Osteoblasts differentiated from mesenchymal stem cells and compositions for treating bone disease comprising same

Abstract

To provide osteoblasts and compositions comprising the osteoblasts for treating bone diseases.SOLUTION: Disclosed is an osteoblast having both proliferation ability and differentiation ability, where the osteoblast has higher expression levels of connexin 43, RUNX2 and collagen type 1A compared with those of undifferentiated stem cells and mature osteocytes; higher expression levels of angiopoietin and alkaline phosphatase compared with those of undifferentiated stem cells; and lower expression levels of osterix, osteocalcin and osteopontin compared with those of mature osteocytes. Also, the osteoblast has a lower Ki-67 expression level compared with that of undifferentiated stem cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application is Korean Patent Application No. 10-2020-1002 filed on October 8, 2020. No. 0130138, the entire disclosure of which is incorporated herein by reference. .

[0002] The present invention relates to a method for differentiating mesenchymal stem cells into osteoblasts, a method for differentiation of osteoblasts into osteoblasts, and a method for the differentiation of osteoblasts into osteoblasts. The present invention relates to a cell therapeutic agent for treating bone diseases and a method for producing the same.

[0003] The present invention also relates to a method for treating bone disease, comprising administering osteoblasts obtained by the above method to a patient with a bone disease. It relates to treatment methods. [Background technology]

[0004] Stem cells can differentiate into various types of cells. Stem cells are a collective term for undifferentiated cells that have the ability to undergo specific differentiation. They can differentiate into specific cells depending on factors and / or the environment. Types of stem cells include embryonic stem cells. embryonic stem cell, embryonic germ cell germ cell), adult stem cell, cancer stem cell ( In recent years, stem cells that can differentiate into various types of cells have become It can be used to regenerate damaged tissue, treat cartilage damage, diabetes, leukemia, neurological disorders, heart disease, spinal cord There is active research being conducted to treat various diseases, including trauma and fibrotic disorders. This has led to research being conducted to try to differentiate stem cells into specific cell types. In addition, we have developed induced pluripotent stem cells ( Cell differentiation such as induced pluripotent stem cells (iPS) In particular, mesenchymal stem cells (MSCs) are used to The ESCC Cell line has the ability to differentiate into various mesodermal cell types, including bone, cartilage, fat, and muscle cells. Due to this ability, mesenchymal stem cells are useful in treating bone diseases and tissue damage. It is believed to be of value as a therapeutic agent in the field of regenerative medicine. Activating the inherent repair mechanisms of tissues and organs that would otherwise be unable to recover or The purpose of this treatment is to regenerate damaged areas by replacing damaged tissue. To grow tissues or organs in a laboratory that the body cannot repair itself and then safely transplant them into the body. This includes attempts to inject or transplant cells into the body as a regenerative medicine-based treatment for intractable diseases. Stem cell therapy using the self-renewal and differentiation capabilities of stem cells is considered to be the next generation of treatment. It has been in the spotlight for this.

[0005] However, this depends on various factors such as the origin, site of origin, degree of culture, and degree of differentiation of the stem cells used. Due to the risk factors involved, safety and effectiveness standards are strict and approval is difficult. In addition, we can differentiate pluripotent stem cells, which have the ability to differentiate into a variety of cells, into specific cells and sell them commercially. For practical use, mass production is necessary, but the stem cells can be rapidly and safely differentiated into bone cells. It is difficult to achieve this.

[0006] Korean Patent 10-2016-00345 for a method of differentiating stem cells into specific cells In issue 41, we report the development of a hydrogel-coated porous membrane using the sol-gel phase transition. A method for differentiating cells into bone cells has been disclosed and is currently under Korean Patent No. 10-2018-01. In issue 14307, hexanoyl glycol was added to the medium to promote differentiation of mesenchymal stem cells. In US8,580,757B2, a method for the preparation of mesenchymal stem cells containing chitosan was disclosed. We have developed a method to regulate cell differentiation using miRNA or siRNA. Thus, various attempts have been made to differentiate mesenchymal stem cells into bone cells. However, the method allows differentiation of stem cells without administering any external substances to the stem cells or adding any expensive components to the differentiation medium. Methods for rapidly differentiating cells into large numbers of bone cells remain to be developed. This is not the case.

[0007] However, osteoblasts can be derived from stem cells so that they can be used as cell therapy for bone-related diseases. Further research into stable and rapid differentiation and culture methods is still required. The reality is that there are Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors have developed a method for regenerative medicine to treat bone diseases using mesenchymal stem cells. The present invention was created while considering the optimal method for producing a therapeutic agent for bone cells differentiated from bone marrow cells. The inventors have provided a method for producing osteoblasts for producing a cell therapy agent for treating bone diseases. When the differentiation method of the present invention is used, the conventional method of differentiating stem cells for use in cell therapy is Compared with other cells, it can differentiate into osteoblasts stably and quickly, and the efficacy of the osteoblasts in bone reconstruction is very excellent. Thus, the present invention has been completed.

[0009] Therefore, the object of the present invention is to provide a method for differentiating mesenchymal stem cells into osteoblasts, The present invention provides a cell therapy agent for treating bone diseases, which comprises transformed osteoblasts, and a method for producing the same. . [Means for solving the problem]

[0010] The present invention involves the steps of: i) coating an air-permeable polymeric membrane with a surfactant bubble; ii) Add 1×10 mesenchymal stem cells to the bubble of step i) 3 ~1×10 5 pieces / cm 2 Density of The step of inoculating with iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts from a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); The present invention provides a method for differentiating mesenchymal stem cells, comprising the steps of:

[0011] According to a preferred embodiment of the present invention, the surfactant in step i) is a poloxamer (po loxamer).

[0012] According to a preferred embodiment of the invention, the bubble of step i) is made of the air-permeable polymer It is produced by adding a surfactant on a membrane and moving it to generate bubbles. It's fine.

[0013] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) are derived from umbilical cord, umbilical cord blood, Any one selected from the group consisting of placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis. It may be derived from one or more.

[0014] The present invention further provides osteoblasts obtained by the above method.

[0015] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome 2 (Runt-related transcriptome on factor 2, RUNX2) and collagen type 1A (Collagen The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature bone cells. The levels of angiopoietin 1 (ANGPT1) and amyloidosis were high in all patients. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (tin, OPN) may be lower than that of mature bone cells.

[0016] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0017] The present invention further provides a cell therapeutic agent for treating bone diseases, comprising osteoblasts obtained by the above method. .

[0018] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome 2 (Runt-related transcriptome on factor 2, RUNX2) and collagen type 1A (Collagen The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature bone cells. The levels of angiopoietin 1 (ANGPT1) and amyloidosis were high in all patients. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (tin, OPN) may be lower than that of mature bone cells.

[0019] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0020] According to a preferred embodiment of the present invention, the bone disease is a bone fracture, avascular necrosis of the femoral head, spinal fusion, Delayed or non-union, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta It may be any one or more selected from the group consisting of:

[0021] The present invention further relates to a method for treating bone disease, comprising administering osteoblasts obtained by the above method to a patient with a bone disease. Methods of treatment are provided.

[0022] The mesenchymal stem cells of the present invention are pluripotent (m ultipotent) is used in the same sense as undifferentiated cells, but it is also used for fat cells, osteoblasts, and cartilage cells. Ectocytes such as various mesodermal or neural cells, including bone cells, cardiac cells, or muscle cells This refers to adult stem cells that have the ability to differentiate into mesenchymal stem cells. Not only are they free from the risk of developing cancer and ethical issues, but they also have immune protection after transplantation. There may be no rejection reaction.

[0023] The term "osteoblast" as used herein refers to a bone cell of a vertebrate. These cells produce bone matrix (osteoblasts), also known as osteoblasts. They synthesize and secrete bone matrix to produce bone. They can also become normal bone cells by embedding themselves in the bone tissue they have created. It can deposit substances such as Ca and Mg ions necessary for bone mineralization in the bones. Depending on the difference in the internal substances and their activity, they are divided into resting and forming stages, and although they have a high division ability, they are old. In bone, their numbers decrease.

[0024] In the present invention, the term "bone disease" refers to a condition in which damage occurs to the bone, causing bone This refers to a condition in which the structure and density of bones change, making them more susceptible to fracture. Bones provide stability to the skeletal system. It is the toughest tissue in the body that maintains the bones and serves as a lever for muscles and protects internal organs. It not only stores calcium, magnesium and other minerals, but also Such bone diseases are mostly caused by trauma such as fractures, or metabolic diseases such as avascular necrosis and osteoporosis. Regardless of the cause, it is caused by a problem with the mechanical support of the body, which reduces mobility. In this specification, bone disease refers to osteoporosis, osteoporosis, etc. These conditions include osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease or osteogenesis imperfecta.

[0025] The term "osteoporosis" refers to a condition in which bone mass is reduced and bone quality changes It is a condition in which bones become weaker and more susceptible to fractures, and is mainly caused by genetics, early menopause, and overweight. The main causes are an unhealthy diet or steroid medication.

[0026] The above-mentioned "osteonecrosis" is a disease in which the bone tissue dies due to lack of blood supply to the bone. It can occur anywhere, but most commonly occurs on the upper thigh, upper arm, shoulder, knee, or It occurs in the spine, etc.

[0027] The above-mentioned "pseudoarthrosis" is also called "pseudoarthrosis" and occurs when the bone does not join together after it is broken ( Nonunion) refers to the movement of the bone as if it were a joint. Alternatively, it may occur due to a bacterial infection at the fracture site.

[0028] The above-mentioned "Paget's disease" refers to the process of bone remodeling ( Localized, in which excessive bone remodeling occurs and affects various parts of the skeletal system. It is a bone disease that mainly affects the pelvis, thighs or skull.

[0029] The above-mentioned "osteogenesis imperfecta" refers to a condition in which bones are congenitally weak and easily broken without any particular cause. Collectively, they are also called skeletal dysplasias.

[0030] The term "differentiation" as used herein refers to the process by which cells divide and grow. The phenomenon in which the structures and functions of living cells and tissues become specialized during the process of life. It refers to the change in form and function of an organism in order to perform the role it is assigned to. The occurrence or result of qualitative differences between parts of a biological system that were originally homogeneous The state in which the system is divided into qualitatively distinct subsystems is called differentiation.

[0031] The "cell therapeutic agent" of the present invention is a The cells and tissues isolated, cultured, and specially engineered from the human body are used for therapeutic, diagnostic, or preventive purposes. A pharmaceutical product that is used by living subjects to restore the function of cells or tissues. or heterologous cells are expanded or selected in vitro or otherwise altered in biological properties of the cells. It may also be used through a series of actions, such as

[0032] As mentioned above, conventional cell therapy using stem cells has not been widely available due to issues such as high unit price. However, it has been difficult to commercialize the technology, especially since it takes too much time and money to differentiate stem cells into osteoblasts. There was a drawback.

[0033] On the other hand, the differentiation method according to the present invention stably and quickly differentiates stem cells at the early stage of subculture into osteoblasts. Conventional stem cell differentiation methods can differentiate stem cells that have been subcultured eight or more times. In contrast, osteoblasts could not be obtained unless the cells were differentiated for more than 20 days. So, if you differentiate stem cells that have been subcultured five times for about three days, you can obtain differentiated osteoblasts. In particular, when the differentiation method of the present invention is used, the source of stem cells (donor Since all of the cells can be differentiated into osteoblasts without any reaction variation due to the nature of the allogeneic It can be used as a cell therapy agent.

[0034] Thus, the present invention provides a method for preparing a surfactant-based polymeric membrane by: i) coating an air-permeable polymeric membrane with a surfactant bubble; The stage, ii) Add 1×10 mesenchymal stem cells to the bubble of step i) 3 ~1×10 5 pieces / cm 2 Density of The step of inoculating with iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts from a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); A method for differentiating mesenchymal stem cells containing the stem cells into osteoblasts, and a method for providing osteoblasts differentiated by the method. Can provide.

[0035] The polymer membrane of step i) may be the porous membrane of a Hyperflask.

[0036] The density of step ii) is preferably 1×10 3 ~1×10 5 pieces / cm 2 Even if More preferably, 1×10 4 pieces / cm 2 may be also possible.

[0037] According to a preferred embodiment of the present invention, the surfactant in step i) is a poloxamer (po Other surfactants than poloxamer may be cytotoxic to cells. The poloxamer is cytotoxic (Example 4) and is not suitable for the present invention. Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 124, Poloxamer Poloxamer 237, Poloxamer 338, and Poloxamer 407. It may be one or more of them.

[0038] The surfactant in step i) may be up to 10% surfactant.

[0039] The bubbles in step i) are generated by adding and moving a surfactant on the air-permeable polymer membrane. For example, the bubble in step i) may be formed by the air-permeable polymer. - Generated by shaking detergent onto the membrane or through pipetting However, the present invention is not limited to this.

[0040] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) are derived from umbilical cord, umbilical cord blood, Any one selected from the group consisting of placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis. More preferably, the mesenchymal stem cells of step ii) are Alternatively, the stem cells may be umbilical cord-derived mesenchymal stem cells.

[0041] In the case of the umbilical cord-derived mesenchymal stem cells, the umbilical cord tissue discarded after birth is used. The advantage of this method is that it is easy to extract and obtain a large amount of stem cells. The proliferation and differentiation potential of the cells are limited by the age and health condition of the donor from whom they are isolated and extracted. Although there are limitations and variability, umbilical cord-derived stem cells are the earliest available adult stem cells. As stem cells that can be used for transplantation, variables such as the donor's age have little effect on stem cell potency. Umbilical cord-derived mesenchymal stem cells are not affected by the immune system and have excellent proliferation and differentiation capabilities. The advantage of this method is that it can isolate stem cells that can be used to treat various diseases, including liver disease and musculoskeletal diseases. be.

[0042] In the step ii), the mesenchymal stem cells are seeded into the bubble by forming an air-permeable polymer membrane on the interface. After coating with surfactant bubbles, wait at least 2 hours for the bubbles to begin to disappear. The vaccine may be administered at the time of vaccination.

[0043] The mesenchymal stem cells in step ii) may be stem cells that have been subcultured for 5 to 8 passages. Alternatively, the stem cells may be those that have been subcultured for 5 to 6 passages.

[0044] The mesenchymal stem cells seeded in step ii) are cultured in a differentiation medium to obtain α- A culture medium which may contain one or more selected from the group consisting of MEM, DMEM and FBS. The culture may be performed in a nutrient solution for 12 to 48 hours. The culture may be carried out at 4° C. for 20 to 30 hours.

[0045] The differentiation in the step iii) may be carried out for 24 hours to 120 hours. Preferably, the differentiation may be carried out for 60 to 80 hours.

[0046] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome 2 (Runt-related transcriptome on factor 2, RUNX2) and collagen type 1A (Collagen The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature bone cells. The levels of angiopoietin 1 (ANGPT1) and amyloidosis were high in all patients. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of oxidative stress hormone (oxidative stress hormone, tin, OPN) may be lower than that of mature bone cells.

[0047] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0048] The osteoblasts of the present invention express Connexin 43 (Connexin 4 3, CX43), Runt 2 (Runt-related transcription factor 2, RUNX2), collagen type 1A 1A1, COL1A1) and alkaline phosphatase (Alkaline Phosphatase The expression of AP, a cell proliferation marker, is significantly higher than that of undifferentiated stem cells. Although the expression of Ki-67, which is a marker of differentiation, is lower than in undifferentiated cells, it is still expressed and can form colonies. In contrast, fully mature osteoblasts are the early stage of osteoblast proliferation. Osterix (OSX), whose expression increases with increasing cell size, and Osteocal Osteocalcin (OCN) and Osteopontin The expression levels of OPN in osteoblasts were lower than those in mature osteocytes (NHOst). Angiopoietin is distinctly expressed in early osteoblasts. 1. The expression of ANGPT1 also increased several hundred times, resulting in early bone formation. They clearly showed the characteristics of blast cells (Table 1).

[0049] [Table 1]

[0050] The CX43 gene is encoded by the GJA1 gene and is involved in the function of chondrocytes, osteoblasts, osteoclasts, and osteoblasts. It is the most common gap junction protein expressed in bone cell types such as CX43. It plays an important role in regulating signaling between various bone cell types and is involved in bone development, differentiation, and differentiation. In particular, CX43 regulates osteoblastic bone formation, modeling, and remodeling, as well as pathology. Necessary for cell survival, proliferation and differentiation, and enhances the expression of various osteogenic markers can be done.

[0051] RUNX2 is a master regulator of bone cell differentiation and an early phenotype of osteoblast differentiation Alkaline phosphatase (ALP) and the late phenotype osteocalcin (Osteocalcin) calcin, OCN) expression. osteo-progenitors, immature osteoblasts) RUNX2+, proliferate, differentiate into mature bone cells, and mineralize bone. The osteoblasts stop dividing for a certain period of time. As differentiation occurs, the cells gradually lose their ability to divide and differentiate into osteocytes. (Figures 17a and 17b).

[0052] Collagen type 1A (COL1A1) is a protein that is expressed during differentiation into osteoblasts. It is a bone formation marker.

[0053] The Osterix (OSX) corresponds to a differentiation factor related to bone cell formation. , increasing bone mattress expression by enhancing COL1A1 promoter activity It plays an important role in the differentiation of osteoblasts into mature osteoblasts.

[0054] The osteocalcin (OCN) corresponds to a differentiation factor related to bone cell formation. is formed by osteoblasts and deposited in the bone matrix, and then part of the newly formed bone is is released into the blood, so measuring the blood concentration can indicate the degree of bone formation.

[0055] Osteopontin (OPN) is a differentiation factor related to bone cell formation and is an osteogenic marker. This corresponds to the Car protein.

[0056] The present invention further provides a cell therapeutic agent for treating bone diseases, comprising osteoblasts obtained by the above method. do.

[0057] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome 2 (Runt-related transcriptome on factor 2, RUNX2) and collagen type 1A (Collagen The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature bone cells. The levels of angiopoietin 1 (ANGPT1) and amyloidosis were high in all patients. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (tin, OPN) may be lower than that of mature bone cells.

[0058] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0059] According to a preferred embodiment of the present invention, the bone disease is a bone fracture, avascular necrosis of the femoral head, spinal fusion, Delayed or non-union, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta It may be any one or more selected from the group consisting of:

[0060] The present invention further relates to a method for treating bone disease comprising administering osteoblasts obtained by the above method to a patient with a bone disease. It may provide a method of treatment.

[0061] The administration may be parenteral, and the osteoblasts may be administered alone or following surgery, radiation, or other suitable route. and in combination with therapeutic, hormonal, chemotherapy and biological response modifiers. This may also be the case. Effect of the Invention

[0062] The differentiation method according to the present invention is capable of stably and rapidly differentiating mesenchymal stem cells into osteoblasts. The differentiated osteoblasts have excellent angiogenic and osteogenic potentials. Therefore, the method of differentiating stem cells into osteoblasts according to the present invention and the osteoblasts obtained by said method The blast cells can also be effectively used as a cell therapy agent for bone diseases. [Brief description of the drawings]

[0063] [Figure 1] FIG. 1 shows the process for differentiating and obtaining umbilical cord-derived osteoblasts of the present invention. [Diagram 2] Figure 2 shows the results of observing whether the poloxamer bubbles remaining on the porous membrane were retained for 7 hours (0H~7H) after removing the poloxamer solution after poloxamer bubble coating. It was confirmed that the bubbles disappeared 2 hours (2H) after removing the poloxamer solution. [Diagram 3] Figure 3 shows 3D fluorescence microscopy images of poloxamer bubble coating followed by loading of QD-treated cells. [Figure 4] Figure 4 shows the results of a cytotoxicity experiment for each type of surfactant. It was confirmed that the surfactants (DIAPON K-SF and Tween-20) other than P407 (poloxamer 407), which is the result of CCK-8 (cell counting kit-8) on the first day, were toxic to cells. [Diagram 5] Figure 5 shows the results of a cytotoxicity experiment for each type of surfactant. As a result of checking cell proliferation using CCK-8 (cell counting kit-8) from 1 to 3 days, it was confirmed that the remaining surfactants (DIAPON K-SF and Tween-20) except for P407 (poloxamer 407) were cytotoxic and did not cause cell proliferation. [Figure 6]Figure 6 shows the results of a cytotoxicity experiment for each type of surfactant. It was confirmed that the remaining surfactants (Methylprednisolone and Tween-20) except for P407 (Poloxamer 407) showed toxicity to cells, as shown in the results of CCK-8 (cell counting kit-8) on the first day. [Figure 7] Figure 7 shows the results of a cytotoxicity experiment for each type of surfactant. As a result of checking cell proliferation using CCK-8 (cell counting kit-8) from 1 to 3 days, it was confirmed that the remaining surfactants (Methylprednisolone and Tween-20) except for P407 (Poloxamer 407) were cytotoxic and did not cause cell proliferation. [Figure 8] FIG. 8 shows that the expression level of a gene (COL1A) was significantly higher in cells differentiated from bubble foam surfactant than in undifferentiated cells or cells differentiated from gel surfactant. [Figure 9] FIG. 9 shows that the expression level of a protein (VEGF) was significantly higher in cells differentiated from bubble foam surfactant than in undifferentiated cells or cells differentiated from gel surfactant. [Figure 10] Figure 10 shows the results of comparing the expression levels of bone induction genes in differentiated cell therapeutic agents of the present invention (RCB001-DP1 to RCB005-DP5) to undifferentiated cells (RCB001, RCB002, RCB005). It was confirmed that the expression levels of Connexin 43 (CX43) and Runt-related transcription factor 2 (RUNX2) secreted from the cell therapeutic agents of the present invention were significantly increased in undifferentiated cells. [Figure 11]11 shows the results of comparing the expression levels of osteoinductive genes of the differentiated cell therapy of the present invention (RCB005-DP1) with undifferentiated cells (RCB005), bone marrow-derived mesenchymal stem cells (BM-MSCs) and mature osteoblasts (NHOst). The expression levels of early osteoblast markers Connexin 43 (CX43), Runt-related transcription factor 2 (RUNX2) and Collagen type 1A1 (COL1A1) were higher than those of undifferentiated cells and mature osteoblasts, and the expression levels of Osterix (OSX), Osteocalcin (OCN) and Osteopontin (OPN) were lower than those of mature osteoblasts (NHOst). [Figure 12] 12 shows that the osteogenic marker protein and the angiogenic marker protein were increased in the differentiated bone cell therapeutic agent of the present invention compared to undifferentiated cells. (A) represents COL1A1, (B) represents osteopontin (OPN), and (C) represents angiopoietin. [Figure 13] 13 shows that the differentiated cell therapeutic agent of the present invention maintains bone formation marker proteins and angiogenesis marker proteins even 3 to 7 days after thawing. (A) represents COL1A1, (B) represents osteopontin (OPN), and (C) represents angiopoietin. [Figure 14] 14 shows the vascular endothelial vessel formation ability of the umbilical cord-derived osteoblasts of the present invention. It was confirmed that angiogenesis was induced by the umbilical cord-derived raw cell (Undifferentiated UCMSC) and osteogenic differentiation cell therapeutic agent to the same extent as the VEGF positive control group, which is known as an angiogenesis-inducing factor, and in particular, it was confirmed that the blood vessels formed by the proteins secreted by the cell therapeutic agent were thicker and stronger. [Figure 15]Figures 15a and 15b show the results of efficacy of the cell therapeutic agent of the present invention in large animals (goats). [Figure 16] FIG. 16 shows the results of efficacy of the cell therapeutic agent of the present invention in small animals (rats). [Figure 17] 17a and 17b show the differentiation stages of mesenchymal stem cells into bone cells. The area indicated by the dotted line represents the stage of the cell therapy agent of the present invention. [Figure 18] Figure 18 shows the results of confirming the cell division ability of two batches of undifferentiated UCMSCs (raw material) and differentiated osteoblasts (DP) using Ki-67 (an indicator of cell division). It was confirmed that Ki-67 expression was consistently and rapidly decreased to less than 1% from DP, which means that CF-M801 is an early stage osteoblast of the osteoblast model. [Figure 19] Figures 19a and 19b show the results of measuring CFU-F using two batches of undifferentiated stem cells BMMSC and UCMSC (raw material) and differentiated osteoblasts (DP). It was confirmed that the differentiated osteoblasts (DP, CF-M801) showed a decrease in CFU-F in a pattern similar to that of Ki-67 (an indicator of cell division) expression. [Figure 20] Figure 20 shows the results of measuring absorbance after staining with alkaline phosphatase after colonies were formed using two batches of undifferentiated stem cells UCMSC (raw material) and differentiated osteoblasts (DP). Colonies formed by differentiated osteoblasts (DP) were stained with alkaline phosphatase compared to undifferentiated cells, and the absorbance value increased, confirming that the colonies formed by proliferation of this cell therapy were osteoblasts. [Figure 21] 21a and 21b show that the differentiated osteoblasts (DP1 to DP3) of the present invention express CD10 at 80% or more, whereas undifferentiated mesenchymal stem cells express CD10 at 15% or less. This means that the osteoblasts of the present invention are controlled by confirming the purity by confirming osteoblast-specific marker factors that are not conventional stem cell marker factors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] [Example 1] Isolation and harvesting of umbilical cord-derived stem cells First, the arteries and veins were removed from the isolated umbilical cord, and the remaining tissue was finely chopped and then transferred to AdiCo. l TM After reacting with CEFO at 37°C for 30 minutes or more, the cells were extracted. The cells are TM Culture in medium at 37℃ and 5% CO2 to obtain mesenchymal stem cells. did.

[0065] [Example 2] Coating an air-permeable polymer membrane with surfactant bubbles 8% Poloxamer 407 (poloxamer 407) completely dissolved in PBS was placed on the porous membrane of the Hyperflask. Shake the loxamer 407) to generate bubbles, then pour in the remaining poloxamer solution. The air-permeable polymer membrane was coated with poloxamer bubbles at 37°C for 2 hours. After the poloxamer 407 bubbles were generated, the solution was removed through 3D microscopic imaging. After removing the water, it was observed whether the bubbles were still there. After 2 hours (2H), the bubbles disappeared. After 5 hours (5H), it was confirmed that the bubbles had completely disappeared (Figure 2).

[0066] In addition, the stem cells were labeled with quantum dot-coding RNA (QD-PCR) to enable tracking of umbilical cord-derived mesenchymal stem cells. Nucleated silica nanoparticles (QDs) were incubated for 24 hours. After the poloxamer 407 bubbles were generated, the solution was removed and the cells were loaded. The sections were then imaged in 3D using a microscope Z-stack (Figure 3).

[0067] [Example 3] Differentiation and culture of osteoblasts In the above [Example 2], 2 hours after coating with poloxamer bubbles, The umbilical cord-derived stem cells obtained in Example 1 were cultured at 1 × 10 4 pieces / cm 2 The cells were inoculated at 100% density and then incubated in DMEM and The cultured stem cells were cultured in a culture medium containing 100% ethanol and FBS for 24 hours. The cultured stem cells were then cultured in a bone differentiation medium for 72 hours to differentiate into osteoblasts. The cells were differentiated.

[0068] [Example 4] Screening of surfactants suitable for coating air-permeable polymeric membranes Poloxamer is a representative biocompatible surfactant. 407, P407), Sodium Methyl Cocoyl Taurate yl Taurate, DIAPON K-SF), Twin 20 (polyoxyeth ylene sorbitan monolaurate, Tween 20) or The toxicity of methylprednisolone was confirmed.

[0069] Specifically, the upper chamber is placed in a transwell containing a three-dimensional porous membrane. The chamber contains surfactant P407, DIAPON K-SF or Twe en 20 at 0, 0.51, 5 or 15% (v / v), respectively. After coating for 2 hours in the same manner as in [Example 2], the umbilical cord obtained in [Example 1] was Mesenchymal stem cells derived from the cervix were used at 20,000 cells / cm 2 I seeded it with the following. The bar was filled with cell growth medium and cultured for 3 days. On days 1, 2, and 3, CCK-8 (Cat. CK04, DOJINDO) solution and react for 3 hours in a 37℃ CO2 incubator. Then, the absorbance was measured at 450 nm to analyze the cytotoxicity (Figure 4) and cell proliferation (Figure 5). Ta.

[0070] In addition, the upper chamber ( The chamber contains surfactant P407, methylprednisolone, or The cells were treated with Tween 20 at a concentration of 4 mM and coated for 2 hours in the same manner as in Example 2. After the incubation, the umbilical cord-derived mesenchymal stem cells obtained in the above [Example 1] were cultured at 20,000 ce ll / cm 2 The lower chamber was filled with cell growth medium and cultured for 3 days. On the first, second, and third days, CCK-8 solution was added and the cells were incubated in a 37°C CO2 incubator for 3 days. After the reaction time, the absorbance was measured at 450 nm to evaluate the cytotoxicity (Figure 6) and cell proliferation (Figure 7). Analyze.

[0071] As a result, as shown in [Figures 4] to [Figures 7], the remaining DIAPON K except for P407 -SF, Tween 20 and methylprednisolone were all confirmed to be toxic to cells. came.

[0072] [Example 5] Selection of surfactant coating methods Poloxamer is bubbled or gelled onto the Hyperflask porous membrane. The progress of osteogenic differentiation of umbilical cord-derived stem cells differentiated by the method of [Example 3] after coating with the above-mentioned cellulose acetate solution was observed. The degrees were compared.

[0073] Specifically, the differentiated osteoblasts and undifferentiated cells were collected and incubated with Trizol. TM The mixture was treated with chloroform (Sigma) and centrifuged to separate the layers, obtaining only the mRNA. ranscriptor Universal cDNA Master Kit(Ro The mRNA obtained using che was synthesized into cDNA. The DNA was amplified by RT-PCR and the number of DNA copies was measured at the gene level. The polymerase chain reaction conditions were 95°C for 10 seconds, 54°C for 10 seconds, and 72°C for 30 seconds. The cycle time was 50 cycles per second (Figure 8).

[0074] In addition, the osteoblasts and undifferentiated cells produced and induced to differentiate by the method of [Example 3] were cultured. The liquid was collected and vascular endothelial growth factor (VAG) was detected. The secretion level of VEGF was measured by enzyme immunoassay. A ELISA (detected immunosorbent assay) was performed (Figure 9).

[0075] As a result, as shown in [Figures 8] and [Figure 9], the undifferentiated or gel-like poloxamer was separated into The cells differentiated by the method of the present invention have higher osteoinductive and angiogenic genes than the cells differentiated by the method of the present invention. It was confirmed that the expression level of the protein was very good.

[0076] [Example 6] Evaluation of bone formation ability of the obtained osteoblasts <6-1> Bone formation ability of osteoblasts (gene level) RNA was isolated from osteoblasts collected during the differentiation process of osteoblasts, cDNA was synthesized, and then the Real Time Polymerase Chain Reaction Using real-time reverse transcription-polymerase chain reaction (RT-PCR), we identified the bone formation gene marker Lance 2 (Run-related transcription factor 2, RUN The gene expression levels of X2 and connexin 43 (CX43) were Compared with undifferentiated cells.

[0077] Specifically, osteoblasts and undifferentiated cells produced and induced to differentiate by the method of [Example 3] above were Each was collected and treated with Trizol TM and chloroform (Sigma), and then centrifuged. The layers were separated to obtain only mRNA. The mRNA obtained using the cDNA Master Kit (Roche) was converted to cDNA. Then, DNA was synthesized by RT-PCR for RUNX2 and CX43. The PCR conditions were 95°C, 1 The incubation period was 50 cycles of 0 s, 54°C for 10 s, and 72°C for 30 s.

[0078] In addition, osteoblasts, undifferentiated cells, and bone marrow-derived cells produced and induced to differentiate by the method of [Example 3] were We collected BM-MSC and mature osteocytes (NHOst) respectively. Obtain mRNA and use the Transcriptor Universal cDNA Master The obtained mRNA was synthesized into cDNA using the ER Kit (Roche). DNA was amplified by RT-PCR for RUNX2, CX43, and COL1A. The gene expression level was confirmed. The polymerase chain reaction conditions were 95°C for 10 seconds, 5 The incubation was performed for 50 cycles at 4°C for 10 seconds and 72°C for 30 seconds.

[0079] As a result, as shown in [Figures 10] and [Figures 11], in the osteoblasts of the present invention, X2, CX43, or COL1A promotes differentiation of undifferentiated stem cells and mature bone cells (NHOst) In Figure 10, RCB001 and RCB002 were highly expressed. and RCB005 means undifferentiated cells, RCB001-DP1 to RCB005-DP 5 means a differentiated cell therapeutic agent of the present invention.

[0080] On the other hand, in the case of OSX, OCN, or OPN, the present invention was expressed in mature osteocytes (NHOst). The expression level of the IL-1 protein was higher than that of the normal osteoblasts, which indicates that the osteoblasts of the present invention were significantly higher than NHOst. In Fig. 11, RCB001, RCB0 RCB001‐DP1 to RCB005‐DP1 represent undifferentiated cells. DP5 refers to the differentiated cell therapeutic agent of the present invention.

[0081] <6-2> Bone formation ability of osteoblasts (protein level) In the culture medium and osteoblasts collected during the osteoblast differentiation process in [Example 3], bone formation or Collagen type 1A (Collagen type 1A), known as an angiogenic protein marker, en type 1A, COL1A), osteopontin, or angiopoietin (ANGPT-1) protein concentration Furthermore, the frozen cell therapy agent of the present invention showed no change in bone morphogenetic protein even after thawing. We also examined whether the cells could maintain vascular induction proteins.

[0082] Specifically, osteoblasts, undifferentiated cells, and The differentiation culture medium and the differentiation medium were collected and analyzed by enzyme-linked immunosorbent assay. We performed immunosorbent assay (ELISA) and COL1A was The degree of change in protein expression in differentiated and undifferentiated cells was examined by lysing the cells. We confirmed that osteopontin and angiopoietin were proteins in the culture medium up to 72 hours after differentiation. The degree of protein secretion was confirmed.

[0083] As a result, as shown in FIG. 12, the COL1A protein was expressed in the osteoblasts of the present invention. The expression of phospholipids was increased, and the secretion of osteopontin and angiopoietin was all highly expressed. In addition, as shown in Figure 13, even after 3 to 7 days from thawing, It has been confirmed that the bone morphogenetic protein or blood vessel inducing protein of the cell therapy agent of the present invention is maintained. In Fig. 12 and Fig. 13, (A) shows COL1A1 and (B) shows osteosarcoma. OPN and (C) stand for angiopoietin. Taste.

[0084] [Example 7] Evaluation of angiogenic potential of the obtained osteoblasts Human umbilical vein endothelial cells (HUVECs) secreted angiogenic proteins by osteoblasts. Human umbilical vein endothelial cell,HU We confirmed the angiogenic ability of VECs.

[0085] Specifically, a 12-well transformer containing a porous membrane with a pore size of 8.0 μm was used. On the well plate, 4 × 10 undifferentiated stem cells or osteoblasts were 4 In cell / well Inoculation and incubation of HUVEC cells with quantum dots for 24 hours After incubation, the cells were harvested and plated under the transwell at 25,000 / cm 2 Density inoculated After seeding, they were co-cultured for one day and then subjected to an analysis of HUVEC vascular tube formation. The cells were seeded into a 12-well plate and then transferred to the medium using a transwell. After the material exchange occurred, the cells were cultured for 12 hours to confirm the induction of blood vessels in HUVEC cells. The osteogenic differentiation cells and undifferentiated cells were placed on the Transwell for comparison. The negative control group was HUVEC cells were seeded on the coated Matrigel without VEGF. The positive control group was cultured in the same HUVEC medium for 12 hours. The cells were cultured under the same conditions, with the exception that VEGF was added at 20 ng / ml.

[0086] As a result, as shown in Figure 14, VEGF-positive mice, which are known to be angiogenesis-inducing factors, The umbilical cord-derived source cells (Undi ferentiation During co-culture of UC-MSCs and bone cell therapeutic agents, In addition, it was confirmed that osteogenic differentiation cell therapy (Osteogen When co-cultured with vascular endothelial cells (VECs), thick and robust blood vessels were formed. In particular, it was confirmed that the proteins secreted by the cell therapy agent were It was confirmed that blood vessels were formed to be thicker and stronger.

[0087] [Example 8] Confirmation of bone regeneration ability of the obtained osteoblasts - in vivo After inducing bone defects in large animal (goat) or small animal (rat) models, the cells of the present invention were The bone regeneration ability of the cell treatment agent was confirmed.

[0088] Specifically, after inducing femoral defects in an immunosuppressed goat model, 1×10 7 The bone regeneration effect was confirmed by treating osteoblasts for 26 weeks (Table 2). To test the efficacy of the dermal endothelial cell, the bone tissue was decalcified for 2.5 months, and H&E and Masson's staining were performed. The efficacy of this cell therapy was evaluated histologically using Trichrome staining.

[0089] In the case of an immunosuppressed rat model, after induction of radial bone defect, 1 × 10 6 The bone regeneration effect was confirmed for 12 weeks by treating osteoblasts (Table 3). Only the scaffold composed of luginate was treated. The degree of bone regeneration was evaluated using μCT images. The bone tissue was fixed, decalcified, sectioned, and stained to prepare slides. New bone regeneration was confirmed.

[0090] [Table 2]

[0091] [Table 3]

[0092] As a result of the efficacy test in the goat model, as shown in [Figure 15a] and [Figure 15b], 26 In both males and females, new bone formation in the proximal and distal parts of the control group was was very insufficient and only partially confirmed, indicating that the wound was in the process of healing. On the other hand, in the proximal and distal areas of the cell-injected group, cells were packed into the defect site and increased in number. The newly formed bone trabeculae are organically connected without any breaks, and the thickness is It was also confirmed that the endothelium had become considerably thicker (white arrow in the lower left of Figure 15a). The formation of new bone at both the 13th and 26th weeks was significantly higher than that at the time of cell injection. It was confirmed that this was significantly observed only in the feeding group.

[0093] As a result of the efficacy test on the rat model, as shown in [Figure 16], the G3 group had pa The G5 group was differentiated for 3 days at passage 5, while the G6 group was differentiated for 2 days at passage 7. In both groups, bone volume and bone mass were significantly increased compared to the control group. The bone mineral density and BMD tended to increase, especially at passage 5. It was confirmed that the bone volume of the treatment group was significantly increased.

[0094] [Example 9] Confirmation of cell therapy stage The steps of the cell therapy agent of the present invention were confirmed.

[0095] <9-1> Confirmation of Ki-67 expression Human Ki-67 is not expressed during the arrest phase (G0) of the cell cycle, but is expressed during the proliferation phase (G1, S, G2 It is known that the IL-1 gene is expressed during the M phase (phase I and II), and cell therapy agents are effective in preventing the proliferation of cells after differentiation into osteocytes. Therefore, the undifferentiated UCSMC (raw material) and the differentiated osteoblasts of the present invention were The expression of Ki-67, an indicator of cell division, was evaluated in two batches of DP. The results were confirmed by immunocytochemistry (ICC) and nuclear staining. The cells were stained with DAPI, a coloring agent, and the expression level of Ki-67 was corrected for cell number.

[0096] Specifically, prepare a slide plate and place 3 × 10 5 Undifferentiated in quantity, After inoculation of the differentiated cells, they were cultured in a CO2 incubator for 24 hours. Fix the cells in 1% Triton X-100 for 10 min at room temperature. The cells were permeabilized and blocked with BSA for 30 minutes at room temperature. After that, the Ki-67 primary antibody was incubated at room temperature for 1 hour, and the fluorescently linked secondary antibody was incubated in the dark. The reaction was allowed to proceed at room temperature for 1 hour. TM Gold A Mounting using ntifade Mountant (Invitrogen) The cells were then observed using a fluorescence microscope.

[0097] As a result, as shown in Figure 18, Ki-67 expression was consistently increased in osteoblasts (DP). It was confirmed that the difference in the

[0098] <9-2> Confirmation of CFU-F expression Two types of undifferentiated stem cells, BMMSC and UCMSC (raw material) and differentiated osteoblasts (DP) of the present invention CFU-F was measured using a batch. In addition, when mesenchymal stem cells were cultured in vitro, they increased in number. It is known that the cells begin to grow and form characteristic cell colonies, and the cells in the colonies are fibroblasts. They show a cell-like shape and each cell colony is called a colony forming unit. This is called t-fibroblast, and is known to be an important characteristic of stem cells. The colonies formed were stained with 2% crystal violet and then visually and microscopically examined. Observe the stained colonies by X-ray photography and select those with a size of 3 mm or more and a density of 80% or more. The amount was quantified by counting.

[0099] As a result, as shown in [Figure 19a] and [Figure 19b], differentiated osteoblasts (DP, CF-M 801) showed a decrease in CFU-F in a pattern similar to that of Ki-67 (an indicator of cell division) expression. It was also confirmed that bone marrow-derived stem cells and umbilical cord-derived stem cells were used as raw materials. Although a large amount of Ronnies were formed, the cell therapy agent hardly allowed colony formation. This confirmed that umbilical cord-derived stem cell source cells differentiate into bone cells, From these results, it was concluded that the cell therapy agent had lost its colony-forming ability. It was confirmed that the tondo had differentiated.

[0100] <9-3> Confirmation of ALP expression Colonies that appeared on average less than 1% in CFU-F using the cell therapy agent CF-M801 The cells that form the bone are either undifferentiated mesenchymal stem cells or cells that have been induced to differentiate into bone (Osteoblasts). st).

[0101] Specifically, the colonies that appear when cultured in the same manner as CFU-F in Example <9-2> above are Ronnie was stained with ALP (Alkaline Phosphatase) to detect osteogenic differentiation. After inoculating 10,000 cells and maintaining them for one week, ALP staining was performed. After ALP staining, the stain was thoroughly dissolved by reacting with dimethylsulfoxide. After collecting only the supernatant, the absorbance was measured using a microplate reader. Ta.

[0102] As a result, as shown in [Figure 20], undifferentiated UCMSCs, which are allogeneic umbilical cord-derived mesenchymal stem cells, Although ALP staining was hardly observed, CF-M801 cells that were induced to differentiate into bone showed different It was confirmed that all colonies in the three lots were ALP positive. The absorbance of the transduced CF-M801 was higher than that of undifferentiated cells. The results showed that the values ​​were 1.5 times higher in all cases than in undifferentiated UCMSCs. As a result, differentiated osteoblasts (DP) continue to divide for a certain period of time. As differentiation progresses, they gradually lose their ability to divide and differentiate into osteocytes. Ta.

[0103] Overall, the cell therapy agent of the present invention is a master regulator of bone cell differentiation. RUNX2, which is involved in the differentiation of mesenchymal stem cells into osteoblasts, is a key factor in the differentiation of osteoblasts. (osteo-progeniotrs, immature osteoblasts) are RUNX2+, have proliferation potential, differentiate into mature bone cells, and are involved in mineralization. ation was further observed (Figures 17a and 17b).

[0104] <9-4> Confirmation of CD-10 expression To confirm the differentiation of cell therapy drugs, the expression level of CD10 was measured by flow cytometry (Fl Flow cytometry (FACS) and immunofluorescence The results were confirmed by ICC (International Cooperative Clinical Trials).

[0105] Specifically, osteoblasts and undifferentiated cells produced and induced to differentiate by the method of [Example 3] above Cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) were suspended in 2% BSA / DPBS solution. The CD101 primary antibody was reacted at room temperature for 1 hour, washed, and then FITC fluorescence was The secondary antibody linked to β-lactam was reacted at room temperature for 30 minutes. After washing the cells, the supernatant was After removing the cells, the cells were fixed in 3.7% formaldehyde at room temperature for 20 minutes and then analyzed by flow cytometry. The expression level of CD10 was confirmed using a cytochrome P450 (BD Accuri C6 Plus) mouse model. .

[0106] The ICC prepared slide plates and placed 3 × 10 5 Undifferentiated and differentiated by quantity After inoculation, the cells were cultured in a CO2 incubator for 24 hours. The cells were fixed in hyaluronan at room temperature for 10 minutes and then lysed in 1% Triton X-100 at room temperature for 10 minutes. The cells were permeabilized and blocked with BSA at room temperature for 30 minutes. After that, the CD101 primary antibody was incubated at room temperature for 1 hour, and the FITC fluorescent-linked secondary antibody was incubated in the dark. After that, the mixture was incubated at room temperature for 1 hour. TM Gold Antifade Mountant (Invitrogen) was used for mounting. The cells were then cultured and observed using a fluorescent microscope.

[0107] As a result, as shown in [Figure 21a] and [Figure 21b], the differentiated osteoblasts of the present invention (DP1-DP3) express CD10 at 80% or more, whereas undifferentiated mesenchymal stem cells It was confirmed that 15% or less of the osteoblasts expressed CD10. We confirmed osteoblast-specific markers that are not conventional stem cell markers, and checked and controlled their purity. It meant being. [Industrial Applicability]

[0108] The differentiation method according to the present invention is capable of stably and rapidly differentiating mesenchymal stem cells into osteoblasts. The differentiated osteoblasts have excellent angiogenic and osteogenic potentials. Therefore, the method of differentiating stem cells into osteoblasts according to the present invention and the osteoblasts obtained by said method The blast cells can also be effectively used as a cell therapy or method for treating bone diseases. The present invention has industrial applicability.

Claims

1. An osteoblast having both proliferation and differentiation capabilities, The osteoblasts have higher expression levels of connexin 43 (CX43), runt-related transcription factor 2 (RUNX2) and collagen type 1A1 (COL1A1) than undifferentiated stem cells and mature bone cells, higher expression levels of angiopoietin 1 (ANGPT1) and alkaline phosphatase (AP) than undifferentiated stem cells, and lower expression levels of osterix (OSX), osteocalcin (OCN) and osteopontin (OPN) than mature bone cells. The osteoblasts have a lower expression level of Ki-67 compared to undifferentiated stem cells. Osteoblasts.

2. The osteoblasts are i) coating an air-permeable polymeric membrane with poloxamer bubbles; ii) Add 1×10 mesenchymal stem cells to the bubble of step i) ３ ~1×10 ５ pieces / cm ２ and inoculating the oocytes at a density of iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts in a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); obtained by a method comprising: The bubbles of step i) are produced by adding and moving a surfactant on the air-permeable polymeric membrane to generate bubbles. The osteoblast of claim 1.

3. The osteoblast of claim 2, wherein the mesenchymal stem cells in step ii) are derived from any one or more of the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp and skin dermis.

4. containing osteoblasts having both proliferation and differentiation capabilities, The osteoblasts have higher expression levels of connexin 43 (CX43), runt-related transcription factor 2 (RUNX2) and collagen type 1A1 (COL1A1) than undifferentiated stem cells and mature bone cells, higher expression levels of angiopoietin 1 (ANGPT1) and alkaline phosphatase (AP) than undifferentiated stem cells, and lower expression levels of osterix (OSX), osteocalcin (OCN) and osteopontin (OPN) than mature bone cells. The osteoblasts have a lower expression level of Ki-67 compared to undifferentiated stem cells. A cell therapy for treating bone diseases.

5. The osteoblasts are i) coating an air-permeable polymeric membrane with poloxamer bubbles; ii) Add 1×10 mesenchymal stem cells to the bubble of step i) ３ ~1×10 ５ pieces / cm ２ and inoculating the oocytes at a density of iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts in a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); obtained by a method comprising: The bubbles of step i) are produced by adding and moving a surfactant on the air-permeable polymeric membrane to generate bubbles. The cell therapy agent according to claim 4.

6. The cell therapy agent according to claim 5, wherein the mesenchymal stem cells in step ii) are derived from any one or more selected from the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis.

7. The cell therapy agent according to claim 4, characterized in that the bone disease is one or more selected from the group consisting of fracture, femoral head osteonecrosis, spinal fusion, delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta.

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