Composition for the treatment of cartilage-related diseases and method for manufacturing the same

Abstract

The present invention relates to a pharmaceutical composition for treating cartilage-related diseases prepared by applying electrical stimulation and a method for preparing the same, and the composition of the present invention can be prepared by applying electrical stimulation only to stem cell aggregates without introducing external growth factors, and has the advantage of being able to drastically reduce medical costs since it can be prepared without using expensive growth factors. In addition, the composition for treating cartilage-related diseases according to the present invention does not express COL2, a marker of mature chondrocytes, compared to cells prepared for existing cartilage-related disease treatment, and therefore has the advantage of having fewer immune-related side effects compared to mature cells.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating cartilage-related diseases produced by applying electrical stimulation and a method for producing the same.

Background Art

[0002] Damage to articular cartilage is a very common problem experienced by millions of people. Since articular cartilage tissue is avascular and does not have stem cells, the cartilage regeneration ability of adults is limited. Defects extending to subchondral bone induce the formation of fibrous or fibrocartilaginous tissue, and the repaired tissue is biochemically and biomechanically different from hyaline cartilage and will experience immature degeneration. When problems occur in the joints, it will cause pain and many restrictions in movement. In the past, there were no appropriate treatment methods, and due to a relatively short average lifespan compared to now, there was no significant difference between the lifespan of joints and the actual lifespan. However, currently, although lifespan has increased due to aging, the lifespan of joints has not kept up with the actual lifespan due to various environmental factors. Excessive use of the knee wears down cartilage, and at the same time, inflammation occurs in the knee joint and ligaments, leading to degenerative arthritis. Eight out of every ten elderly people aged 65 and above in the Republic of Korea suffer from degenerative arthritis. Degenerative arthritis, which was only known as an elderly disease, has now become a factor causing arthritis in the younger generation due to incorrect lifestyle habits and an increase in traumatic arthritis caused by excessive exercise.

[0003] To treat such cartilage-related diseases, methods such as using artificial cartilage containing chondrocytes separated from rib cartilage (Korean Application No.: 10-2006-0106812) or utilizing stem cells for treatment are being studied. However, the method of utilizing stem cells mainly uses a method of differentiating into chondrocytes through the addition of expensive growth factors, so the cost problem must be solved for clinical application. Since such problems as cost and immunity have not yet been solved, there is a need to develop a therapeutic agent for treating cartilage-related diseases that solves such problems. [Overview of the project] [Problems that the invention aims to solve]

[0004] Against the background described above, the inventors conducted research and efforts to develop a therapeutic agent that can treat cartilage-related diseases without the addition of growth factors. As a result, they completed the present invention by confirming that when electrical stimulation is applied to stem cells, they do not express COL2, a marker for mature chondrocytes, and can differentiate into chondrocyte precursor cells that possess the characteristics of chondrocytes.

[0005] Therefore, the present invention aims to provide a pharmaceutical composition for the treatment or prevention of cartilage-related diseases, comprising chondrocytes or aggregates thereof having the following characteristics as an active ingredient: (a) not expressing Col2; (b) the chondrocytes are stained with one or more of the following substances selected from the group consisting of Alcian Blue, Safranin O, and Toluidine Blue.

[0006] Furthermore, the present invention also aims to provide a method for producing a pharmaceutical composition for the treatment or prevention of cartilage-related diseases, comprising the step of producing chondrocytes or aggregates thereof by applying electrical stimulation to stem cells; the chondrocytes having the following characteristics: (a) not expressing Col2; (b) the chondrocytes being stained with one or more of the substances selected from the group consisting of Alcian blue, safranin O, and toluidine blue.

[0007] Furthermore, the present invention aims to provide a method for treating or preventing cartilage-related diseases, comprising the step of administering chondrocytes or aggregates thereof having the following characteristics to an individual in need: (a) not expressing Col2; (b) the chondrocytes are stained with one or more of the following substances selected from the group consisting of Alcian blue, safranin O, and toluidine blue.

[0008] Furthermore, the present invention provides the use of chondroprogenitor cells or aggregates thereof having the following characteristics for the manufacture of agents for the treatment or prevention of cartilage-related diseases: (a) They do not express Col2; (b) The chondroprogenitor cells are subjected to substrate staining with one or more selected from the group consisting of Alcian blue, safranin O, and toluidine blue.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] To achieve the above-described objectives of the present invention, the present invention provides a pharmaceutical composition for the treatment or prevention of cartilage-related diseases, comprising chondrocytes or aggregates thereof having the following characteristics as an active ingredient.

[0011] (a) Not expressing Col2;

[0012] (b) The chondrocytes are stained with one or more of the following substances: Alcian blue, safranin O, and toluidine blue.

[0013] In one embodiment of the present invention, the cartilage-related disease may be selected from the group consisting of osteoarthritis, arthritis, meniscus derangements, rheumatoid arthritis, tear of meniscus, triangular fibrocartilage complex injury, traumatic cartilage injury, and degenerative arthritis.

[0014] In other embodiments of the present invention, the active ingredient of the pharmaceutical composition may contain 90% or more of cells that are homogeneous with the chondrogenic cells.

[0015] In yet another embodiment of the present invention, the chondrogenic cells may be differentiated from stem cells.

[0016] In yet another embodiment of the present invention, the stem cells may be mesenchymal stem cells.

[0017] In yet another embodiment of the present invention, the differentiation induction may be induced by electrical stimulation.

[0018] In yet another embodiment of the present invention, the electrical stimulation is It may have a frequency greater than 0 and less than or equal to 20 Hz; an amplitude of -20 V or more and less than or equal to 20 V; and a duty cycle greater than 0 and less than or equal to 80%.

[0019] In yet another embodiment of the present invention, the chondrogenic progenitor cells may have a reduced expression level of one or more genes selected from the group consisting of COL1 and COL5, or proteins encoded by those genes, compared to mesenchymal stem cells; and an increased expression level of the COL6 gene or proteins encoded by that gene, compared to mesenchymal stem cells.

[0020] In yet another embodiment of the present invention, the chondrogenic progenitor cells may have increased expression levels of one or more genes selected from the group consisting of GJB2, GJC1, PECAM1, CLDN2, CLDN7, CLDN10, and CLDN19, or proteins encoded by those genes, compared to mesenchymal stem cells.

[0021] In yet another embodiment of the present invention, the pharmaceutical composition may be in the form of a dosage form that is easily implanted directly into the cartilage site.

[0022] In yet another embodiment of the present invention, the aggregates of chondrogenic cells may be aggregated in the form of spheroids.

[0023] In still another embodiment of the present invention, the diameter of the spheroid may be from 0.5 mm to 1.5 mm.

[0024] The present invention also provides a method for producing a pharmaceutical composition for treating or preventing cartilage-related diseases, comprising the step of applying an electrical stimulus to stem cells to produce chondrogenic progenitor cells or aggregates thereof; wherein the chondrogenic progenitor cells have the following characteristics: (a) They do not express Col2; (b) The chondrogenic progenitor cells are subjected to substrate staining with one or more selected from the group consisting of alcian blue, safranin O, and toluidine blue.

[0025] The present invention also provides a method for treating or preventing cartilage-related diseases, comprising the step of administering to an individual in need thereof a chondrogenic progenitor cell or an aggregate thereof having the following characteristics: (a) They do not express Col2; (b) The chondrogenic progenitor cells are subjected to substrate staining with one or more selected from the group consisting of alcian blue, safranin O, and toluidine blue.

[0026] The present invention also provides the use of a chondrogenic progenitor cell or an aggregate thereof having the following characteristics for the production of a medicament for treating or preventing cartilage-related diseases: (a) They do not express Col2; (b) The chondrogenic progenitor cells are subjected to substrate staining with one or more selected from the group consisting of alcian blue, safranin O, and toluidine blue.

Advantages of the Invention

[0027] The composition for treating cartilage-related diseases according to the present invention can be produced by applying only an electrical stimulus to stem cell aggregates without introducing growth factors or the like derived from the outside, and can be produced without using expensive growth factors, so it has the advantage of significantly reducing medical costs. In addition, the composition for treating cartilage-related diseases according to the present invention does not express Col2, which is a marker of mature chondrocytes, compared to cells produced for the treatment of existing cartilage-related diseases, so it has the advantage of having lower immune-related side effects compared to mature cells.

[0028] However, the effects of the present invention are not limited to those described above, but include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention as described in the claims. [Brief explanation of the drawing]

[0029] [Figure 1a] A diagram relating to the application of electrical stimulation to cells, showing the conditions for electrical stimulation and the general state in which the cells are electrically stimulated. [Figure 1b] Results of observing cells that were subjected to electrical stimulation using a phase-contrast microscope [Figure 1c] Observation of cells stained with Alcian blue and safranin-O yielded the following results. [Figure 1d] Results of confirming cell viability using the Live / Dead viability / cytotoxicity kit [Figure 1e] This shows the number of cells in a single micromass. [Figure 1f] Results after checking the cell membrane antigen levels [Figure 2a] This figure shows the results of confirming calcium oscillation in cells induced by electrical stimulation, with calcium transport confirmed through Fluo-4 staining. [Figure 2b] The results of observing the changes in intracellular Ca2+ concentration over time [Figure 2c] Results of checking the FITC strength of cells [Figure 3a] This diagram shows the changes in gene expression caused by electrical stimulation, and is a schematic diagram of the workflow for single-cell RNA-seq analysis. [Figure 3b] The results of clustering gene expression profiles of 2D (cultured ADSCs), cell micromasses without electrical stimulation for 6 hours, cell micromasses with electrical stimulation for 6 hours, and cell micromasses with electrical stimulation for 72 hours are shown. [Figure 3c]The heatmap shows the expression of differentially expressed genes (DEGs) in four samples (the top 10 markers, regulated upwards, are shown on the right). [Figure 4a] This figure shows the results of gene ontology enrichment analysis of the top 20 genes that were clearly regulated upward in four samples, confirming the gene expression of cartilage formation markers induced by electrical stimulation. [Figure 4b] This figure shows the results of gene ontology enrichment analysis of the top 20 genes that were clearly regulated upward in four samples, confirming the gene expression of cartilage formation markers induced by electrical stimulation. [Figure 4c] This figure shows the results of gene ontology enrichment analysis of the top 20 genes that were clearly regulated upward in four samples, confirming the gene expression of cartilage formation markers induced by electrical stimulation. [Figure 4d] Using NCBI's GEO (gene expression omnibus), we compared the gene expression profiles of articular cartilage (left) and developing chondrocytes (intermediate, BM-MSCs undergoing chondrogenic differentiation, right). [Figure 4e] Using NCBI's GEO (gene expression omnibus), we compared the gene expression profiles of articular cartilage (left) and developing chondrocytes (intermediate, BM-MSCs undergoing chondrogenic differentiation, right). [Figure 4f] Using NCBI's GEO (gene expression omnibus), we compared the gene expression profiles of articular cartilage (left) and developing chondrocytes (intermediate, BM-MSCs undergoing chondrogenic differentiation, right). [Figure 4g] By utilizing the gene ontology data of GO0060591, a chondrocyte (or chondrocyte), we confirmed the expression levels of genes involved in chondrocyte development. [Figure 5a] This figure shows the results of confirming changes in cell viability and karyotype induced by electrical stimulation. After applying electrical stimulation for 3 days, condensed cell micromass and fragmented cells were transferred to a 96-well plate, and cell viability was confirmed using the CCK-8 viability analysis kit. [Figure 5b] The results of examining the expression levels of SHARPIN mRNA, a gene associated with cell death, in different cell samples to which electrical stimulation was applied were as follows. [Figure 5c] The degree of cell death at the single-cell level of micromass was confirmed through AO / PI (acridine orange / propidium iodide) staining. [Figure 5d] This shows the expression heatmaps of genes associated with stem cell function, such as MKI67, HMMR, and TOP2A. [Figure 5e] Results of confirming the normal karyotype in micromass cells to which electrical stimulation was applied [Figure 6a] This figure shows the changes in collagen expression levels in cells due to the application of electrical stimulation, illustrating the changes in the expression levels of all collagen in cells, including even minute changes in the number of cells. [Figure 6b] Among these, we confirmed significant changes in the expression of COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, and COL6A3. [Figure 6c] Results of confirming COL1A1 expression levels by RT-PCR [Figure 6d] The expression level of COL1A1 was confirmed by Western blotting. [Figure 7a] This figure shows the results of confirming the genetic regulation of type 1 collagen by applying electrical stimulation, specifically the expression of transcription factor genes such as RBFOX2, NFIC, YBX1, and ID3, which are associated with TGFβ signaling. [Figure 7b] The results of examining the expression levels of transcription factors that bind to the promoter / enhancer of COL1A1 [Figure 7c]This heatmap shows the expression levels of the TGFβ superfamily and its receptors. [Figure 7d] Using the Gene Ontology Data Portal, we confirmed the expression levels of tissue formation-related genes GO0007043 (cell-cell junction assembly) and GO0051495 (positive regulation of cytoskeletal tissue) by observing changes in cells subjected to electrical stimulation. [Figure 7e] Using the Gene Ontology Data Portal, we confirmed the expression levels of tissue formation-related genes GO0007043 (cell-cell junction assembly) and GO0051495 (positive regulation of cytoskeletal tissue) by observing changes in cells subjected to electrical stimulation. [Figure 8a] This diagram shows the overall progress of the experiment, illustrating the therapeutic effect after implanting a spheroid, to which electrical stimulation was applied, into the femoral cartilage of a rabbit's hind leg, and confirming its therapeutic effect. [Figure 8b] Sixteen weeks after implantation, the thigh was removed, and cartilage regeneration was confirmed by imaging using micro-CT. [Figure 8c] Sixteen weeks after implantation, the thigh was removed, and cartilage regeneration was confirmed by imaging using micro-CT. [Figure 8d] We prepared histopathology slides and confirmed histological regeneration using representative cartilage staining methods. [Figure 8e] We prepared histopathology slides and confirmed histological regeneration using representative cartilage staining methods. [Figure 8f] We prepared histopathology slides and confirmed histological regeneration using representative cartilage staining methods. [Figure 8g] We prepared histopathology slides and confirmed histological regeneration using representative cartilage staining methods. [Figure 8h] The results of evaluating the regeneration of spheroids treated with electrical stimulation in cartilage defects in five rabbits through scoring. [Figure 8i] The results of evaluating the regeneration of spheroids treated with electrical stimulation in cartilage defects in five rabbits through scoring. [Figure 8j]The results of evaluating the regeneration of spheroids treated with electrical stimulation in cartilage defects in five rabbits through scoring. [Figure 8k] The results of evaluating the regeneration of spheroids treated with electrical stimulation in cartilage defects in five rabbits through scoring. [Figure 8l] The results of evaluating the regeneration of spheroids treated with electrical stimulation in cartilage defects in five rabbits through scoring. [Figure 8m] By checking the body weight of the experimental animals, we confirmed that they were not affected by inflammatory responses or health problems caused by the creation of the defects. [Modes for carrying out the invention]

[0030] The inventors of this invention have conducted research and made efforts to develop a therapeutic agent that can treat cartilage-related diseases without the addition of expensive growth factors. As a result, they have confirmed that when electrical stimulation is applied to stem cells, the cells do not express Col2, a marker for mature chondrocytes, and can differentiate into chondrocyte precursor cells that possess the characteristics of chondrocytes. This has led to the completion of the present invention.

[0031] Therefore, the present invention provides a pharmaceutical composition for the treatment or prevention of cartilage-related diseases, comprising chondrogenic cells or aggregates thereof having the following characteristics as an active ingredient.

[0032] (a) The cells that do not express Col2; (b) The chondrogenic progenitor cells are stained with one or more of the following substances selected from the group consisting of Alcian blue, safranin O, and toluidine blue.

[0033] In this invention, Col2 (Collagen 2) forms the basis of articular cartilage and hyaline cartilage, accounting for 50% of the total cartilage protein and 85% to 90% of the collagen in articular cartilage. Due to these characteristics, Col2 is used as a typical marker for mature chondrocytes. However, the chondrocyte progenitor cells of this invention do not express Col2 at all, thus confirming that they are not mature chondrocytes.

[0034] In the present invention, the chondrocytes are produced by applying electrical stimulation to stem cells, preferably mesenchymal stem cells, and do not express proliferation markers such as MKI67, TOP2A, and HMMR, which are factors associated with the stem cell function of mesenchymal stem cells, and are therefore different from mesenchymal stem cells themselves. The chondrocytes exhibit similar characteristics to chondrocytes, as they can be stained with Alcian blue, safranin-O, and other substrates characteristic of chondrocytes. However, since they do not express COL2, a typical marker of mature chondrocytes, they represent cells in the preliminary stage of differentiation into mature chondrocytes. Because they spontaneously aggregate into spheroid form without the addition of growth factors or external factors such as centrifugation, they can be used as an easily digestible dosage form for therapeutic transplantation and other applications.

[0035] In this invention, the term "spheroid" refers to a cell aggregate in the form of a spheroid, and methods such as 96-well culture and hanging drop are generally used to produce it. For the treatment of cartilage-related diseases, which is the application of this invention, a step of implanting cells or their aggregates is necessary, and the spheroid form is suitable for such an implantation process. Methods such as centrifugation are used to create cell aggregates in the form of spheroids for implantation, but it has been confirmed that the cell aggregates of this invention form a spheroid form that is easy to transplant simply by applying electrical stimulation to induce differentiation into chondrocytes, without going through such a separate step.

[0036] The aforementioned cartilage-related diseases may be selected from the group consisting of, but are not limited to, osteoarthritis, arthritis, meniscus derangements, rheumatoid arthritis, tear of meniscus, triangular fibrocartilage complex injury, traumatic cartilage injury, and degenerative arthritis.

[0037] As used in this invention, the term "prevention" refers to all actions that suppress symptoms of cartilage-related diseases or delay the onset of the disease by administering the pharmaceutical composition according to the present invention.

[0038] As used in this invention, the term "treatment" means all actions in which the symptoms of cartilage-related diseases are improved or beneficially altered by the administration of the pharmaceutical composition according to the present invention.

[0039] The active ingredient of the pharmaceutical composition may contain 90% or more cells identical to the chondrogenic progenitor cells, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. It may also be differentiated from stem cells, and preferably the stem cells may be mesenchymal stem cells.

[0040] The mesenchymal stem cells (MSCs) of this invention are cells that can differentiate into chondrocytes, osteocytes, adipocytes, and muscle cells, and can be induced to differentiate into cartilage, bone, muscle, ligaments, and adipose tissue under specific culture conditions in vitro. Mesenchymal stem cells are easily extracted from bone marrow, and much research is underway on their potential use as cell therapies for various intractable diseases. Cartilage has insufficient regenerative capacity, and once damaged, it is very difficult to treat. Degenerative arthritis occurs due to degenerative changes in the joints, and it is not possible to completely stop it, so treatment currently relies on drug therapy and physical therapy. However, there is no definitive drug to treat arthritis, and long-term use of steroids and lubricants results in accelerated cartilage degeneration. In recent years, autologous chondrocyte transplantation has been developed, but limitations in cartilage tissue, dedifferentiation of chondrocytes during in vitro culture, and limitations in cell proliferation due to age remain problems. Therefore, the use of mesenchymal stem cells, which have abundant regenerative capacity, can be applied as an effective cell therapy agent for biological restoration to regenerate damaged cartilage. Cartilage regeneration using cell therapy agents can be applied not only to musculoskeletal diseases but also to diseases of the digestive and urinary systems. In other words, by locally regenerating cartilage tissue, it can be applied to the treatment of diseases such as reflux esophagitis and urethral reflux.

[0041] The differentiation induction of the present invention may be by electrical stimulation, but is not limited thereto. The frequency of the electrical stimulation may be greater than 0 and less than or equal to 20 Hz, preferably greater than 3 and less than or equal to 15 Hz or greater than 5 and less than or equal to 12 Hz, and more preferably greater than or equal to Hz. The voltage of the electrical stimulation may have an amplitude of -20 V or more and less than or equal to 20 V, preferably greater than or equal to -15 V and less than or equal to 15 V, and more preferably greater than or equal to -10 V and less than or equal to 10 V.

[0042] The chondrogenic progenitor cells of the present invention are not limited to those described herein, but may have a reduced expression level of one or more genes selected from the group consisting of COL1 and COL5, or proteins encoded by those genes, compared to mesenchymal stem cells; an increased expression level of the COL6 gene, or proteins encoded by that gene, compared to mesenchymal stem cells; or an increased expression level of one or more genes selected from the group consisting of GJB2, GJC1, PECAM1, CLDN2, CLDN7, CLDN10, and CLDN19, or proteins encoded by those genes, compared to mesenchymal stem cells.

[0043] The inventors have confirmed, through specific examples, that cells to which the electrical stimulation of the present invention is applied can differentiate into chondrocytes, and that this can be used to treat cartilage-related diseases.

[0044] In one embodiment of the present invention, the inventors confirmed that cell aggregation occurs when canine mesenchymal stem cells are subjected to electrical stimulation under specific conditions. Upon confirming the cell aggregates, they confirmed that differentiation into chondrocytes occurs even without the addition of exogenous factors, and confirmed that substrate staining is possible when stained with Alcian blue and safranin-O, confirming that these cells possess the characteristics of chondrocytes (see Example 2-1). In the case of cells produced in this manner, it was confirmed that calcium oscillations occurring during the differentiation process increase compared to cells not subjected to electrical stimulation, confirming that cell aggregation occurs through the electrical stimulation described above, and that differentiation of mesenchymal stem cells into chondrocytes occurs through this (see Example 2-2).

[0045] In other embodiments of the present invention, when mesenchymal stem cells were subjected to electrical stimulation under specific conditions, no significant difference in viability could be observed despite the application of electrical stimulation (see Example 3-2). Furthermore, the cells subjected to electrical stimulation did not express markers such as MKI67, TOP2A, and HMMR, which are factors associated with stem cell potential, thus distinguishing them from mesenchymal stem cells (see Example 3-3). Additionally, they did not express Col2, which is used as a typical marker for mature chondrocytes, thus specifically confirming their difference from mature chondrocytes (see Example 3-4).

[0046] In yet another embodiment of the present invention, when chondrogenic cells produced by applying electrical stimulation as described above were transplanted to the cartilage defect site of a rabbit after inducing a defect in the cartilage site of the rabbit, it was confirmed that the defective site could be regenerated to an effective level (see Example 5).

[0047] Through the results described above, the inventors have confirmed that, by applying electrical stimulation under specific conditions, mesenchymal stem cells can be differentiated into chondroprogenitor cells that are completely different from mesenchymal stem cells and chondrocytes, even without the addition of expensive growth factors. The chondroprogenitor cells of the present invention differ in that they do not express Col2, a marker for mature chondrocytes. Therefore, they are immature cells and are expected to be more free from immune-related problems that mainly occur when transplanting mature cells.

[0048] On the other hand, in the present invention, "pharmaceutical composition" means a product manufactured for the purpose of preventing or treating a disease, and each may be used in various dosage forms by conventional methods. For example, it can be made into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be used in the form of topical preparations, suppositories, and sterile injection solutions.

[0049] The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one that is commonly used in formulation and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further contain other common additives such as antioxidants and buffers as needed. Diluents, dispersants, surfactants, binders, lubricants, etc. may be added additionally, and the composition can be formulated into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated using the methods disclosed in Remington's Pharmaceutical Sciences (19th edition, 1995). The pharmaceutical composition of the present invention is not limited in dosage form, but can be formulated as an injection, inhalation, topical skin preparation, or oral administration preparation, and may be in a dosage form that is easy to implant, preferably in the form of a spheroid. The diameter of the spheroid is not limited thereto, but may be 0.5 mm to 1.5 mm, preferably 0.8 mm to 1.2 mm, and more preferably 1.0 mm.

[0050] The pharmaceutical compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) by the method of intent, and the dosage will vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0051] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose may be determined by factors including the type and severity of the patient’s disease, the activity of the drug, the patient’s sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0052] The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple agent. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0053] Furthermore, the present invention provides a method for preventing or treating cartilage-related diseases, which includes the step of administering the aforementioned pharmaceutical composition to an individual.

[0054] As used in this invention, the term "administration" means providing a given composition of the present invention to an individual in any suitable manner.

[0055] As used in this invention, the term "individual" means an object requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, dog, cat, horse, and cow.

[0056] Furthermore, the present invention provides the pharmaceutical composition for use in the prevention or treatment of cartilage-related diseases.

[0057] Furthermore, the present invention provides a method for producing a pharmaceutical composition for the treatment or prevention of cartilage-related diseases, comprising the step of producing chondrocytes or aggregates thereof by applying electrical stimulation to stem cells; the chondrocytes provided are characterized by the following: (a) not expressing Col2; and (b) the chondrocytes are stained with one or more of the substances selected from the group consisting of Alcian blue, safranin O, and toluidine blue.

[0058] The following are preferred embodiments to facilitate understanding of the present invention. However, these embodiments are provided only to make the present invention easier to understand, and the scope of the present invention is not limited by these embodiments. [Examples]

[0059] Example 1. Experimental materials and experimental method 1-1. Primary cell culture Three batches of canine adipose-derived stem cells (ADSCs) were obtained from the abdominal adipose tissue of three 4-month-old female Beagle dogs. The cells were stored in passage 0, and their differentiation potential into adipocytes, chondrocytes, and osteoblasts was evaluated. 2 Flask (5 × 10 5 Cells were plated onto cells / flask (BD Falcon) and cultured in low-glucose DMEM (Dulbecco's modified Eagle medium, GIBCO) containing 10% FBS (GIBCO) and 1X antibiotic-antimycotic (GIBCO).

[0060] 1-2. Micromass culture and electrical stimulation After isolating canine ADSCs from 3 to 5 passages, they were incubated in serum-free advanced DMEM / F12 medium (GIBCO), which contains 1x antibiotic-antifungal agent and 1x GlutaMax (GIBCO), at high density (2.5 × 10⁻¹⁴). 7 The cells were suspended in 35 ml of water (cells / ml). To generate micromass, 10 μl of the cell suspension was added to 35 mm of water. 2The cells were placed in a Corning dish and cultured in an incubator (N-Biotek, Korea) at 37°C and 5% CO2. One hour after culturing, serum-free advanced DMEM / F12 medium (GIBCO) was added. The cells were placed in a multi-channel stimulator capable of providing chronic cell stimulation. Micromass cells of primary canine ADSCs were cultured with and without electrical stimulation (ES) at a frequency of 2.0 Hz, 10 V / cm, and 10 ms. After applying electrical stimulation for 3 days, the formation of condensed cell clumps was observed using a phase-contrast microscope (Eclipse Ti2, Nikon, Japan).

[0061] 1-3. Single-cell preparation and RNA sequencing Single cells were prepared at room temperature using the MACS tissue dissociation kit (Miltenyi Biotech) and the gentleMACS dissociator (Miltenyi Biotech). After transferring the cell suspension to a cell filter, it was washed with complete medium. Live / dead assays were performed to analyze cell viability (using a molecular probe, cutting off groups with cell viability below 90%). RNA sequencing was performed via Macrogen's (Korea) RNA-seq analysis service.

[0062] To prepare the single-cell library, we used Next GEM Single Cell 3' Library & Single Cell 3' V3.1 gel beads, which can capture approximately 500 cells according to the manufacturer's guidelines, and processed the cells on a 10x Genomics platform. The uniquely barcoded cDNA library can be prepared from RNA via reverse transcription, purification, and PCR in single-cell droplets. The library was sequenced using HiSeqX (Illumina) with read 1 (cell barcode and unique molecular identifier [UMI]), 8bp index read (sample barcode), and 91bp read 2 (RNA read) and a read length of 28bp.

[0063] Cell Ranger v3.1.0 (10XGenomics) was used to generate FASTQ files for data analysis. For this purpose, data were aligned with a canine reference genome (CanFam 3.1 release 100), gene expression was measured using UMI and cell barcodes, cell clusters were determined, and differential gene expression analysis was performed. To normalize across multiple datasets, the final aggregated dataset was brought in using Seurat 3.1.3. Cell populations with a mitochondrial ratio >0.2 were filtered to remove cells containing low UMI content. UMAP (Uniform Manifold Approximation and Projection) analysis was performed based on statistically significant principal components. Specific marker comparisons between all clusters and remaining cells were determined using the minimum percentage of cells and the minimum fraction, and a Wilcox rank sum test was performed; only significant results were reported.

[0064] 1-4. Genome analysis using online databases To evaluate chondrogenesis in canine ADSCs, we analyzed transcriptome changes using the NCBI GEO (Gene Expression Omnibus). For this purpose, we used transcripts from GSE32398 (top 250 genes altered in human articular cartilage compared to growth plate cartilage), GSE51812 (top 239 genes altered in human articular chondrocytes 17 weeks after development, compared weekly to chondrocytes for 6 embryonic development), and GSE19664 (top 128 genes altered over time in healthy human BMSCs (Bone marrow-derived stem cells) during chondrogenesis). GO annotation (Gene Ontology annotation) and the PANTHER classification system were used to analyze important probe lists.

[0065] 1-5. Measurement of calcium oscillation Canine primary ADSC 2-2.5 × 10 7 Suspend the cells at a density of cells / ml, and apply a 10 μl droplet of the cell suspension to the CellBIND surface at a density of 35 mm. 2 The cells were placed in Corning dishes. Each micromass of canine ADSCs was cultured with or without electrical stimulation (stimulation conditions: 10V / cm over 10ms at a frequency of 2.0Hz). Fourteen hours after ES application, probenecid-free Fluo-4 reagent (Molecular Probe) was loaded into the culture medium at a 1:1 ratio for 30 minutes according to the manufacturer's protocol. Calcium oscillation was stimulated by exposure to 488nm for 1 second without 10 minutes of incubation, and then recorded at 1 fps. Time-lapse analysis of fluorescence intensity was performed using NIS-Elements Advanced Research Imaging software (Eclipse Ti2, Nikon, Japan).

[0066] 1-6. Measurement of total glycosaminoglycans To measure glycosaminoglycans (GAGs), cells were washed with PBS, fixed with paraformaldehyde (Biosesang) for 20 minutes, and stored at 4°C until staining. Cells were then cultured at room temperature for 30 minutes using Alcian Blue (IHC World) solution, or at room temperature for 1 hour using Safranin-O (IHC World) solution, followed by multiple rinses with distilled water. GAG accumulation was captured using a digital USB camera (My first lab, USA). 1-7.Flow Cytometry The single cells prepared in Examples 1-3 above were labeled Alexa488 anti-dog CD44 (MCA1041A488, Bio-Rad), PE anti-dog CD90 (12-5900-42, BD), PerCP-Cy5.5 anti-dog CD29 (303024, BioLegend), Alexa488 anti-dog CD45 (MCA1042F, Bio-Rad), Alexa647 anti-dog CD73 (Bs-4834R-A647, Bioss), FITC anti-dog CD54 (GTX76274, GeneTex), APC anti-dog CD49d (304308, BioLegend), PE anti-dog CD34 (559369, BD), PE anti-hu / dog HLA-DR (361606, BioLegend), or APC anti-dog The cells were stained with CD80 (104714, BD) by treatment at 4°C for 20 minutes. Fluid cell analysis was performed using the BD LSRII analysis service at Yonsei University's Clinical Research Institute.

[0067] 1-8.RT qPCR analysis Total RNA was isolated from canine ADSCs cultured under diverse conditions for 3 days using GentleMACS dissociator (Miltenyi Biotech) and Direct-zol RNA MiniPrep (Zymo Research) according to the manufacturer's protocol. RNA concentration was measured using a biospectrometer (Eppendorf), and reverse transcription was performed using a TOPscript cDNA synthesis kit (Enzynomics) with 0.3 μg to 0.5 μg of total RNA. Real-time PCR for GAPDH and COL1A1 was performed using a SYBR 2x Mix (Bio-Rad) containing 10 ng of cDNA / tube and a CFX-coupled Real-Time PCR detection system (Bio-Rad). Specifically, samples were maintained at 95°C for 15 minutes, followed by 40 amplification cycles including a denaturation phase at 95°C for 10 seconds, and an expansion and annealing phase at 60°C for 30 seconds.

[0068] The expression level of COL1A1 was normalized to the expression level of GAPDH, and the relative gene expression levels were calculated using the 2-△△CT method. Primer sequences were independently determined using Primer-BLAST as follows: Canine GAPDH forward primer 5'-GGTGATGCTGGTGCTGAGTA, reverse primer 5'-GGCATTGCTGACAATTCTGA; Canine COL1A1 forward primer 5'-CCGCTTCACCTACAGTGTCA, reverse primer 5'-CAGACAGGGCCAATATCCAT (Bioneer, Korea).

[0069] 1-9. Western blot analysis Cells were washed three times with ice-cold PBS and harvested in RIPA cell lysis buffer (genDEPOT) containing phosphatase inhibitor cocktails (genDEPOT). Protein concentrations were determined using a BCA protein analysis kit (Pierce). Protein samples were separated from SDS-PAGE gels and electrotransferred onto PVDF membranes (ATTO) using standard procedures. The membranes were blocked with 5% skim milk powder dissolved in 0.05% TBST and incubated with primary antibody on a 4°C rocking platform for 12 hours. The membranes were washed three times with TBST buffer for 15 minutes each and incubated with 1% skim milk in TBST containing HRP-conjugated secondary antibody (GeneTex) for 1 hour. The hybridized membranes were washed with TBST buffer and visualized using an enhanced chemiluminescence detection kit (Merk) and a LAS500 imaging system (GE Healthcare).

[0070] 1-10.Karyotyping For sampling, ES-induced cell condensates were disrupted using a 70 μm pore size nylon mesh in a homogeneous single-cell suspension. The cell suspension was collected in a 15 ml tube and centrifuged at 450 xg for 5 minutes. After aspiration, the pellet was suspended in fresh medium, and the karyotype was analyzed using G-banding staining and a chromosome imaging analyzer system from the GenDix Karyotyping Service.

[0071] 1-11.Statistical analysis All data are presented as mean ± standard deviation (n = number of individual samples). All statistical analyses were performed using MS Excel software (Microsoft 365), and a p-value of <0.05 was considered to indicate a statistically significant difference.

[0072] Example 2. Production of chondrogenic cells by applying electrical stimulation to stem cells. 2-1. Cartilage of ADSCs by electrical stimulation Canine ADSCs (MSCs) were stimulated with ES (10V / cm, 10ms, 2Hz frequency), and the cells that aggregated in response to the stimulation were observed. Micromass culture of canine ADSCs was performed without the addition of serum or exogenous factors (Figure 1a). Three days after ES application, the results of immunohistochemical staining of cartilage matrix-related molecules in the aggregates, with and without ES application, were examined using a phase-contrast microscope. As shown in Figure 1b, sheet-like cell aggregation and even larger aggregations were observed in cells treated with ES compared to cells not treated with ES. Furthermore, when cells stimulated with ES were stained with Alcian blue and safranin-O and then observed, proteoglycan deposition was confirmed, as shown in Figure 1c.

[0073] To confirm the effect of ES on cell death, viability was evaluated using a Live / Dead reagent. As shown in Figure 1d, it was confirmed that there was no significant difference in cell viability with or without ES application. Furthermore, as shown in Figure 1e, the aggregates were approximately 10 5 The presence of individual cells was confirmed. To confirm the changes in cell membrane proteins due to ES application, fluid cell analysis was performed using antibodies against surface molecules. When comparing the expression of membrane proteins in ADSCs grown in single phase with that of ADSCs, as shown in Figure 1f, it was confirmed that CD44, CD90, CD29, CD73, CD54, CD34, CD49d, CD45, HLA-DR, and CD80 showed similar expression.

[0074] Through the results described above, the inventors were able to confirm that ES induces very dense prechondrogenic condensation in canine ADSCs.

[0075] 2-2. Confirmation of calcium oscillation during the cartilage formation process. It has been previously reported that ES-induced chondrogenesis reproduces the spontaneous intracellular calcium oscillations observed in cartilage development. The inventors monitored whether similar calcium oscillations occurred in the canine ADSC micromass of the present invention when ES was applied. Calcium fluorescence measurements using Fluo-4 Direct, as shown in Figure 2a, confirmed that the ES-treated cell aggregates exhibited more regular frequencies and higher amplitudes compared to the control group without ES application. More specifically, as shown in Figures 2b and 2c, the electrically stimulated cell aggregates exhibited typical calcium oscillations. 2+ The results show a variation pattern. These results indicate that even under ES application conditions without exogenous factors, pre-chondrogenesis condensation is induced within ADSC cells during the micromass aggregation process.

[0076] Example 3. Characterization of chondrogenic cells produced by electrical stimulation 3-1. Confirmation of the transfer profile Single cells were isolated from clusters of ES-stimulated cells. Single-cell RNA-seq libraries were prepared on the 10X Genomics Chromium platform, and data were retrieved using the standard Seurat toolkit for data analysis. The entire experiment to confirm the transcriptional profiles of the cells is shown in Figure 3a. Specifically, the normalized datasets were analyzed using the Loupe Cell Browser to identify highly variable genes and subdivide 12 clusters into 4 datasets (Figure 3b). Furthermore, as shown in Figure 3c, we found that 2D ADSCs exhibited significant differences in phenotypic and functional characteristics between ES-treated cell micromasses and transcriptional regions.

[0077] Using open-source GO annotation and PANTHER database analysis tools, we explored gene expression patterns in four datasets. In ADSC micromasses treated with ES for 6 hours, we confirmed upward regulation of genes associated with proton ion transport, ornithine metabolism, biogenesis, and calcium ion import / release (Figure 4a). In cell micromasses without ES treatment, we confirmed upward regulation of genes associated with cartilage condensation, calcium ion transmembrane transport, skeletal muscle tissue development, the NF-κB pathway, and chemotaxis of immune cells (Figure 4b). Cellular micromasses treated with ES over 72 hours showed upward regulation of genes associated with prostaglandin synthesis, multicellular organism development, arginine catabolism, and negative regulation of apoptosis (Figure 4c).

[0078] To understand the role of the gene expression patterns described above, we compared them with publicly available prechondrocyte differentiation gene expression data cells. As a result, as shown in Figures 4d to 4f, cell micromasses treated with ES over 72 hours showed that a significant number of genes were upwardly regulated between embryonic chondrogenic condesation and articular chondrocyte differentiation, inducing prechondrogenic changes. When we conducted studies to identify core factors associated with osteochondral progenitor cell differentiation, we confirmed that the balance between RUNX2 and SOX9 in osteochondral progenitor cells plays an essential role in the differentiation of chondrocytes and osteoblasts. Current data for ES-treated micromasses show that the levels of RUNX2 and its inhibitor NKX3.2 were specifically altered to typical levels in chondrocyte progenitor cells (Figure 4e).

[0079] Through the results described above, the inventors confirmed that condensation of canine ADSCs stimulated by ES induces a phenotype similar to that of chondrocyte developmental stages.

[0080] 3-2. Confirmation of Survival Ability Since physiological stress induced by ES affects many cellular processes and can lead to diverse physiological and pathological consequences, we examined the cell viability of ADSCs treated with ES stimulation for three days using the CCK-8 viability kit. As shown in Figure 5a, we confirmed that ES stimulation did not affect the viability of canine ADSC micromass. GO database analysis also confirmed, as shown in Figure 5b, that electrical exposure did not affect the expression of SHARPIN, a gene associated with cell death. Single ADSC suspensions prepared by mechanically and enzymatically dissociating cell micromass were stained with PI (propidium iodide). As shown in Figure 5c, the number of PI-stained cells was found to be unrelated to the presence or absence of ES treatment.

[0081] 3-3. Confirmation of differentiation into chondrocytes Adult stem cells express representative proliferation markers such as MKI67, TOP2A, and HMMR when compared to typical growing cells. As shown in Figure 5d, these proliferation markers were not expressed in aggregated cells, regardless of whether or not ES cells were applied. Observation of the karyotype in cells treated with ES cells confirmed that no karyotype changes occurred, as shown in Figure 5e.

[0082] Through the results described above, the inventors confirmed that when cells aggregate, further differentiation into mature cell types that are not stem cells proceeds.

[0083] 3-4. Confirmation of changes in collagen production levels Since the main function of chondrocytes is to synthesize extracellular matrix such as collagen types 2, 4, 6, 10, 11, 12, and 14, the collagen production level was checked to confirm whether or not cells treated with the ES of the present invention were differentiated. As a result, as shown in Figure 6a, it was confirmed that the expression of COL3A1, COL4A1, COL4A2, COL4A5, COL5A3, COL6A1, COL6A3, COL6A5, COL8A1, COL13A1, COL14A1, COL15A1, COL16A1, COL21A1, COL23A1, COL24A1, COL24A1, and COL27A1 increased in canine ADSC micromasses, regardless of the presence or absence of ES stimulation. Of these, the expression of COL3A1, COL5A2, and COL6A1 / 3 was confirmed to increase strongly and uniformly in ADSC micromasses. In cells treated with ES, we confirmed that COL6A3 and COL16A1 were suppressed overall in micromass compared to cells not treated with ES.

[0084] However, in the case of Col2, which is expressed along with chondrogenesis in MSCs and is known as a marker of mature chondrocytes, we were able to confirm that it is not expressed regardless of whether or not ES is applied, as shown in Figure 6b. In an existing patent application (KR10-2015-0047361) disclosing a method for differentiating existing mesenchymal stem cells into chondrocytes by applying electrical stimulation, it was confirmed through comparative examples that the expression of Col2, which is used as a marker of mature chondrocytes, increased to a significant level in all mesenchymal stem cells, including those to which growth factors were added and those to which electrical stimulation was applied. However, in contrast to this, it was confirmed that the cell aggregates to which electrical stimulation was applied in the present invention did not express Col2 at all.

[0085] On the other hand, to confirm the effect of ES on collagen type 1, RT-qPCR (Real-time quantitative PCR) was performed. As a result, as shown in Figure 6c, it was confirmed that COL1A1 expression increased significantly in cells treated with ES compared to cells that were not treated. COL1A1 expression also increased significantly when confirmed by Western blotting, as shown in Figure 6d.

[0086] 3-5. Confirmation of collagen regulation-related factor expression by electrical stimulation. Collagen type 1 was selected, and the mechanism by which its expression level changes upon electrical stimulation was analyzed. For the analysis of this mechanism, an open-source database provided by GeneHancer, which integrates enhancers from diverse sources, was utilized. The levels of transcription factors NR4A1, RFBOX2, NFIC, ID3, HDGF, BMI1, YBX1, SMARCE1, HLTF, and SMC3, known to be associated with COL1A1 expression in ES-treated cells, were found to decrease considerably over time (Figure 7a). Conversely, transcription factors such as TCF12, POLR2A, ATF4, ARID4B, SMARCA5, GTF2F1, LARP7, HDAC2, and YY1 were found to increase in cellular micromass over 72 hours after ES application (Figure 7b).

[0087] Since each of the aforementioned factors is associated with TGFβ signaling, it leads to changes in COL1A1 expression levels. Despite increased expression of transcription factors associated with COL1A1, COL1A1 expression was significantly decreased. Through these results, we confirmed that factors other than the aforementioned transcription factors influence COL1A1 expression.

[0088] Example 4. Confirmation of the tissue-forming ability of the chondrocytes of the present invention. To confirm the role of ES in the cytoskeleton, we examined the expression of genes that specifically regulate cytoskeleton tissue, including cell-cell junctions. As a result, as shown in Figures 7d and 7e, we confirmed that the expression of connexins (GJB2 / GJC1), cell adhesion proteins (PECAM1), and claudins (CLDN2 / CLDN7 / CLDN10 / CLDN19), which have been reported to be essential for the function of the cartilage extracellular matrix, was significantly regulated upward.

[0089] These results are consistent with the GAG ​​staining results of Example 2-1. When ES is applied to cellular micromass, it induces significantly high expression of genes related to the biosynthesis of chondroitin sulfate and keratan sulfate, two GAGs that constitute aggrecan. These data suggest that electrical stimulation can directly induce differentiation into chondrocytes, stimulate plasticity of the cartilage matrix and chondrocytes, and restore hyaline cartilage.

[0090] Example 5. Confirmation of the therapeutic effect of the chondrogenic progenitor cells of the present invention in vivo. To confirm the histological regeneration and restoration of cartilage through differentiation and engraftment of electrically stimulated chondrocyte aggregates into cartilage tissue in vivo, a small cartilage defect site with a diameter of 4 mm (average cartilage thickness of rabbits is 0.3 mm) was created in the femoral cartilage of experimental New Zealand white rabbits (NZW rabbits) weighing 4 kg or more that had completed bone maturation. Eight electrically stimulated aggregates were implanted, and the aggregates were fixed by applying fibrin glue. The overall progress of this experiment is shown in Figure 8a. Since the implanted electrically stimulated aggregates were derived from mesenchymal stem cells derived from canine fatty oil, interspecies transplantation was considered, and cyclosporine, an immunosuppressant, was administered intravenously once daily (10 mg / kg). This was applied within two weeks, or immediately discontinued if the animal did not show normal health status. Sixteen weeks after implantation, the femoral cartilage was excised by necropsy, and the defect and regeneration of the femoral cartilage were evaluated using images obtained through micro-CT imaging with micro-resolution. As a result, as shown in Figures 8b and 8c, it was confirmed that the defect site to which the electrically stimulated aggregates were implanted recovered to the extent that it could be evaluated as a cartilage layer after 4 months.

[0091] To confirm the results described above, cartilage matrix staining was performed. As shown in Figures 8c to 8g, when aggregates subjected to electrical stimulation were implanted, histological evaluation of the implanted aggregates in relation to the defect using Alcian blue, trichrome, and safranin O staining slides confirmed differentiation into chondrocytes and recovery of the cartilage matrix, similar to the characteristics of normal cartilage tissue. Furthermore, long-term engraftment in vivo, connection with surrounding normal cartilage, and smooth tissue integration with the underlying bone tissue were confirmed. These results are consistent with the histopathological staining results confirmed above.

[0092] In the scoring chart, significant regeneration and recovery were observed on average in five rabbits, and the results are shown in Figures 8h to 8m. In these data, no inflammation was observed at the cartilage defect sites or implanted aggregate sites in the experimental animals, and no abnormal symptoms were observed when health monitoring was performed through changes in the experimental animals' body weight and macroscopic observation. No additional influences were considered when evaluating the efficacy of the implanted aggregates.

[0093] The description of the present invention described above is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, each embodiment described above is illustrative in all respects and not limiting.

Claims

1. The following features: (a) Not expressing Col2; (b) Chondrocytes can be stained with one or more of the following: Alcian blue, safranin O, and toluidine blue. It contains aggregates of chondrogenic progenitor cells as an active ingredient, The aforementioned chondrogenic progenitor cells were differentiated from mesenchymal stem cells. The differentiation induction described above is induced by electrical stimulation. The aforementioned electrical stimulation is Frequencies between 0 and 20 Hz; -20V or greater, and 20V or less in amplitude; and, It has a duty cycle that is greater than 0 and less than or equal to 80%. A pharmaceutical composition for the treatment or prevention of cartilage-related diseases, characterized by the following features.

2. The aforementioned cartilage-related diseases are selected from the group consisting of osteoarthritis, arthritis, meniscus disorders, rheumatoid arthritis, tear of meniscus, triangular fibrocartilage complex injury, traumatic cartilage injury, and degenerative arthritis. The pharmaceutical composition according to claim 1.

3. The active ingredient of the pharmaceutical composition contains 90% or more cells that are identical to the chondrocytes. The pharmaceutical composition according to claim 1.

4. Compared to mesenchymal stem cells, the chondrogenic progenitor cells have reduced expression levels of one or more genes selected from the group consisting of COL1 and COL5, or proteins encoded by those genes; Compared to mesenchymal stem cells, the expression level of the COL6 gene or the protein encoded by that gene is increased. The pharmaceutical composition according to claim 1.

5. The aforementioned chondrogenic progenitor cells exhibit increased expression levels of one or more genes selected from the group consisting of GJB2, GJC1, PECAM1, CLDN2, CLDN7, CLDN10, and CLDN19, or proteins encoded by those genes, compared to mesenchymal stem cells. The pharmaceutical composition according to claim 1.

6. The aforementioned pharmaceutical composition is in a dosage form that is easily implanted directly into the cartilage site. The pharmaceutical composition according to claim 1.

7. The aggregates of the aforementioned chondrocyte progenitor cells are aggregated in the form of spheroids. The pharmaceutical composition according to claim 1.

8. The diameter of the spheroid is 0.5 mm to 1.5 mm. The pharmaceutical composition according to claim 7.

9. The following features: (a) Not expressing Col2; (b) The chondrocytes are stained with one or more of the following substances: Alcian blue, safranin O, and toluidine blue. The step includes administering aggregates of chondrogenic progenitor cells containing to individuals (excluding humans) who require them, The aforementioned chondrogenic progenitor cells were differentiated from mesenchymal stem cells. The differentiation induction described above is induced by electrical stimulation. The aforementioned electrical stimulation is Frequencies between 0 and 20 Hz; -20V or greater, and 20V or less in amplitude; and, It has a duty cycle that is greater than 0 and less than or equal to 80%. A method for treating or preventing cartilage-related diseases, characterized by the following features.

10. The following characteristics for the manufacture of drugs for the treatment or prevention of cartilage-related diseases: (a) Not expressing Col2; (b) The chondrocytes are stained with one or more of the following substances: Alcian blue, safranin O, and toluidine blue. It has, The aforementioned chondrogenic progenitor cells were differentiated from mesenchymal stem cells. The differentiation induction described above is induced by electrical stimulation. The aforementioned electrical stimulation is Frequencies between 0 and 20 Hz; -20V or greater, and 20V or less in amplitude; and, It has a duty cycle that is greater than 0 and less than or equal to 80%. Uses of aggregates of chondrocyte precursor cells characterized by these features.

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