Composition for promoting ciliogenesis containing mesenchymal stem cells or mesenchymal stem cell culture medium as an active ingredient

Abstract

The present invention provides a composition for ciliogenesis comprising mesenchymal stem cells or mesenchymal stem cell cultures. The mesenchymal stem cells or mesenchymal stem cell cultures according to the present invention have the effect of increasing not only the number but also the length of primary cilia of cells. The mesenchymal stem cells or mesenchymal stem cell cultures according to the present invention can be used as pharmaceutical compositions for the prevention or treatment of ciliary dysplasia or diseases caused by cilia damage. The mesenchymal stem cells or mesenchymal stem cell cultures according to the present invention can be used as cosmetic compositions.

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0036871, filed on March 26, 2020, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a composition for promoting ciliogenesis, the active ingredient of which is mesenchymal stem cells or a mesenchymal stem cell culture medium. [Background technology]

[0003] Stem cells can differentiate into various types of cells, and extensive research is being conducted into the possibility of utilizing these stem cell properties in cell therapy. Embryonic stem cells, which have pluripotency, have been attracting attention as a cell therapy agent due to their ability to differentiate into various types of cells, but safety and ethical issues have made their practical use difficult.

[0004] To avoid these safety and ethical issues, many researchers have become interested in adult stem cells as an alternative to embryonic stem cells. Adult stem cells are cells that can self-proliferate in an undifferentiated state and differentiate into multiple tissue cells. The earliest known adult stem cells were bone marrow stem cells (BMSCs), which were derived from bone marrow. They have since been found to exist in various parts of the body, including the umbilical cord, blood, and fat.

[0005] Among these adult stem cells, mesenchymal stem cells have a high self-renewal capacity and can differentiate into various tissues, and research utilizing these stem cells is currently being actively conducted.

[0006] Mesenchymal stem cells have the ability to regenerate muscle, cartilage, bone tissue, ligaments, bone marrow matrix, etc. through differentiation. Furthermore, through the action of various proteins secreted by mesenchymal stem cells themselves, they induce homeostasis restoration (inflammation suppression, angiogenesis, cell attraction, immunomodulation, etc.) at the lesion site. This is called the paracrine action of mesenchymal stem cells. Because of this paracrine action, mesenchymal stem cells are also called "drug stores" or "drug factories."

[0007] Cilia are hair-like organelles that protrude from the cell surface. They are present in most cells of animals, including humans, but have long been known as vestigial organs with no essential functions. However, interest in cilia has been growing as they are now known to regulate the cell cycle and signal transduction of growth factors crucial for maintaining tissue homeostasis, such as sonic hedgehog (SHH), platelet-derived growth factor (PDGF), Wnt, and Notch. They also act as a hub for biosignal input and output, sensing chemical senses such as smell, taste, and osmotic pressure, and physical senses such as sight and hearing, and controlling the signaling system.

[0008] The specific roles of cilia are listed in Table 1 below. [Table 1]

[0009] Ciliopathy, a disorder of cilia, is a general term for diseases caused by abnormal cilia function or structure.

[0010] Specifically, it manifests in various forms, including genetic disorders such as Bardet-Biedl syndrome (BBS), Joubert syndrome, Meckel-Gruber syndrome, Leber congenital amaurosis, and Alstrom syndrome, as well as polycystic kidney disease (PKD), renal cysts, infertility, respiratory disorders, situs inversus, cancer, and metabolic disorders such as diabetes, obesity, and hypertension. However, no therapeutic agents have yet been developed for these diseases.

[0011] Specific symptoms of ciliogenesis disorders include obesity in Bardet-Biedl syndrome and Alström syndrome, and just like in human hereditary ciliopathy disorders, mice with ciliogenesis disorders also develop obesity and hyperphagia. The reason for this is known to be a decrease in the action of leptin, an appetite-suppressing hormone, in neurons in the hypothalamus.

[0012] It has also been reported that the absence of cilia in the skin inhibits hair production and keratin differentiation, and it is known that when primary cilia are activated by SHH (sonic hedgehog)-smoothened-GLI2 signaling in relation to skin pigment production, melanin production is inhibited.

[0013] Recently, as the diverse functions of cilia have become known, it is believed that cilia disorders are one of the causes of imbalances in whole-body metabolic homeostasis in the pathogenesis of intractable metabolic diseases such as diabetes, obesity, and hypertension. Restoring cilia function is also emerging as a target for the treatment of various diseases, including metabolic diseases. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Republic of Korea 10-2020-0000415 [Patent Document 2] Republic of Korea 10-2056172 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention provides a composition for promoting ciliogenesis using mesenchymal stem cells or a mesenchymal stem cell culture medium.

[0016] The present invention provides a composition for treating or preventing diseases caused by abnormalities in cilia production, using mesenchymal stem cells or a mesenchymal stem cell culture medium.

[0017] Another object of the present invention is to provide a cosmetic composition that promotes hair production, promotes keratin differentiation, and induces the suppression of melanin production through the promotion of ciliogenesis using mesenchymal stem cells or mesenchymal stem cell culture medium. [Means for solving the problem]

[0018] To achieve the above object, the present invention provides a composition for promoting ciliogenesis, which contains a mesenchymal stem cell culture medium as an active ingredient.

[0019] The present invention also provides a method for treating or preventing a disease caused by ciliogenesis abnormalities by administering a mesenchymal stem cell culture medium to an individual.

[0020] The present invention provides a composition for ciliogenesis comprising mesenchymal stem cells.

[0021] The present invention also provides a method for treating or preventing a disease caused by dyscilia formation by administering mesenchymal stem cells to an individual.

[0022] The present invention provides a method for inducing ciliogenesis by co-culturing mesenchymal stem cells with cells capable of developing cilia.

[0023] The present invention also provides a method for treating diseases caused by abnormal ciliogenesis by inducing ciliogenesis by co-culturing mesenchymal stem cells with cells capable of generating cilia or cells with abnormalities in ciliogenesis in vitro, and administering the co-cultured cells capable of generating cilia or cells with abnormalities in ciliogenesis to an individual.

[0024] The present invention provides a cosmetic composition for skin whitening and / or inducing ciliogenesis in skin keratinocytes, which contains a mesenchymal stem cell culture medium as an active ingredient.

[0025] The present invention also provides a method for whitening skin and / or inducing ciliogenesis in keratinocytes by applying or treating skin with a cosmetic composition containing a mesenchymal stem cell culture medium. [Effects of the Invention]

[0026] It has been confirmed that the length of cilia increases when cells are treated with a composition containing the mesenchymal stem cell culture medium of the present invention as an active ingredient, and thus the culture medium can be used to prevent or treat ciliary disorders.

[0027] When the mesenchymal stem cells of the present invention are co-cultured with cells having ciliary disorders, the cilia of the cells are elongated, and the cells and / or mesenchymal stem cells with restored function can be used to treat ciliary disorder diseases.

[0028] It has been confirmed that the length of cilia increases when cells are treated with a composition containing the mesenchymal stem cell culture medium of the present invention as an active ingredient, and thus the culture medium can be used as a cosmetic composition that induces ciliogenesis in skin keratinocytes. [Brief explanation of the drawings]

[0029] [Figure 1]This shows the results of primary cilia development when RPE cell lines were treated with culture medium (10X) of mesenchymal stem cells of various origins isolated and prepared by the method of Example 1. Figure 1A shows the results of observing the shape of cilia developed in each treatment group under a fluorescence microscope after fluorescent staining (double staining of nuclei and cilia), and Figure 1B shows the results of measuring cilia development and cilia length in each treatment group. [Figure 2] Figure 2 shows the results of primary cilia development when the RPE / Smo-GFP cell line was treated with various concentrations (1X, 2X, 5X) of culture medium containing mesenchymal stem cells of various origins, isolated and prepared according to the method of Example 1. Figure 2A shows the results of observing the cell morphology in each treatment group using a fluorescence microscope, and Figure 2B shows the results of measuring cilia development and cilia length in each treatment group. [Figure 3] This shows the results of primary cilia development in RPE cell lines when co-cultured with mesenchymal stem cells of various origins isolated in Example 1. Figure 3A shows the results of observing the shape of cilia generated in co-culture with each mesenchymal stem cell under a fluorescence microscope after fluorescent staining (double staining for nuclei and cilia), and Figure 3B shows the results of measuring cilia development and cilia length in each treatment group. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be described in more detail below. In the following description, descriptions of well-known and well-known technologies that are well-known to those skilled in the art may be omitted. Furthermore, in describing the present invention, if a detailed description of related known technologies or configurations is deemed to unnecessarily limit the scope of the present invention, such description may be omitted. Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intentions of users and implementers, the practices in the field to which the present invention pertains, etc.

[0031] Therefore, the definitions of these terms should be based on the contents throughout this specification. Throughout this specification, when a part "encompasses" a certain element, this does not mean that other elements are excluded, and means that other elements may also be included, unless otherwise specified.

[0032] The present invention provides a composition for ciliogenesis, comprising a mesenchymal stem cell culture medium.

[0033] The present invention provides a composition for ciliogenesis comprising mesenchymal stem cells.

[0034] The present invention provides a pharmaceutical composition for preventing or treating ciliary dysplasia or ciliary damage, comprising a mesenchymal stem cell culture medium.

[0035] The present invention provides a pharmaceutical composition for preventing or treating ciliary dysplasia or ciliary damage, comprising mesenchymal stem cells.

[0036] The present invention provides a method for inducing ciliogenesis in abnormally ciliated cells by co-culturing mesenchymal stem cells and abnormally ciliated cells ex vivo.

[0037] The present invention provides a cosmetic composition containing a mesenchymal stem cell culture medium.

[0038] The term "ciliopathy" refers to any disease caused by abnormal cellular cilia or the function or structure of cilia. In this specification, "ciliopathy" and "disease caused by ciliogenesis abnormalities" are used interchangeably.

[0039] Ciliary dysplasias are generally known to be associated with proteins localized in motile or non-motile cilia or centrosomes, and are known to be caused by genetic defects. However, the associated disease genes are not fully known, and no therapeutic agents for the disease have yet been developed.

[0040] The ciliary dysplasia is not particularly limited as long as it is a disease caused by abnormal cellular cilia function or structure, and may be, for example, Joubert syndrome, Bardet-Biedl syndrome, nephronophthisis, Meckel-Gruber syndrome, Leber congenital amaurosis, polycystic kidney disease (PKD), renal cysts, infertility, respiratory disorders, situs inversus, obesity, hypertension, cancer, etc.

[0041] The ciliated or damaged cells may be selected from, but not limited to, ciliated epithelial cells, retinal cells, skin cells, and auditory cells.

[0042] The term "stem cell" as used herein refers to a cell that has the ability to self-renew and differentiate into two or more types of cells, including adult stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells. The term "adult stem cell" refers to a stem cell that appears at the stage when various organs are formed in the embryo or at the adult stage during the developmental process.

[0043] As used herein, the term "mesenchymal stem cells" refers to multipotent progenitor cells derived from the mesenchyme prior to differentiation into cells of specific organs, such as bone, cartilage, fat, tendon, neural tissue, fibroblasts, and muscle cells. In the present invention, mesenchymal stem cells are contained in a composition in an undifferentiated state. The mesenchymal stem cells of the present invention may be derived from human umbilical cord, Wharton's jelly, umbilical cord blood, placenta, adipose tissue, bone marrow, tonsil, human embryonic yolk sac, umbilical cord, skin, peripheral blood, muscle, liver, neural tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, crescentic cartilage, anterior cruciate ligament, articular chondrocytes, deciduous teeth, perivascular cells, trabecular bone, subpatellar fat pad, splenomegaly, and thymus. Preferably, the tissue can be derived from one or more selected from the group consisting of human umbilical cord, Wharton's jelly, umbilical cord blood, placenta, fat, bone marrow, and tonsils, and is preferably derived from human umbilical cord, Wharton's jelly, umbilical cord blood, or adipose tissue, but the origin is not limited thereto.

[0044] The mesenchymal stem cells are preferably derived from humans, but may also be derived from fetuses or other mammals other than humans, such as canines, felines, primates, cows, sheep, pigs, horses, rats, mice, or guinea pigs, and are not limited to specific origins.

[0045] In the present invention, "adipose tissue-derived mesenchymal stem cells (AM)" may be isolated from human adipose tissue. Suitable human adipose tissue may refer to cells containing mature adipocytes and the connective tissue surrounding them. Regardless of the location in an individual's body, any adipose tissue obtained by any method used to harvest fat may be used.

[0046] In the present invention, "placenta-derived mesenchymal stem cells (PLM)" includes all stem cells isolated from the placenta, and preferably includes four types of stem cells isolated from ex vivo human placenta: (1) human amniotic epithelial cells (hAECs), (2) human amniotic mesenchymal stromal cells (hAMSCs), (3) human chorionic mesenchymal stromal cells (hCMSCs), and (4) human chorionic trophoblastic cells (hCTCs).

[0047] In the present invention, "umbilical cord blood derived mesenchymal stem cells (UCBM)" refers to only mesenchymal stem cells, excluding blood stem cells, among the stem cells present in the umbilical cord after birth.

[0048] In the present invention, "umbilical cord-derived stem cells (Wharton's Jelly-derived mesenchymal stem cells; WJM)" includes all stem cells isolated from the umbilical cord, and may refer to the cord that connects the mother's body to the abdomen so that a mammalian fetus can grow in the placenta. Generally, it may refer to tissue composed of three blood vessels, namely, two umbilical arteries and one umbilical vein, surrounded by Wharton's Jelly.

[0049] The active ingredient of the composition for cilia generation of the present invention is, but is not limited to, mesenchymal stem cells, mesenchymal stem cell culture medium or a concentrate thereof, or an extract of the active ingredient.

[0050] The term "culture media" as used herein refers to a culture medium capable of supporting the growth and survival of stem cells under in vitro culture conditions, and includes all commonly used media suitable for stem cell culture. Furthermore, the medium and culture conditions can be selected depending on the type of cells. Media used for culture include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle's Medium (BME), RPMI 1640, F-10, F-12, α-modified Minimum Essential Media (α-MEM), Glasgow's Minimal Essential Medium (GMEM), and Iscove's Modified Dulbecco's Medium (IMDM).

[0051] The present invention is based on α-MEM (α-modified Minimum Essential Media) which is used in the industry for cell culture, and contains serum commonly used for cell culture.

[0052] The serum used is preferably 0.1 to 20% fetal bovine serum (FBS), and it is preferable to use antibiotics, antifungals, and substances commonly used in the relevant industry that prevent the growth of mycoplasma that causes contamination.

[0053] As antibiotics, all antibiotics commonly used in cell culture, such as penicillin and streptomycin, can be used. As antifungal agents, it is preferable to use commonly used substances such as amphotericin B, and as mycoplasma inhibitors, gentamicin, ciprofloxacin, and azithromycin.

[0054] In the present invention, mesenchymal stem cells are isolated from various mesenchymal tissues such as human umbilical cord, placenta, umbilical cord blood, and adipose tissue by a known method suitable for each tissue, and then each mesenchymal stem cell is subcultured in α-MEM (α-modified Minimum Essential Media) medium containing 10% fetal bovine serum (FBS) and 0.5% gentamicin (10 mg / ml).

[0055] To prepare the mesenchymal stem cell culture medium for cilia development, mesenchymal stem cells from the fourth passage (P4) were cultured in α-MEM medium containing 10% fetal bovine serum and 0.5% gentamicin (10 mg / ml). When the cells reached 70% confluency, they were cultured in serum-free α-MEM medium alone for 24 hours. The culture medium was then concentrated (2X, 5X, or 10X) or used at the original concentration (1X).

[0056] The mesenchymal stem cell culture media (5X, 10X) of the present invention, derived from various sources, were shown to induce the development of primary cilia in retinal pigment epithelial cells (RPE cells) at levels equal to or higher than those of the positive controls SAG and GSK-503.

[0057] In the present invention, a co-culture method with mesenchymal stem cells can be used to induce the development of primary cilia in cells. The mesenchymal stem cells of the present invention and the target cells for inducing cilia development can be cultured in a commonly used Transwell culture medium. TM Co-culture is performed using a chamber system. Specifically, target cells that induce cilia formation are cultured in the lower well, with a permeable membrane as the boundary, and mesenchymal stem cells are cultured in the transwell. Direct contact between the two types of cells is prevented, and the target cells receive factors secreted by the mesenchymal stem cells, causing them to produce cilia.

[0058] Co-culture of mesenchymal stem cells of various origins and retinal pigment epithelial cells (RPE cells) of the present invention also showed the induction of primary cilia at the same or higher levels than the positive control groups SAG and GSK-503.

[0059] The present invention provides a method for treating or preventing ciliopathy, a disease caused by cilia abnormalities, by administering a mesenchymal stem cell culture medium or mesenchymal stem cells to an individual.

[0060] In order to treat ciliary dysplasia, the present invention provides a cell therapy agent that can be injected back into a patient after isolating cells with abnormal ciliogenesis from the patient and improving ciliogenesis by treating them with a mesenchymal stem cell culture medium or by ex-vivo co-culturing them with mesenchymal stem cells.

[0061] The present invention also provides a method for treating ciliary dysplasia, which includes isolating cells with abnormalities in ciliogenesis from a patient, treating them with a mesenchymal stem cell culture medium or by ex-vivo co-culturing them with mesenchymal stem cells to improve ciliogenesis, and then injecting the cells back into the patient.

[0062] The present invention provides a cosmetic composition that uses a mesenchymal stem cell culture medium to promote hair production, keratin differentiation, and melanin production inhibition through the promotion of ciliogenesis.

[0063] The present invention also provides a method for whitening skin and / or inducing ciliogenesis in keratinocytes by applying or treating skin with a cosmetic composition containing a mesenchymal stem cell culture medium.

[0064] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier, which includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0065] In addition to the above-mentioned carrier components, the pharmaceutical composition of the present invention may additionally contain lubricants, wetting agents, binders, glidants, surfactants, fillers, disintegrants, thickeners, stabilizers, diluents, preservatives, sweeteners, etc. Suitable pharmaceutically acceptable carriers and other formulations are within the level of those skilled in the art.

[0066] The pharmaceutical composition according to the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injection solutions by conventional methods and used.

[0067] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, or the like.

[0068] Determining the appropriate dosage of the pharmaceutical composition of the present invention is within the level of a person of ordinary skill in the art, and may vary depending on the formulation method, administration method, age, weight, disease, health condition of the patient, etc. The effective dosage can be estimated from dose-response curves obtained from in vitro experiments or animal model tests.

[0069] The pharmaceutical composition of the present invention may be used to treat diseases associated with abnormal cilia expression, which manifest in various forms, including human hereditary ciliopathy disorders such as Bardet-Biedl syndrome (BBS) and Alstrom syndrome, as well as polycystic kidney disease (PKD), renal cysts, infertility, respiratory disorders, situs inversus, cancer, and metabolic disorders such as diabetes, obesity, and hypertension.

[0070] When the composition of the present invention is prepared as a cosmetic composition, it may be prepared in any dosage form commonly prepared in the art, such as, but not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, etc. More specifically, it may be prepared in the dosage form of a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

[0071] When the dosage form of the present invention is a paste, cream or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or the like.

[0072] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, hydroxyaluminum, or calcium silicate can be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be additionally contained.

[0073] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, aliphatic esters of glycerol, polyethylene glycol or fatty acid esters of sorbitan.

[0074] When the dosage form of the present invention is a suspension, the carrier component may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant.

[0075] When the dosage form of the present invention is a surfactant-containing cleanser, the carrier component may be fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0076] The components contained in the cosmetic composition of the present invention include, in addition to the mesenchymal stem cell culture medium as an active ingredient and a carrier component, components commonly used in cosmetic compositions, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0077] The content of mesenchymal stem cells or their culture medium contained in the cosmetic composition of the present invention is, but is not limited to, 0.001 to 10 wt%, more preferably 0.01 to 5 wt%, based on the total weight of the entire composition. If the content is less than 0.001 wt%, the desired effects of promoting hair growth, whitening, or promoting differentiation of keratinocytes cannot be expected, while if it exceeds 10 wt%, safety or formulation issues may arise.

[0078] Hereinafter, preferred examples will be presented to aid in understanding the present invention. However, the following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope of the scope and technical idea of ​​the present invention, and it is natural that such changes and modifications also fall within the scope of the appended claims.

[0079] Example 1: Preparation of culture medium for umbilical cord tissue-derived mesenchymal stem cells Human umbilical cord, placenta, and umbilical cord blood were collected with the consent of the mother after approval by the Institutional Review Board of Samsung Medical Center (IRB), and adipose tissue was also collected with the consent of the patient after approval by the IRB of Samsung Medical Center.

[0080] Human placenta-derived mesenchymal stem cells (PLM) were isolated from placentas by the previously reported method of CHOI et al. (PLoS ONE, (2017) 12(2): e0172642). Umbilical cord blood-derived mesenchymal stem cells (UCBM) were isolated from umbilical cord blood by the method of KIM et al. (FEBS Letters, (2010) 584: 3601-3608). Umbilical cord-derived mesenchymal stem cells (WJM) were isolated from umbilical cords by the method of PARK et al. (Arch. Pharm. Res. (2016) 39: 1171-1179).

[0081] Adipose tissue-derived mesenchymal stem cells (AMs) were isolated from adipose tissue by the previously reported method of Palumbo et al. (Int. J. Mol. Sci. (2018) 19:1987).

[0082] Each of the obtained mesenchymal stem cells was subcultured in α-MEM (α-modified Minimum Essential Media) medium containing 10% fetal bovine serum (FBS) and 0.5% gentamicin (10 mg / ml).

[0083] To prepare the mesenchymal stem cell culture medium for primary cilia development, mesenchymal stem cells from the fourth passage (P4) were cultured in α-MEM medium containing 10% fetal bovine serum and 0.5% gentamicin (10 mg / ml). When the cells reached 70% confluency, they were cultured in serum-free α-MEM medium alone for 24 hours. The resulting culture medium was then concentrated or used at the original concentration.

[0084] The mesenchymal stem cell culture medium was concentrated 2-fold, 5-fold, or 10-fold using an Amicon Ultra 3K filter unit (MERCK).

[0085] Example 2: Ciliogenesis in RPE cells upon treatment with mesenchymal stem cell culture medium (10X) To analyze the effect of mesenchymal stem cell culture medium on primary cilia growth, retinal pigment epithelium cells (human telomerase-immotalized retinal pigment epithelium cells, hTERT RPE-1 cell line, CRL-4000; RPE cell line) purchased from ATCC were used.

[0086] RPE cell lines were cultured in a 5% (v / v) CO2 incubator for 37 o The cells were cultured in α-MEM medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 0.5% gentamicin under the temperature condition of 0°C.

[0087] Cultured RPE cell lines were seeded into 12-well plates (8x10 4 The RPE cells were cultured at 100x the concentration of 1000 cells / well for 24 hours to stabilize, and then the medium was replaced with the 10x concentrated mesenchymal stem cell culture medium prepared in Example 1. After 24 hours, the RPE cells were observed and the primary cilia were analyzed. As a positive control for cilia development in the RPE cell line, 1 mM SAG and 10 mM GSK-503 were added to α-MEM medium, and as a negative control, α-MEM medium alone was used for culture.

[0088] After culturing, the primary cilia generated in the RPE cell lines were fluorescently stained with ARL13B for the primary cilia and Hoechst33342 for the nuclei. The number of cells and primary cilia were then directly counted under a fluorescence microscope, and the length of the primary cilia was directly measured to analyze the number and length of the primary cilia.

[0089] After staining, the cells were observed under a fluorescence microscope (Figure 1A). The percentage of cells with primary cilia and the length of the cilia were analyzed (Figure 1B).

[0090] The results in Figure 1B confirm that when mesenchymal stem cell culture medium isolated from adipose tissue (AM), placenta (PLM), umbilical cord blood (UCBM), and umbilical cord (Wharton's jelly (WJM)) tissue was treated with a 10-fold concentrated medium under ciliogenesis-inducing conditions in RPE cell lines, primary cilia were generated at levels equal to or higher than those of the positive controls SAG and GSK-503 (3-5 times higher than the negative control group, and approximately 1.2 times higher than the positive control group).

[0091] In addition, under the same conditions, the length of the cilia was also found to be longer than that of the negative and positive control groups (1.5 to 2 times longer than that of the negative control group, and approximately 1.2 times longer than that of the positive control group).

[0092] Example 3: Ciliogenesis in RPE cells when treated with mesenchymal stem cell culture medium (1X, 2X, 5X) at different concentrations To directly analyze the actual cilia formed, we used the RPE / Smo-GFP cell line, which expresses the Smo (smoothened protein)-GFP fusion gene, which is specifically expressed in cilia, in an RPE cell line.

[0093] The 1X, 2X, and 5X concentrated solutions of each mesenchymal stem cell (adipose tissue (AM), placenta (PLM), umbilical cord blood (UCBM), and umbilical cord (WJM)) culture medium prepared in Example 1 were applied to the RPE / Smo-GFP cell line under the same ciliogenesis induction conditions as in Example 2, and cilia formation and length were analyzed using a fluorescence microscope.

[0094] After the culture was completed, the cells were observed under a fluorescent microscope (Figure 2A). The percentage of cells that had primary cilia and the length of the cilia were analyzed (Figure 2B).

[0095] The results in Figure 2B confirm that the promotion of cilia development and the increase in cilia length are proportional to the concentration of mesenchymal stem cell culture medium. In particular, a 5X culture medium concentration promoted cilia development in the RPE / Smo-GFP cell line at a level equal to or higher than that of the positive control (1.5-2.5 times higher than the negative control, and approximately 1.2 times higher than the positive control). Furthermore, the 5X culture medium concentration also increased cilia length (1.5-2 times higher than the negative control).

[0096] Example 4: Ciliogenesis in RPE cells when co-cultured with mesenchymal stem cells Co-culture with RPE cell lines and mesenchymal stem cells was performed using Transwell in a serum-free environment. TM Using a chamber, RPE cell lines were cultured in the lower wells and mesenchymal stem cells were cultured in the transwells. In the positive control group, RPE cells were cultured in the lower wells and in α-MEM medium supplemented with 1 mM SAG and 10 mM GSK-503 in the transwells. In the negative control group, RPE cell lines were cultured in the lower wells and in α-MEM medium in the transwells.

[0097] At this time, the same number of RPE cell lines were cultured in the negative control group, the positive control group, and the mesenchymal stem cell co-culture group, and cultured in the same amount of culture medium.

[0098] After co-culture, the RPE cells in the lower well were fluorescently stained in the same manner as in Example 2, and the development and length of cilia were analyzed.

[0099] After the culture was completed, the cells were observed under a fluorescent microscope (Figure 3A). The percentage of cells that had primary cilia and the length of the cilia were analyzed (Figure 3B).

[0100] Co-culture with mesenchymal stem cells promoted cilia development in RPE cell lines at levels equal to or higher than those of the positive controls SAG and GSK-503 (2-2.5 times higher than the negative control, and approximately 1.1 times higher than the positive control), and it was confirmed that cilia also increased in length (1.5-2 times higher than the negative control, and 1.2-1.3 times higher than the positive control).

[0101] Although the present invention has been described above using limited examples and drawings, the present invention is not limited thereto, and it goes without saying that various modifications and variations can be made by a person having ordinary knowledge in the technical field to which the present invention pertains within the technical spirit of the present invention and the equivalent scope of the claims set forth below.

Claims

1. A pharmaceutical composition for preventing or treating primary ciliary dysplasia or primary cilia damage, comprising a mesenchymal stem cell culture medium.

2. The pharmaceutical composition of claim 1 , wherein the mesenchymal stem cells are derived from bone marrow matrix, adipose tissue, umbilical cord, cartilage, placenta, or umbilical cord blood.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the primary ciliogenesis disorder is selected from Bardet-Biedl syndrome (BBS), Alstrom syndrome, polycystic kidney disease (PKD), renal cysts, respiratory diseases, infertility, situs inversus, cancer, and metabolic diseases such as obesity, diabetes, and hypertension.

4. A pharmaceutical composition for preventing or treating primary ciliary dysplasia or primary cilia damage, comprising mesenchymal stem cells.

5. The pharmaceutical composition of claim 4, wherein the mesenchymal stem cells are derived from bone marrow matrix, adipose tissue, umbilical cord, cartilage, placenta, or umbilical cord blood.

6. 6. The composition according to claim 4 or 5, wherein the primary ciliogenesis disorder is selected from Bardet-Biedl syndrome (BBS), Alstrom syndrome, polycystic kidney disease (PKD), renal cysts, respiratory diseases, infertility, situs inversus, obesity, diabetes, hypertension, and cancer.

7. A method for inducing primary cilia development in cells having abnormal primary cilia by co-culturing mesenchymal stem cells and cells having abnormal primary cilia ex vivo.

8. The method described in claim 7, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow matrix, adipose tissue, umbilical cord, cartilage, placenta, or umbilical cord blood.

9. The method described in claim 7 or 8, wherein the cells having abnormal primary cilia are cells having an abnormality in primary cilia development selected from ciliated epithelial cells, retinal cells, skin cells, and auditory cells.

10. A cosmetic composition for primary cilia development, comprising a mesenchymal stem cell culture medium.

11. The cosmetic composition according to claim 10, wherein the mesenchymal stem cells are derived from bone marrow matrix, adipose tissue, umbilical cord, cartilage, placenta, or umbilical cord blood.

12. The cosmetic composition according to claim 10 or 11, which is used for promoting hair growth, whitening skin, or protecting cells through skin keratinization.

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