay kit (#S1000, Sircol TM ) was used to measure the collagen content according to the manual.

[0067] As a result, it was confirmed that the collagen content when cultured two-dimensionally including CM60 was 43,632 ng / ㎖, whereas the collagen content when cultured three-dimensionally including CM60 was 70,838 ng / ㎖, thereby confirming that the collagen content when cultured three-dimensionally increased significantly (p<0.05) by about 62.3% or more compared to when cultured two-dimensionally (Fig. 4).

[0068] Based on the above results, it was found that when stem cells were cultured in a three-dimensional stirred culture method using biocompatible microcarriers in a medium containing CM60 according to the present invention, proliferation capacity, telomere length, VEGF synthesis capacity, and collagen synthesis capacity were significantly increased.

Claims

1. A method for extending the length of telomeres of stem cells, comprising a step of three-dimensionally stirring and culturing stem cells in a medium containing CM60.

2. A method for extending the length of telomeres of stem cells, characterized in that in the first paragraph, the CM60 comprises 1 to 100 ng / ml of epidermal growth factor (EGF), 1 to 100 ng / ml of basic fibroblast growth factor (bFGF), 10 to 200 mM of L-ascorbic acid, and 100 to 900 mM of N-acetyl-cysteine, and improves proliferation ability.

3. A method for extending the length of telomeres of stem cells, characterized in that in the first paragraph, the three-dimensional stirred culture is cultured by attaching stem cells to microcarriers.

4. A method for extending the length of telomeres of stem cells, characterized in that the stem cells are three-dimensionally stirred and cultured in DMEM / F12 medium containing CM60 consisting of 1 to 10 ng / ml of epidermal growth factor, 1 to 10 ng / ml of basic fibroblast growth factor, 10 to 70 mM of L-ascorbic acid, and 200 to 600 mM of N-acetyl-cysteine ​​in the first paragraph.

5. Stem cells with extended telomere length and increased VEGF (vascular endothelial growth factor) synthesis ability and collagen synthesis ability, cultured using any one of the methods in clauses 1 to 4.

6. A cosmetic composition for preventing or improving skin aging containing the culture solution of stem cells of Article 5 as an active ingredient.

7. A medium composition for culturing stem cells with extended telomere length, containing CM60 as an active ingredient.

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