KR20130008293A - Large scale production of growth promoting factors using fetus-derived mesenchymal stem cells from amniotic fluids with hypoxic condition and composition for regenerating skin using the same - Google Patents

Abstract

PURPOSE: A method for increasing a large amount of mesenchymal stem cells, a conditioned medium composition, and a composition for skin regeneration are provided to ensure cell growth and regeneration abilities. CONSTITUTION: A method for increasing a large amount of mesenchymal stem cells derived from a fetus in amniotic fluid comprises: a step of isolating fetus-derived cells; a step of sub-culturing the cells in a medium containing FBS(fetal bovine serum) and bFGF(basic fibroblast growth factor) and collecting the mesenchymal stem cells; and a step of culturing the mesenchymal stem cells in a hypoxic state for 24-72 hours. A composition for skin regeneration contains a conditioned medium as an active ingredient, which is prepared by culturing the mesenchymal stem cells in a serum-free medium for 3 days.

Description

Large scale production of growth promoting factors using fetus-derived mesenchymal stem cells from amniotic fluids with hypoxic condition and composition for regenerating skin using the same}

The present invention relates to a fetus-derived mesenchymal stem cells in amniotic fluid cultured under hypoxic conditions, and more particularly, comprising the step of culturing in a hypoxic state, a method for mass proliferation of mesenchymal stem cells, and a conditioned medium prepared using the same A composition, a method for mass production of human growth factors, and a composition for skin regeneration using the composition.

Stem cells are cells that have the ability to differentiate into various cells and propagation through appropriate environment and stimulation. They are embryonic stem cells (ES cells) isolated from early embryos, Three types of embryonic germ cells (EG cells) isolated from primordial germ cells and multipotent adult progenitor cells (MAP cells) isolated from adult bone marrow are best known. Since stem cells have the potential to develop into cells having characteristic phenomena and specialized functions, they have been the subject of research as a cell resource for function recovery of various organs.

As reported so far, adult stem cells are known to have the ability to differentiate into various cells. Adult stem cells are bone marrow (Science 276, 71-74, 1997; Science 284, 143-147, 1999; Science 287, 1442-1446, 2000), skeletal muscle (Proc. Natl Acad. Sci USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999), and Fat tissue (Tissue Eng 7, 211-228, 2001; J. Cell. Physiol. 206, 229-237, 2006, each of which can differentiate into similar lines.

Bone marrow-derived mesenchymal stem cells, one of the types of adult stem cells, have been used for a long time and have proved effective. However, since isolation and large amounts of cells are difficult to obtain, there is an urgent need for other sources to replace them. Studies have shown that cells isolated from adipose tissue as well as other tissues have similar characteristics to human bone marrow-derived mesenchymal stem cells. Recently, a new hypothesis exists that stem cells exist in the amniotic fluid protecting fetuses. .

Thus, the present inventors focused on easily detachable amniotic fluid without harming the mother and the fetus.

After the fertilized egg is fertilized and implanted on the wall of the uterus, after several days, the embryo is surrounded by amniotic sac filled with amniotic fluid. Amniotic fluid not only keeps the fetus free to move, but also protects the fetus from external shocks and stimuli, prevents bacterial infections, and helps to regulate the body's body temperature. In addition, since the amniotic fluid is mixed with various substances from the fetus's body, the amniotic fluid can be analyzed to determine whether the fetus's chromosome is abnormal or not infected with bacteria.

Before the baby is born, the amniotic fluid is tested to find out various information about the health of the fetus. At this time, the amniotic fluid can be extracted without harming the mother from the beginning of pregnancy until after the birth. The cells used in the test are discarded after the test. If the patient's consent is obtained, the cells can be used for research purposes without discarding. Therefore, the amniotic fluid cells have an advantage in that a large amount of cells can be easily obtained as compared to other adult stem cells. However, there are few reports on the efficient method of mass-proliferating amniotic stem-derived mesenchymal stem cells and the conditioned medium prepared by using the medium extracted therefrom.

Under these backgrounds, the present inventors have made intensive efforts to develop a method for mass proliferation of amniotic stem-derived mesenchymal stem cells. As a result, the amniotic fetal stem cells cultured under hypoxic conditions may be adipocytes or It was confirmed that the cells have differentiation capacity to osteoblasts, a method of producing a conditioned medium using amniotic stem-derived mesenchymal stem cells, and a conditioned medium prepared by the method is used for growth of other cells (fibroblasts). The effect was confirmed. Mouse in The effect of wound regeneration was confirmed through in vivo experiments, and the present invention was completed by developing a composition having cell growth, wound healing, and skin regeneration ability using conditioned medium.

One object of the present invention is to provide a method for mass proliferation of fetal derived mesenchymal stem cells in amniotic fluid, which comprises culturing in a hypoxic state.

Another object of the present invention to provide a conditioned medium composition using the fetal-derived mesenchymal stem cells.

Still another object of the present invention is to provide a method for mass production of human growth factors using the embryo-derived mesenchymal stem cells.

Another object of the present invention to provide a composition for skin regeneration comprising the culture solution of the fetal-derived mesenchymal stem cells as an active ingredient.

In order to achieve the above object, the present invention as one embodiment provides a method for mass proliferation of fetal-derived mesenchymal stem cells in amniotic fluid, comprising culturing in a hypoxic state.

More specifically, (a) isolating fetal derived cells in amniotic fluid obtained from the mother; (b) subcultured fetal-derived cells isolated in step (a) in a medium containing fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF) to obtain fetal-derived mesenchymal stem cells in amniotic fluid; And (c) culturing the obtained mesenchymal stem cells in a hypoxic state.

In the present invention, the term "mesenchymal stem cells (MSCs)" is a cell that helps to make bones, cartilage, fat, myeloid epilepsy, muscle, nerves, etc., but in adults generally stay in the bone marrow In the umbilical cord blood, peripheral blood, other tissues and the like, it means a cell that can be obtained from them. Amniotic fluid from the mother contains many chemicals from the fetus's body that can produce most of the cells in the body and are easy to harvest. In addition, hepatogenous cells are present in the amniotic fluid, and the present inventors have found that there are homogenous mesenchymal stem cells having a fibroblast-like shape, which is characteristic of mesenchymal stem cells. Confirmed. In addition, it was confirmed that the growth and proliferation of mesenchymal stem cells are increased by culturing these mesenchymal stem cells in a hypoxic state.

As used herein, the term "hypoxia" refers to the presence of less oxygen than the amount of oxygen in general cell culture conditions, and conditions that can proliferate the mesenchymal stem cells of the present invention in large quantities include without limitation. However, preferably, the obtained mesenchymal stem cells of step (c) are preferably incubated in about 5% carbon dioxide for 24 hours to 72 hours under oxygen conditions of 1% to 5%, most preferably 1% It is preferable to incubate for 72 hours in conditions containing oxygen.

In the present invention, the term "culture medium" is in Means a medium capable of supporting the growth and survival of fetal derived cells in amniotic fluid in vitro, and includes all conventional media used in the art suitable for the culture of fetal derived cells in amniotic fluid. Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and IMEM (Iscove's Modified Dulbecco's Medium), but are not limited thereto. In addition, the medium may include antibiotics such as penicillin, streptomycin, or gentamicin.

In the present invention, the fetal mesenchymal stem cells derived from amniotic fluid can be obtained by culturing cells isolated from amniotic fluid in basal medium containing FBS and bFGF, preferably in low glucose DMEM medium containing 10% FBS. It can be obtained by incubating by adding 4 ng / ml bFGF. In a preferred embodiment of the present invention, the low glucose DMEM medium may further comprise 4 ng / ml bFGF solution with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin. According to a preferred embodiment of the present invention, the obtained mesenchymal stem cells were cultured for 3 days at 1% or 5% hypoxic conditions, it was confirmed that the cell growth is increased compared to normal oxygen conditions (Figs. 5A and B) , It was confirmed that the cell growth is promoted when cultured for 3 days at 1% hypoxic conditions (Figs. 6A to C). These results support the massive proliferation of mesenchymal stem cells when cultured in amniotic fluid-derived mesenchymal stem cells under hypoxic conditions.

The present invention is characterized in that the amniotic cells are derived from the fetus.

As a preferred embodiment of the present invention, it is possible to confirm that the cells in the amniotic fluid are cells derived from the fetus through karyotyping. In the amniotic fluid, not only fetal cells but also maternal cells may be present. In the present invention, it was possible to confirm that the amniotic cells are male-derived cells through chromosome analysis (FIG. 2).

In addition, amniotic fetal-derived mesenchymal stem cells prepared by the method of the present invention (a) show positive immunological characteristics for CD13, CD29, CD44 and CD90, and (b) adhere to and grow in cell culture vessels. It shows the morphological characteristics of the spine-shape, a typical shape of fibroblasts, and (c) has the ability to differentiate into mesodermal lineage cells. According to one embodiment of the present invention, the fetal-derived mesenchymal stem cells of the amniotic fluid of the present invention are homologous to fibroblasts (FIG. 1B), flow cytometry (Flourescence-activated cell sorter; FACS) results, mesenchymal stem cells It was confirmed that the markers specific for CD13, CD29, CD44 and CD90 are expressed (Fig. 3). These results support that the cells isolated from amniotic fluid of the present invention is mesenchymal stem cells.

In the present invention, the term "differentiation" refers to a phenomenon in which the structure or function of the cell is specialized while the cell divides and grows. Multipotent mesenchymal stem cells can differentiate into lineage-defining progenitor cells (eg, mesodermal cells), and then further differentiate into other forms of progenitor cells (eg, osteoblasts, etc.), followed by specific Can differentiate into terminal differentiated cells (eg, adipocytes, bone cells, chondrocytes, etc.) that play a characteristic role in tissue (eg bone). In a preferred embodiment, the amniotic fluid-derived mesenchymal stem cells of the present invention have the ability to differentiate into adipocytes or osteoblasts.

In a preferred embodiment of the present invention, the differentiation medium into adipocytes is high glucose DMEM, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-caffeine (3-isobutyl-1-methyl-xanthine). , 10 ng / ml insulin, 100 mM indomethacin and 10% FBS.

In a preferred embodiment of the present invention, the differentiation medium into osteoblasts may comprise high glucose DMEM, 100 mM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM ascorbate and 10% FBS. have. As a preferred embodiment of the present invention, the culture in each differentiation medium may be performed for 7 days to 28 days.

According to one embodiment of the present invention, after culturing mesenchymal stem cells derived from the amniotic fluid in each medium for differentiation for 7 days to 28 days, lipoprotein lipase (lipoprotein lipase; LPL), which is an adipocyte-specific marker ), Fatty acid binding protein 2 (aP2) and peroxysome proliferator-activated receptor gamma (PRAR) expression and lipid droplets are produced. (FIG. 4A), osteopontin (osteopontin), osteocalcin (osteocalcin) and osteocalcin (osteocalcin) expression and confirm that the calcium salt is accumulated (Fig. 4B), the mesenchymal stem cells of the present invention is mesenchymal stem cells It was confirmed that the ability to differentiate into human bone cells and adipocytes. Therefore, in the present invention, the fetal-derived cells in the amniotic fluid are cells having the properties of mesenchymal stem cells, and it is possible to use them as a source of other cells other than bone marrow.

In another aspect, the present invention provides a method for treating a fetus, comprising: (a) isolating fetal derived cells in amniotic fluid obtained from a mother; (b) subcultured in medium containing FBS and bFGF to obtain fetal mesenchymal stem cells derived from amniotic fluid; (c) culturing the obtained mesenchymal stem cells in a hypoxic state; And (d) culturing the obtained intrauterine fetal derived mesenchymal stem cells in a serum-free medium for 3 days to prepare a conditioned medium. Provide a way to produce.

Specifically, the present invention is a human growth factor, for example, Apo-1 / Fas, epidermal growth factor (EGF), IP-10 by culturing fetal derived mesenchymal stem cells extracted from human amniotic fluid in an appropriate medium and hypoxic conditions (Interferon-γ inducible protein-10), leptin, MIP4, MMP3, Rantes, interferon-gamma (IFNγ), human transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNFα), tumor necrosis factor receptor I (TNFRI), tumor necrosis factor receptor II (TNFRII), cell adhesion factor-1 (ICAM-1), vascular cell adhesion factor-1 (VCAM-1), vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), interleukin-1 receptor alpha (IL-1Rα), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL -7, IL-8, IL-12, IL-15 and the like is provided to manipulate the method to synthesize. Preferably VEGF. Hypoxic conditions are as described above. Using conditioned medium of fetal-derived mesenchymal stem cells in amniotic fluid, components can be identified through a two-dimensional antibody array and confirmed in vitro .

In the present invention, in order to obtain a conditioned medium of fetal-derived mesenchymal stem cells, the mesenchymal stem cells obtained from amniotic fluid containing Ham's F-12 nutrient mixture containing amino acids or their analogues and vitamins or their analogs It is preferable to use serum medium. Serum-free medium containing Ham's F-12 nutrient mixture according to the present invention is based on DMEM without a pH indicator such as phenol red and Ham's F-12 nutrient mixture is added at a ratio of about 1: 0.5-2. . At this time, it is possible to add an oxidizing source such as L-glutamine, an energy metabolite such as sodium pyruvate, and a carbon regulator such as sodium bicarbonate. This mixed solution is a growth factor secreted from a variety of minerals and amino acids and fetal-derived mesenchymal stem cells that help cell growth and homeostasis and enhance cell stability and maintenance in subcultures after initial culture of mesenchymal stem cells. It is formed by mixing a certain proportion of vitamin-based nutrients and other factors that can promote higher production of nutrients.

Conditioned medium of the present invention is preferably incubated for 24 to 72 hours in a hypoxic state containing 1% to 5% oxygen of step (c), and incubated for 3 days in a serum-free medium of step (d) It is preferable to prepare, more preferably incubated for 3 days in a low oxygen state containing 1% oxygen, it may be prepared by culturing for 3 days in a serum-free medium.

According to one embodiment of the present invention, the embryo-derived cells isolated from the amniotic fluid obtained from the mother are passaged in medium containing FBS and bFGF to obtain the fetal-derived mesenchymal stem cells in the amniotic fluid, followed by 3 days at 1% hypoxic state. After incubation, the conditioned medium was prepared by centrifugation and filtration of the culture solution obtained by culturing for 3 days with different numbers of amniotic cells in DMEM / F-12 serum-free medium.

In another aspect, the present invention provides a conditioned medium composition obtained by culturing in fetal mesenchymal stem cells in amniotic fluid in a serum-free medium after culturing in hypoxic conditions.

The conditioned medium composition of the present invention comprises the steps of (a) isolating fetal derived cells in amniotic fluid obtained from the mother; (b) subcultured in medium containing FBS and bFGF to obtain fetal mesenchymal stem cells derived from amniotic fluid; And (c) culturing the obtained mesenchymal stem cells in a hypoxic state.

As used herein, the term "conditioned medium" refers to a cell growth suspension in which the cells are subjected to liquid suspension culture, and then, when the apoptotic growth phase is reached, the dividing cells are removed by centrifugation or filtration, and only the culture medium is collected and mixed with the culture substrate. Say one badge. This uses an unknown growth factor that is extracted in the medium from the dividing cells, and is widely used for low density cell plating or protoplast culture.

For the purposes of the present invention, the conditioned medium composition of the present invention is a composition comprising a solution in which fetal-derived mesenchymal stem cells are removed from a medium in which fetal-derived mesenchymal stem cells are cultured. Refers to a composition containing abundant growth factors, such as Apo-1 / Fas, epidermal growth factor (EGF), IP-10 (Interferon-γ inducible protein-10), leptin, MIP4, MMP3, Rantes, Interferon-gamma (IFNγ), human transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNFα), tumor necrosis factor receptor I (TNFRI), tumor necrosis Factor Receptor II (TNFRII), Cell Attachment Factor-1 (ICAM-1), Vascular Attachment Factor-1 (VCAM-1), Vascular Endothelial Growth Factor (VEGF), Interleukin-1 Beta (IL-1β), Interleukin -1 receptor alpha (IL-1Rα), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 and IL-15 Include. Most preferably VEGF.

Thus, as another embodiment, the present invention relates to a composition containing a large amount of growth factors, preferably, ApoI / Fas, EGF, IP-10, Leptin, MIP4, MMP3, Rantes, IFNγ, TGFβ, TNFα, TNFRI, TNFRII, ICAM-1, VCAM-1, VEGF, IL-1β, IL-1Rα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL- 7, IL-8, IL-12 and IL-15.

According to a preferred embodiment of the present invention, in order to confirm the effect of the conditioned medium of the present invention, as a result of culturing the skin-derived fibroblasts in the conditioned medium prepared by the above method, S during the growth cycle of the cells Expression of genes associated with the phase was increased (FIG. 6), and also an increase in VEGF was observed (FIG. 8). Therefore, the composition of the present invention was found to be useful as a medium for cell growth.

In another aspect, the present invention provides a method for treating a fetus, comprising: (a) isolating fetal derived cells in amniotic fluid obtained from a mother; (b) subcultured in medium containing FBS and bFGF to obtain fetal mesenchymal stem cells derived from amniotic fluid; (c) culturing the obtained mesenchymal stem cells in a hypoxic state; And (d) conditioned medium produced by culturing the obtained amniotic fluid-derived mesenchymal stem cells in a serum-free medium for 3 days as an active ingredient.

Hypoxic conditions of step (c) and serum-free medium incubation period of step (d) are as described above.

The compositions of the present invention are effective for skin regeneration, in particular for improving wrinkles, healing skin damage caused by ultraviolet light, restoring skin elasticity, shrinking pores, treating skin scars or wounds.

According to one specific embodiment of the present invention, after artificially wounding the skin-derived fibroblasts and treating the composition of the present invention, the degree of cell migration is higher, and the wound healing gene is Since it was expressed quantitatively high, it was confirmed that the wound healing effect was excellent (FIG. 8).

In addition, after artificial wounds were applied to the mouse skin, the composition of the present invention was applied, and thus the wound area was gradually reduced, and thus the wound closure and skin regeneration effects were excellent (FIG. 9).

The composition for skin regeneration of the present invention may be a cosmetic composition.

The components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to the culture medium of amniotic fluid-derived mesenchymal stem cells as active ingredients, for example, antioxidants, stabilizers, solubilizers, vitamins, Conventional adjuvants such as pigments and perfumes, and carriers. In addition, the cosmetic composition may further include a skin absorption promoting material to enhance the effect.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder. When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether. When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan. In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

In addition, the compositions of the present invention may be prepared as pharmaceutical compositions.

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. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as oral or parenteral, for example, oral, rectal or intravenous, muscle, subcutaneous, intrauterine dural or It can be administered by cerebrovascular injection. It is preferably applied in the form of topical application during parenteral administration, more preferably by application.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Can be.

The dosage of the pharmaceutical composition of the present invention may be administered once or several times a day in an oral dosage form of 0.1 to 100 mg / kg on an adult basis. It is recommended to apply 1 to 5 times a day in an amount of 3.0 ml to continue for 1 month or more. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation can be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may further comprise a dispersant or stabilizer.

The present invention enables culturing of fetal derived cells in amniotic fluid to obtain multipotent mesenchymal stem cells, thereby enabling another source other than bone marrow-derived mesenchymal stem cells. And a method for producing a large amount of growth factors in the conditioned medium prepared by culturing the amniotic fluid-derived mesenchymal stem cells in hypoxic condition containing 1% oxygen for 3 days and further cultured in serum-free medium for 3 days Can provide. That is, the present invention suggests the possibility of multipotent fetal-derived cells as multipotent mesenchymal stem cells, and supplies 1% hypoxia to amniotic fetal-derived mesenchymal stem cells for 3 days to obtain a conditioned medium using the same. This enables the development of a composition having cell growth and regeneration ability.

Figure 1A is a result showing that the form of the amniotic fetal-derived cell line is heterogeneous (heterogenous), Figure 1B is when the passage of the amniotic fetal-derived cell line 2-3 times (passage 4), the cell type is homogeneous (homogenous) This is the result showing the change in mesenchymal stem cell form.
2 shows karyotype analysis of fetal stem cells derived from amniotic fluid isolated from mothers. The sex chromosome came out as XY, indicating that it came from the amniotic fetus, not from the mother.
Figure 3 shows the immunological phenotype of the cells through flow cytometry that the fetal stem cells in the amniotic fluid have similar characteristics to the human bone marrow-derived mesenchymal stem cells as a control.
4A shows that the amniotic fluid-derived cells (b) have differentiation capacity similar to that of human bone marrow-derived mesenchymal stem cells (a). Induction of differentiation into adipocytes, which is similar to the control group, was confirmed by immunostaining and RT-PCR analysis. FIG. 4B shows that the amniotic fluid-derived fetal cells (b) are bone marrow-derived mesenchymal stem cells (a Induced differentiation into bone cells that had differentiation ability similar to), and confirmed through tissue staining and RT-PCR analysis.
Figure 5A is a result of comparing the cell shape of the amniotic fetal-derived mesenchymal stem cells in the amniotic fluid fetal derived mesenchymal stem cells when the hypoxic conditions (1%, 5% oxygen concentration) is incubated, Figure 5B Is a result of charting the growth level of amniotic fluid-derived mesenchymal stem cells, Figure 5C is a result of comparing the expression level of HIF1-a. The results support that 1% hypoxia is better than 5% hypoxia.
Figure 6A is a result showing the cell shape of the fetal-derived mesenchymal stem cells in the amniotic fluid at different feed times of the established hypoxic concentration, Figure 6B is a result of the growth level of fetal-derived mesenchymal stem cells in the amniotic fluid , FIG. 6C is a result of quantifying the growth of fetal-derived mesenchymal stem cells in amniotic fluid through PI staining. As a result, it was confirmed that the growth of cells in the hypoxic state for 3 days is better.
Figure 7A is a result of comparing the growth level of the normal oxygen conditioned medium and the conditioned medium produced from the established hypoxic conditions to other fibroblasts, Figure 7B is a result of comparing the BrdU assay, 7C is a chart of the BrdU assay. As a result, it was confirmed that cell growth increased under established hypoxic conditions.
FIG. 8 shows the results of comparing VEGF present in DMEM / F12 medium, normal oxygen conditioned medium, and conditioned medium produced from established hypoxic conditions by VEGF ELISA analysis. As a result, it was confirmed that the production of VEGF increased under hypoxic conditions.
FIG. 9A shows wounds with an interstitial gap decrease over time when artificial wounds were applied to fibroblasts and treated with DMEM / F12 medium, normal oxygen conditioned medium, and conditioned medium produced from established hypoxic conditions. It is a result showing the therapeutic effect, FIG. 9B is a result of graphing it, and FIG. 9C is a result of comparing the expression levels of p-SMAD2, p-SMAD3 and SMAD2 / 3 by Western blot analysis, FIG. 9D Is a result of comparing the relative expression levels of genes related to wound regeneration through real-time PCR (real-time PCR) analysis.
FIG. 10A shows wound healing recovery when artificially inducing wounds on mouse skin and treating wound sites with DMEM / F12 medium, normal oxygen conditioned medium and conditioned medium produced from established hypoxic conditions. ) Is an in vivo result showing the degree, Figure 10B is a result of the degree of wound healing recovery.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: Homologous Culture Using Amniotic and Fetal Cell Lines Intermediate lobe  Stem cell shape confirmation

There are a lot of suspended solids in the amniotic fluid from the mother. Amniotic fluid was placed in T-flask and cultured in a 37 ° C incubator to separate amniotic fetal derived cells from the suspension. The next day, all but the cells on the bottom were removed. Cells attached to the bottom were separated from the bottom using trypsin and centrifuged to obtain the cells, and low glucose DMEM containing 10% FBS, 1% L-glutamine, 1% penicillin-streptomycin and 4 ng / ml bFGF Cells were seeded in 100 mm cell culture vessels by resuspension in basal medium. 12 to 24 hours after seeding was replaced with fresh medium.

As a result, it was confirmed that the amniotic fetal-derived cell line had a heterogeneous shape (FIG. 1A), and exhibited one fibroblast-like homogenous population after two or three passages. (FIG. 1B). In the cell form as shown in FIG. 1, the medium was changed every 2 to 3 days, and passaged when the cell density was about 80 to 90%.

Example  2: Identification of Amniotic Fetal Cells by Karyotyping

Karyotyping was performed to confirm that the cells of Example 1 obtained from amniotic fluid were cells derived from fetuses.

The number, size, and shape of chromosomes are called karyotypes, and chromosome mutations and sex can be identified by examining the chromosomes of the fetus. In order to perform karyotyping, the cells obtained from amniotic fluid were treated with Colcemid for 1-2 hours to stop cell division in the metaphase, and G-banding staining was performed. The chromosome was analyzed.

As a result, the chromosome was normal, the sex chromosome was shown as XY, and from this result, it was confirmed that the cells obtained from amniotic fluid were cells derived from the fetus rather than the mother (FIG. 2).

Example  3: Amniotic Cells of Fetal Origin Intermediate lobe  Stem Cell Characterization

It was confirmed that the fetal-derived cells in the amniotic fluid of Example 1 exhibited multipotent mesenchymal stem cell characteristics.

Comparison of immunological phenotypes of fetal-derived cells and human bone marrow-derived mesenchymal stem cells using a flow cytometer showed that CD13, CD29, CD44, and CD90, markers specific for mesenchymal stem cells, were found It was confirmed that this is expressed (Fig. 3). However, because different characteristics may exist for each cell, the differentiation ability to osteoblasts and adipocytes, which are representative characteristics of mesenchymal stem cells, was confirmed to more reliably verify.

After differentiating fetal stem cells from the amniotic fluid in the same manner as in the differentiation conditions of human bone marrow-derived mesenchymal stem cells, osteopontin, osteocalcin, lipoprotein lipase (LPL), The degree of expression of Patty acid binding protein 2 (aP2) and Peroxysome proliferator-activated receptor gamma (PPARγ) was confirmed.

As a result, in the case of bone cell differentiation, the expression of osteopontin and osteocalcin, which are genes specifically expressed as bone was formed, was observed, and in the case of adipocyte differentiation, lipo, a gene related to adipose formation, was observed. Expression of protein lipase (LPL), faty acid binding protein 2 (aP2) and peroxysome proliferator-activated receptor gamma (PPARγ) was observed. (Right panel of Figures 4A and B).

Alizarin red S staining was performed to identify calcium salts generated by differentiation into osteoblasts, and oil red O staining (Liil droplets) to identify lipid droplets generated during differentiation into adipocytes. red O staining) (left panel of FIGS. 4A and B). From the above results, it was confirmed that the fetal-derived cells in the amniotic fluid are cells having characteristics similar to those of the bone marrow-derived mesenchymal stem cells.

Example  4: Hypoxia  Amniotic fetal origin by conditions Intermediate lobe  Effects of Stem Cells on Cell Growth

Cell growth was observed when 1% and 5% hypoxic conditions were supplied to fetal mesenchymal stem cells in amniotic fluid established in Example 1.

First, it was isolated from the bottom by trypsin treatment to the amniotic stem-derived mesenchymal stem cells growing attached to the 100 mm cell culture vessel. Separated cells were obtained and placed in 15 ml tubes and centrifuged. The obtained cells were released in 5-10 ml medium and mixed well. Measure the cell number using a hematocytometer by picking 20 μl of cell suspension in the tube, and seed 2.5 × 10 ⁵ cells in a 100 mm cell culture vessel to prepare 10% FBS, 1% L- in low glucose DMEM. Cultures were carried out in basal medium containing glutamine, 1% penicillin-streptomycin and 4 ng / ml bFGF.

Selected cells were resuspended in ice-cold cell lysis buffer, RIPA buffer (containing proteinase inhibitor cocktail and phosphatase inhibitor), and then incubated on ice for 30 minutes. vortexing). After centrifugation at 14000 rpm for 30 minutes, the supernatant was obtained, and the protein concentration was measured using Bradford assay reagents (Bio-rad), and then 50% to 100 μg of 10% or 4 to 12% was prepared in advance. Running on a precasted SDS-PAGE NuPAGE gel (Invitrogen). This was transferred to a PVDF membrane (Millipore), and then blocked (tris buffered saline with 0.1% Tween 20) containing 3 to 5% skim milk (Skimmed milk). Shaking overnight at 4 ° C. using anti-HIF1-a (BD Bioscience), α-tubulin (Sigma), followed by Hoseradish peroxidase-conjugated anti-secondary IgG for 1 hour at room temperature. horseradish peroxidase-conjugated anti-secondary IgG (Invitrogen) and detected by Super Signal West Pico Chemiluminescent Substrate kit (Pierce).

As a result, growth changes of fetal-derived mesenchymal stem cells in amniotic fluid incubated for 12 hours at 1% and 5% hypoxic conditions were observed (FIG. 5A), and the cell growth was better at 1% hypoxic conditions (FIG. 5A). 5B), it was confirmed that HIF1-a is activated at 1% hypoxic conditions (FIG. 5C).

The results show that 1% hypoxic conditions can stimulate the growth of fetal-derived mesenchymal stem cells in the amniotic fluid and can express cytokines such as VEGF, a growth factor associated with angiogenesis by activating HIF1-a.

Example  5: Hypoxia  Amniotic fetal origin when treated hourly Intermediate lobe  Effects of Stem Cells on Cell Growth

When the 1% hypoxic supply to the fetal-derived mesenchymal stem cells in the amniotic fluid was confirmed that the cell growth is promoted through the results of Example 4 above. Next, cell shape and growth changes were observed when 1% hypoxia was supplied to amniotic fluid-derived mesenchymal stem cells over time (FIGS. 6A to B).

As a result, growth changes of fetal-derived mesenchymal stem cells in amniotic fluid cultured for 3 days at 1% hypoxic condition were observed (FIG. 6A), and it was found that 3 days culture at 1% hypoxia was better for cell growth. (FIG. 6B) and PI staining confirmed that cell growth was promoted (FIG. 6C). From the above results, it was confirmed that culturing for 3 days at 1% hypoxia is the most suitable condition for promoting the growth of fetal mesenchymal stem cells in amniotic fluid. These results support the mass proliferation of fetal-derived mesenchymal stem cells in amniotic fluid under hypoxia.

Example  6: in vitro  On Conditioned  Check the effect of the badge

After supplying 1% hypoxic cells to fetal mesenchymal stem cells in amniotic fluid for 3 days and replacing with DMEM / F12 serum-free medium for 3 more days to produce a conditioned medium, the medium was further fibroblasts. An experiment was conducted to determine the effect on the growth of.

After 3 days of 1% hypoxic condition, the produced conditioned medium was treated with other fibroblasts to compare the cell morphology and the degree of growth.The cells were treated with 1% hypoxic conditioned medium compared to the control medium. It was confirmed that the growth was higher (Fig. 7A), and the BrdU assay, which acts only in the DNA replication process of the S phase of the cell growth cycle, showed that the cells in the conditioned medium produced after supplying 1% hypoxic acid for 3 days It was confirmed that the growth cycle is high (Fig. 7C). From the above results, it was possible to establish that the optimum culture condition capable of producing a conditioned medium effective for cell growth was produced after supplying 1% hypoxic acid for 3 days.

Example  7: Amniotic fetal origin Intermediate lobe  1% on stem cells Hypoxia  Generated after a three-day supply Conditioned  Present in the badge Increased VEGF  Confirm

After supplying 1% hypoxic cells to fetal mesenchymal stem cells in amniotic fluid for 3 days, VEGF ELISA was performed to confirm whether VEGF, a target substance of hypoxia, in the conditioned medium produced was increased than that of the control group.

As a result, it was confirmed that a large amount of VEGF was present in the conditioned medium produced after supplying 1% hypoxic acid for 3 days compared to the negative control medium DMEM / F12 (FIG. 8). These results support that the conditioned medium of the present invention can produce human growth factors in large quantities.

Example  8: for skin-derived fibroblasts Conditioned  Wound Healing Effect of Medium

Artificial wounds on skin-derived fibroblasts to determine whether the conditioned medium produced after supplying 1% hypoxia for 3 days to amniotic stem-derived mesenchymal stem cells is effective for wound healing. After addition, the conditioned medium prepared in Example 6 was treated. After 12 hours, it was confirmed that the degree of cell migration was higher in the skin-derived fibroblasts treated with the conditioned medium, in particular, when the conditioned medium produced after supplying 1% low oxygen for 3 days was treated. It was found that this tendency appeared more prominently (FIG. 9A). As a result of comparing the migration rate at this time, it was confirmed that the distance between cells was the smallest in the group treated with the conditioned medium produced after supplying 1% hypoxic for 3 days. It was confirmed that the conditioned medium produced after supplying 1% hypoxic cells to the derived mesenchymal stem cells for 3 days was effective for wound healing (FIG. 9B). In addition, the expression levels of genes related to wound healing (Collagen3, Vitronectin, Syndecan2, Syndecan4, Fibronectin, SPP1, MMP3, and Elastin) were confirmed using real-time PCR. Real-time PCR was carried out through an elongation reaction at 72 ° C for 30 seconds after annealing the DNA at 62 ° C for 30 seconds after DNA denaturation at 94 ° C for 30 seconds. Quantitative analysis was also quantified by GAPDH and amplified using SYBR Green Super Mix.

As a result, it was confirmed that the expression was significantly higher in the medium produced after supplying 1% hypoxic for 3 days compared to the medium produced in the control group, and from the results 1% hypoxic to the fetal-derived mesenchymal stem cells in the amniotic fluid It was confirmed that a large amount of genes related to skin regeneration exist in the conditioned medium produced after feeding for 3 days (FIG. 9D).

Example  9: mouse in vivo  On Conditioned  Skin regeneration effect of badge

We examined the effect of conditioned medium produced after supplying 1% hypoxic cells to fetal-derived mesenchymal stem cells for 3 days on amniotic fluid.

29 to 30 mm ² on mouse skin After punching in size to artificially make the wound site, supply the medium produced in the control group and 1% hypoxic acid for 3 days, and then apply the produced medium to the wound site to seal the wound ( Wound closure was measured to confirm the skin regeneration effect.

As a result, it was confirmed that the wound area was reduced when the conditioned medium produced after supplying 1% hypoxic acid for 3 days compared to the medium produced in the control group (FIG. 10A), and numerically indexed the wound closure measurement value. (FIG. 10B). From the above results, it was confirmed that the conditioned medium produced after supplying 1% hypoxia for 3 days to amniotic embryo-derived mesenchymal stem cells had a skin regeneration effect.

Claims (13)

(a) isolating fetal derived cells in amniotic fluid obtained from the mother;
(b) subcultured fetal-derived cells isolated in step (a) in a medium containing fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF) to obtain fetal-derived mesenchymal stem cells in amniotic fluid; And
(c) culturing the obtained mesenchymal stem cells in a hypoxic state, mass propagation method of fetal mesenchymal stem cells derived from amniotic fluid.
The method of claim 1, wherein the low oxygen state of step (c) comprises 1% to 5% oxygen.
The method of claim 1, wherein the hypoxic state of step (c) is 24 hours to 72 hours.
(a) isolating fetal derived cells in amniotic fluid obtained from the mother;
(b) subcultured in medium containing FBS and bFGF to obtain fetal mesenchymal stem cells derived from amniotic fluid;
(c) culturing the obtained mesenchymal stem cells in a hypoxic state; And
(d) culturing the obtained intrauterine fetal derived mesenchymal stem cells in a serum-free medium for 3 days to produce a conditioned medium, and a large amount of human growth factors are produced from the fetal derived mesenchymal stem cells in the amniotic fluid. How to.
The method of claim 4, wherein the hypoxic state of step (c) is incubated for 24 to 72 hours in a low oxygen state containing 1% to 5% oxygen.
The method of claim 4, wherein the growth factor is vascular endothelial growth factor (VEGF).
Conditioned medium composition of amniotic fluid-derived mesenchymal stem cells obtained by the method of claim 4.
(a) isolating fetal derived cells in amniotic fluid obtained from the mother;
(b) subcultured in medium containing FBS and bFGF to obtain fetal mesenchymal stem cells derived from amniotic fluid;
(c) culturing the obtained mesenchymal stem cells in a hypoxic state; And
(D) a composition for skin regeneration comprising a conditioned medium produced by culturing the obtained amniotic fluid-derived mesenchymal stem cells in a serum-free medium for 3 days.
The composition of claim 8, wherein the hypoxic state of step (c) is incubated for 24 hours to 72 hours in a low oxygen state containing 1% to 5% oxygen.
The composition of claim 8, wherein the skin regeneration is wrinkle improvement, healing skin damage by ultraviolet light, restoring skin elasticity, shrinking pores, treating skin scars or wounds.
The composition of claim 8, wherein the composition is a cosmetic composition.
The group of claim 11 wherein the composition consists of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray A composition characterized by having a formulation selected from.
According to claim 8, wherein the composition is a pharmaceutical composition for the treatment of skin damage, wrinkles or skin scars caused by UV containing a culture medium of the amniotic fluid-derived fetal mesenchymal stem cells prepared as an active ingredient.

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