Mesenchymal stem cells with enhanced bone differentiation potential and their uses

Abstract

The mesenchymal stem cells with enhanced bone differentiation function provided in one embodiment have excellent bone differentiation ability, adipocyte differentiation ability, chondrocyte differentiation ability, and neural cell differentiation ability, and in particular, the bone differentiation ability is significantly superior to mesenchymal stem cells of other origins. In addition, the mesenchymal stem cells with enhanced bone differentiation function have less immune rejection and can be effective in preventing or treating bone diseases and estrogen deficiency syndrome. Furthermore, the mesenchymal stem cells with enhanced bone differentiation function have the advantages of maintaining proliferation ability and karyotype stability despite continuous subculture, being safe for commercialization, and being mass-produced.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2021-0059516, filed on May 7, 2021, the entirety of which is incorporated herein by reference.

[0002] The present invention relates to mesenchymal stem cells with enhanced bone differentiation function, and uses thereof. [Background technology]

[0003] Fetal-derived mesenchymal stem cells have many advantages for commercial development based on their own cellular characteristics, but there is a diversity of cells that can be isolated depending on the rapidly changing fetal developmental stages and anatomical and histological location. In addition, there are some ethical issues with obtaining stem cells from fetuses. Even in research using ectopic pregnancies or stillborn fetuses, it is difficult to secure ectopic pregnancies or stillborn fetuses and obtain tissue donations, so research and development of fetuses themselves is currently minimal. Summary of the Invention

[0004] Compared to adult stem cells, fetal-derived mesenchymal stem cells (1) express the omnipotent stem cell marker SSEA4, (2) have superior differentiation potential, and (3) maintain telomeres of 20 kb or more, allowing them to maintain karyotype stability and proliferation rate.

[0005] In addition, fetal-derived stem cells secrete immunomodulatory cytokines such as TGF-β, have low expression of HLA type I and costimulatory molecules, (4) and are known to be less immunogenic than mesenchymal stem cells from other sources.

[0006] As a result, we have developed stem cells with enhanced bone differentiation function using mesenchymal stem cells derived from donated fetal tissue from ectopic pregnancy or stillborn fetuses, and compositions using these stem cells for the prevention or treatment of bone diseases and / or estrogen deficiency syndromes that mainly appear after menopause.

[0007] One aspect provides mesenchymal stem cells with enhanced bone differentiation ability, which express at least one gene selected from the group consisting of Osx (SP7), c-kit, CD24, CD70, and SFRP2.

[0008] Another aspect provides a pharmaceutical composition for preventing or treating bone diseases, comprising the mesenchymal stem cells with enhanced bone differentiation potential.

[0009] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating estrogen deficiency syndrome, comprising the mesenchymal stem cells with enhanced osteogenic differentiation potential.

[0010] In still another aspect, there is provided a health functional food for preventing or ameliorating bone diseases, which contains the mesenchymal stem cells with enhanced bone differentiation potential.

[0011] In still another embodiment, there is provided a health functional food for preventing or improving estrogen deficiency syndrome, which contains the mesenchymal stem cells with enhanced bone differentiation potential.

[0012] In yet another aspect, there is provided a method for mass production of mesenchymal stem cells with enhanced bone differentiation potential, comprising the step of continuously subculturing the mesenchymal stem cells with enhanced bone differentiation potential.

[0013] In yet another aspect, there is provided a method for preventing or treating a bone disease, comprising the step of administering the mesenchymal stem cells with enhanced bone differentiation potential to an individual in need thereof.

[0014] In yet another aspect, there is provided a method for preventing or treating estrogen deficiency syndrome, comprising administering the mesenchymal stem cells with enhanced osteogenic differentiation potential to an individual in need thereof. [Means for solving the problem]

[0015] One aspect provides mesenchymal stem cells with enhanced osteogenic differentiation potential, in which at least one gene selected from the group consisting of Osx(SP7), c-kit, CD24, CD70, and SFRP2 is expressed.

[0016] As used herein, the term "mesenchymal stem cells (MSCs)" refers to cells that maintain self-renewal and stemness maintenance and can differentiate into various mesenchymal tissues, and includes mesenchymal stem cells of animals, including mammals, such as humans.

[0017] In addition, the term "mesenchymal stem cells with enhanced bone differentiation ability" refers to mesenchymal stem cells with enhanced ability to differentiate into bone cells, and may be used interchangeably with bone progenitor cells or mesenchymal progenitor cells.

[0018] In one aspect, the mesenchymal stem cells with enhanced bone differentiation potential may be derived from fetal tissue from an ectopic pregnancy or stillborn fetus, preferably fetal tissue from the fetal skull or calvaria tissue, which is the top of the head, more preferably fetal tissue from the fetal skull excluding the fetal neural tube, or calvaria tissue, and even more preferably calvaria tissue.

[0019] The ectopic fetus may have died as a result of the ectopic pregnancy, or may have been diagnosed with an ectopic pregnancy and subsequently died after being surgically separated.

[0020] The mesenchymal stem cells with enhanced bone differentiation function express at least one gene selected from the group consisting of Osx(SP7), c-kit, CD24, CD70, and SFRP2, and specifically, can express all of Osx(SP7), c-kit, CD24, CD70, and SFRP2.

[0021] More specifically, the mesenchymal stem cells with enhanced bone differentiation potential may express at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% of the following markers: Osx (SP7), a marker specifically expressed only in bone-forming mesenchymal stem cells; c-kit, a fetal mesenchymal stem cell marker; CD24 and CD70, cell surface markers; and SFRP2, a bone differentiation marker.

[0022] As used herein, the term "positive" in relation to a stem cell marker can refer to a marker being present in greater amounts or at higher concentrations than other reference stem cells (e.g., umbilical cord Wharton's jelly-derived mesenchymal stem cells (CordSTEM) and adult bone marrow-derived mesenchymal stem cells (BM-MSC)). That is, a cell is positive for a marker if the marker is present inside or on the cell, allowing the cell to be distinguished from one or more other cell types. It can also refer to a cell possessing the marker in an amount sufficient to produce a signal, e.g., a signal from a cell measuring instrument, greater than background. For example, if a cell is detectably labeled with an antibody specific for CD90 and the signal from the antibody is detectably greater than a control (e.g., background), the cell is "CD90+."

[0023] As used herein, the term "negative" means that a particular cell surface marker cannot be detected relative to background levels using an antibody specific for that marker. For example, if a cell cannot be detectably labeled with an antibody specific for CD34, the cell is "CD34-."

[0024] In one aspect, the mesenchymal stem cells with enhanced bone differentiation function may overexpress at least one gene selected from the group consisting of KITLG, CXCL12, HES4, TWIST1, BMP2, BMPR1B, NOG, and ALPL compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow; specifically, KITLG, CXCL12, HES4, TWIST1, BMP2, BMPR1B, NOG, and ALPL may all be overexpressed compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow.

[0025] More specifically, the mesenchymal stem cells with enhanced bone differentiation function may overexpress fetal mesenchymal stem cell markers KITLG and CXCL12, and bone differentiation markers HES4, TWIST1, BMP2, BMPR1B, NOG, and ALPL, compared to mesenchymal stem cells derived from umbilical Wharton's jelly or mesenchymal stem cells derived from adult bone marrow.

[0026] Alternatively, the mesenchymal stem cells may be used in the form of a culture, lysate, or extract thereof. The culture, lysate, or extract is a useful alternative when it is difficult to use the cells as is, and since it contains cellular components including proteins, it can exhibit biological activity similar to or equivalent to that of the original cells. The lysate or extract can be obtained using a commercially available cell lysis kit or cell extraction kit.

[0027] The mesenchymal stem cells with enhanced osteogenic differentiation ability maintain their proliferation potential and karyotypic stability even after repeated subculture, compared to other mesenchymal stem cells (e.g., umbilical cord Wharton's jelly-derived mesenchymal stem cells (CordSTEM), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and embryonic stem cell-derived mesenchymal stem cells (ES-MSC)), making them safe for commercialization and easy to mass-produce. Specifically, the mesenchymal stem cells can maintain their proliferation potential even after being cultured for at least 20 passages and can maintain karyotypic stability even after being cultured for at least 24 passages.

[0028] Furthermore, the mesenchymal stem cells with enhanced bone differentiation function in a single phase also have excellent chondrocyte differentiation ability, adipocyte differentiation ability, and neuronal differentiation ability, and in particular, their bone differentiation ability is significantly superior to other mesenchymal stem cells. Specifically, their bone differentiation ability is significantly superior to that of umbilical cord Wharton's jelly-derived mesenchymal stem cells (CordSTEM), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and embryonic stem cell-derived mesenchymal stem cells (ES-MSC).

[0029] In one embodiment, the mesenchymal stem cells with enhanced bone differentiation potential may not express HLA class I.

[0030] Specifically, when the mesenchymal stem cells with enhanced osteogenic differentiation ability are cultured in a medium containing bFGF, the percentage of cells expressing HLA class I can be reduced to 5% or less. Therefore, the mesenchymal stem cells produced by this method have fewer cells killed by immune rejection, resulting in a high cell survival rate during allogeneic cell therapy.

[0031] In one embodiment, the mesenchymal stem cells with enhanced osteogenic differentiation ability are those which, compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow, over-secrete at least one polypeptide selected from the group consisting of ABL1, IGFBP-7, MMP-1, GLO-1, progranulin, amylin, BMX, ALPP, FAP, ADAMTS-L2, SPARC, TLR1, galectin-3, beta IG-H3, HB-EGF, 11b-HSD1, PTHLP, ACTH, activin A, GDF11, and GDF3. Specifically, the mesenchymal stem cells with enhanced osteogenic differentiation ability are those which over-secrete at least one polypeptide selected from the group consisting of ABL1, IGFBP-7, MMP-1, GLO-1, progranulin, amylin, BMX, ALPP, FAP, ADAMTS-L2, SPARC, TLR1, galectin-3, beta IG-H3, HB-EGF, 11b-HSD1, PTHLP, ACTH, activin A, GDF11, and GDF3. IG-H3, HB-EGF, 11b-HSD1, PTHLP, ACTH, activin A, GDF11, and GDF3 may all be hypersecreted compared to mesenchymal stem cells derived from umbilical Wharton's jelly or mesenchymal stem cells derived from adult bone marrow.

[0032] Furthermore, the hypersecretion may specifically be hypersecretion into the extracellular culture medium.

[0033] The term "polypeptide" refers to a polymer consisting of two or more amino acids linked by amide bonds (or peptide bonds). The term may include polypeptides having sequence homology of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% with the amino acid sequence of the polypeptide.

[0034] The term "homology" indicates the degree of similarity to a wild-type amino acid sequence. Such homology comparison can be performed using a comparison program well known in the art, and the homology between two or more sequences can be calculated as a percentage (%). In one embodiment, the stem cells may heterogeneously express CD90.

[0035] Specifically, the mesenchymal stem cells with enhanced osteogenic differentiation potential are derived from fetal bone tissue, which may be the skull or calvaria, and may contain a cell population with high CD90 expression and a cell population with low CD90 expression, which can be further characterized by the addition of bFGF to the culture medium.

[0036] Another aspect provides a pharmaceutical composition for preventing or treating a bone disease, comprising the mesenchymal stem cells with enhanced bone differentiation potential.

[0037] The "mesenchymal stem cells with enhanced bone differentiation potential" may be within the above-mentioned range.

[0038] The bone disease may be, for example, at least one bone disease selected from the group consisting of osteoporosis, bone fracture, nonunion, osteogenesis imperfecta, osteopenia, osteolytic metastasis, senile kyphosis, Paget's disease, bone atrophy, osteomalacia, osteoarthritis, periodontal disease, rickets, aplastic bone disease, bone damage caused by bone metastasis of cancer cells, fibrous dysplasia, McCune-Albright syndrome, bone malformation, and osteodysplasia, preferably at least one bone disease selected from the group consisting of osteoporosis, bone fracture, fibrous dysplasia, McCune-Albright syndrome, nonunion, and osteogenesis imperfecta, and more preferably osteoporosis.

[0039] The term "prevention" may refer to any action that inhibits or delays the onset of bone disease in an individual by administering a pharmaceutical composition by one modality.

[0040] The term "treatment" may refer to any action in which the symptoms of an individual's bone disease are improved or favorably altered by administering a pharmaceutical composition by one modality.

[0041] In one aspect, the mesenchymal stem cells with enhanced osteogenic differentiation have superior proliferation ability, and as a result of proliferation, they have excellent karyotype stability, excellent bone differentiation ability, and remarkable osteogenic ability, compared to other mesenchymal stem cells. Based on the aforementioned remarkable osteogenic ability, they can increase bone mineral density, bone cell number, and bone volume, and exhibit remarkable therapeutic effects for bone diseases.

[0042] In one embodiment, the pharmaceutical composition may further comprise a pharmaceutical composition for preventing or treating other bone diseases in addition to the mesenchymal stem cells with enhanced bone differentiation potential.

[0043] That is, the pharmaceutical composition may be provided in combination with other known compositions for preventing or treating bone diseases or other newly developed compositions for preventing or treating bone diseases.

[0044] When the pharmaceutical composition contains a composition for preventing or treating another bone disease, it is important that the amount of the compound to be mixed is such that the maximum effect can be obtained with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0045] When the pharmaceutical composition further comprises a composition for preventing or treating another bone disease, a synergistic effect may be exhibited, which results in a more pronounced effect in preventing or treating bone diseases than when the pharmaceutical composition contains only the mesenchymal stem cells with enhanced bone differentiation potential function as an active ingredient.

[0046] In one embodiment, the pharmaceutical composition may be administered alone or in combination with other compositions for the prevention or treatment of bone diseases.

[0047] The aforementioned composition for preventing or treating other bone diseases may be a conventionally known composition for preventing or treating other bone diseases, or a newly developed composition for preventing or treating other bone diseases.

[0048] The pharmaceutical composition may be administered in combination with other compositions for the prevention or treatment of bone diseases, and may be administered simultaneously, separately, or sequentially, and may be administered as a single dose or multiple doses. Taking into consideration all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0049] When the pharmaceutical composition is administered in combination with other compositions for preventing or treating bone diseases, a synergistic effect may be exhibited, in which the effect of preventing or treating bone diseases is more pronounced than when the pharmaceutical composition is administered alone.

[0050] The term "administration" means introducing a predetermined substance into an individual by an appropriate method, and "individual" means any living organism, such as a rat, mouse, or livestock, including humans, that may have a bone disease. Specific examples include mammals, including humans.

[0051] In one embodiment, the route of administration of the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intra-arterial, intraperiosteal or intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0052] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the age, sex, condition, and weight of the patient, the degree of absorption of the active ingredient into the body, the inactivation rate, and excretion rate, the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time, route, and excretion rate of administration, the treatment period, the severity of obesity, concurrently used drugs, and other factors well known in the medical field. For example, the pharmaceutical composition is administered at a dose of 0.001 mg / kg / day to 1,000 mg / kg / day, and the effective dose is 10 mg / kg / day based on the mesenchymal stem cells with enhanced bone differentiation potential contained in the pharmaceutical composition and the individual to be administered. 2 cells / kg to 10 9 cells / kg, 10 3cells / kg to 10 9 cells / kg, 10 3 cells / kg to 10 8 cells / kg, 10 4 cells / kg to 10 8 cells / kg, or 10 4 cells / kg to 10 7 It may be administered at the level of cells / kg.

[0053] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating estrogen deficiency syndrome, comprising the mesenchymal stem cells with enhanced osteogenic differentiation potential.

[0054] The "mesenchymal stem cells with enhanced bone differentiation potential" may be within the above-mentioned range.

[0055] The estrogen deficiency syndromes include, for example, facial flushing, sweating, insomnia, irritability, depression, dizziness, impaired concentration, impaired short-term memory, anxiety, decreased memory, palpitations, muscle pain, joint pain, dry and atrophic skin, vaginal dryness, vaginal atrophy, atrophy of the lower urethra, vaginitis, uterine atrophy, cystitis, painful urination, urgent urination, obesity, type 2 diabetes, hyperlipidemia, arteriosclerosis, fatty liver, sudden changes in heart rate, sleep disorders, loss of confidence and sexual desire, osteoporosis, atherosclerotic heart disease, venous thrombosis, irregular menstrual cycles, and irregular menstrual cycles. The patient may exhibit at least one symptom selected from the group consisting of menstrual flow, irregular menstrual periods, hyperhidrosis, tinnitus, high blood pressure, gastrointestinal disorders, headache, cognitive dysfunction, fatigue, mood swings, Alzheimer's disease, pain during intercourse, short-term memory impairment, decreased libido, blood flow disorders, and skin aging, and preferably exhibits at least one symptom selected from the group consisting of obesity and type 2 diabetes caused by estrogen deficiency, and more preferably exhibits osteoporosis and obesity symptoms caused by estrogen deficiency.

[0056] The term "prevention" may refer to any action that suppresses or delays the onset of estrogen deficiency symptoms in an individual by administering a pharmaceutical composition in one modality.

[0057] The term "treatment" may refer to any action in which the symptoms of an individual suffering from estrogen deficiency syndrome are improved or favorably altered by administering a pharmaceutical composition by one modality.

[0058] According to one aspect, the mesenchymal stem cells with enhanced osteogenic differentiation have superior proliferation ability, and as a result of proliferation, they have excellent karyotype stability, excellent osteogenic differentiation ability, and suppress the increase of adipocytes, compared to other mesenchymal stem cells, and therefore, the mesenchymal stem cells with enhanced osteogenic differentiation ability can be effective in preventing or treating estrogen deficiency syndrome.

[0059] In one embodiment, the pharmaceutical composition may further comprise a pharmaceutical composition for preventing or treating other estrogen deficiency syndromes in addition to the mesenchymal stem cells with enhanced osteogenic differentiation potential.

[0060] That is, the pharmaceutical composition may be provided in admixture with other compositions for the prevention or treatment of other known estrogen deficiency syndromes or other newly developed compositions for the prevention or treatment of other estrogen deficiency syndromes.

[0061] When the pharmaceutical composition contains a composition for preventing or treating other estrogen deficiency syndromes, it is important that the amount of the compound that can achieve the maximum effect with the minimum amount without side effects is mixed, which can be easily determined by a person skilled in the art.

[0062] When the pharmaceutical composition further comprises a composition for preventing or treating other estrogen deficiency syndromes, a synergistic effect may be exhibited, which results in a more pronounced effect in preventing or treating estrogen deficiency syndromes than when the pharmaceutical composition contains only the mesenchymal stem cells with enhanced bone differentiation potential as an active ingredient.

[0063] In one embodiment, the pharmaceutical composition may be administered alone or in combination with other compositions for the prevention or treatment of estrogen deficiency syndromes.

[0064] The aforementioned composition for preventing or treating other estrogen deficiency syndromes may be a conventionally known composition for preventing or treating other estrogen deficiency syndromes, or a newly developed composition for preventing or treating other estrogen deficiency syndromes.

[0065] The pharmaceutical composition may be administered in conjunction with other compositions for the prevention or treatment of estrogen deficiency syndromes, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0066] When the pharmaceutical composition is administered in combination with other compositions for preventing or treating estrogen deficiency syndromes, a synergistic effect may be exhibited, which may result in a more pronounced effect in preventing or treating estrogen deficiency syndromes than when the pharmaceutical composition is administered alone.

[0067] The term "administration" means introducing a substance into an individual by an appropriate method, and "individual" means any living organism, such as rats, mice, and livestock, including humans, that may have estrogen deficiency syndrome. Specific examples include mammals, including humans.

[0068] The pharmaceutical composition may comprise the active ingredient alone or may be provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients or diluents.

[0069] Specifically, the carrier may be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, a microsphere, or a nanosphere, which may be complexed or associated with a delivery vehicle and delivered in vivo using delivery systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0070] When the pharmaceutical composition is formulated, it may be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, may be used. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may be prepared using 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, lauric butter, and glycerogelatin. When prepared as eye drops, known diluents or excipients may be used.

[0071] In one embodiment, the route of administration of the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0072] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the age, sex, condition, and weight of the patient, the degree of absorption of the active ingredient into the body, the inactivation rate, and excretion rate, the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time, route, and excretion rate of administration, the treatment period, the severity of obesity, concurrently used drugs, and other factors well known in the medical field. For example, the pharmaceutical composition is administered at a dose of 0.001 mg / kg / day to 1,000 mg / kg / day, and the effective dose is 10 mg / kg / day based on the mesenchymal stem cells with enhanced bone differentiation potential contained in the pharmaceutical composition and the individual to be administered. 2 cells / kg to 10 9 cells / kg, 10 3 cells / kg to 10 9 cells / kg, 10 3 cells / kg to 10 8 cells / kg, 10 4 cells / kg to 10 8 cells / kg, or 10 4 cells / kg to 10 7 It may be administered at the level of cells / kg.

[0073] In yet another aspect, there is provided a health functional food for preventing or ameliorating bone diseases, which contains the mesenchymal stem cells with enhanced bone differentiation potential.

[0074] The "mesenchymal stem cells with enhanced bone differentiation potential," "bone disease," "prevention," and the like can be within the scope described above.

[0075] The term "improvement" may refer to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms. In this case, the health functional food may be used for the prevention or improvement of bone disease before or after the onset of the disease, simultaneously with or separately from a therapeutic drug.

[0076] In one embodiment, the health functional food may further include a health functional food for preventing or improving other bone diseases.

[0077] The functional health food may be provided in a mixture with other conventionally known functional health foods for preventing or improving bone diseases, or newly developed functional health foods for preventing or improving other bone diseases.

[0078] When the health functional food contains a health functional food for the prevention or improvement of other bone diseases, it is important that the amount mixed is such that the maximum effect can be obtained with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0079] When the functional health food further contains a functional health food for preventing or improving other bone diseases, a synergistic effect can be observed in which the effect of preventing or improving bone diseases is more pronounced than when the functional health food contains only mesenchymal stem cells with enhanced bone differentiation ability as an active ingredient.

[0080] The functional health food may be taken in conjunction with other conventionally known functional health foods for preventing or ameliorating bone diseases, or newly developed functional health foods for preventing or ameliorating other bone diseases, and may be taken simultaneously, separately, or sequentially, and may be taken singly or multiple times. Taking all of the above factors into consideration, it is important to take an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0081] When the functional health food is taken in combination with other functional health foods for preventing or improving bone diseases, a synergistic effect may be observed, making the effect of preventing or improving bone diseases more pronounced than when the functional health food is taken alone.

[0082] In yet another embodiment, there is provided a health functional food for preventing or ameliorating estrogen deficiency syndrome, comprising the mesenchymal stem cells with enhanced bone differentiation potential.

[0083] The "mesenchymal stem cells with enhanced bone differentiation potential," "estrogen deficiency syndrome," "prevention," etc. may fall within the scope described above.

[0084] The term "improvement" can refer to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms. In this case, the health functional food can be used to prevent or improve estrogen deficiency syndrome before or after the onset of the disease, simultaneously with or separately from a therapeutic drug.

[0085] In one embodiment, the health functional food may further include a health functional food for preventing or improving other estrogen deficiency syndromes.

[0086] The health functional food may be provided in a mixture with other health functional foods for the prevention or improvement of other previously known estrogen deficiency syndromes, or newly developed health functional foods for the prevention or improvement of other estrogen deficiency syndromes.

[0087] When the health functional food contains a health functional food for preventing or improving other estrogen deficiency syndromes, it is important that the amount mixed is such that the maximum effect can be obtained with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0088] When the functional health food further contains a functional health food for preventing or improving other estrogen deficiency syndromes, a synergistic effect can be observed in which the effect of preventing or improving estrogen deficiency syndromes is even more pronounced than when the functional health food contains only the mesenchymal stem cells with enhanced bone differentiation ability as an active ingredient.

[0089] The health functional food may be taken in parallel with other conventionally known health functional foods for preventing or improving estrogen deficiency syndromes, or other newly developed health functional foods for preventing or improving estrogen deficiency syndromes, and may be taken simultaneously, separately, or sequentially, and may be taken singly or multiple times. Taking all of the above factors into consideration, it is important to take an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0090] When the health functional food is taken in combination with other health functional foods for preventing or improving estrogen deficiency syndromes, a synergistic effect can be observed, making the effect of preventing or improving estrogen deficiency syndromes even more pronounced than when the health functional food is taken alone.

[0091] In the health functional foods, the active ingredient can be added to the food directly or used together with other foods or food ingredients, and can be used appropriately by common methods. The amount of the active ingredient to be mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when producing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, when taking the food or beverage for long-term purposes such as health and hygiene or health regulation, the amount can be less than the above range.

[0092] The health functional food may further include one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which mesenchymal stem cells may be added in one aspect include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

[0093] Specific examples of the carrier, excipient, diluent, and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0094] The health functional food may contain other essential ingredients in addition to the active ingredient, without any particular limitation. For example, it may contain various flavorings or natural carbohydrates as additional ingredients in addition to a regular beverage. Examples of the natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as common sugars like dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Other flavorings that can be advantageously used include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.). The proportion of the natural carbohydrates can be appropriately determined by the skill of a person skilled in the art.

[0095] In addition to the above, health functional foods according to one aspect may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. Such ingredients may be used independently or in combination, and the ratio of such additives may also be appropriately selected by those skilled in the art. In yet another aspect, there is provided a method for mass production of mesenchymal stem cells with enhanced bone differentiation potential, comprising the step of continuously subculturing the mesenchymal stem cells with enhanced bone differentiation potential.

[0096] The "mesenchymal stem cells with enhanced bone differentiation potential" and the like may fall within the above-mentioned range.

[0097] The term "subculture" originally refers to culturing new cells or microorganisms by transferring some cells in a culture to a new culture medium, and through this subculture, the number of cells or microbial cells can be increased or the lifespan of the cell line can be extended. The above-mentioned "successive subculture" means that the original cells or microorganisms are subcultured without changing their phenotype.

[0098] Unlike general mesenchymal stem cells, the mesenchymal stem cells with enhanced bone differentiation function described above can be continuously subcultured without losing their original traits, i.e., bone differentiation ability, adipocyte differentiation ability, chondrocyte differentiation ability, neural cell differentiation ability, reduced immune rejection, and preventive or therapeutic effects against bone diseases and / or estrogen deficiency syndromes, and can maintain karyotype stability.

[0099] According to the mass production method using a single method, mesenchymal stem cells with enhanced bone differentiation function can be mass-produced, which can contribute to the commercialization of mesenchymal stem cells with enhanced bone differentiation function.

[0100] In yet another embodiment, there is provided a method for preventing or treating estrogen deficiency syndrome, comprising administering the mesenchymal stem cells with enhanced osteogenic differentiation potential to an individual in need thereof. The aforementioned "mesenchymal stem cells with enhanced bone differentiation potential," "individual," "administration," "estrogen deficiency syndrome," "prevention," "treatment," and the like may fall within the scope mentioned above.

[0101] In yet another aspect, there is provided a use of the mesenchymal stem cells with enhanced bone differentiation potential for the prevention or treatment of bone diseases.

[0102] The aforementioned "mesenchymal stem cells with enhanced bone differentiation potential," "bone disease," "prevention," "treatment," and the like may fall within the scope of the aforementioned.

[0103] Yet another embodiment provides a use of the mesenchymal stem cells with enhanced bone differentiation potential for the prevention or treatment of estrogen deficiency syndrome. The aforementioned "mesenchymal stem cells with enhanced bone differentiation potential," "estrogen deficiency syndrome," "prevention," "treatment," etc. may fall within the scope of the aforementioned terms. [Effects of the Invention]

[0104] Mesenchymal stem cells with enhanced osteogenic differentiation potential have excellent bone differentiation, adipocyte differentiation, chondrocyte differentiation, and neural cell differentiation capabilities, and their osteogenic differentiation is particularly superior to mesenchymal stem cells of other origins. Furthermore, the mesenchymal stem cells with enhanced osteogenic differentiation have low immune rejection and can be effective in preventing or treating bone diseases and estrogen deficiency syndromes. Furthermore, the mesenchymal stem cells with enhanced osteogenic differentiation maintain their proliferation ability and karyotype stability despite repeated subculture, and are advantageous in that they are safe for commercial use and can be mass-produced. [Brief explanation of the drawings]

[0105] [Figure 1] FIG. 1 is a diagram showing the proliferation of mesenchymal stem cells derived from fetal mesenchymal stem cells (Fetal MSCs) with enhanced bone differentiation potential, specifically showing the morphology of mesenchymal stem cells derived from Fetal MSCs with enhanced bone differentiation potential. [Figure 2] FIG. 1 is a diagram showing the proliferation of mesenchymal stem cells derived from fetal mesenchymal stem cells (Fetal MSCs) with enhanced bone differentiation potential, specifically showing the growth curve and predicted cell number of mesenchymal stem cells derived from Fetal MSCs with enhanced bone differentiation potential. [Figure 3]FIG. 1 shows the differentiation potential of mesenchymal stem cells derived from fetal MSCs with enhanced bone differentiation potential. Specifically, A shows bone differentiation of mesenchymal stem cells derived from fetal MSCs with enhanced bone differentiation potential, B shows adipocyte differentiation of mesenchymal stem cells derived from fetal MSCs with enhanced bone differentiation potential, C shows chondrocyte differentiation of mesenchymal stem cells derived from fetal MSCs with enhanced bone differentiation potential, and D shows neuronal differentiation of mesenchymal stem cells derived from fetal MSCs with enhanced bone differentiation potential. [Figure 4] FIG. 1 is a diagram comparing the differentiation potential of fetal skull-derived mesenchymal stem cells with enhanced bone differentiation potential and fetal limb-derived mesenchymal stem cells with enhanced bone differentiation potential. [Figure 5] FIG. 1 is a diagram comparing bone differentiation potential, specifically bone differentiation of MSCs of various origins. [Figure 6] FIG. 1 is a diagram comparing bone differentiation ability, specifically, a diagram comparing the bone differentiation ability of fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation ability and BM-MSC (bone marrow-mesenchymal stem cells). [Figure 7] FIG. 1 is a diagram comparing bone differentiation potential, specifically showing gene expression of bone formation markers during the bone differentiation process of mesenchymal stem cells with enhanced bone differentiation potential derived from fetal MSCs. [Figure 8] FIG. 1 shows chromosome analysis of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs. [Figure 9] FIG. 1 shows the surface marker expression of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs by FACS analysis. Specifically, FACS analysis was performed using mesenchymal stem cell markers. [Figure 10] FIG. 1 shows the surface marker expression of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs by FACS analysis. Specifically, FACS analysis was performed using pluripotent stem cell markers. [Figure 11]FIG. 1 shows the surface marker expression of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs by FACS analysis. Specifically, FACS analysis was performed using hematopoietic stem cell markers. [Figure 12] FIG. 1 shows surface marker expression of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs by FACS analysis. Specifically, FACS analysis was performed using human leukocyte antigen. [Figure 13] FIG. 1 shows RNA sequencing analysis of various sources of mesenchymal stem cells, specifically showing clustering heat maps and principal component analysis. [Figure 14] FIG. 1 shows RNA sequencing analysis of various sources of mesenchymal stem cells, specifically differentially expressed gene analysis. [Figure 15] FIG. 1 shows RNA sequencing analysis of various sources of mesenchymal stem cells, specifically mRNA expression. [Figure 16] FIG. 1 shows RNA sequencing analysis of various sources of mesenchymal stem cells, specifically mRNA expression. [Figure 17] FIG. 1 shows secretome analysis, specifically clustering heat map and principal component analysis. [Figure 18A] FIG. 1 shows a scatter plot for secretome analysis, comparing fetal MSC(+) with adult bone-derived MSC. [Figure 18B] FIG. 1 shows a scatter plot for secretome analysis, comparing fetal MSC(+) with umbilical cord Wharton's jelly-derived mesenchymal stem cells (CordSTEM). [Figure 18C] FIG. 1 shows a scatter plot for secretome analysis, comparing fetal MSC(+) with fetal MSC(−). [Figure 19]FIG. 1 shows the heterogeneity of CD90 confirmed through FACS analysis, specifically, FACS analysis. [Figure 20-1] FIG. 1 shows the heterogeneity of CD90 confirmed by FACS analysis, specifically the results of FACS analysis of fetal bone tissue and the level of bone differentiation. [Figure 20-2] FIG. 1 shows the heterogeneity of CD90 confirmed by FACS analysis, specifically showing the FACS analysis results and bone differentiation level for fetal skin tissue. [Figure 21] FIG. 1 shows an experimental design for confirming the osteoprotective ability of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs. [Figure 22A] FIG. 22A shows bone protection of mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs. Specifically, FIG. 22A shows an image obtained via micro-computerized tomography. [Figure 22B] FIG. 22B shows the bone protection of mesenchymal stem cells with enhanced bone differentiation function derived from fetal MSCs, specifically the results of calculating bone volume, cancellous bone marrow, and bone density from the image obtained in FIG. 22A. [Figure 23] FIG. 1 shows the protection of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential from estrogen deficiency. [Figure 24] FIG. 1 shows the protection of estrogen deficiency by mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs, and is a diagram showing the analysis of adipose tissue. DETAILED DESCRIPTION OF THE INVENTION

[0106] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. Example

[0107] Example 1. Proliferation of fetal mesenchymal stem cells (Fetal MSCs) with enhanced bone differentiation potential in the presence and absence of bFGF (basic fibroblast growth factor) additive

[0108] Proliferation of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential was confirmed in the presence and absence of bFGF (basic fibroblast growth factor) additive. Fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential were expanded in culture medium in the presence (lower panel of Figure 1) and absence (upper panel of Figure 1) of bFGF additive. Fetal MSCs were subcultured and counted every 3 to 4 days as indicated. As a result, the morphology of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential was confirmed, as shown in Figure 1.

[0109] In addition, two other fetal MSC-derived mesenchymal stem cell lines with enhanced osteogenic differentiation potential (fMSC_001, fMSC_002) were identified, and the growth curve (upper row of Figure 2) and expected cell number (lower row of Figure 2) of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential are shown in Figure 2.

[0110] Referring to Figures 1 and 2, it was confirmed that fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation function had a spindle-shaped morphology, which is a morphological characteristic of mesenchymal stem cells, regardless of whether bFGF was added or not.

[0111] However, compared to adult-derived MSCs (BM-MSCs, AD-MSCs) and neonatal-derived MSCs (umbilical cord MSCs, placental MSCs, UC blood MSCs), they have superior proliferation ability, and it was confirmed that this proliferation ability is maintained for up to 20 passages. In terms of mass production, after 18 passages, 6 This indicates that it is possible to produce more than 6 billion vials on a cell basis.

[0112] Example 2. Differentiation ability of mesenchymal stem cells with enhanced bone differentiation ability derived from fetal MSCs To confirm the differentiation potential of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation ability in the presence and absence of bFGF additive, we examined their osteogenic differentiation potential (Figure 3A), adipogenic differentiation potential (Figure 3B), chondrogenic differentiation potential (Figure 3C), and neurogenic differentiation potential (Figure 3D).

[0113] Specifically, fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation ability were expanded in culture medium in the presence (lower panel) and absence (upper panel) of bFGF.

[0114] To confirm bone differentiation potential, fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential were cultured in osteogenic differentiation medium (StemPro® Osteogenesis Differentiation Kit) for 14 days. After differentiation, extracellular calcium deposits were stained with Alizarin Red (Figure 3A).

[0115] To confirm their adipogenic potential, fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential were cultured in adipogenic differentiation medium (StemPro® Adipogenesis Differentiation Kit) for 10 days. After differentiation, lipid droplets were stained with Oil Red (Figure 3B).

[0116] To confirm the chondrocyte differentiation potential, cell pellets (2.5 × 10 5 The cells / pellet were cultured for 21 days in chondrocyte differentiation medium (DMEM / high-glucose supplemented with 1% FBS, 1X ITS+Premix, 10 ng / mL TGF-β1, 10 μM dexamethasone, 1 μM ascorbate-2-phosphate, 1% sodium pyruvate, 1X NEAA, and 1% penicillin / streptomycin). After differentiation, paraffin sections were stained with H&E (Figure 3C).

[0117] To confirm their neuronal differentiation potential, fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential were cultured for 14 days in neuronal differentiation medium supplemented with B-27 serum-free supplement and GlutaMAX-I. After differentiation, differentiated neurons were stained by immunocytochemistry using nestin or GFAP-specific antibodies.

[0118] As a result, it was confirmed that fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation ability maintained the differentiation characteristics of mesenchymal stem cells, including bone differentiation ability, adipocyte differentiation ability, chondrocyte differentiation ability, and some neuronal differentiation ability, regardless of whether bFGF was added.

[0119] Example 3. Comparison of differentiation potential between mesenchymal stem cells with enhanced bone differentiation potential derived from skull tissue or calvaria tissue of ectopic pregnancy or stillborn human fetuses and mesenchymal stem cells with enhanced bone differentiation potential derived from limb tissue This study compares the differentiation potential of mesenchymal stem cells with enhanced bone differentiation potential derived from skull tissue or calvaria tissue of ectopic pregnancy or stillborn human fetuses, with mesenchymal stem cells with enhanced bone differentiation potential derived from limb tissue of ectopic pregnancy or stillborn human fetuses.

[0120] Specifically, to confirm bone differentiation potential, fetal MSC (limb tissue)-derived mesenchymal stem cells with enhanced bone differentiation potential were cultured in osteogenic differentiation medium (StemPro® Osteogenesis Differentiation Kit) for 14 days. After differentiation, extracellular calcium deposits were stained with Alizarin Red.

[0121] To confirm adipogenic differentiation, fetal MSC (limb tissue)-derived mesenchymal stem cells with enhanced osteogenic differentiation were cultured in adipogenic differentiation medium (StemPro® Adipogenesis Differentiation Kit) for 10 days. After differentiation, lipid droplets were stained with Oil Red.

[0122] To confirm the chondrocyte differentiation potential, cell pellets (2.5 × 10 5 The cells / pellet were cultured in chondrocyte differentiation medium (DMEM / high-glucose supplemented with 1% FBS, 1X ITS+Premix, 10 ng / mL TGF-β1, 10 μM dexamethasone, 1 μM ascorbate-2-phosphate, 1% sodium pyruvate, 1X NEAA, and 1% penicillin / streptomycin) for 21 days. After differentiation, paraffin sections were stained with H&E.

[0123] As a result, we were able to confirm that the adipocyte differentiation potential of fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential isolated from fetal limbs was similar to that of fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential isolated from fetal skull tissue or calvaria tissue, and that the chondrocyte differentiation potential was slightly increased, but the bone differentiation potential was slightly decreased (Figure 4).

[0124] In the remaining examples except for Example 3, mesenchymal stem cells with enhanced osteogenic differentiation ability derived from fetal MSCs isolated from fetal head tissue or calvaria tissue were used.

[0125] Example 4. Comparison of bone differentiation potential This study compares the osteogenic differentiation potential of MSCs from various sources.

[0126] Specifically, mesenchymal stem cells, including umbilical cord Wharton's jelly-derived mesenchymal stem cells (CordSTEM), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and embryonic stem cell-derived mesenchymal stem cells (ES-MSC), were expanded in culture medium. The mesenchymal stem cells were cultured for 14 days in osteogenic differentiation medium (StemPro® Osteogenesis Differentiation Kit). After differentiation, extracellular calcium deposits and basic phosphatase enzyme activity were stained with Alizarin Red and AP staining, respectively (Figure 5).

[0127] To compare the bone differentiation potential of fetal MSC-derived mesenchymal stem cells with that of BM-MSCs, the mesenchymal stem cells were cultured in osteogenic differentiation medium (StemPro® Osteogenesis Differentiation Kit) for the indicated periods (7, 14, and 21 days). After differentiation, extracellular calcium deposits were stained with Alizarin Red (Figure 6).

[0128] To confirm the expression of osteogenic marker genes, total RNA was prepared during osteogenic differentiation, and RT-PCR was performed using primers for osteogenic marker genes (ALP: Alkaline phosphatase; S100A4: S100 Calcium Binding Protein A4; COL1A1: Collagen Type I Alpha 1 Chain; COL1A2: Collagen Type I Alpha 2 Chain) as indicated (Figure 7).

[0129] As a result, through comparative analysis of bone differentiation potential, it was confirmed that fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential had superior bone differentiation potential compared to adult-derived MSCs (BM-MSCs), neonatal-derived MSCs (umbilical cord MSCs), and embryonic stem cell-derived MSCs, particularly fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential cultured in bFGF-free medium (Figure 5).

[0130] Compared to BM-MSCs, which are currently known to have the highest bone differentiation potential, fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential began bone differentiation 7 days after induction and reached saturation by 14 days, whereas BM-MSCs had not yet reached saturation even after 21 days. This was also confirmed by the expression of various bone differentiation marker genes.

[0131] Example 5. Chromosome analysis (karyotyping) To perform karyotyping of fetal MSC-derived osteogenic MSCs, fetal MSC-derived osteogenic MSCs were expanded in culture medium with or without bFGF supplementation. Fetal MSC-derived osteogenic MSCs were cultured for up to 8 passages without bFGF (Figure 8A) and for up to 24 passages with bFGF (Figure 8B), as indicated. At least 10 cells were analyzed, and representative Giemsa band metaphase images are shown.

[0132] Karyotyping confirmed karyotype stability, demonstrating that karyotype stability was maintained even at passages 8 and 24. This indicates that fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation function are safe for commercialization.

[0133] Example 6. Surface marker expression FACS analysis was used to confirm the expression of surface markers in fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential. Fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential were expanded in culture medium in the presence or absence of bFGF additive. At the indicated passages, cells were cultured and subjected to FACS analysis using mesenchymal stem cell markers (Figure 9), pluripotent stem cell markers (Figure 10), hematopoietic stem cell markers (Figure 11), and human leukocyte antigen (HLA) (Figure 12).

[0134] To confirm the mesenchymal stem cell characteristics of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential, the expression of mesenchymal stem cell surface markers was examined by FACS analysis. The results confirmed that fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential uniformly expressed CD73, CD44, CD29, CD105, and CD166, which are representative surface markers of mesenchymal stem cells, at the initial culture passage (P9), middle culture passage (P13), and late culture passage (P17), regardless of whether bFGF was added (Figure 9).

[0135] Furthermore, to confirm the mesenchymal stem cell characteristics of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential, we examined the expression of surface markers on all differentiation-potential cells by FACS analysis. Regardless of the presence or absence of bFGF, fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential did not express SSEA-3 or TRA1-81, which are representative surface markers of all differentiation-potential cells, at the initial culture passage (P9), middle culture passage (P13), or late culture passage (P17). However, in particular, cells expressing the fetal-derived cell surface marker c-kit were observed in the initial culture conditions without bFGF. Therefore, we were able to confirm that c-kit expression is one of the characteristics of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential (Figure 10).

[0136] Furthermore, to confirm the mesenchymal stem cell characteristics of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential, the expression of hematopoietic stem cell surface markers was examined by FACS analysis. The results confirmed that fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential did not express CD14, CD24, CD34, CD45, or CD70, which are typical surface markers of hematopoietic stem cells, at the initial culture passage (P9), intermediate culture passage (P13), or late culture passage (P17), regardless of whether bFGF was added (Figure 11).

[0137] Furthermore, we investigated the immunogenicity of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential due to HLA expression. Similar to other MSCs, we found that HLA class II (HLA-DR) expression was completely absent. Uniquely, HLA class I expression was almost absent in cells cultured with bFGF. Therefore, we found that reduced HLA class I expression in bFGF-supplemented medium is one of the characteristics of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential, which may be extremely useful in the development of allogeneic cell therapy agents, as it increases cell survival rates due to immune rejection (Figure 12).

[0138] Example 7. Analysis of gene expression RNA sequencing analysis of mesenchymal stem cells of various origins was performed, and a clustering heat map and principal component analysis (PCA) were obtained (FIG. 13).

[0139] Clustering heatmap and PCA show the relative expression (z-score) of differentially expressed genes between BM-MSCs, umbilical cord MSCs (CordSTEMs), and fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential in the presence or absence of bFGF in culture medium.

[0140] Additionally, differentially expressed gene analysis was performed, and a Venn diagram of differentially expressed genes between MSCs of various origins was obtained (Figure 14). Table 1 in Figure 14 summarizes mesenchymal stem cell-specific marker genes that were exclusively (red) or highly (black) expressed in fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential.

[0141] In addition, total RNA was prepared from MSCs of various origins, and RT-PCR was performed using specific primers for (1) osteogenic MSC marker genes, (2) fetal MSC marker genes, (3) cell surface markers, and (4) osteogenic MSC markers in Table 1 of Figure 14 to confirm mRNA expression (Figures 15 and 16).

[0142] Using RNA sequencing, gene expression analysis of adult-derived MSCs (BM-MSCs) and neonatal-derived MSCs (umbilical cord MSCs) revealed genes (markers) specifically expressed in fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential. The fetal MSC-specific genes, Osx (SP7), c-kit, CD24, CD70, and SFRP2, were not expressed in MSCs of other origins but were expressed exclusively in fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential. Therefore, it was demonstrated that fetal MSCs are differentiated from other MSCs through the expression of these markers.

[0143] Among these, Osx (SP7), which is related to bone differentiation function, is known as an osteogenic MSC-specific marker, and SFRP2 is known to be involved in cell signaling that plays an important role in bone differentiation.

[0144] Furthermore, C-kit is a specific marker for fetus-derived cells, and other fetus-derived genes KITLG and CXCL12 were confirmed to be overexpressed in fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential compared to MSCs from other sources.

[0145] Furthermore, CD24 and CD70 are cell surface markers, and these markers allowed us to distinguish the specificity of mesenchymal stem cells with enhanced bone differentiation function derived from fetal MSCs.

[0146] Other transcription factors, ligands, cell signaling components, and enzymes involved in bone differentiation were confirmed to be overexpressed in mesenchymal stem cells with enhanced bone differentiation potential derived from fetal MSCs (e.g., HES4, TWIST1, BMP2, BMPR1B, NOG, ALPL, etc.).

[0147] Example 8. Secretome analysis To perform secretome analysis, we first performed clustering heatmap and principal component analysis (PCA) (Figure 17). RayBiotech human cytokine arrays (RayBiotech #L-507, #L-493) were analyzed using serum-free medium samples obtained from MSCs of various origins. The clustering heatmap and PCA show the relative secretion of differentially secreted cytokines and growth factors among BM-MSCs, umbilical cord MSCs (CordSTEM), and fetal MSC-derived osteogenic-enriched mesenchymal stem cells in the presence or absence of bFGF in culture medium.

[0148] A scatter plot was also performed (Figure 18). The scatter plot showed differentially secreted proteins, with increased protein secretion shown in red and decreased protein secretion shown in green compared to Fetal MSC(+). The table in Figure 18 shows a list of highly secreted genes in the scatter plot and their bone metabolism-related functions.

[0149] Using secretome analysis, we analyzed protein secretion into the culture medium of adult-derived MSCs (BM-MSCs) and neonatal-derived MSCs (umbilical cord MSCs). As a result, we were able to confirm the secretion of proteins specific to mesenchymal stem cells with enhanced osteogenic differentiation potential derived from fetal MSCs. Compared to MSCs of other origins, we confirmed the secretion of numerous proteins in excess from mesenchymal stem cells with enhanced osteogenic differentiation potential (Figure 18).

[0150] In particular, functional proteins such as numerous cytokines and growth factors related to MSC proliferation, bone differentiation, and bone metabolism were found to be secreted in large amounts by fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential, as summarized in the table in Figure 18. Therefore, it was found that fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation potential have excellent bone differentiation function.

[0151] Example 9. CD90 Non-uniform heterogeneity and osteogenic potential Fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential express CD90 Non-uniform To confirm this, FACS analysis was performed (Figure 19). Specifically, mesenchymal stem cells (ES-MSCs, CordSTEM) and fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential were expanded in culture medium with or without bFGF supplementation. Cells were cultured at the indicated passages (P4, P6, P11, and P12) and subjected to FACS analysis using specific antibodies against CD90, CD105, SSEA-3, and SSEA-4.

[0152] Specifically, FACS analysis was performed on mesenchymal stem cells derived from the calvaria of the head of an 8.5-week-old fetus (Figure 20A), and mesenchymal stem cells derived from the calvaria, limbs, and skin tissue of a 7-week-old fetus (Figure 20B). Non-uniform In the case of mesenchymal stem cells derived from the hands, feet, and skin, CD90 expression was not detected, whereas in mesenchymal stem cells derived from bone tissue, CD90 expression was detected. Non-uniform We were able to confirm this.

[0153] Furthermore, it was confirmed that calvarial-derived mesenchymal stem cells had the most pronounced bone differentiation potential (Fig. 20C).

[0154] Based on the above results, it was confirmed that mesenchymal stem cells with enhanced osteogenic differentiation function can be divided into cells with high CD90 expression and cells with low CD90 expression during the subculture process. Non-uniform It was confirmed that this ability was strongly exhibited in mesenchymal stem cells derived from bone tissue (head region). It was confirmed that the phenomenon described above was even more evident when bFGF was added.

[0155] Example 10. Bone protection and regeneration of mesenchymal stem cells with enhanced bone differentiation potential derived from fetal MSCs in an osteoporosis animal model Experiments were conducted to confirm the bone protective and bone regenerative effects of mesenchymal stem cells with enhanced bone differentiation function derived from fetal MSCs, and Figure 21 shows the experimental design.

[0156] Specifically, the ovaries of 8-week-old female C57BL / 6 mice were removed. Two weeks later, 1x10 6 Mice were injected with fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential or an equal volume of PBS (100 μl) into the tail vein. Six weeks after transplantation of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic potential, the mice were sacrificed. In the immunosuppressed group, 10 mg / kg of cyclosporine A (CsA) was administered subcutaneously daily.

[0157] The results of micro-computerized tomography and bone density are shown in Figure 22. After sacrifice, femurs and bones were excised and fixed in 10% PFA solution. Bone structure variables were analyzed using a micro-CT system and H&E staining. For the analysis of bone density and bone composition, femurs and bones were scanned at 1 mm intervals using a micro-computerized tomography system (SkyScan 1173, Bruker-CT, ​​Belgium). Bone volume (cm 3 ), cancellous bone count and bone mineral density (mg / cm 3) was calculated using CT analyzer software (CTAnMicro-CT software) (BV / TV: bone volume per tissue volume; Tb.N: trabecular bone number; BMD: bone mineral density).

[0158] Specifically, to confirm the efficacy of mesenchymal stem cells with enhanced osteogenic differentiation potential in animal models of bone disease, an ovariectomy-mediated osteoporosis model was created. Fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential were administered intravenously to the osteoporosis model, resulting in increased bone mineral density, osteocyte numbers, and bone volume. Therefore, it was confirmed that fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential can be used as a cell therapy for bone diseases such as osteoporosis, fractures, bone nonunion, osteogenesis imperfecta, and fibrodysplasia.

[0159] Furthermore, as one of the mechanisms of action, it was confirmed that in an ovariectomy-induced osteoporosis model, adipocyte differentiation of bone marrow mesenchymal stem cells was reduced by administering mesenchymal stem cells with enhanced osteogenic differentiation function derived from fetal MSCs.

[0160] Example 11. Protection against estrogen deficiency syndrome An experiment was conducted to confirm the protective effect of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential on estrogen deficiency syndrome. The experimental design is shown in Figure 23.

[0161] Specifically, the ovaries of 8-week-old female C57BL / 6 mice were removed. Two weeks later, 1x10 6 Fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential or an equal volume of PBS (100 μl) was injected into the tail vein. Mice were sacrificed 6 weeks after transplantation of fetal MSC-derived mesenchymal stem cells with enhanced osteogenic differentiation potential. In the immunosuppression group, 10 mg / kg of cyclosporine A (CsA) was administered using an osmotic pump (ALZET, USA).

[0162] The results of adipose tissue analysis are shown in Figure 24. After sacrifice, the morphology and weight of the fat pads (gonadal fat, retroperitoneal fat) were analyzed (one-way ANOVA test; P value ****; <0.0001, P value **; 0.0018, P value *; 0.0139). Blood glucose and total glyceride levels were analyzed using serum (one-way ANOVA test; P value **; 0.0025).

[0163] Specifically, to confirm the efficacy of fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation function in an animal model of estrogen deficiency syndrome, an animal model involving ovariectomy was created, and it was confirmed that obesity due to an increase in adipocytes, a type of metabolic disorder, occurred in one of the estrogen deficiency syndrome groups caused by ovariectomy.

[0164] In an ovariectomy model, intravenous administration of fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation capacity demonstrated a significant reduction in obesity due to increased adipocytes. Therefore, it was confirmed that fetal MSC-derived mesenchymal stem cells with enhanced bone differentiation capacity can be used as a cell therapy for metabolic diseases (obesity, type 2 diabetes, etc.), which are one of the estrogen deficiency syndromes that occur after menopause.

[0165] Reference Example Reference Example 1. Isolation and culture of human fetal mesenchymal stem cells (Fetal MSCs) After removing the neural tube from the skull or limb tissue of an ectopic or stillborn human fetus, the skull tissue was placed in a 50ml tube, and HBSS was added and mixed thoroughly to remove blood. The fetal tissue was cut into thin strips using a razor blade and transferred to a 50ml tube along with 10ml of HBSS. The skull pieces were gently placed in a 10ml pipette and centrifuged at 1,200 rpm for 5 minutes at room temperature. The supernatant was removed, and 10ml of collagenase solution was added. The mixture was placed in a 37°C shaking incubator and incubated for 15 minutes. Centrifuge at 1,200 rpm for 3 minutes at room temperature to settle the tissue chunks. Pipette 9 ml of the supernatant into a new 50 ml tube, add the same volume of DPBS solution containing 10% FBS, and mix thoroughly with a pipette to block the collagenase enzyme. Strain through a 100 μm strainer and centrifuge at 1,500 rpm for 5 minutes at room temperature. Mix the pellet thoroughly with 1 ml of culture medium (DMEM high glucose with 10% FBS, 1% penicillin and streptomycin). 5 x 10 per T-25 flask. 5 The cells are cultured.

[0166] Reference Example 2: Method for differentiating human fetal mesenchymal stem cells (1) Osteogenic differentiation After culturing the cells in a 24-well plate, the basal medium and supplement from the StemPro osteogenesis differentiation kit (A10072-01 (Gibco)) were mixed at a ratio of 9:1 and replaced with medium containing 1% penicillin / streptomycin. The medium was replaced every 3-4 days. After fixation with 4% PFA, the cells were stained with 2% Alizarin Red solution for 30 minutes, and calcium was confirmed by red staining.

[0167] (2) Adipogenic differentiation After culturing the cells in a 12-well plate, the medium was replaced with a 9:1 mixture of basal medium and supplement from the StemPro adipogenesis differentiation kit (A10070-01) and supplemented with 1% penicillin / streptomycin. The medium was replaced every 3-4 days. After fixation with 4% PFA, the cells were stained with Oil Red solution for 15 minutes to confirm the presence of oil drops.

[0168] (3)chondrogenic differentiation The cell pellets were cultured for 21 days in chondrocyte differentiation medium (DMEM-high glucose + 1% FBS, 1X ITS + Premix, 10 ng / mL TGF-β1, 10 μM dexamethasone, 1 μM ascorbate-2-phosphate, 1% sodium pyruvate, 1X NEAA, 1% penicillin / streptomycin), then paraffin sections were prepared and the degree of chondrocyte differentiation was confirmed by H&E staining or PAS staining.

[0169] Reference Example 3: Experiment to confirm the differentiation potential of human fetal mesenchymal stem cells (1) RNA production and cDNA synthesis Place the harvested cells in 1 ml of TRIzol and let them sit for 2-3 minutes. Then, add 200 μl of chloroform and mix thoroughly. Leave at room temperature for 2-3 minutes, then centrifuge at 12,000 g for 15 minutes at room temperature. Transfer the colorless upper portion to a new tube. Add an equal volume of 100% ethanol and mix 5-6 times. Transfer the contents to a MiniSpin Column of the GeneAll kit (#304-150 (GeneAll Biotech)) and centrifuge at 10,000 g for 30 seconds at room temperature. Discard the contents that have escaped, add 500 μl of GW1 buffer, and centrifuge under the same conditions. Add 700 μl of RNW buffer, centrifuge, transfer to a new tube, and extract RNA using 50 μl of water. The mixture was centrifuged at 10,000G for 1 minute to extract RNA, and its concentration was confirmed using a spectrometer (NanoDrop). After that, a total of 500ng of the mixture was added to the LaboPass cDNA synthesis kit (#CMRTK002 (Cosmogenetic)) along with oligo dT / random primer, dNTP, RNA, and nuclease-free water, and the mixture was incubated at 42°C for 1 hour to synthesize cDNA.

[0170] (2) Real-time PCR SYBR green mix (#RT500S (Enzynomics)), cDNA, forward and reverse primers, and RNase-free water were added to each well of a 96-well reaction plate (#N8010560 (Applied Biosystems)), and the PCR cycles were run in an RT-PCR machine (viiA7 (Applied Biosystems)). The expression levels of each gene were then compared using the comparative delta-delta-Ct method or the standard curve method.

[0171] (3)FACS analysis The cells were detached using trypsin and collected by centrifugation. After washing twice with PBS, the cells were collected at 1x10 5The cells were placed in FACS buffer at a concentration of 200 μl per cell. Antibodies were added at 100x concentration and placed in a dark place at 4°C for 30 minutes. The cells were washed with FACS buffer, then placed in 400 μl of FACS buffer and analyzed using a FACS machine.

[0172] (4) Secretome analysis Prepare 100% confluence dishes and wash twice with DPBS. Add 8 ml of DMEM medium supplemented with 1% penicillin / streptomycin to each dish and incubate for 24 hours. Transfer the culture medium from each dish to a 15 ml tube and centrifuge at 2,000 rpm for 10 minutes. Use an antibody array (Raybiotech Human L1000 Antibody Array) to check and compare the concentration of the target substance in the supernatant.

[0173] (5) Ovariectomized model (OVX model) Eight-week-old female C57BL / 6 mice were anesthetized via respiratory anesthesia, and then an incision was made on the back. The fat-filled ovaries were removed and the incision sutured. The weight and condition were then monitored weekly. Two weeks after surgery, stem cells of various conditions were injected via the tail vein in a volume of 100 μl. Six weeks after intravenous administration, the animals were sacrificed, and tissue and blood samples were collected for analysis.

[0174] (6) Karyotype analysis Fetal MSCs were cultured in culture medium with or without bFGF for 8 passages (Figure 8A) or 24 passages (Figure 8B), respectively, and then isolated using trypsin. G-banding of at least 10 metaphase cells was observed under a microscope using Giemsa staining. The present disclosure includes the following embodiments. [1] Mesenchymal stem cells with enhanced bone differentiation ability, which express at least one gene selected from the group consisting of Osx(SP7), c-kit, CD24, CD70, and SFRP2. [2] The mesenchymal stem cells with enhanced bone differentiation potential according to embodiment 1, wherein the stem cells heterogeneously express CD90. [3] The mesenchymal stem cells having enhanced bone differentiation potential function described in embodiment 1, wherein the stem cells do not express HLA class I. [4] The mesenchymal stem cells with enhanced bone differentiation potential described in embodiment 1, wherein the stem cells overexpress at least one gene selected from the group consisting of KITLG, CXCL12, HES4, TWIST1, BMP2, BMPR1B, NOG, and ALPL compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow. [5] The mesenchymal stem cells with enhanced osteogenic differentiation potential according to embodiment 1, wherein the stem cells hypersecrete at least one polypeptide selected from the group consisting of ABL1, IGFBP-7, MMP-1, GLO-1, progranulin, amylin, BMX, ALPP, FAP, ADAMTS-L2, SPARC, TLR1, galectin-3, beta IG-H3, HB-EGF, 11b-HSD1, PTHLP, ACTH, activin A, GDF11, and GDF3, compared to mesenchymal stem cells derived from umbilical Wharton's jelly or mesenchymal stem cells derived from adult bone marrow. [6] The mesenchymal stem cells with enhanced bone differentiation potential according to embodiment 1, wherein the stem cells are derived from tissue of an ectopic pregnancy or a stillborn fetus. [7] The mesenchymal stem cells with enhanced bone differentiation potential function described in embodiment 6, wherein the fetal tissue is fetal head tissue or fetal calvarial tissue. [8] A pharmaceutical composition for preventing or treating a bone disease, comprising mesenchymal stem cells with enhanced bone differentiation potential as described in embodiment 1. [9] The pharmaceutical composition for preventing or treating a bone disease according to embodiment 8, wherein the bone disease is at least one bone disease selected from the group consisting of osteoporosis, bone fracture, nonunion, osteogenesis imperfecta, osteopenia, osteolytic metastasis, senile kyphosis, Paget's disease, bone atrophy, osteomalacia, osteoarthritis, periodontal disease, rickets, aplastic bone disease, bone damage caused by bone metastasis of cancer cells, fibrodysplasia, McCune-Albright syndrome, bone malformation, and bone dysplasia.

[10] The pharmaceutical composition for preventing or treating a bone disease according to embodiment 8, wherein the bone disease is at least one bone disease selected from the group consisting of osteoporosis, bone fracture, fibrodysplasia, McCune-Albright syndrome, nonunion, and osteogenesis imperfecta.

[11] A pharmaceutical composition for preventing or treating estrogen deficiency syndrome, comprising mesenchymal stem cells with enhanced bone differentiation potential according to embodiment 1.

[12] The above-mentioned estrogen deficiency syndromes include facial flushing, sweating, insomnia, irritability, depression, dizziness, concentration problems, short-term memory problems, anxiety, decreased memory, palpitations, muscle pain, joint pain, dry and atrophic skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, uterine atrophy, cystitis, painful urination, urgent urination, obesity, type 2 diabetes, hyperlipidemia, arteriosclerosis, fatty liver, sudden changes in heart rate, sleep disorders, loss of confidence and sexual desire, and osteoporosis. 12. The pharmaceutical composition for the prevention or treatment of estrogen deficiency syndrome according to embodiment 11, wherein the patient exhibits at least one symptom selected from the group consisting of: eczema, atherosclerotic heart disease, venous thrombosis, irregular menstrual cycles, irregular menstrual flow, irregular menstrual periods, hyperhidrosis, tinnitus, high blood pressure, gastrointestinal disorders, headache, cognitive dysfunction, fatigue, mood swings, Alzheimer's disease, painful intercourse, short-term memory impairment, decreased libido, impaired blood flow, and skin aging.

[13] The pharmaceutical composition for preventing or treating estrogen deficiency syndrome according to embodiment 11, wherein the estrogen deficiency syndrome exhibits at least one symptom selected from the group consisting of obesity and type 2 diabetes caused by estrogen deficiency.

[14] A health functional food for preventing or improving bone diseases, comprising mesenchymal stem cells with enhanced bone differentiation ability as described in embodiment 1.

[15] A health functional food for preventing or improving estrogen deficiency syndrome, comprising mesenchymal stem cells with enhanced bone differentiation function as described in embodiment 1.

Claims

1. A mesenchymal stem cell with enhanced bone differentiation ability, which expresses at least one gene selected from the group consisting of Osx (SP7) and SFRP2, wherein the stem cell is derived from fetal tissue.

2. The mesenchymal stem cell described in claim 1, which further expresses at least one gene selected from the group consisting of c-kit, CD24, and CD70.

3. The mesenchymal stem cells with enhanced bone differentiation potential according to claim 1 , wherein the stem cells heterogeneously express CD90.

4. The mesenchymal stem cells having enhanced bone differentiation function according to claim 1, wherein the stem cells do not express HLA class I.

5. The mesenchymal stem cells with enhanced bone differentiation function according to claim 1, wherein the stem cells overexpress at least one gene selected from the group consisting of KITLG, CXCL12, HES4, TWIST1, BMP2, BMPR1B, NOG, and ALPL compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow.

6. 2. The mesenchymal stem cells with enhanced bone differentiation potential according to claim 1, wherein the stem cells hypersecrete at least one polypeptide selected from the group consisting of ABL1, IGFBP-7, MMP-1, GLO-1, progranulin, amylin, BMX, ALPP, FAP, ADAMTS-L2, SPARC, TLR1, galectin-3, beta IG-H3, HB-EGF, 11b-HSD1, PTHLP, ACTH, activin A, GDF11, and GDF3, compared to mesenchymal stem cells derived from umbilical cord Wharton's jelly or mesenchymal stem cells derived from adult bone marrow.

7. The mesenchymal stem cells with enhanced bone differentiation potential according to claim 1, wherein the stem cells are derived from tissue of an ectopic pregnancy or a stillborn fetus.

8. The mesenchymal stem cells having enhanced bone differentiation potential function according to claim 7, wherein the fetal tissue is fetal head tissue or fetal calvarial tissue.

9. A pharmaceutical composition for preventing or treating bone diseases, comprising the mesenchymal stem cells with enhanced bone differentiation potential according to claim 1.

10. 10. The pharmaceutical composition for preventing or treating a bone disease according to claim 9, wherein the bone disease is at least one bone disease selected from the group consisting of osteoporosis, bone fracture, nonunion, osteogenesis imperfecta, osteopenia, osteolytic metastasis, senile kyphosis, Paget's disease, bone atrophy, osteomalacia, osteoarthritis, periodontal disease, rickets, aplastic bone disease, bone damage caused by bone metastasis of cancer cells, fibrodysplasia, McCune-Albright syndrome, bone malformation, and bone dysplasia.

11. 10. The pharmaceutical composition for preventing or treating a bone disease according to claim 9, wherein the bone disease is at least one bone disease selected from the group consisting of osteoporosis, bone fracture, fibrodysplasia, McCune-Albright syndrome, nonunion, and osteogenesis imperfecta.

12. A pharmaceutical composition for preventing or treating estrogen deficiency syndrome, comprising the mesenchymal stem cells with enhanced bone differentiation potential according to claim 1.

13. The estrogen deficiency syndromes include facial flushing, sweating, insomnia, irritability, depression, dizziness, concentration problems, short-term memory problems, anxiety, decreased memory, palpitations, muscle pain, joint pain, dry and atrophic skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, vaginitis, uterine atrophy, cystitis, painful urination, urgent urination, obesity, type 2 diabetes, hyperlipidemia, arteriosclerosis, fatty liver, sudden changes in heart rate, sleep disorders, loss of confidence and sexual desire, and osteoporosis.

13. The pharmaceutical composition for preventing or treating estrogen deficiency syndromes according to claim 12, wherein the estrogen deficiency syndromes exhibit at least one symptom selected from the group consisting of atherosclerotic heart disease, venous thrombosis, irregular menstrual cycles, irregular menstrual flow, irregular menstrual periods, hyperhidrosis, tinnitus, high blood pressure, gastrointestinal disorders, headache, cognitive dysfunction, fatigue, mood swings, Alzheimer's disease, pain during intercourse, short-term memory impairment, decreased libido, blood flow disorders, and skin aging.

14. The pharmaceutical composition for preventing or treating estrogen deficiency syndrome according to claim 12, wherein the estrogen deficiency syndrome exhibits at least one symptom selected from the group consisting of obesity and type 2 diabetes caused by estrogen deficiency.

15. A functional health food for preventing or ameliorating bone diseases, comprising the mesenchymal stem cells with enhanced bone differentiation function according to claim 1.

16. A health functional food for preventing or improving estrogen deficiency syndrome, comprising the mesenchymal stem cells with enhanced bone differentiation function according to claim 1.

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