Mammalian dedifferentiated adipocyte-derived osteochondral progenitor cell

Abstract

The present invention relates to osteochondral progenitor cells derived from mammalian-derived dedifferentiated adipocytes.

Description

Mammalian dedifferentiated adipocyte-derived osteochondral progenitor cells

[0001] This invention relates to osteochondral progenitor cells induced from dedifferentiated adipocytes of mammalian origin. Furthermore, this invention relates to a culture medium for inducing differentiation from dedifferentiated adipocytes of mammalian origin into osteochondral progenitor cells.

[0002] Musculoskeletal disorders cause pain and movement impairment, reducing the patient's quality of life. However, musculoskeletal function involves a complex interplay of mesodermal tissues such as bone, cartilage, and tendons, and it is known that these tissues have a low capacity for recovery.

[0003] In recent years, the development of treatments aimed at restoring musculoskeletal function has progressed through the transplantation of osteochondral progenitor cells created using pluripotent stem cells such as human iPS cells. However, when human iPS cells are used to create osteochondral progenitor cells, the necessary genetic manipulation carries risks such as cell tumorigenesis, gene mutations, and excessive immune responses. Therefore, there is a need for a simple and efficient method to produce osteochondral progenitor cells in vitro without genetic manipulation.

[0004] Non-patent document 1 reports on the effects of the interaction between the Hedgehog signaling pathway and the bone morphogenetic protein (BMP) signaling pathway on bone formation. Non-patent document 2 reports that cartogenin (KGN) promotes the cartilage differentiation of mesenchymal stem cells (MSCs).

[0005] Hojo H, Ohba S, Taniguchi K, Shirai M, Yano F, Saito T, Ikeda T, Nakajima K, Komiyama Y, Nakagata N, Suzuki K, Mishina Y, Yamada M, Konno T, Takato T, Kawaguchi H, Kambara H, Chung UI. Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium. J Biol Chem. 2013 Apr 5;288(14):9924-9932. doi: 10.1074 / jbc.M112.409342. Epub 2013 Feb 19. PMID: 23423383; PMCID: PMC3617292.Chen CY, Li C, Ke CJ, Sun JS, Lin FH. Kartogenin Enhances Chondrogenic Differentiation of MSCs in 3D Tri-Copolymer Scaffolds and the Self-Designed Bioreactor System. Biomolecules. 2021 Jan 16;11(1):115. doi: 10.3390 / biom11010115. PMID: 33467170; PMCID: PMC7829855.

[0006] Osteochondragon progenitor cells induced from dedifferentiated adipocytes have not been reported to date. The present invention aims to provide osteochondragon progenitor cells from dedifferentiated adipocytes in a simple and efficient manner.

[0007] As a result of diligent research, the present inventors discovered that, although artificially synthesized proteins were an essential component in the method described in Non-Patent Document 2, dedifferentiated adipocytes of mammalian origin can be differentiated into osteochondral progenitor cells by culturing them in a medium substantially free of artificially synthesized proteins, thus completing the present invention. The present invention is as follows: [1] Osteochondral progenitor cells induced from dedifferentiated adipocytes of mammalian origin. [2] Osteochondral progenitor cells according to [1], which are positive for the RUNX1 and RUNX2 markers. [3] A method for producing osteochondral progenitor cells, comprising the step of culturing dedifferentiated adipocytes of mammalian origin in a medium containing an osteochondral progenitor cell differentiation inducer and substantially free of artificially synthesized proteins. [4] The method according to [3], wherein the osteochondral progenitor cell differentiation inducer is cartogenin. [5] The method according to [3] or [4], wherein the concentration of the osteochondral progenitor cell differentiation inducer in the medium is 100 μM or less. [6] The method according to any one of [3] to [5], wherein the mammal is human. [7] A culture medium for inducing differentiation of dedifferentiated adipocytes of mammalian origin into osteochondral progenitor cells, comprising an osteochondral progenitor cell differentiation inducer and substantially free of artificially synthesized proteins. [8] The culture medium according to [7], wherein the osteochondral progenitor cell differentiation inducer is Kartogenin. [9] The culture medium according to [7] or [8], wherein the concentration of the osteochondral progenitor cell differentiation inducer in the culture medium is 50 μM or less.

[10] A kit for inducing differentiation of dedifferentiated adipocytes of mammalian origin into osteochondral progenitor cells, comprising the culture medium according to any one of [7] to [9].

[0008] According to the present invention, osteochondral progenitor cells can be easily and efficiently provided from dedifferentiated adipocytes.

[0009] Figure 1A is a graph showing the mRNA expression level of the osteochondral progenitor cell marker RUNX1 in cells (cell number = 3) cultured using differentiation induction medium. "control" indicates the control group, and the numbers 0, 1d, 3d, and 6d following "control" and "KGN" indicate the number of days elapsed since the start of culture, respectively (the same applies to Figure 1B). Figure 1B is a graph showing the mRNA expression level of the osteochondral progenitor cell marker RUNX2 in cells (cell number = 3) cultured using differentiation induction medium. Figure 2A is a graph showing the mRNA expression level of the osteochondral progenitor cell marker RUNX1 in cells (cell number = 3) cultured for 3 days using differentiation induction medium containing different concentrations of osteochondral progenitor cell differentiation inducer (KGN). Figure 2B is a graph showing the mRNA expression levels of the osteochondral progenitor cell marker RUNX2 in cells (cell number = 3) cultured for 3 days using differentiation induction medium containing different concentrations of osteochondral progenitor cell differentiation inducer (KGN). Figure 3A is a graph showing the mRNA expression levels of the osteochondral progenitor cell marker SOX9 in cells (cell number = 3) cultured for 3 days using differentiation induction medium containing different concentrations of osteochondral progenitor cell differentiation inducer (KGN). Figure 3B is a graph showing the mRNA expression levels of the osteochondral progenitor cell marker BGLAP in cells (cell number = 3) cultured for 3 days using differentiation induction medium containing different concentrations of osteochondral progenitor cell differentiation inducer (KGN).

[0010] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the embodiments described below, and can be implemented in various ways within the scope of its essence.

[0011] In this embodiment, osteochondral progenitor cells induced from dedifferentiated adipocytes of mammalian origin are provided.

[0012] (Osteochondrocytes) In this specification, “osteochondrocytes” means cells that have the ability to differentiate into osteoblasts and / or chondrocytes.

[0013] In this embodiment, osteochondral progenitor cells are positive for at least one mesenchymal stem cell marker. For example, osteochondral progenitor cells are positive for at least one of RUNX1, RUNX2, CD29, CD44, CD73, CD90, and CD105, and are preferably positive for RUNX1 and RUNX2. Osteochondral progenitor cells may also be negative for molecules not expressed in mesenchymal stem cells, for example, they may be negative for at least one of CD45, CD34, CD14, CD11b, CD79, CD19, and HLA-DR. Furthermore, osteochondral progenitor cells in this embodiment may be negative for SOX9 and / or BGLAP.

[0014] The positivity of osteochondral progenitor cells to mesenchymal stem cell markers can be detected by known methods; for example, positivity can be confirmed by the expression level of the mesenchymal stem cell marker mRNA. In addition to the methods described above, osteochondral progenitor cells may also be identified by the expression of conventionally known stem cell markers (e.g., microtubule-associated protein 2 (MAP2)).

[0015] (Dedifferentiated Adipocytes) The dedifferentiated adipocytes used in this embodiment are obtained by dedifferentiating mature adipocytes using a known method developed by the present inventors (see, for example, Japanese Patent No. 5991687 and Japanese Patent No. 5055613). Specifically, first, mature adipocytes are isolated from adipose tissue by collagenase treatment or the like. Then, the isolated mature adipocytes are cultured using the ceiling culture method to obtain dedifferentiated adipocytes.

[0016] In this embodiment, the dedifferentiated adipocytes are derived from mammals, and examples of mammals include humans, monkeys, dogs, cats, chickens, rabbits, pigs, cows, goats, sheep, mice, rats, guinea pigs, and hamsters. Among these, humans are preferred as the mammal.

[0017] (Method for producing osteochondral progenitor cells) In this embodiment, a method for producing osteochondral progenitor cells is provided, which includes a step of culturing dedifferentiated adipocytes of mammalian origin using a culture medium containing an osteochondral progenitor cell differentiation inducer and substantially free of artificially synthesized proteins (hereinafter sometimes referred to as the "differentiation induction medium").

[0018] In this specification, "substantially free" of synthetic proteins in the differentiation induction medium may mean that the concentration of synthetic proteins in the differentiation induction medium is less than 0.1 μM, 0.05 μM or less, or 0.01 μM or less, or it may mean that the differentiation induction medium is completely free of synthetic proteins.

[0019] According to this embodiment, it is also possible to provide a drug screening method that corresponds to the activity of osteochondral progenitor cells, using the obtained osteochondral progenitor cells. Furthermore, it is possible to produce a large number of differentiated adipocytes from an individual's adipocytes and use the manufacturing method of this embodiment to produce a large number of osteochondral progenitor cells. In addition, the osteochondral progenitor cells obtained by the manufacturing method of this embodiment may be used as a cell source for personalized regenerative medicine for musculoskeletal diseases (e.g., osteoporosis, sarcopenia, osteoarthritis, etc.).

[0020] (Culturing process) The culturing process is the process of culturing dedifferentiated adipocytes using differentiation induction medium.

[0021] The culture temperature can be, for example, 25°C to 40°C, or 30°C to 37°C.

[0022] The culture time can be, for example, 1 to 20 days, or 2 to 15 days, and preferably 3 to 10 days.

[0023] CO2 in the culture process ２ For example, a concentration of 5% CO2 ２ It can be done this way.

[0024] (Differentiation Induction Medium) The differentiation induction medium is a medium that contains a differentiation inducer for osteochondral progenitor cells and is substantially free of artificially synthesized proteins.

[0025] The osteochondral progenitor cell differentiation inducer is not particularly limited, but it is preferably a low-molecular-weight compound having the ability to induce osteochondral progenitor cell differentiation. Here, "ability to induce osteochondral progenitor cell differentiation" means the property of inducing dedifferentiated adipocytes into osteochondral progenitor cells having the above-described functions. Specifically, such low-molecular-weight compounds include, for example, kartogenin (KGN), TGF-β (Transforming Growth Factor-β), bone morphogenetic protein (BMP), and their derivatives. Among these, kartogenin is preferred as the osteochondral progenitor cell differentiation inducer. Kartogenin is C ２０ H １５ NO ３ TGF-β is a small molecule compound that promotes the differentiation of mesenchymal stem cells into chondrocytes (CAS registration number: 4727-31-5, molecular weight: 317.34). TGF-β is a cytokine that regulates cell proliferation, cell differentiation, and other functions. TGF-β exists in at least three isoforms called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibin, activin, anti-Müllerian hormone, osteomorphic proteins, decapentaplesic, and Vg-1. Osteomorphic proteins are proteins that induce the differentiation of osteoblasts and other cells, forming bone, cartilage, tendons, and other tissues present in bone. Approximately 20 families of osteomorphic proteins are known.

[0026] The concentration of the osteochondral progenitor cell differentiation inducer in the differentiation induction medium is preferably 100 μM or less, more preferably 0.5 μM to 90 μM, and even more preferably 1 μM to 80 μM. The concentration of the osteochondral progenitor cell differentiation inducer may also be 10 μM to 50 μM.

[0027] (Other components) In addition to cartogenin, the differentiation induction medium may further contain other components, provided that it does not substantially contain artificially synthesized proteins.

[0028] Other components are not particularly limited and include, for example, components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins), etc.

[0029] The inorganic salts included in the differentiation induction medium are not particularly limited, but are, for example, used to help maintain osmotic equilibrium in cells and to help regulate membrane potential.

[0030] Inorganic salts are not particularly limited and include, for example, salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are not particularly limited and are used in the form of, for example, chlorides, phosphates, sulfates, nitrates, and bicarbonates.

[0031] The weight osmolality of the differentiation induction medium is not particularly limited and may be, for example, 200 mOsm / kg or more and 400 mOsm / kg or less, 290 mOsm / kg or more and 350 mOsm / kg or less, 280 mOsm / kg or more and 310 mOsm / kg or less, or 280 mOsm / kg or more and less than 300 mOsm / kg (specifically, 280 mOsm / kg).

[0032] There are no particular limitations on what constitutes a carbohydrate; examples include glucose, galactose, maltose, and fructose.

[0033] The concentration of carbohydrates (preferably D-glucose) in the differentiation induction medium is not particularly limited, but is preferably, for example, 0.5 g / L or more and 2 g / L or less.

[0034] There are no particular limitations on the amino acids used; for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof are examples.

[0035] The concentration of glutamine contained in the differentiation induction medium is not particularly limited, and may be, for example, 0.05 g / L or more and 1 g / L or less (usually 0.1 g / L or more and 0.75 g / L or less). The concentration of each amino acid other than glutamine contained in the differentiation induction medium is not particularly limited, and may be, for example, 0.001 g / L or more and 1 g / L or less (usually 0.01 g / L or more and 0.15 g / L or less). The amino acids may be synthetically derived.

[0036] There are no particular limitations on the vitamins, and examples include thiamine (vitamin B1), riboflavin (vitamin B2), niacinamide (vitamin B3), D-pantothenic acid hemicalcium (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α tocopherol (vitamin E), biotin (vitamin H), menadione (vitamin K), choline chloride, myo-inositol, and the like.

[0037] The differentiation induction medium may further contain an antibiotic, serum, hormone, or the like.

[0038] There are no particular limitations on the antibiotics, and examples include gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentacin, tyrosin, aureomycin, and the like, which are used in the culture of normal animal cells. These antibiotics may be contained alone or in combination.

[0039] The concentration of the antibiotic contained in the differentiation induction medium is not particularly limited, and may be, for example, 0.1 μg / mL or more and 100 μg / mL or less.

[0040] There are no particular limitations on the serum, and examples include FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf serum), CS (Calf Serum), HS (Horse Serum), and the like.

[0041] The concentration of serum contained in the differentiation induction medium is not particularly limited, and may be, for example, 2% by mass or more and 10% by mass or less.

[0042] The hormones are not particularly limited, and examples include insulin, glucagon, triiodothyronine, adrenal cortical hormones (such as hydrocortisone), etc. These hormones may be contained alone or in combination of a plurality.

[0043] The concentration of the hormone contained in the differentiation induction medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

[0044] Also, as a medium additive containing a hormone, there is no particular limitation, and for example, bovine pituitary extract (Bovine Pituitary Extract: BPE) may be used.

[0045] The differentiation induction medium may be prepared by mixing the above components, or may be prepared by adding the above chondro-osseous progenitor cell differentiation inducer and, if necessary, the above other components, etc. to a medium for culturing chondro-osseous progenitor cells.

[0046] The basic medium for culturing chondro-osseous progenitor cells is not particularly limited, and examples include α-MEM (alpha Modified Eagle Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), complete medium for culturing mesenchymal stem cells (manufactured by Funakoshi), etc., and preferably α-MEM. The differentiation induction medium used in the culturing process may contain a basic medium. ’ s Modified Eagle Medium), complete medium for culturing mesenchymal stem cells (manufactured by Funakoshi), etc., and preferably α-MEM. The differentiation induction medium used in the culturing process may contain a basic medium.

[0047] The fact that cells produced by the method according to this embodiment are osteochondral progenitor cells may be confirmed by the cells being positive for at least one mesenchymal stem cell marker. For example, osteochondral progenitor cells are positive for at least one of RUNX1, RUNX2, CD29, CD44, CD73, CD90, and CD105, and preferably positive for RUNX1 and RUNX2. Osteochondral progenitor cells may also be negative for molecules not expressed in mesenchymal stem cells, for example, they may be negative for at least one of CD45, CD34, CD14, CD11b, CD79, CD19, and HLA-DR. Furthermore, osteochondral progenitor cells produced by the method according to this embodiment may be negative for SOX9 and / or BGLAP.

[0048] The osteochondral progenitor cells produced by the method according to this embodiment have an mRNA expression level of the mesenchymal stem cell marker that is 1.2 times or more, preferably 1.5 times or more, more preferably 1.7 times or more, and even more preferably 2 times or more, compared to cells similarly cultured in a medium that does not contain an osteochondral progenitor cell differentiation inducer. The osteochondral progenitor cells produced by the method according to this embodiment have an mRNA expression level of the mesenchymal stem cell marker that is 1.8 times or more, preferably 2 times or more, more preferably 2.5 times or more, and even more preferably 3 times or more, compared to cells on day 0 of culture. The osteochondral progenitor cells produced by the method according to this embodiment have an mRNA expression level of the mesenchymal stem cell marker that is 1.5 times or more, preferably 1.7 times or more, more preferably 2 times or more, and even more preferably 2.5 times or more, compared to the mRNA expression level of the internal standard GAPDH.

[0049] The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of RUNX1, RUNX2, CD29, CD44, CD73, CD90, and CD105 that is 1.2 times or more, preferably 1.5 times or more, more preferably 1.7 times or more, and even more preferably 2 times or more, compared to cells cultured in the same manner using a medium that does not contain an osteochondral progenitor cell differentiation inducer. The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of RUNX1, RUNX2, CD29, CD44, CD73, CD90, and CD105 that is 1.8 times or more, preferably 2 times or more, more preferably 2.5 times or more, and even more preferably 3 times or more, compared to cells cultured on day 0. The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of RUNX1, RUNX2, CD29, CD44, CD73, CD90, and CD105 that is 1.5 times or more, preferably 1.7 times or more, more preferably 2 times or more, and even more preferably 2.5 times or more, compared to the expression level of the internal standard GAPDH mRNA.

[0050] The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of CD45, CD34, CD14, CD11b, CD79, CD19, HLA-DR, SOX9, and BGLAP that is 0.8 times or less, preferably 0.5 times or less, more preferably 0.3 times or less, and even more preferably 0.2 times or less, compared to cells similarly cultured in a medium that does not contain an osteochondral progenitor cell differentiation inducer. The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of CD45, CD34, CD14, CD11b, CD79, CD19, HLA-DR, SOX9, and BGLAP that is 0.9 times or less, preferably 0.8 times or less, more preferably 0.7 times or less, and even more preferably 0.6 times or less, compared to cells on day 0 of culture. The osteochondral progenitor cells produced by the method according to this embodiment have an expression level of at least one mRNA of CD45, CD34, CD14, CD11b, CD79, CD19, HLA-DR, SOX9, and BGLAP that is 0.8 times or less, preferably 0.5 times or less, more preferably 0.3 times or less, and even more preferably 0.1 times or less, compared to the expression level of the internal standard GAPDH mRNA.

[0051] (Kit for inducing differentiation of dedifferentiated adipocytes from mammals into osteochondral progenitor cells) In this embodiment, a kit for inducing differentiation of dedifferentiated adipocytes from mammals into osteochondral progenitor cells is provided. The kit for inducing differentiation of dedifferentiated adipocytes from mammals into osteochondral progenitor cells in this embodiment includes the "differentiation induction medium" described above. The kit for inducing differentiation of dedifferentiated adipocytes from mammals into osteochondral progenitor cells in this embodiment allows for the simple and efficient production of osteochondral progenitor cells.

[0052] The kit of this embodiment may further include other components in addition to the differentiation induction medium. Other components included in the kit of this embodiment are not particularly limited and include, for example, dedifferentiated adipocytes, culture medium for dedifferentiated adipocytes, petri dishes, flasks, plastic bags, etc. The kit of this embodiment may also include a basic medium and an osteochondral progenitor cell differentiation inducer. Furthermore, the kit of this embodiment may include, in addition to the differentiation induction medium, an accompanying document describing the protocol, etc.

[0053] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0054] [Example 1] 1. Preparation of dedifferentiated adipocytes First, human dedifferentiated adipocytes were prepared from human mature adipocytes using a known method (see, for example, Japanese Patent No. 5991687). Specifically, 5 g of adipose tissue collected from the buccal fat pad located beneath the human buccal mucosa was first mixed with collagenase (Type II) (manufactured by SIGMA) to a final concentration of 0.02 w / v% in NaHCO3. ３ The cells were placed in Dulbecco's modified Eagle medium (DMEM) (SIGMA Corporation) and treated with collagenase. After collagenase treatment, the cells were filtered through a nylon mesh to obtain a cell suspension. The obtained cell suspension was centrifuged at 700 G for 1 minute, and the unilocular lipid fraction separated into the upper layer was added to fresh 10% fetal bovine serum (FBS) supplemented DMEM. This was repeated three times at 700 G for 1 minute each to obtain mature adipocytes as unilocular adipocytes.

[0055] Next, the obtained unicellular adipocytes were transferred to a tissue culture flask (Falcon, 3107), and the flask was completely filled with DMEM containing 20% ​​FBS, 1% penicillin, and streptomycin, and cultured at 37°C under 5% CO2. ２ The flask was placed upright in a carbon dioxide culture apparatus adjusted to a 95% air gas phase and incubated for 7 days.

[0056] After 4 days of culture, most cells firmly adhered to the flask ceiling surface and underwent morphological changes into multicellular adipocytes with various sizes of lipid droplets around large lipid droplets. After 6 days of culture, the lipid droplets became even smaller, and a large number of cells that changed into a fibroblast-like morphology without any lipid droplets were observed.

[0057] After 7 days of culture, the medium in the flask was replaced with DMEM supplemented with 20% FBS, and the culture was continued for 10 days in a carbon dioxide incubator with the cell adhesion surface facing the bottom. The medium was changed every 4 days. Cells without lipid droplets proliferated actively, and after 10 days of culture, the cells in the flask consisted solely of fibroblast-like cells and reached confluence.

[0058] These fibroblast-like cells have an active proliferative ability and the ability to redifferentiate into adipocytes with lipid droplets by differentiation-inducing agents such as dexamethasone (DEX), insulin (INS), and isobutylmethylxanthine (IBMX). Therefore, they were created as dedifferentiated adipocytes derived from unicellular adipocytes.

[0059] Using 1% penicillin and streptomycin and DMEM supplemented with 20% FBS (high glucose), the created dedifferentiated adipocytes were added at a concentration of 1×10 ６ cells / 5 mL / 75-cm ２ flask and cultured. The medium was changed once every 2 - 3 days.

[0060] Subculturing was performed once a week, and subculture was carried out 4 times. After the 4th subculture, the cells were washed with 5 mL of PBS. The washed cells were treated with 2 mL of trypsin-EDTA at 37°C for 2 minutes. The treated cells were collected and centrifuged at 300 g for 1 minute at room temperature. Cells were seeded at a concentration of 1×10 ６ cells / 5 mL / 75-cm ２ flask and cultured.

[0061] 2. Preparation of Differentiation Induction Medium 10 mL of α-MEM (WAKO) was mixed with cartogenin (Selleck) to final concentrations of 0 μM (control), 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM to prepare a differentiation induction medium for osteochondral progenitor cells. As a control, the mRNA expression level of GAPDH was measured.

[0062] 3. Differentiation induction from dedifferentiated adipocytes to osteochondral progenitor cells The dedifferentiated adipocytes prepared in "1." were washed with 5 mL of PBS. The washed cells were treated with 2 mL of trypsin-EDTA at 37°C for 2 minutes. The treated cells were collected and centrifuged at 300 g for 1 minute at room temperature. 3.0 × 10 ５ Seeds were seeded in a 6-well plate to achieve a cell-per-well ratio and incubated for 24 hours.

[0063] The cultured cells were washed with 1 mL of PBS. The washed cells were then cultured in 1 mL of α-MEM for 24 hours.

[0064] The cultured cells were washed with 1 mL of PBS. The washed cells were then cultured for 6 days in 1 mL of differentiation induction medium prepared in step 2. The culture medium was changed once a day.

[0065] 4. Confirmation of mRNA Expression of Osteochondral Progenitor Cell Markers Total RNA was collected from cells differentiated in "3." (culture days 0, 1, 3, and 6) using 1 mL of Trizole. The mRNA expression of various osteochondral progenitor cell markers (RUNX1 and RUNX2) was confirmed by real-time RT-PCR using the primers shown in Table 2 below. Figure 1A-B shows the mRNA expression levels of various markers on culture days 0, 1, 3, and 6 for cells cultured in a medium containing control and 50 μM cartogenin. Figure 2A-B shows the mRNA expression levels of various markers on culture day 3 for cells cultured in a medium containing 0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM cartogenin. Figures 1A-B and 2A-B show the relative expression levels of various osteochondral progenitor cell markers (RUNX1 and RUNX2) to the expression level of GAPDH mRNA.

[0066] Similar to the method described above, the mRNA expression of various osteochondral progenitor cell markers (SOX9 and BGLAP) was confirmed. Figure 3A-B shows the mRNA expression levels of various markers on days 0, 1, 3, and 6 of culture for cells cultured in a medium containing control and 50 μM cartogenin. Figure 3A-B shows the relative ratio of mRNA expression levels of various osteochondral progenitor cell markers (SOX9 and BGLAP) to the mRNA expression level of GAPDH.

[0067]

[0068] Figures 1A-B show that the mRNA expression of various markers in osteochondral progenitor cells increased on days 3 and 6 of culture. Figure 2A shows that RUNX1 mRNA expression increased in cells cultured in a medium containing 50 μM cartogenin compared to cells cultured in a medium containing 0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 20 μM cartogenin. Furthermore, Figure 2B shows that the expression of RUNX2 mRNA tended to increase with increasing cartogenin concentration in the culture medium. On the other hand, a tendency for SOX9 and BGLAP mRNA expression to decrease was observed.

[0069] From the above, it has been shown that osteochondral progenitor cells can be produced by culturing dedifferentiated adipocytes of mammalian origin in a culture medium that contains an osteochondral progenitor cell differentiation inducer and is substantially free of artificially synthesized proteins.

[0070] According to the manufacturing method and kit of this embodiment, osteochondral progenitor cells can be produced simply and efficiently.

Claims

1. Osteochondragon progenitor cells induced from dedifferentiated adipocytes of mammalian origin.

2. The osteochondral progenitor cells according to claim 1, which are positive for the RUNX1 and RUNX2 markers.

3. A method for producing osteochondral progenitor cells, comprising the step of culturing dedifferentiated adipocytes of mammalian origin in a medium containing an osteochondral progenitor cell differentiation inducer and substantially free of artificially synthesized proteins.

4. The method according to claim 3, wherein the osteochondral progenitor cell differentiation inducer is cartogenin.

5. The method according to claim 3, wherein the concentration of the osteochondral progenitor cell differentiation inducer in the culture medium is 100 μM or less.

6. The method according to any one of claims 3 to 5, wherein the mammal is a human.

7. A culture medium for inducing the differentiation of dedifferentiated adipocytes of mammalian origin into osteochondral progenitor cells, comprising an osteochondral progenitor cell differentiation inducer and substantially free of artificially synthesized proteins.

8. The culture medium according to claim 7, wherein the osteochondral progenitor cell differentiation inducer is Kartogenin.

9. The culture medium according to claim 7, wherein the concentration of the osteochondral progenitor cell differentiation inducer in the culture medium is 50 μM or less.

10. A kit for inducing differentiation of dedifferentiated adipocytes of mammalian origin into osteochondral progenitor cells, comprising the culture medium described in any one of claims 7 to 9.

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