KR102035411B1 - Chondrogenicpellet differentiation using BMP2 and TGFβ3 expression minicircle vectors - Google Patents

Abstract

The present invention relates to chondrogenic pellets differentiated from induced pluripotent stem cells (iPSCs) and their use by transducing a minicircle vector encoding a growth factor. The chondrocyte pellet may express a marker gene of chondrocytes significantly, and express higher levels of chondrocyte marker genes in comparison with the chondrocytes induced by differentiation in a medium containing recombinant growth factor. When the chondrocyte pellets are implanted at the site of cartilage damage in vivo, cartilage regeneration may be effectively induced by differentiated chondrocytes, which is more effective than when transplantation of chondrocytes induced by differentiation-induced chondrocytes by adding a recombinant growth factor. Can be useful in tissue engineering therapeutic methods for cartilage regeneration.

Description

Chondrogenic differentiation using BMP2 and TGFβ3 expression minicircle vectors}

The present invention relates to chondrogenic pellets differentiated from induced pluripotent stem cells (iPSCs) and their use by transducing a minicircle vector encoding a growth factor.

Cartilage (cartilage) is bone tissue consisting of chondrocytes and cartilage substrate, and usually refers to the tissue forming part of the joint. Cartilage has a high elasticity and a very low coefficient of friction, which acts as a cushion to prevent chafing, and helps the joints to move in the absence of friction. It plays a role in structuring the structure of the area that requires elasticity, or resistance to pressure, such as the costal cartilage, interosseous cartilages.

In particular, articular cartilage consists of chondrocytes, which are cells that have been specially differentiated to distribute between the cartilage matrix and the cartilage matrix. Chondrocytes play a role in building and maintaining articular cartilage. Cell division occurs in chondrocytes, but once growth stops, chondrocytes no longer divide under normal conditions.

Therefore, cartilage is difficult to regenerate once damaged. Various regenerative medicine studies have been conducted for the purpose of increasing the regeneration ability of cartilage, such as inducing the differentiation of chondrocytes for the regeneration of cartilage. The most used technique for this is a cell therapy technique using cells. However, there is a disadvantage in that the proliferation efficiency is different depending on the type of cell line and may have a limitation. Accordingly, in recent years, it has been induced to differentiate from adult stem cells, such as adipocyte-derived stem cells or mesenchymal stem cells (MSC), to cartilage cells in vitro , and to use them in cell therapy methods. Known.

However, even in these cell treatment methods, early chondrocytes and adult stem cells quickly lose their characteristics when cultured in vitro and thus have limitations in their continued use even after transplantation. In addition, the differentiation capacity of adult stem cells to chondrocytes may vary depending on the pathological characteristics of the adult stem cells, which may have a number of variables that can be applied to regenerative medicine. do.

Accordingly, studies have been attempted to use human induced pluripotent stem cells (hiPSCs) as cell therapeutic agents of tissue regenerative medicine. hiPSC has a differentiation ability that can be differentiated into a variety of lines has a feature that can be used without limitation to tissue in tissue regenerative medicine (Non-Patent Document 001). Therefore, even in the case of tissues with low differentiation ability when inducing differentiation from stem cells, relatively high regeneration effect can be expected when hiPSC is used.

Among growth factors used to induce differentiation from hiPSC to chondrocytes, bone morphogenetic protein (BMP) or transforming growth factor (TGF) is used recently (Non-Patent Document 002). BMP itself is known to play a major role in the development of bone or cartilage mainly, and is known to affect the survival and proliferation of chondrocytes. Among these, there is a report that the development of cartilage endothelial can be inhibited due to deficiency of BMP2 (Non-Patent Document 003).

TGFβ proteins are known as factors that regulate structural skeleton in various tissues through their involvement in processes such as cell proliferation, differentiation and death. TGFβ protein is divided into three homologous proteins such as TGFβ1, TGFβ2 and TGFβ3, and it has been reported that cartilage formation can be induced through the action between them (Non-Patent Document 004, Non-Patent Document 005). Among them, in particular, TGFβ1 or TGFβ3 can be used to induce cartilage formation, of which TGFβ3 has been reported to have higher differentiation ability (Non-Patent Document 006). In the process of inducing stem cells to differentiate into target tissue cells, the use of recombinant human growth factors may act as an important factor. However, this requires the frequent addition of growth factor molecules during differentiation, which has the disadvantage of incurring expensive costs.

In order to overcome these disadvantages, attempts have been made to induce differentiation into target tissue cells by inducing overexpression of growth factors through gene transfer without adding growth factors to the medium during the differentiation of stem cells. Regarding differentiation into chondrocytes, gene transfer of TGFβ1 to viral vectors in synovial-derived MSCs has been reported to increase cell proliferation and speed up cartilage differentiation (non-patent literature). 007). In addition, overexpression of SOX9 in mouse mesenchymal stem cells (MSC) and umbilical cord blood-derived MSCs promotes differentiation into chondrocytes (Non-Patent Document 008, Non-Patent Document 009), and the expression of type 2 collagen may also increase. (Non-patent document 010).

With regard to gene delivery techniques for cell therapy, non-viral gene delivery techniques have attracted attention as a safe method. This is because commercial DNA vector plasmids contain sequences from bacteria that can elicit an immune response against bacterial proteins. For this purpose, a minicircle vector can be used. The minicircle vector is a vector having a relatively small size by removing an antibiotic resistance gene, a gene encoding a bacterial structural protein, and a transcription unit gene. The minicircle vector has the advantage of being small in size and avoiding the immune response, and having high expression of external genes in vitro and in vivo , which can be used in preclinical gene therapy research. It is attracting attention as a non-patent document 011. Through this, safe and efficient gene delivery is possible, and the therapeutic effect can be enhanced when the genetically modified stem cells are applied to the treatment (Non-Patent Document 012).

Therefore, the present inventors have studied to develop a method of inducing the differentiation of chondrocyte pellets from stem cells and applying them to cartilage regeneration treatment efficiently, producing a minicircle vector expressing growth factors BMP2 and TGFβ3, When transduced into the stem cells and induced differentiation into chondrocytes, it was confirmed that they can effectively differentiate into chondrocyte pellets. The differentiated chondrocytes were able to significantly express the marker genes of chondrocytes, and when they were transplanted in vivo, the present invention was completed by confirming that the chondrocytes showed significant cartilage regeneration effect at the site of chondral defect.

 Kim Y, Rim YA, Yi H, Park N, Park SH, Ju JH: The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells Int 2016, 2016: 1329459.  Nam Y, Rim YA, Jung SM, Ju JH: Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration. Stem Cell Res Ther 2017, 8:16.  Shu B, Zhang M, Xie R, Wang M, Jin H, Hou W, Tang D, Harris SE, Mishina Y, O'Keefe RJ, et al: BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development. J Cell Sci 2011, 124: 3428-3440.  Tuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, Danielson KG, Hall DJ, Tuan RS: Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J Biol Chem 2003, 278: 41227-41236.  Mueller MB, Tuan RS: Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum 2008, 58: 1377-1388.  Barry F, Boynton RE, Liu B, Murphy JM: Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001, 268: 189-200.  Kim YI, Ryu JS, Yeo JE, Choi YJ, Kim YS, Ko K, Koh YG: Overexpression of TGF-beta1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells. Biochem Biophys Res Commun 2014, 450: 1593-1599.  Tsuchiya H, Kitoh H, Sugiura F, Ishiguro N: Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun 2003, 301: 338-343.  Wang ZH, Li XL, He XJ, Wu BJ, Xu M, Chang HM, Zhang XH, Xing Z, Jing XH, Kong DM, et al: Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model. Braz J Med Biol Res 2014, 47: 279-286.  Kim JH, Do HJ, Yang HM, Oh JH, Choi SJ, Kim DK, Cha KY, Chung HM: Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment. Exp Mol Med 2005, 37: 261-268.  Gill DR, Pringle IA, Hyde SC: Progress and prospects: the design and production of plasmid vectors. Gene Ther 2009, 16: 165-171.  Bandara N, Gurusinghe S, Chen H, Chen S, Wang LX, Lim SY, Strappe P: Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells. Stem Cell Res Ther 2016, 7:48.

As described above, cartilage is difficult to regenerate, and an effective regeneration treatment method is required. Accordingly, it is an object of the present invention to provide chondrocytes induced by differentiation from stem cells and a method for producing the same.

In addition, another object of the present invention to provide a pharmaceutical composition for preventing or treating cartilage defect disease comprising the differentiation-induced chondrocytes as an active ingredient.

In order to achieve the above object, the present invention provides a method for producing chondrocytes induced differentiation from stem cells consisting of the steps i) to v):

i) culturing induced pluripotent stem cells (iPSCs) to form an embryoid body (EB);

ii) culturing the EB formed in step i) in gelatin coating medium to obtain outgrowth cells (OG);

iii) transduction of the OG cells obtained in step ii) with a minicircle vector containing a base sequence encoding BMP2 or a minicircle vector with a base sequence encoding TGFβ3 Making;

iv) inducing differentiation of OG cells transduced in step iii) into chondrocytes; And

v) obtaining chondrocytes induced differentiation in step iv).

In addition, the present invention provides a method for producing chondrocytes induced differentiation from stem cells, comprising the following steps i) to vi):

i) culturing the iPSC to form EB;

ii) culturing the EB formed in step i) in gelatin coating medium to obtain OG;

iii) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding BMP2;

iv) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding TGFβ3;

v) inducing differentiation into chondrocytes by mixing and culturing the OG cells transduced in step iii) and the OG cells transduced in step iv); And

vi) obtaining chondrocytes induced differentiation in step v).

The present invention also provides chondrocyte pellets prepared by the above method.

In a preferred embodiment of the present invention, the minicircle vector comprising the nucleotide sequence encoding the BMP2 comprises (a) a CMV promoter, a BMP2 gene consisting of the nucleotide sequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence. A gene expression cassette; (b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And (c) a non-viral vector that does not include an origin of replication and an antibiotic resistance gene.

In another preferred embodiment of the present invention, the minicircle vector comprising the nucleotide sequence encoding the TGFβ3 is (a) a CMV promoter, the TGFβ3 gene consisting of the nucleotide sequence of SEQ ID NO: 2, and SV40 polyadenylation sequence A gene expression cassette comprising; (b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And (c) a non-viral vector that does not include an origin of replication and an antibiotic resistance gene.

In addition, in another preferred embodiment of the present invention, the step of inducing differentiation into chondrocytes may be performed by culturing for 3 to 30 days in a medium that does not contain recombinant growth factor.

The present invention also provides a pharmaceutical composition for preventing or treating cartilage damage diseases, comprising the chondrocytes as an active ingredient.

In a preferred embodiment of the present invention, the cartilage damage disease may be a degenerative arthritis, rheumatoid arthritis, fracture, plantar fasciitis, humerus externalitis, calcification myositis, nonunion of fracture, or joint injury by trauma.

Therefore, the present invention provides a chondrogenic pellet differentiated from induced pluripotent stem cells (iPSC) by transducing minicircle vectors encoding growth factors BMP2 and TGFβ3.

The chondrocyte pellet may express a marker gene of chondrocytes significantly, and express higher levels of chondrocyte marker genes in comparison with the chondrocytes induced by differentiation in a medium containing recombinant growth factor.

When the chondrocyte pellets are implanted at the site of cartilage damage in vivo, cartilage regeneration may be effectively induced by differentiated chondrocytes, which is more effective than when transplantation of chondrocytes induced by differentiation-induced chondrocytes by adding a recombinant growth factor. Can be useful in tissue engineering therapeutic methods for cartilage regeneration.

1 shows the preparation and identification of a minicircle vector encoding the human growth factor of the present invention:
Figure 1a is a schematic diagram showing the manufacturing process of the minicircle vector for the expression of BMP2 and TGFβ3; And
FIG. 1B shows the results of checking the size of the minicircle vector (mcBMP2) including the BMP2 coding gene and the minicircle vector (mcTGFβ3) including the TGFβ3 coding gene.
2 shows the expression efficiency of mcBMP2 or mcTGFβ3 transduced in HEK293T cells:
Figure 2a is a fluorescence micrograph showing the degree of RFP expression according to the transduction of mcBMP2 or mcTGFβ3 in HEK293T cells;
Figure 2b is the result confirming the proportion of cells transduced with mcBMP2 or mcTGFβ3;
FIG. 2C shows the results of comparing relative expression levels of BMP2 in HEK293T cells transduced with mcBMP2 or mcTGFβ3; FIG. And
Figure 2d is a result of comparing the relative expression level of TGFβ3 in HEK293T cells transduced with mcBMP2 or mcTGFβ3.
Figure 3 shows the differentiation induction of chondrocyte pellets from iPSC in the present invention:
3A is a schematic diagram of the process of inducing differentiation from iPSC to chondrocyte pellets;
3B to 3E show iPSC colonies (FIG. 3B), embryoid bodies (FIG. 3C), OG cells (FIG. 3D) in which EBs are attached to gelatin containers, and OG cells (FIG. 3E) before transduction. Form; And
3F to 3H are fluorescence micrographs for confirming RFP expression in OG cells transduced with minicircle vectors.
Figure 4 shows the identification of mesenchymal stem cell related factors in OG cells transduced with the minicircle vector of the present invention:
4A shows the percentage of OG cells transduced with minicircle vectors;
4B to 4F show the results of confirming the expression level of mesenchymal stem cell marker gene in OG cells transduced with a minicircle vector; And
4G to 4I show the results of azizanine red staining (FIG. 4G), oil red O staining (FIG. 4H) and alcian blue staining (FIG. 4I) of OG cells transduced with minicircle vectors.
5 is a result of confirming the characteristics of differentiation-induced chondrocyte pellets by transducing a minicircle vector:
5A is the cell morphology 5 days after transduction of mcBMP2 or mcTGFβ3; And
5B-5E show RFPs 5 days after start of differentiation induction (FIG. 5B), 10 days (FIG. 5C), 20 days (FIG. 5D) and 30 days (FIG. 5E) after transduction of mcBMP2 or mcTGFβ3. The result of confirming the expression level. GFP results were confirmed to confirm the fluorescence intensity expressed spontaneously in three-dimensionally cultured pellets.
Figure 6 shows the results of confirming the expression level of chondrocyte marker genes in differentiated chondrocyte pellets by transducing the minicircle vector of the present invention.
7 is a result of confirming the characteristics of the differentiated chondrocyte pellets by transducing the minicircle vector of the present invention:
7a to 7c show the results of alcian blue staining (FIG. 7a), safranin O staining (FIG. 7b) and toluidine blue staining (FIG. 7c) of the chondrocyte pellets; And
Figure 7d and 7e is a result of confirming the production of collagen expressed in chondrocyte pellets.
8 shows the results of confirming the in vivo cartilage regeneration ability of chondrocytes differentiated by minicircle vector transduction:
Figure 8a is a schematic diagram showing the process of transplanting chondrocyte pellets into cartilage defect model mice to confirm the cartilage regeneration ability in vivo;
8B to 8D are deficient with alcian blue (FIG. 8B), toluidine blue (FIG. 8C) and safranin O (FIG. 8D) staining 4 weeks after transplantation of chondrocytes induced by differentiation with minicircle vector transduction. The site is identified; And
Figure 8e is the result of confirming the degree of cartilage regeneration according to the minicircle vector transduced chondrocyte transplantation by ICRS score.

Hereinafter, the present invention will be described in detail.

As described above, cartilage is difficult to regenerate, and an effective regeneration treatment method is required. To this end, a method of inducing chondrocyte differentiation from stem cells and displaying cartilage regeneration effect as a cell therapeutic agent is used, but when the recombinant growth factor is used to induce differentiation into chondrocytes, there is a limit in the differentiation efficiency, to overcome this Research is needed.

Accordingly, the present invention provides a method for preparing chondrocytes induced by differentiation from stem cells comprising the following steps i) to v):

i) culturing induced pluripotent stem cells (iPSCs) to form an embryoid body (EB);

ii) culturing the EB formed in step i) in gelatin coating medium to obtain outgrowth cells (OG);

iii) transduction of the OG cells obtained in step ii) with a minicircle vector containing a base sequence encoding BMP2 or a minicircle vector with a base sequence encoding TGFβ3 Making;

iv) inducing differentiation of OG cells transduced in step iii) into chondrocytes; And

v) obtaining chondrocytes induced differentiation in step iv).

In addition, the present invention provides a method for producing chondrocytes induced differentiation from stem cells, comprising the following steps i) to vi):

i) culturing the iPSC to form EB;

ii) culturing the EB formed in step i) in gelatin coating medium to obtain OG;

iii) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding BMP2;

iv) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding TGFβ3;

v) inducing differentiation into chondrocytes by mixing and culturing the OG cells transduced in step iii) and the OG cells transduced in step iv); And

vi) obtaining chondrocytes induced differentiation in step v).

The present invention also provides chondrocyte pellets prepared by the above method.

In the method for producing chondrocytes of the present invention, the iPSC of step i) may be derived from cells derived from patients, and commercially available ones may be used. However, in the point of increasing the biocompatibility in transplanting chondrocytes of the present invention to the cartilage defect site, the iPSC is more preferably derived from cells derived from patients.

In the method for producing chondrocytes of the present invention, the OG cells obtained in step ii) are more preferably separated and obtained in a single cell unit, but are not limited thereto. In order to obtain a single cell unit, the embryonic body can be separated by a cell strainer or the like, and the OG of the single cell obtainable in the production method of the present invention preferably exhibits a fibrous form similar to that of mesenchymal stem cells.

In the method for producing chondrocytes of the present invention, OG cells transduced with a minicircle vector are preferably cultured in chondrogenic differentiation medium for 3 to 30 days to induce differentiation into chondrocyte pellets. Specifically, the differentiation induction is more preferably carried out by culturing for 5 to 20 days, but is not limited thereto. In the differentiation induction, it is preferable that the recombinant growth factor is not further included in the cartilage differentiation medium.

In a specific embodiment of the present invention, the inventors prepared a minicircle vector (mcBMP2 or mcTGFβ3) expressing BMP2 or TGFβ3 by the same process as the schematic diagram of Figure 1a to produce a minicircle vector encoding a human growth factor ( 1).

In addition, the present inventors confirmed that the prepared mcBMP2 and mcTGFβ3 can express a significant growth factor in the cells, it was confirmed that the expression of significant growth factor in HEK293T cells transduced with mcBMP2 or mcTGFβ3 (Fig. 2). ).

Thus, the inventors induced differentiation into chondrocytes from iPSC via EB and OG cells (FIG. 3A). iPSCs were cultured to form EBs, which were cultured in gelatin coated vessels to obtain OG cells (FIGS. 3B-3D). MCBMP2 or mcTGFβ3 were transduced into the obtained OG cells and induced to differentiate into chondrocytes (FIGS. 3F to 3H). OG cells transduced with mcBMP2 or mcTGFβ3 significantly expressed marker genes of mesenchymal stem cells (FIGS. 4A to 4F), and when confirmed the differentiation potential, it was confirmed that they could be differentiated into chondrocyte lineages (FIGS. 4G to 4G). 4i).

Next, we induced differentiation of OG cells transduced with mcBMP2 or mcTGFβ3 into chondrocytes (FIG. 5). In the chondrocyte pellets obtained after differentiation induction, chondrocyte marker genes were significantly expressed, indicating that the marker genes were expressed at a higher level than the control group induced by differentiation by adding a recombinant growth factor to the medium (FIG. 6). Differentiation-induced chondrocyte pellets can accumulate extracellular matrix (ECM) and produce both type 1 collagen and type 2 collagen (FIG. 7).

Finally, we transplanted and bred differentiated chondrocyte pellets into the joints of cartilage-deficient model mice to see if the differentiated chondrocyte pellets showed significant regeneration at cartilage defect sites in vivo (FIG. 8A). As a result, it was confirmed that the chondrocytes were significantly regenerated at the site where the chondrocyte pellets of the present invention were transplanted to show the cartilage regeneration effect (FIG. 8).

Therefore, chondrocyte pellets differentiated from induced pluripotent stem cells (iPSCs) by transducing minicircle vectors encoding growth factors BMP2 and TGFβ3 provided by the present invention are significantly expressed while expressing marker genes of chondrocytes. It is possible to express higher levels of marker genes of chondrocytes compared to differentiation-induced chondrocytes in the medium to which the growth factor is added.

When the chondrocyte pellets are implanted at the site of cartilage damage in vivo, cartilage regeneration may be effectively induced by differentiated chondrocytes, which is more effective than when transplantation of chondrocytes induced by differentiation-induced chondrocytes by adding a recombinant growth factor. Can be useful in tissue engineering therapeutic methods for cartilage regeneration.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cartilage damage diseases, comprising chondrocytes prepared by the method of the present invention as an active ingredient.

In the pharmaceutical composition of the present invention, the cartilage damage disease is any one or more selected from the group consisting of degenerative arthritis, rheumatoid arthritis, fracture, plantar fasciitis, humerus periarthritis, calcification myositis, fracture nonunion and joint injury due to trauma. Preferably, but not limited to, any disease known in the art as a disease of the cartilage region that can be caused by a defect or damage to the cartilage may be applied without limitation.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the site of interest, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such considerations when determining the effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives can be added. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Manufacturing of minicircle vectors encoding human growth factors

<1-1> Preparation of a minicircle vector expressing BMP2 or TGFβ3

In order to induce the expression of BMP2 and TGFβ3 in the present invention, a minicircle vector was prepared by the same process as in FIG. 1A.

Specifically, the human BMP2 and TGFβ3 genes were optimized by using codons to synthesize cDNA sequences as base sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The synthesized cDNA sequence was inserted into the parent plasmid (CMV-MCS-EF1-RFP-SV40-PolyA; manufacturer: System Biosciences, Mountain View, CA, USA) which is an empty vector. Upon insertion, a BMP2 and / or TGFβ3 sequence was inserted into the sequence between BamHI and XbaI in a multiple cloning site present under the CMV promoter to prepare a parent vector comprising growth factor genes. Each parent vector (ppBMP2, pp TGFβ3) containing BMP2 or TGFβ3 was transformed into ZYCY10P3S2T E. coli cells, respectively. Transformed cells were separated into single colony units and seeded in 2 ml of LB medium containing 500 μg / ml kanamycin and initially cultured at 30 for 2 hours. Then, 200 ml terrific broth (TB) was added to the 1 L culture flask, and 100 µl of the initial culture medium was inoculated and incubated for 15 hours while shaking culture at 30 to 200 rpm. To convert the parental vector plasmid into minicircle vector after incubation, 200 ml of LB medium containing 200 μl of 4% 1N NaOH and 20% L-arabinose was added to the culture flask. The flask to which the medium was added was further incubated at 30 to 200 rpm for 5 hours. After incubation, cells were obtained and plasmid DNA was extracted using NucleoBond Xtra plasmid purification kit (Macherey-Nagel, Duren, Germany). The extracted DNA was double-cut into XbaI and BamHI to confirm the size of the minicircle and the inserted BMP2 or TGFβ3 gene. Each vector into which BMP2 and TGFβ3 was inserted was named mcBMP2 and mcTGFβ3. To prepare a negative control, a minicircle vector (mcMock) was prepared in the same manner using the parental plasmid (ppMock) without BMP2 and TGFβ3 inserted.

As a result, as shown in Figure 1b mcBMP2 size of about 7.3 kb, when double-cut with restriction enzymes appeared to appear in two bands of about 1.1 kb BMP2 gene and about 5 kb minicircle. In addition, the size of mcTGFβ3 was about 7.5 kb, and when double-cut by restriction enzyme, it was confirmed that two bands of about 1.3 kb of TGFβ3 gene and about 5 kb of minicircle were shown.

<1-2> Transduction efficiency of mcBMP2 and mcTGFβ3

In order to determine whether mcBMP2 and mcTGFβ3 can express expression activity when transduced into cells, the expression level of RFP coexisting in mcBMP2 and mcTGFβ3 was measured.

Specifically, each of mcBMP2 or mcTGFβ3 prepared in Example <1-1> was mixed with lipofectamine (lipofectamine) in Opti-MEM medium (Thermo Fisher Scientific) for 20 minutes. The mixed DNA-lipopeptamine mixture was then added to HEK293T cell culture medium and incubated for 6 hours in a 37% 5% CO ₂ incubator. After incubation, the cells were checked for morphology by phase contrast microscopy, followed by fluorescence microscopy to observe RFP expression levels in HEK293T cells. In addition, the protein expression level was confirmed by a Bradford assay in the medium in which the cells were cultured to confirm the protein level expressed in HEK293T cells transformed with mcBMP2 or mcTGFβ3. For quantitative analysis, the absorbance values of the recombinant BMP2 protein (rhBMP2) or the recombinant TGFβ3 protein solution were compared based on the absorbance values of 1.0.

As a result, as shown in FIG. 2, compared with HEK293T cells transduced with mcBMP2 and mcTGFβ3, mcMock showed the highest level of RFP expression in the cells transduced with mcMock, confirming the highest transduction efficiency of mcMock (FIG. 2a and 2b). In addition, when confirming the expression level of the protein secreted in the cell culture medium, the absorbance of the supernatant of the HEK293T cells transformed with mcBMP2 showed a significantly high level, it was confirmed that the expression level of about 0.1 mg / ㎖ (FIG. 2C). Compared with this, mcTGFβ3 expression was found to show a relatively low level, but compared with mcMock culture supernatant, it was confirmed that the expression level of protein can be expressed as a significant level of transduction with mcTGFβ3 (Fig. 2d).

Induction of Chondrocyte Differentiation from Human iPSC Using mcBMP2 and mcTGFβ3

The method of differentiating human cartilage from iPSC (hiPSC) into chondrocytes was performed according to the schematic diagram shown in FIG. 3a. All experiments were repeated three times for the same experimental group.

i) iPSC preparation step: The method for obtaining iPSC from cord blood mononuclear cells (PBMC) was performed by a reprogramming method according to a conventional reverse differentiation induction method (Non Patent Literature 001). The obtained iPSCs were cultured in a vitronectin coated vessel (Thermo Fisher Scientific, Waltham, Mass., USA), and the culture medium was incubated using E8 medium (STEMCELL Technologies) with one cycle daily. The prepared iPSC is as shown in Figure 3b.

ii) Generation of Embryos (EBs) from iPSCs: The prepared iPSCs were detached from the bottom of the vessel, counted to 2 × 10 ⁶ cells and seeded in new plates. The culture medium was used by mixing 1: 1 with TeSR-E8 medium and Aggrewell medium (STEMCELL Technologies). IPSC cells inoculated in mixed media were incubated for 24 hours in 37 5% CO ₂ incubator. Then, the medium was removed and replaced with fresh E8 medium to incubate for 3 days, and then the medium was replaced with E7 medium for another 3 days to obtain an embryo (EB) (FIG. 3C).

iii) induction from EB to outgrowth cells (OG cells): EB was then transferred to gelatin coated containers. To this end, the culture vessel was coated with 0.1% gelatin for 30 minutes on the bottom of the vessel and dried thoroughly. The obtained EB was obtained and suspended in OG induction medium. OG induction medium was used with DMEM (Thermo Fisher Scientific) medium containing 20% fetal bovine serum (FBS, Thermo Fisher Scientific) and 10% penicillin / streptomycin (Thermo Fisher Scientific). EBs were inoculated in gelatin-coated vessels at a density of 50 to 70 EB / cm ₂ , incubated for 3 days in 37% 5% CO ₂ incubators and incubated with eggplant-protruding cells (embryonic overgrowth cells, OG cells). Induced. The cell morphology of the induced OG is shown in Figure 3d.

iv) Transducing OG Cells with Minicircle Vectors: The OG cells are then detached and the remaining EB clumps are removed with a 40 μm cell strainer (BD Technologies, Franklin Lakes, NJ, USA) OG cells in cell units were obtained (FIG. 3E). OG cells exhibit a fibrous form similar to mesenchymal stem cells. The obtained OG cells were inoculated again in a new gelatin coated container at a density of 1 to 5 × 10 ⁴ cells / cm 2 and transduced with the minicircle vector prepared in Example <1-1>. The day before transduction, the culture medium was exchanged with DMEM medium containing no serum and antibiotics and incubated overnight, followed by transduction with mcMock, mcBMP2 or mcTGFβ3 using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Cells transduced with the minicircle vector were checked for transduction by confirming the expression level of RFP in the cells using a fluorescence microscope (FIGS. 3F to 3H). OG cells transduced with mcMock show high levels of RFP expression, and similar to RFP expression in HEK293T cells, RFP expression levels are lower in OG cells transduced with mcBMP2 or mcTGFβ3 than mcMock. Confirmed.

v) Inducing Differentiation of OG Cells Transduced with Minicircle Vector into Chondrocyte Pellets: Cultured overnight and transduced OG cells were prepared in 15 ml conical tubes to be 3 × 10 ⁵ cells per pellet to differentiate cartilage Cultured in the medium. Cartilage differentiation medium (CDM) contains 20% knockout serum replacement, 1 × non-essential amino acids, 1 mM L-glutamine, 1% sodium pyruvate, 1% ITS + premix, 10 ^-7 M The composition of DMEM medium containing dexamethasone, 50 mM ascorbic acid and 40 μg / ml L-proline was used. The CDM was not added with recombinant growth factors such as BMP2 and TGFβ3. OG cells were suspended in CDM medium, precipitated by centrifugation at 750 × g for 5 minutes, and then cultured for 30 days to differentiate into chondrocyte pellets. Medium was changed every three days. After the end of the culture, differentiated chondrocyte pellets were obtained. The obtained chondrocyte pellets were stored frozen at -80 until use.

Characterization of Differentiated Induced Cells Using Minicircle Vectors

<3-1> Characterization of OG cells derived from iPSC

Iii) In order to confirm the characteristics of the cultured OG cells in the induction of the OG cells in the EB, the expression level of the mesenchymal stem cell (MSC) marker was confirmed.

The OG cells induced in step iii) of <Example 2> were obtained and suspended in Trizol (Trizol, Thermo Fisher Scientific) to disrupt the cells and extract the mRNA. Using the extracted mRNA as a template, cDNA was synthesized with RevertAid ^™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Using the synthesized cDNA as a template, PCR was performed for MSC marker genes CD44, CD73, CD90, CD105 and CD45 to confirm the expression level of each gene. After quantitative analysis was repeated three times for the same gene, each gene expression level value was corrected based on the GAPDH expression level in the same cell. Together with OG cells, iPSCs were identified for the same gene expression level.

First, when the efficiency of the minicircle vector transduced into the OG cells was confirmed, the efficiency of the minicircle vector transduced into the OG cells was confirmed to appear similar to the HEK293T cells (Fig. 4a). In addition, as a result of confirming the expression of the MSC marker gene, the expression levels of CD44, CD73 and CD105 in OG cells were significant, showing a slightly lower expression level compared to MSC, but significantly higher than in hiPSC cells. It confirmed that it shows (FIG. 4B, FIG. 4C, FIG. 4E). In comparison, CD90 was confirmed to be expressed at a somewhat higher level with MSC in OG cells (FIG. 4D). In addition, CD45, known as a marker that is negatively expressed in mesenchymal stem cells, was confirmed to be expressed at low levels in both MSC and OG cells (FIG. 4F).

In addition, the possibility of differentiation of MSCs was determined. MSCs are known to be able to differentiate into three lineages: adipocytes, chondrocytes and osteoblasts. The inventors performed alizarin red staining, oil red O staining and alkian blue staining to confirm the lineage differentiation capacity of OG cells derived from iPSC.

As a result, as shown in Figures 4g to 4i OG cells can be differentiated into chondrocyte lineages (Fig. 4g), not only can be differentiated into adipocyte lineages (Fig. 4h), but also can be differentiated into chondrocyte pellets It was confirmed to represent (Fig. 4i).

<3-2> Confirmation of Differentiation-Induced Growth Factor Protein Expression Efficiency Using mcBMP2 and mcTGFβ3

In order to confirm the expression efficiency of the minicircle vector in the chondrocyte differentiation induced in step v) of <Example 2>, the expression of RFP and GFP was confirmed in the cells of the differentiation induction process. mcBMP2-OG and mcTGFβ3-OG as well as OG (mcBMP2-OG) and mcTGFβ3 transduced mcBMP2 transduced (mcBOTH-OG) in a 1: 1 ratio by mixing Co-culture induced differentiation into chondrocytes. OG cells transduced with minicircle vectors were started to induce differentiation in chondrocyte differentiation medium, and then pellets were formed by centrifugation at 5, 10, 20 and 30 days to form condensates. Cell morphology, intracellular RFP expression, and GFP expression levels were determined.

As a result, as shown in FIG. 5, the amount of cells condensed first was confirmed that the amount of mcTGFβ3-OG was somewhat lower than other experimental groups (FIG. 5A). From the 20th day after induction of differentiation, the size of the cell condensate of mcTGFβ3-OG proliferated compared to mcBMP2-OG and mcBOTH-OG, and the proliferation rate of the cells was significantly higher than that of the other experimental groups (FIG. 5D). .

When confirming the expression level of RFP, it was confirmed that the RFP expression level in chondrocyte pellets increased from 5 days after the start of differentiation induction in all experimental groups (FIG. 5B). Then, it was confirmed that the morphology of the aggregated cells continued even on day 10 (FIG. 5C). In all experimental groups of mcBMP2-OG, mcTGFβ3-OG and mcBOTH-OG, the expression of RFP showed a tendency to increase continuously until 20 days after the induction of differentiation (FIG. 5D). After 30 days it showed a tendency to decrease (Fig. 5e). In the OG cell control group (NA) transduced with mcMock, RFP expression was continuously increased after initiation of differentiation, and was confirmed to be maintained even after 30 days.

<3-3> Confirmation of differentiation efficiency of differentiation-induced chondrocyte pellets using mcBMP2 and mcTGFβ3

Then, in order to analyze the characteristics of the differentiated chondrocyte pellets, the expression levels of marker genes SOX9, ACAN, COL2A1, COL1A1 and COL10A1 in chondrocytes were confirmed. In addition, the chondrocyte osteoblast formation ability of the chondrocyte pellet was confirmed by confirming the expression level of the bone formation marker RUNX2. In order to compare the differentiation efficiency using the minicircle vector of the present invention with the prior art, to the chondrocyte differentiation-induced positive control (Both rhGF) in a medium containing both growth factors of BMP2 and TGFβ3 without transducing the minicircle vector The same marker gene expression was also confirmed.

As a result, as shown in Figure 6, all the experimental group of mcBMP2-OG, mcTGFβ3-OG and mcBOTH-OG confirmed that the chondrocyte marker gene is expressed at a high level compared to the control of hiPSC, OG cells and mcMock-transduced OG cells It was. SOX9, a marker expressed in early chondrocytes, was found to be expressed at a significant level in chondrocyte pellets derived from cells transduced with minicircle vectors (FIG. 6A). In the case of the ACAN and COL10A1 markers, the absolute expression level was expressed at a lower level than SOX9, but was confirmed to be expressed at a high level compared to the positive control Both rhGF group (FIGS. 6D and 6E). In addition, COL2A1 and COL1A1 were also expressed at significant levels in mcBMP2-OG, mcTGFβ3-OG and mcBOTH-OG, and it was confirmed that chondrocyte marker genes were expressed at significantly higher levels than both rhGF positive controls (FIG. 6c and 6d). In comparison, the bone formation marker RUNX2 was confirmed to be expressed at a lower level than the addition of the recombinant growth factor, it was confirmed that the expression at the lowest level in mcBOTH-OG.

In addition to the expression of chondrocyte marker genes, the level of accumulation of extracellular matrix (ECM) was confirmed. ECM accumulation confirmation was determined by alcian blue, safranin O and toluidine blue staining on chondrocytes derived from differentiation from mcBMP2-OG, mcTGFβ3-OG and mcBOTH-OG. Confirmed.

The experimental process for this is as follows. Chondrocyte pellet cells were first washed with phosphate-buffered saline (PBS). The washed samples were fixed by treating 4% paraformaldehyde at room temperature for 2 hours. After fixation, dehydration was performed using an ethanol solution and washed again using an ethanol-xylene mixed solution. Washed samples were embedded in paraffin overnight. The obtained paraffin pieces were fixed and cut into 7 μm units using a microtome to prepare sample pieces. Prior to staining each piece, the piece sections were placed in 60 ovens for at least 10 minutes to raise the temperature. The fragment sections were immediately deparaffinized with xylene, hydrated with decreasing ethanol concentration and washed with running tap water for 1 minute.

For alcian blue staining, the slices were immersed in 1% alcian blue solution (Sigma Aldrich, St. Louis, MO, USA) and incubated for 30 minutes at room temperature. After incubation, the pieces were again washed with tap water and contrast stained using a nuclear fast red solution, and then the samples were observed under a microscope.

For safranin O staining, the fragment sections were treated with Weigert's hematoxylin (Sigma Aldrich) solution, stained for 10 minutes at room temperature, and washed again with running tap water for 10 minutes. The washed pieces were again stained with 0.001% Fast Green solution (Sigma Aldrich) and 0.1% Safranin O solution (Sigma Aldrich) for 5 minutes each, and the samples were observed under a microscope.

Toluidine blue staining was performed by immersing the dehydrated fragment pieces in 0.04% toluidine blue solution and incubating for 10 minutes. The stained sections were washed with running tap water and dried for 10 minutes until complete drying. At the end of the staining process, the slices were dehydrated by increasing the ethanol concentration. Ethanol was removed by two treatments with 100% xylene, mounted on VectaMount ^™ Permanent mounting medium (vector Laboratories, Calif., USA) and observed microscopically.

In addition, in order to confirm the type of collagen constituting the generated ECM, it was confirmed by immunochemical staining whether collagen formation of chondrocyte pellets through type 1 collagen and type 2 collagen staining. Prior to staining each piece, the piece sections were placed in 60 ovens for at least 10 minutes to raise the temperature. The fragment sections were immediately deparaffinized with xylene, hydrated with decreasing ethanol concentration and washed with running tap water for 1 minute.

The sample sections were then immersed in boiling citrate buffer and hydrated again to unmask the antigenic protein. After the antigen exposure was completed, the sample pieces were cooled, and then treated with 3% hydrogen peroxide solution to block the activity of the peroxidase expressed in the tissue. The sample pieces were then washed again and blocked with TBS containing 1% BSA. The primary antibody was diluted with blocking solution and treated with anti-type collagen antibody (1/200 dilution; Abcam) or anti-type collagen antibody (1/100 dilution; Abcam) to the sample for 4 nights. Incubated. The next day, the sample pieces were washed with TBS containing 0.1% Tween-20 and treated with secondary antibody. Secondary antibodies (1/200 dilution; vector Laboratories) were treated at room temperature for 40 minutes and then washed. After washing, ABC reagent drops (vector Laboratories) were treated for 30 minutes. Then, it was immersed in DAB solution and incubated for 5 minutes and counterstained by treatment with Mayer's hematoxylin (Sigma Aldrich) for 1 minute. Contra-stained samples were mounted and microscopically observed with bright illumination.

As a result, as shown in Figure 7, it was confirmed that ECM was significantly formed in all experimental groups, of which chondrocytes derived from mcBMP2-OG showed homogeneous overall ECM expression, while chondrocytes derived from mcTGFβ3-OG In the pellet, it was confirmed that ECM was accumulated and expressed in some regions (FIGS. 7A to 7C).

Examples of collagen constituting cartilage in vivo include type 1 collagen and type 2 collagen. It is known that type 1 collagen forms fibrous cartilage and type 2 collagen forms hyaline cartilage. Expression of collagen was found to be significantly expressed in all experimental groups, but it was confirmed that the type 1 collagen and type 2 collagen at high levels in chondrocyte pellets derived from mcBOTH-OG (FIGS. 7D and 7E). In this way, the present inventors in the process of inducing chondrocyte differentiation using the minicircle vector of the present invention, inducing chondrocyte differentiation is more effective when chondrocytes are differentiated by culturing mcBMP2-OG and mcTGFβ3-OG together. It was expected to be possible.

In vivo cartilage regeneration of differentiation-induced chondrocyte pellets by minicircle vector transduction

In order to confirm that the differentiation-induced chondrocyte pellets effectively express cartilage regeneration ability in vivo, the differentiation-induced chondrocyte pellets from mcBOTH-OG were transplanted in a cartilage-deficient mouse model to confirm the regeneration effect. .

First, model mice with cartilage defects were prepared (FIG. 8A). To this end, anesthesia was used to anesthetize Sprague-Dawley and use a microdrill to articular cartilage of the trochlear groove of the distal femur, using a 1.5 mm x 1.5 mm x 1.5 mm cartilage. Caused a defect. Then, chondrocyte pellets induced for differentiation for 10 days in Example 2 were implanted at the site of cartilage defect. The skin at the surgical site opened for arthrotomy was closed with nylon thread. Four weeks after the operation, the animal model was fed with sufficient feed and drinking water, and after 4 weeks, the mice were sacrificed and their cartilage defects were examined by tissue immunity. For the cartilage regeneration effect, the degree of regeneration was confirmed using the recovery score established by the International Cartilage Repair Society (ICRS) and compared.

As a result, as shown in FIG. 8, it was confirmed that ECM formation was induced by chondrocytes transplanted at the site where the cartilage defect was induced through alcian blue, toluidine blue, and safranin O staining. In the control group that maintained the defect for 4 weeks (defect only) and the control group transplanted with mcMock-OG-derived chondrocytes, ECM accumulation was not observed and the empty space was increased. This result shows that despite the transplantation of the cells of mcMock-OG, growth factors are not provided, and thus, no significant differentiation of chondrocytes has progressed, indicating that cell death was induced.

In comparison, in the experimental group transplanted with chondrocyte pellets derived from mcBOTH-OG, chondrocytes were densely induced in different areas and induced differentiation, indicating that cartilage regeneration was significantly induced and that ECM accumulation was induced (FIGS. 8B to 8D). ). In addition, it was confirmed that the cartilage regeneration was effectively performed by showing a significantly higher level of scores compared to the control group when confirming histological scores (FIG. 8E).

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Claims (8)

i) culturing induced pluripotent stem cells (iPSCs) to form an embryoid body (EB);
ii) culturing the EB formed in step i) in gelatin coating medium to obtain outgrowth cells (OG);
iii) transduction of the OG cells obtained in step ii) with a minicircle vector containing a base sequence encoding BMP2 or a minicircle vector with a base sequence encoding TGFβ3 Making;
iv) inducing differentiation of OG cells transduced in step iii) into chondrocytes; And
v) obtaining chondrocytes induced differentiation in step iv);
The minicircle vector containing the nucleotide sequence encoding the BMP2 is
(a) a gene expression cassette comprising a CMV promoter, a BMP2 gene consisting of the nucleotide sequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence;
(b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And
(c) is a non-viral vector that does not include an origin of replication and an antibiotic resistance gene,
The minicircle vector containing the nucleotide sequence encoding the TGFβ3 is
(a) a gene expression cassette comprising a CMV promoter, a TGFβ3 gene consisting of the nucleotide sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence;
(b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And
(c) A method for producing differentiation-induced chondrocytes from stem cells, characterized in that it is a non-viral vector containing no starting point of replication and an antibiotic resistance gene.
i) culturing the iPSC to form EB;
ii) culturing the EB formed in step i) in gelatin coating medium to obtain OG;
iii) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding BMP2;
iv) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding TGFβ3;
v) inducing differentiation into chondrocytes by mixing and culturing the OG cells transduced in step iii) and the OG cells transduced in step iv); And
vi) obtaining differentiation-induced chondrocytes in step v);
The minicircle vector containing the nucleotide sequence encoding the BMP2 is
(a) a gene expression cassette comprising a CMV promoter, a BMP2 gene consisting of the nucleotide sequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence;
(b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And
(c) A method for producing differentiation-induced chondrocytes from stem cells, which is a non-viral vector containing no starting point of replication and an antibiotic resistance gene.
delete i) culturing the iPSC to form EB;
ii) culturing the EB formed in step i) in gelatin coating medium to obtain OG;
iii) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding BMP2;
iv) transducing the OG cells obtained in step ii) with a minicircle vector comprising a nucleotide sequence encoding TGFβ3;
v) inducing differentiation into chondrocytes by mixing and culturing the OG cells transduced in step iii) and the OG cells transduced in step iv); And
vi) obtaining differentiation-induced chondrocytes in step v);
The minicircle vector containing the nucleotide sequence encoding the TGFβ3 is
(a) a gene expression cassette comprising a CMV promoter, a TGFβ3 gene consisting of the nucleotide sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence;
(b) an att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); And
(c) A method for producing differentiation-induced chondrocytes from stem cells, characterized in that it is a non-viral vector containing no starting point of replication and an antibiotic resistance gene.
The method according to any one of claims 1, 2 and 4, wherein the step of inducing differentiation into chondrocytes is performed by culturing for 3 to 30 days in a medium containing no recombinant growth factor. Method for producing chondrocytes induced differentiation from stem cells.
Chondrocytes prepared by the method of any one of claims 1, 2 and 4.
Claim 6 cartilage cells comprising as an active ingredient, cartilage damage disease prevention or treatment pharmaceutical composition.
8. The prevention of cartilage damage disease according to claim 7, wherein the cartilage injury disease is degenerative arthritis, rheumatoid arthritis, fracture, plantar fasciitis, humerus external surgery, calcific myositis, nonunion of fracture, or joint injury due to trauma. Or therapeutic pharmaceutical compositions.

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