Osteoblasts differentiated from mesenchymal stem cells and compositions containing the same for treating bone diseases

Abstract

The present invention relates to a method for differentiating mesenchymal stem cells into osteoblasts, a cell therapy agent for treating bone diseases comprising osteoblasts differentiated by the above method, or a method for preparing the same. Furthermore, the present invention relates to a method for treating bone diseases, comprising administering osteoblasts obtained by the above method to a patient with a bone disease. The differentiation method of the present invention can stably and rapidly differentiate mesenchymal stem cells into osteoblasts. The above-mentioned differentiated osteoblasts have excellent angiogenesis ability and excellent bone formation ability. Therefore, the method of differentiating stem cells into osteoblasts of the present invention or the osteoblasts obtained by the above method can be effectively used as a cell therapy agent or treatment method related to bone diseases.

Description

Technical Field

[0001] This application claims priority from Korean Patent Application No. 10-2020-0130138, filed on October 8, 2020, which is hereby incorporated by reference into this application in its entirety.

[0002] The present invention relates to a method for differentiating mesenchymal stem cells into osteoblasts, a cell therapeutic agent for treating bone diseases comprising osteoblasts differentiated by the above method, or a method for preparing the same.

[0003] Furthermore, the present invention relates to a method for treating bone diseases, comprising the step of administering osteoblasts obtained by the above method to a patient with the bone disease. Background Art

[0004] Stem cells are a general term for undifferentiated cells that possess the pluripotency to differentiate into various cell types. Stem cells can differentiate into specific cell types under the influence of specific differentiation factors and / or environmental factors. Types of stem cells include embryonic stem cells, embryonic germ cells, adult stem cells, and cancer stem cells. Recently, research is underway to utilize stem cells, which can differentiate into various cell types, to treat a variety of diseases, including tissue regeneration, cartilage damage, diabetes, leukemia, neurological diseases, heart disease, spinal cord injury, and fibrotic disorders. This research is focused on differentiating stem cells into specific cell types. Furthermore, methods such as induced pluripotent stem cells (iPS), which convert fully differentiated cells into stem cells through reverse differentiation, are also being used for cell differentiation. In particular, mesenchymal stem cells (MSCs) are pluripotent stem cells with the ability to differentiate into various mesenchymal cells, including bone, cartilage, fat, and muscle cells. Due to these abilities, mesenchymal stem cells are considered valuable therapeutic agents in the field of regenerative medicine for bone diseases and tissue damage. The goal of regenerative medicine is to regenerate damaged areas by activating the inherent recovery mechanisms of previously unrecoverable tissues or organs or by replacing damaged tissue. This includes attempts to cultivate tissues or organs that the body cannot heal on its own in the laboratory and then safely transplant or inject them into the body. As a treatment based on regenerative medicine and a treatment for intractable diseases, stem cell therapies that exploit the self-replication and differentiation abilities of stem cells are attracting attention as next-generation therapeutics.

[0005] However, due to risk factors based on various factors, such as the source of the stem cells used, the site of origin, the degree of culture, and the degree of differentiation, strict safety and efficacy standards have made it difficult to obtain approval. Furthermore, in order to differentiate stem cells, which have the potential to differentiate into various cell types, into specific cells for commercial use, large-scale production is required, but it is difficult for stem cells to differentiate quickly and safely into bone cells.

[0006] Regarding the method of differentiating stem cells into specific cells, Korean Patent No. 10-2016-0034541 discloses a method of differentiating stem cells into bone cells in a porous membrane coated with a hydrogel using a sol-gel phase transition, and Korean Patent No. 10-2018-0114307 discloses a method of including hexanoyl glycol chitosan in a culture medium to promote the differentiation of mesenchymal stem cells. US 8580757 B2 discloses a method of adjusting the differentiation of mesenchymal stem cells, which is a method using miRNA or siRNA. As described above, various attempts have been made to differentiate mesenchymal stem cells into bone cells, but the following method has not yet been developed: no external substances are added to the stem cells, and no expensive components are added to the differentiation medium, thereby rapidly and massively differentiating stem cells into bone cells.

[0007] However, in order to use them as cell therapeutic agents for bone-related diseases, further research is still needed on methods for stably and rapidly differentiating and culturing stem cells into osteoblasts. Summary of the Invention

[0008] Technical issues

[0009] Therefore, the present inventors have sought to optimize methods for producing osteoblasts differentiated from mesenchymal stem cells, a regenerative medicine approach for treating bone diseases, leading to the completion of the present invention. The present inventors sought to provide a method for producing osteoblasts, which is used to produce cell therapeutics for treating bone diseases. Using the differentiation method of the present invention, they confirmed that, compared to conventional methods for differentiating stem cells for cell therapeutics, these osteoblasts exhibit significantly superior bone remodeling efficacy, leading to the completion of the present invention.

[0010] Therefore, an object of the present invention is to provide a method for differentiating mesenchymal stem cells into osteoblasts, a cell therapy agent for treating bone diseases comprising osteoblasts differentiated by the above method, or a method for preparing the same.

[0011] Technical Solution

[0012] The present invention provides a method for differentiating mesenchymal stem cells into osteoblasts, comprising:

[0013] Step i), coating the breathable polymer film with surfactant bubbles;

[0014] Step ii), mesenchymal stem cells were cultured at a rate of 1×10 3 pieces / cm 2 to 1×10 5 pieces / cm 2 The density of is seeded in the bubbles of step i) above;

[0015] Step iii), differentiating the mesenchymal stem cells from step ii) into osteoblasts in a differentiation medium; and

[0016] Step iv), isolating and obtaining the osteoblasts differentiated in the above step iii).

[0017] According to a preferred embodiment of the present invention, the surfactant in step i) may be poloxamer.

[0018] According to a preferred embodiment of the present invention, the bubbles in step i) may be prepared by adding a surfactant to the breathable polymer film and moving the surfactant to generate bubbles.

[0019] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) can be derived from at least one selected from the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicles, teeth, dental pulp and dermis.

[0020] The present invention also provides osteoblasts obtained by the above method.

[0021] According to a preferred embodiment of the present invention, in the above-mentioned osteoblasts, the expression levels of connexin 43 (CX43), Runt-related transcription factor 2 (RUNX2), and collagen type 1A1 (COL1A1) may be higher than those in undifferentiated stem cells and mature bone cells; the expression levels of angiopoietin 1 (ANGPT1) and alkaline phosphatase (AP) may be higher than those in undifferentiated stem cells; and the expression levels of osteoblast-specific transcription factor Osterix (OSX), osteocalcin (OCN), and osteopontin (OPN) may be lower than those in mature bone cells.

[0022] According to a preferred embodiment of the present invention, the expression level of Ki-67 in the osteoblasts may be lower than that in the undifferentiated stem cells.

[0023] The present invention also provides a cell therapeutic agent for treating bone diseases, comprising osteoblasts obtained by the above method.

[0024] According to a preferred embodiment of the present invention, in the above-mentioned osteoblasts, the expression levels of connexin 43, Runt-related transcription factor 2 and type 1A collagen may be higher than those of undifferentiated stem cells and mature bone cells; the expression levels of angiogenin and alkaline phosphatase may be higher than those of undifferentiated stem cells; and the expression levels of osteoblast-specific transcription factor, osteocalcin and osteopontin may be lower than those of mature bone cells.

[0025] According to a preferred embodiment of the present invention, the expression level of Ki-67 in the osteoblasts may be lower than that in the undifferentiated stem cells.

[0026] According to a preferred embodiment of the present invention, the bone disease may be one or more selected from the group consisting of fracture, femoral head necrosis, spinal union, delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta.

[0027] The present invention also provides a method for treating bone diseases, comprising the step of administering osteoblasts obtained by the above method to a patient with the bone disease.

[0028] In the present invention, the term "mesenchymal stem cells" is used synonymously with multipotent, undifferentiated cells and refers to adult stem cells that possess the ability to differentiate into various mesenchymal cells, including adipocytes, osteoblasts, chondrocytes, cardiac cells, and muscle cells, as well as ectodermal cells, neural cells. Furthermore, these cells are free of the cancer-causing and ethical issues often associated with embryonic stem cells and, upon transplantation, do not induce immune rejection.

[0029] The "osteoblasts" of this invention are cells that produce osteoblasts (bone cells) in vertebrates, also known as osteoblasts. They form bone by synthesizing and secreting bone matrix, and they also become ordinary osteoblasts by burying themselves in the bone tissue they create. Furthermore, they deposit substances such as calcium and magnesium ions, which are essential for bone formation, into bone, calcifying the tissue. Osteoblasts are divided into two phases, quiescent and formative, depending on the differences in their internal substances and their activity. While their ability to divide is high, their number decreases in older bones.

[0030] "Bone disease" in the present invention refers to a condition in which bone damage alters its structure and density, making it susceptible to fracture. Bone is the hardest tissue in the body, maintaining the stability of the skeletal system. It serves as a lever for muscles, protecting internal organs and storing minerals such as calcium and magnesium. Most bone diseases occur through trauma, such as fractures, or metabolic diseases, such as avascular necrosis and osteoporosis. These diseases cause problems with the body's mechanical support, regardless of the cause. This not only reduces mobility but also causes persistent pain due to the formation of false joints. For the purposes of this specification, bone disease includes osteoporosis, osteonecrosis, false joints, Paget's disease, or osteogenesis imperfecta.

[0031] The above-mentioned "osteoporosis" refers to a state in which bone mass decreases, bone strength weakens due to quantitative changes, and the probability of fracture is high. It is mainly caused by genetics, early menopause, excessive diet or steroid drugs.

[0032] Osteonecrosis is a condition in which insufficient blood supply to the bones causes bone tissue to die. It can occur anywhere in the body, but it most commonly occurs in the upper thigh (femur), upper arm, shoulder, knee, or spine.

[0033] Pseudarthrosis, also known as false joints, is a condition in which a broken bone does not properly connect (nonunion) and moves like a joint. This condition can be caused by errors in post-fracture treatment or bacterial infection at the fracture site.

[0034] Paget's disease is a localized bone disease that affects various parts of the skeletal system due to excessive bone remodeling (bone remodeling), a process of bone regeneration and growth, and resorption. It primarily occurs in the pelvis, thigh, or skull.

[0035] The above-mentioned "osteogenesis imperfecta" is a general term for symptoms of congenital weak bones that are prone to fractures without a specific cause, and is also called brittle bone disease.

[0036] "Differentiation" in the present invention refers to the phenomenon of structural or functional specialization during the process of cell division and proliferation, that is, the change in form or function of cells, tissues, etc. in an organism in order to perform a given task. For example, during ontogeny, the differentiation of initially homogeneous parts of a biological system into qualitatively different states can be described as differentiation.

[0037] The "cell therapeutic agent" of the present invention is a drug used for therapeutic, diagnostic, or preventive purposes by isolating, culturing, and preparing cells and tissues from an individual through specialized procedures. To restore the function of cells or tissues, living individuals can use these agents through a series of procedures, such as in vitro proliferation and screening of allogeneic or xenogeneic cells or other methods to alter the biological properties of the cells.

[0038] As described above, existing cell therapy agents utilizing stem cells have been difficult to commercialize due to problems such as high unit prices. In particular, they have drawbacks such as the considerable time and expense required to differentiate stem cells into osteoblasts.

[0039] In contrast, the differentiation method of the present invention can stably and rapidly differentiate stem cells in the early stage of subculture into osteoblasts in a short period of time. In conventional stem cell differentiation methods, it is necessary to differentiate stem cells that have been subcultured more than 8 times for about 20 days or more before osteoblasts can be obtained. In contrast, in the present invention, if stem cells that have been subcultured 5 times are differentiated for about 3 days, differentiated osteoblasts can be obtained ( Figure 1 In particular, when the differentiation method of the present invention is used, all stem cells can be differentiated into osteoblasts without causing response variation due to the source (donor) of the stem cells, and therefore, can be used as an allogeneic cell therapy agent.

[0040] Therefore, the present invention can provide a method for differentiating mesenchymal stem cells into osteoblasts or osteoblasts differentiated by the above method, wherein the above method comprises:

[0041] Step i), coating the breathable polymer film with surfactant bubbles;

[0042] Step ii), mesenchymal stem cells were cultured at a rate of 1×10 3 pieces / cm 2 to 1×10 5 pieces / cm 2 The density of is seeded in the bubbles of step i) above;

[0043] Step iii), differentiating the mesenchymal stem cells from step ii) into osteoblasts in a differentiation medium; and

[0044] Step iv), isolating and obtaining the osteoblasts differentiated in the above step iii).

[0045] The polymer membrane in step i) can be a porous membrane of a super flask.

[0046] Preferably, the density of step ii) can be 1×10 3 pieces / cm 2 to 1×105 pieces / cm 2 , more preferably, it can be 1×10 4 pieces / cm 2 .

[0047] According to a preferred embodiment of the present invention, the surfactant in step i) can be a poloxamer. Surfactants other than poloxamer are cytotoxic (Example 4) and are therefore not suitable for use in the present invention. The poloxamer can be at least one selected from the group consisting of poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 124, poloxamer 237, poloxamer 338, and poloxamer 407.

[0048] The surfactant in step i) may be a maximum of 10% of a surfactant.

[0049] The bubbles in step i) can be generated by adding a surfactant to the breathable polymer membrane and then moving the surfactant. For example, the bubbles in step i) can be generated by shaking the surfactant on the breathable polymer membrane or by using a pipette, but are not limited thereto.

[0050] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) above may be derived from one or more of the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, adipose tissue, hair follicles, teeth, dental pulp, and dermis. More preferably, the mesenchymal stem cells in step ii) above may be umbilical cord-derived mesenchymal stem cells.

[0051] In the case of the aforementioned umbilical cord-derived mesenchymal stem cells, the use of umbilical cord tissue, which is discarded after childbirth, has the advantage of being easy to collect and easily secure a large number of stem cells. Stem cells derived from adipose tissue or bone marrow are affected by factors such as the age and health status of the donor from whom they are isolated and extracted, and therefore have limitations and variability in terms of proliferation and differentiation capacity. In the case of umbilical cord-derived stem cells, they are the earliest stem cells that can be obtained among adult stem cells, and their stem cell capacity is hardly affected by variables such as the age of the donor, and they have excellent proliferation and differentiation capacity. Furthermore, umbilical cord-derived mesenchymal stem cells have the advantage of being able to isolate stem cell populations that can be used for various diseases such as nervous system diseases, liver diseases, and musculoskeletal system diseases.

[0052] In the above step ii), the mesenchymal stem cells are seeded in the air bubbles, which can be formed by coating the air-permeable polymer membrane with surfactant bubbles, at least 2 hours later when the bubbles begin to disappear.

[0053] The mesenchymal stem cells in step ii) can be stem cells that have been subcultured for 5 to 8 generations. More specifically, they can be stem cells that have been subcultured for 5 to 6 generations.

[0054] The mesenchymal stem cells seeded in step ii) can be cultured in a culture medium comprising at least one selected from the group consisting of α-MEM, DMEM, and FBS for 12 to 48 hours before differentiating into osteoblasts in the differentiation medium. More preferably, the cells can be cultured in the culture medium for 20 to 30 hours.

[0055] The differentiation in step iii) can be performed for 24 to 120 hours, more preferably for 60 to 80 hours.

[0056] According to a preferred embodiment of the present invention, in the above-mentioned osteoblasts, the expression levels of connexin 43, Runt-related transcription factor 2 and type 1A collagen may be higher than those of undifferentiated stem cells and mature bone cells; the expression levels of angiogenin and alkaline phosphatase may be higher than those of undifferentiated stem cells; and the expression levels of osteoblast-specific transcription factor, osteocalcin and osteopontin may be lower than those of mature bone cells.

[0057] According to a preferred embodiment of the present invention, the expression level of Ki-67 in the osteoblasts may be lower than that in the undifferentiated stem cells.

[0058] In the osteoblasts of the present invention, the expression of connexin 43, Runt-related transcription factor 2, type 1A collagen, and alkaline phosphatase in early osteoblasts was significantly higher than in undifferentiated stem cells. Expression of Ki-67, a marker of cell proliferation, decreased compared to undifferentiated cells, but still remained expressed, and colony formation was observed, indicating that these cells were early osteoblasts undergoing cell proliferation. In contrast, the expression levels of osteoblast-specific transcription factors, osteocalcin and osteopontin, expressed as osteoblasts matured to sufficient maturity, were lower than those in mature osteocytes (NHOst). Furthermore, the expression of angiogenin, which is prominently expressed in early osteoblasts, increased by more than several hundred-fold at both the gene and protein levels, clearly demonstrating the characteristics of early osteoblasts (Table 1).

[0059] Table 1

[0060]

[0061] CX43, encoded by the GJA1 gene, is the most prevalent gap junction protein expressed in bone cell types, including chondrocytes, osteoblasts, osteocytes, and osteoclasts. CX43 plays a crucial role in regulating signaling between various bone cell types, thereby regulating bone development, differentiation, modeling, and remodeling, as well as pathology. In particular, CX43 is essential for the survival, proliferation, and differentiation of osteoblasts and increases the expression of various bone formation markers.

[0062] RUNX2 is a major regulator of osteoblast differentiation and an important transcription factor that regulates the expression of alkaline phosphatase (ALP), an early phenotype of osteoblast differentiation, and osteocalcin, a late phenotype. Early osteoblasts (osteo-progenitors, immature osteoblasts) are RUNX2+, have the ability to proliferate, differentiate into mature osteocytes, and further undergo mineralization. Osteoblasts maintain cell division for a certain period of time, but as differentiation progresses, they gradually lose their ability to divide and differentiate into osteocytes ( Figure 17a and Figure 17b ).

[0063] The above-mentioned type 1A collagen (COL1A1) is a bone formation marker that is characteristically expressed when cells differentiate into osteoblasts.

[0064] The osteoblast-specific transcription factor (OSX) is a differentiation factor involved in bone cell formation. OSX increases COL1A1 promoter activity, thereby increasing the expression of bone matrix and playing a key role in the differentiation of osteoblasts into mature osteoblasts.

[0065] Osteocalcin (OCN) is a differentiation factor involved in bone cell formation. After being formed in osteoblasts, OCN is deposited in the bone matrix. A portion of the newly formed OCN is then released into the blood. Therefore, measuring blood levels can provide insights into the extent of bone formation.

[0066] The above-mentioned osteopontin (OPN) is a differentiation factor associated with bone cell formation, and is equivalent to a bone formation marker protein.

[0067] The present invention also provides a cell therapeutic agent for treating bone diseases, comprising osteoblasts obtained by the above method.

[0068] According to a preferred embodiment of the present invention, in the above-mentioned osteoblasts, the expression levels of connexin 43, Runt-related transcription factor 2 and type 1A collagen may be higher than those of undifferentiated stem cells and mature bone cells; the expression levels of angiogenin and alkaline phosphatase may be higher than those of undifferentiated stem cells; and the expression levels of osteoblast-specific transcription factor, osteocalcin and osteopontin may be lower than those of mature bone cells.

[0069] According to a preferred embodiment of the present invention, the expression level of Ki-67 in the osteoblasts may be lower than that in the undifferentiated stem cells.

[0070] According to a preferred embodiment of the present invention, the bone disease may be one or more selected from the group consisting of fracture, femoral head necrosis, spinal union, delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta.

[0071] The present invention also provides a method for treating bone diseases, comprising the step of providing osteoblasts obtained by the above method to a patient with the bone disease.

[0072] The above-mentioned administration is parenteral administration, and the above-mentioned osteoblasts can be administered alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers.

[0073] Effects of the Invention

[0074] The differentiation method of the present invention can stably and rapidly differentiate mesenchymal stem cells into osteoblasts. These differentiated osteoblasts exhibit excellent angiogenesis and bone formation abilities. Therefore, the method of the present invention for differentiating stem cells into osteoblasts, or the osteoblasts obtained by the method, can be effectively used as a cell therapy for bone diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The process of differentiating and obtaining umbilical cord-derived osteoblasts according to the present invention is shown.

[0076] Figure 2 The results show the results of observing whether the poloxamer bubbles remained on the porous membrane for 7 hours (0H-7H) after the poloxamer solution was removed after application of poloxamer bubbles. The bubbles disappeared 2 hours after the poloxamer solution was removed (2H).

[0077] Figure 3 The results of three-dimensional (3D) image capture using a fluorescence microscope showing the loading of QD-treated cells after poloxamer bubble coating.

[0078] Figure 4 The results of cytotoxicity experiments for each surfactant type are shown. After treatment with the CCK-8 kit (Cell Counting Kit-8) for one day, it was confirmed that the remaining surfactants (DIAPONK-SF and Tween-20) except P407 (Poloxamer 407) were toxic to cells.

[0079] Figure 5 The results of cytotoxicity experiments for each surfactant type are shown. The results of confirming cell proliferation after treatment with the CCK-8 kit for 1 to 3 days confirmed that the remaining surfactants (DIAPON K-SF and Tween 20) other than P407 were cytotoxic and prevented cell proliferation.

[0080] Figure 6 The results of the cytotoxicity test for each surfactant type are shown. After one day of treatment with the CCK-8 kit, it was confirmed that the remaining surfactants (methylprednisolone and Tween 20) except P407 were toxic to the cells.

[0081] Figure 7 The results of cytotoxicity experiments for various surfactant types are shown. Cell proliferation was confirmed after treatment with the CCK-8 kit for 1 to 3 days. It was confirmed that the remaining surfactants (methylprednisolone and Tween 20) except P407 were cytotoxic and did not inhibit cell proliferation.

[0082] Figure 8 The results showed that the gene (COL1A) expression level was significantly higher in cells differentiated in a surfactant with a bubble morphology (bubble foam) than in undifferentiated cells or cells differentiated in a surfactant with a gel morphology (gel).

[0083] Figure 9 It was shown that the expression level of a protein (vascular endothelial growth factor (VEGF)) was significantly higher in cells differentiated in a surfactant with a bubble morphology than in undifferentiated cells or cells differentiated in a surfactant with a gel morphology.

[0084] Figure 10 Comparison of osteoinductive gene expression levels in the differentiated cell therapy agents of the present invention (RCB001-DP1 to RCB005-DP5) compared to undifferentiated cells (RCB001, RCB002, and RCB005) is shown. Significantly increased expression levels of connexin 43 and Runt-related transcription factor 2 secreted by the cell therapy agents of the present invention were confirmed compared to undifferentiated cells.

[0085] Figure 11 Results show comparisons of osteoinductive gene expression levels in the differentiated cell therapy agent (RCB005-DP1) of the present invention relative to undifferentiated cells (RCB005), bone marrow-derived mesenchymal stem cells (BM-MSCs), and mature osteoblasts. These osteoblasts demonstrated higher expression levels of early osteoblast markers such as connexin 43, Runt-related transcription factor 2, and type 1A collagen than undifferentiated and mature osteoblasts, while lower expression levels of the osteoblast-specific transcription factors osteocalcin and osteopontin than mature osteoblasts.

[0086] Figure 12It was confirmed that bone formation marker proteins and angiogenesis marker proteins were increased in the differentiated osteoblast therapeutic agent of the present invention compared to undifferentiated cells. (A) refers to COL1A1, (B) refers to osteopontin, and (C) refers to angiopoietin.

[0087] Figure 13 The results show that the differentiated cell therapy agent of the present invention maintains bone formation marker proteins and angiogenesis marker proteins 3 to 7 days after thawing. (A) indicates COL1A1, (B) indicates osteopontin, and (C) indicates angiogenin.

[0088] Figure 14 The umbilical cord-derived osteoblasts of the present invention demonstrate their endothelial angiogenesis ability. Undifferentiated umbilical cord-derived UCMSCs and osteogenic differentiation cell therapy agents demonstrated similar angiogenesis induction as the positive control group (vascular endothelial growth factor), a well-known angiogenesis-inducing factor. In particular, the proteins secreted by the cell therapy agent demonstrated that the blood vessels formed were thicker.

[0089] Figure 15a and Figure 15b The results of the effectiveness of the cell therapy agent of the present invention in large animals (goats) are shown.

[0090] Figure 16 The results of the effectiveness of the cell therapy agent of the present invention in small animals (rats) are shown.

[0091] Figure 17a and Figure 17b The diagram shows the stages of differentiation of mesenchymal stem cells into osteocytes. The area indicated by the dotted line indicates the stage of the cell therapy agent of the present invention.

[0092] Figure 18 The results show the cell division capacity of two batches of undifferentiated UCMSCs (raw material) and differentiated osteoblasts (DP) as assessed by Ki-67 (a cell division marker). Consistently, Ki-67 expression was significantly reduced to below 1% in DP, indicating that CF-M801 cells are early-stage osteoblasts in the osteoblast model.

[0093] Figure 19a and Figure 19bThe results of CFU-F measurements using two batches of undifferentiated stem cells (BMMSCs and UCMSCs) and differentiated osteoblasts (DP) are shown. A decrease in CFU-F in differentiated osteoblasts (DP, CF-M801) was confirmed, following a pattern similar to that of Ki-67 (a cell division marker).

[0094] Figure 20 The results show absorbance measurements after alkaline phosphatase staining for colonies formed using two batches of undifferentiated stem cells (UCMSCs) and differentiated osteoblasts (DP). Compared to undifferentiated cells, colonies formed by differentiated osteoblasts (DP) showed increased absorbance after alkaline phosphatase staining, confirming that the colonies formed by the proliferation of this cell therapy agent are osteoblasts.

[0095] Figure 21a and Figure 21b The differentiated osteoblasts (DP1-DP3) of the present invention express over 80% CD10, whereas undifferentiated mesenchymal stem cells express less than 15%. This means that the osteoblasts of the present invention use osteoblast-specific markers, rather than conventional stem cell markers, to confirm and manage their purity. DETAILED DESCRIPTION

[0096] Example 1

[0097] Isolation and harvesting of umbilical cord-derived stem cells

[0098] First, remove the arteries and veins from the isolated umbilical cord and mince the remaining tissue to be mixed with AdiCol at 37°C. TM (CEFO) for more than 30 minutes, and then extract the cells. TM The cells were cultured in a culture medium at 37°C and 5% CO2 to obtain mesenchymal stem cells.

[0099] Example 2

[0100] Coating breathable polymer membranes with surfactant bubbles

[0101] After shaking 8% poloxamer 407 completely dissolved in PBS on the porous membrane of the super flask to generate bubbles, the remaining poloxamer solution was poured out. The air-permeable polymer membrane was coated with poloxamer bubbles at 37°C for 2 hours. After the solution was removed after the generation of poloxamer 407 bubbles, the bubbles were observed using three-dimensional microscopic imaging to confirm whether the bubbles were maintained. After 2 hours (2H), the bubbles began to disappear, and after 5 hours (5H), the bubbles had completely disappeared. Figure 2 ).

[0102] Furthermore, quantum dot-conjugated silica nanoparticles (QD) were absorbed into the stem cells for 24 hours to track umbilical cord-derived mesenchymal stem cells. After generating bubbles of poloxamer 407, the solution was removed and the cells were loaded. The Z-stack microscope was used to visualize the cells in three dimensions. Figure 3 ).

[0103] Example 3

[0104] Differentiation and culture of osteoblasts

[0105] After 2 hours of application of poloxamer bubbles in Example 2, the umbilical cord stem cells obtained in Example 1 were added at a concentration of 1×10 4 pieces / cm 2 The cells were seeded at a high density and cultured in a medium containing DMEM and FBS for 24 hours. The cultured stem cells were differentiated into osteoblasts in an osteogenic differentiation medium for 72 hours.

[0106] Example 4

[0107] Screening for suitable surfactants for coating breathable polymer membranes

[0108] The toxicity of representative biocompatible surfactants, such as poloxamer 407 (p407), sodium methyl cocoyl taurate (DIAPON K-SF), polyoxyethylene sorbitan monolaurate (Tween 20), or methylprednisolone, was determined.

[0109] Specifically, in a transwell comprising a three-dimensional porous membrane, surfactant p407, DIAPONK-SF or Tween 20 was treated in the upper chamber at 0% (v / v), 0.5% (v / v), 1% (v / v), 5% (v / v) or 15% (v / v), and coated for 2 hours using the same method as in Example 2. The cells were then plated at 20,000 cells / cm 2Umbilical cord-derived mesenchymal stem cells obtained in Example 1 were inoculated. Cell growth medium was added to the lower chamber and cultured for 3 days. On days 1, 2, and 3, CCK-8 (Cat. CK04, DOJINDO) solution was added and incubated in a 37°C, CO2 incubator for 3 hours. The absorbance was measured at 450 nm and cytotoxicity was analyzed ( Figure 4 ) and cell growth rate ( Figure 5 ).

[0110] Furthermore, in a transwell containing a three-dimensional porous membrane, surfactant p407, methylprednisolone or Tween 20 was treated at a concentration of 4 mM in the upper chamber and coated for 2 hours using the same method as in Example 2. 2 Umbilical cord-derived mesenchymal stem cells obtained in Example 1 were inoculated. Cell growth medium was added to the lower chamber and cultured for 3 days. On days 1, 2, and 3, CCK-8 solution was added and incubated in a 37°C, CO2 incubator for 3 hours. The absorbance was measured at 450 nm and cytotoxicity was analyzed ( Figure 6 ) and cell growth rate ( Figure 7 ).

[0111] Results, such as Figures 4 to 7 As shown, it was confirmed that DIAPON K-SF, Tween 20, and methylprednisolone, excluding P407, were all toxic to cells.

[0112] Example 5

[0113] Screening of surfactant coating methods

[0114] After poloxamer was coated on the porous membrane of the super flask in the form of bubbles or gel, the degree of osteodifferentiation of umbilical cord-derived stem cells differentiated by the method of Example 3 was compared.

[0115] Specifically, differentiated osteoblasts and undifferentiated cells were captured and treated with Trizol TM and chloroform (Sigma), and centrifuged to separate the layers and obtain only mRNA. The mRNA obtained using the Transcriptor Universal cDNA Master Kit (Roche) was synthesized into cDNA. Subsequently, DNA was amplified by RT-PCR for COL1A1 to confirm the difference in DNA copy number at the gene level. The polymerase chain reaction conditions were 50 cycles of 95°C for 10 seconds, 54°C for 10 seconds, and 72°C for 30 seconds ( Figure 8 ).

[0116] Furthermore, the culture medium of osteoblasts and undifferentiated cells prepared and differentiated by the method of Example 3 was collected, and the secretion level of vascular endothelial growth factor (VEGF) was measured by enzyme-linked immunosorbent assay (ELISA). Figure 9 ).

[0117] Results, such as Figure 8 and Figure 9 As shown, it was confirmed that the cells differentiated by the method of the present invention expressed much higher levels of osteoinductive genes and angiogenesis-inducing proteins than cells differentiated from undifferentiated cells or cells differentiated from poloxamer in a gel state.

[0118] Example 6

[0119] Evaluation of the bone-forming ability of the obtained osteoblasts

[0120] 6-1: Bone-forming ability of osteoblasts (gene level)

[0121] RNA was isolated from osteoblasts collected during osteoblast differentiation, and cDNA was synthesized. Real-time polymerase chain reaction (RT-PCR) was then used to compare the expression levels of Runt-related transcription factor 2 and connexin 43, bone formation gene markers, with those in undifferentiated cells.

[0122] Specifically, osteoblasts and undifferentiated cells prepared and differentiated by the method of Example 3 were captured and treated with Trizol TM The mRNA was isolated by centrifugation using chloroform (Sigma) and centrifuged to separate the layers. The mRNA was synthesized into cDNA using the Transcriptor Universal cDNA Master Kit (Roche). DNA was then amplified by RT-PCR to confirm differences in gene expression for RUNX2 and CX43. The polymerase chain reaction (PCR) consisted of 50 cycles of 95°C for 10 seconds, 54°C for 10 seconds, and 72°C for 30 seconds.

[0123] Furthermore, mRNA was collected from osteoblasts, undifferentiated cells, bone marrow-derived mesenchymal stem cells (BM-MSCs), and mature osteocytes prepared and differentiated using the method of Example 3. The mRNA was then synthesized into cDNA using the Transcriptor Universal cDNA Master Kit (Roche). DNA was then amplified by RT-PCR to confirm differences in gene expression for RUNX2, CX43, and COL1A. The polymerase chain reaction conditions were 50 cycles of 95°C for 10 seconds, 54°C for 10 seconds, and 72°C for 30 seconds.

[0124] Results, such as Figure 10 and Figure 11 As shown in the results, it was confirmed that RUNX2, CX43 or COL1A were expressed at a higher level in the osteoblasts of the present invention than in undifferentiated stem cells and mature osteoblasts. Figure 10 In the table, RCB001, RCB002, and RCB005 refer to undifferentiated cells, and RCB001-DP1 to RCB005-DP5 refer to differentiated cell therapeutic agents of the present application.

[0125] In contrast, in the case of OSX, OCN, or OPN, the expression was higher in mature osteocytes than in the osteoblasts of the present invention, which indicates that the osteoblasts of the present invention are in the immature bone differentiation stage compared to NHOst ( Figure 11 ).exist Figure 11 In the table, RCB001, RCB002, and RCB005 refer to undifferentiated cells, and RCB001-DP1 to RCB005-DP5 refer to differentiated cell therapeutic agents of the present application.

[0126] 6-2: Bone-forming ability of osteoblasts (protein level)

[0127] The goal was to examine changes in the protein concentrations of type 1A collagen (COL1A), osteopontin, or angiopoietin (ANGPT-1), known as bone formation or angiogenesis protein markers, in the culture medium and osteoblasts collected during the osteoblast differentiation process described in Example 3. Furthermore, the goal was to examine whether the bone formation protein or angiogenesis protein was retained after thawing the frozen cell therapy of the present invention.

[0128] Specifically, osteoblasts, undifferentiated cells, and differentiation culture medium prepared and induced using the method of Example 3 were collected and subjected to enzyme-linked immunosorbent assays (ELISAs). Changes in protein expression in COL1A-induced and undifferentiated cells were assessed in the lysed cells. Protein secretion levels of osteopontin and angiogenin in the culture medium up to 72 hours of differentiation were also assessed.

[0129] Results, such as Figure 12 As shown in FIG, in the osteoblasts of the present invention, it was confirmed that the expression of COL1A protein increased, and the secretion of osteopontin and angiogenin was high. Figure 13 As shown in FIG, it was confirmed that the bone morphogenetic protein or angiogenic protein of the cell therapy agent of the present invention was maintained even 3 to 7 days after thawing. Figure 12 and Figure 13 In the figure, part (A) refers to COL1A1, part (B) refers to osteopontin, and part (C) refers to angiopoietin.

[0130] Example 7

[0131] Evaluation of the angiogenesis ability of the obtained osteoblasts

[0132] The goal was to confirm the angiogenesis ability of human umbilical vein endothelial cells (HUVECs) induced by angiogenesis proteins secreted by the obtained osteoblasts.

[0133] Specifically, on a 12-well invasion chamber plate comprising a porous membrane with a pore size of 8.0 μm, 4×10 4 Undifferentiated stem cells or osteoblasts were inoculated into the cell / well, and HUVEC cells in culture were used to absorb quantum dots. After 24 hours, the cells were taken out and injected into the invasion chamber at 25,000 / cm 2After seeding, the cells were co-cultured for 1 day, and angiogenesis analysis of HUVEC was performed. After seeding each cell in a 12-well plate, the culture medium and substances were exchanged using an invasion chamber, and then cultured for 12 hours to confirm the angiogenesis of HUVEC cells. Osteodifferentiated cells and undifferentiated cells were added to the invasion chamber for comparison. For the negative control group, only HUVEC cells were seeded on the coated Matrigel and cultured for 12 hours using HUVEC culture medium without vascular endothelial growth factor. For the positive control group, 20 ng / ml of vascular endothelial growth factor was added and cultured under the same conditions as the negative control group.

[0134] Results, such as Figure 14 As shown, when umbilical cord-derived undifferentiated UC-MSCs were co-cultured with a bone cell therapy at levels similar to those in the positive control group, which is known for its angiogenesis-inducing factor, vascular endothelial growth factor (VEGF), tube formation was confirmed. Furthermore, when co-cultured with a bone-differentiating cell therapy, robust blood vessels were observed, with the formation of particularly robust blood vessels confirmed to be caused by proteins secreted by the cell therapy.

[0135] Example 8

[0136] Confirming the bone regeneration ability of the obtained osteoblasts in vivo

[0137] After inducing bone defects in large animal (goat) or small animal (rat) models, the bone regeneration ability of the cell therapy of the present invention was confirmed.

[0138] Specifically, after femoral defects were induced in immunosuppressed goat models, 1×10 7 The bone regeneration effect of this cell therapy was confirmed by the addition of osteoblasts to the 26-week model (Table 2). Furthermore, as a test of its effectiveness in a goat model, bone tissue was demineralized for 2.5 months, and the effectiveness of this cell therapy was histologically evaluated using hematoxylin and eosin (H&E) and Masson's Trichrome staining.

[0139] In the immunosuppressed rat model, after radial bone defect was induced, 1×10 6 The bone regeneration effect was confirmed after 12 weeks of treatment with osteoblasts (Table 3). In the sham control group, only the alginate scaffold was treated. The extent of bone regeneration was confirmed by μCT imaging. Bone tissue was fixed, demineralized, and sectioned to prepare slides, which were then stained to confirm new bone regeneration.

[0140] Table 2

[0141]

[0142] Table 3

[0143]

[0144] The test results of the effectiveness of the goat model, such as Figure 15a and Figure 15b As shown, at 26 weeks, new bone formation in the proximal and distal parts of the control group in both males and females was poor and only partially confirmed, confirming that the wound was in the process of healing. In contrast, in the cell-administered group, cells filled the defect site, proliferated, and differentiated into osteocytes in the proximal and distal parts, resulting in organic connection of the new bone lines without breaking and a thicker thickness. Figure 15a (white arrow at the lower left end of the image). Blood vessels were also well-formed and regular. Significant new bone formation was observed in the cell-administered groups at both weeks 13 and 26.

[0145] For the effectiveness test results of rat model, Figure 16 As shown, the G3 group is a cell therapy group differentiated for 2 days in passage 7, and the G5 group is a therapy group differentiated for 3 days in passage 5. Both groups showed a tendency to increase bone volume, bone volume density, BMD, etc. compared to the control group. In particular, it was confirmed that the bone volume of the therapy group differentiated for 3 days in passage 5 was further significantly increased.

[0146] Example 9

[0147] Stages for Confirming Cell Therapy

[0148] The stage at which the cell therapy agent of the present invention is to be confirmed.

[0149] 9-1: Confirmation of Ki-67 expression

[0150] Human Ki-67 is known to be expressed during the cell cycle's proliferative phases (G1, S, G2, and M) but is absent during the resting phase (G0) of the cell cycle. However, after differentiation into osteoblasts, cells no longer proliferate and are therefore no longer expressed. Therefore, the cell division capacity of two batches of undifferentiated UCMSCs (raw material) and differentiated osteoblasts (DP) of the present invention was assessed by immunofluorescence chemistry (ICC) to determine the expression level of Ki-67, an indicator of cell division. The expression level of Ki-67 was corrected for cell number by staining with DAPI, a nuclear stain.

[0151] Specifically, a slide plate was prepared and 3×10 5 After inoculating undifferentiated and differentiated cells with a mass of 100 μg / ml, the cells were cultured in a CO2 incubator for 24 hours. The cells were fixed with 4% formaldehyde at room temperature for 10 minutes and permeabilized with 1% Triton X-100 at room temperature for 10 minutes. After blocking with BSA at room temperature for 30 minutes, the Ki-67 primary antibody was reacted at room temperature for 1 hour. The fluorescent-linked secondary antibody was shielded from light and reacted at room temperature for 1 hour. ProLong containing DAPI was used. TM The cells were mounted with Gold Antifade Mountant (Invitrogen), and observed under a fluorescence microscope.

[0152] Results, such as Figure 18 As shown in Figure 3, it was confirmed that Ki-67 expression was rapidly reduced to below 1% in osteoblasts (DP).

[0153] 9-2: Confirmation of CFU-F expression

[0154] CFU-F was measured using two batches of undifferentiated stem cells: BMMSCs and UCMSCs (raw material), and the differentiated osteoblasts (DP) of the present invention. Mesenchymal stem cells are known to proliferate and form characteristic cell colonies upon in vitro culture. The cells within these colonies have a fibroblast-like shape, and each colony is referred to as a colony-forming unit (CFU-F), a key characteristic of stem cells. Therefore, the formed colonies were stained with 2% crystal violet and observed with the naked eye and a camera. Quantification was performed by counting the stained colonies that were 3 mm or larger and had a density of 80% or greater.

[0155] Results, such as Figure 19a and Figure 19b As shown, the CFU-F of differentiated osteoblasts (DP, CF-M801) decreased in a pattern similar to Ki-67 (cell division marker) expression. Furthermore, while numerous colonies formed in the starting cells, which are bone marrow-derived stem cells and umbilical cord-derived stem cells, almost no colonies were observed in the cell therapy. This suggests that the umbilical cord-derived stem cell starting cells lose their colony-forming ability as they differentiate into osteoblasts. These results confirm that the cell therapy is primarily differentiated within the starting cells.

[0156] 9-3: Confirmation of ALP expression

[0157] In CFU-F using the cell therapy agent CF-M801, it is to be confirmed whether cells forming colonies showing an average of less than 1% are undifferentiated mesenchymal stem cells or cells induced to undergo osteodifferentiation.

[0158] Specifically, colonies cultured using the same method as for CFU-F in Example 9-2 were stained using ALP staining to confirm bone differentiation. 10,000 cells were seeded and maintained for one week before ALP staining. Following ALP staining, the cells were reacted with dimethylsulfoxide to fully dissolve the stain, and only the supernatant was collected and the absorbance measured using a microplate reader.

[0159] Results, such as Figure 20 As shown, undifferentiated UCMSCs, which are allogeneic umbilical cord-derived mesenchymal stem cells, showed almost no ALP staining. However, colonies of CF-M801 cells, which had undergone bone differentiation, were confirmed to be ALP-positive across three different lots. Furthermore, the absorbance of CF-M801 cells, which had undergone bone differentiation, was higher than that of undifferentiated cells. Analysis of this relative value revealed values ​​that were at least 1.5 times higher than those of undifferentiated UCMSCs in all cases. These results confirm that differentiated osteoblasts (DPs) continue to divide within a specified timeframe, gradually losing their ability to divide as they progress, and ultimately differentiate into osteoblasts.

[0160] In summary, it can be seen that in the cell therapy agent of the present invention, RUNX2, as the master regulator of osteoblast differentiation, can differentiate mesenchymal stem cells into osteoblasts. Osteoblasts in the early stage are RUNX2+, have the ability to proliferate, differentiate into mature osteocytes, and further undergo mineralization ( Figure 17a and Figure 17b ).

[0161] 9-4: Confirmation of CD-10 expression

[0162] To confirm the differentiation of cell therapy, the expression level of CD10 was confirmed by flow cytometry (FACS) and immunofluorescence (ICC).

[0163] Specifically, osteoblasts, undifferentiated cells, and bone marrow-derived mesenchymal stem cells (BM-MSCs), prepared and differentiated according to the method of Example 3, were suspended in a 2% BSA / DPBS solution. The cells were incubated with a CD10 primary antibody for 1 hour at room temperature. After washing, the cells were incubated with a FITC-conjugated secondary antibody for 30 minutes at room temperature. After washing, the supernatant was removed, and the cells were fixed with 3.7% formaldehyde for 20 minutes at room temperature. CD10 expression was then assessed using a flow cytometer (BD Accuri C6 Plus).

[0164] For ICC, prepare slides with 3 × 10 5 After inoculating undifferentiated and differentiated cells with the same mass, the cells were cultured in a CO2 incubator for 24 hours. The cells were fixed with 4% formaldehyde for 10 minutes at room temperature and permeabilized with 1% TritonX-100 for 10 minutes at room temperature. After blocking with BSA for 30 minutes at room temperature, the CD10 primary antibody was reacted for 1 hour at room temperature. The FITC fluorescent-linked secondary antibody was shielded from light and reacted for 1 hour at room temperature. ProLong containing DAPI was used. TM The cells were mounted with Gold Antifade Mountant (Invitrogen), and observed under a fluorescence microscope.

[0165] Results, such as Figure 21a and Figure 21b As shown, the differentiated osteoblasts (DP1-DP3) of the present invention were confirmed to express CD10 at a rate of 80% or more, while undifferentiated mesenchymal stem cells expressed CD10 at a rate of less than 15%. This suggests that the osteoblasts of the present invention were purified using osteoblast-specific markers, rather than conventional stem cell markers, for purity verification and management.

[0166] Industrial applicability

[0167] The differentiation method of the present invention can stably and rapidly differentiate mesenchymal stem cells into osteoblasts. These differentiated osteoblasts exhibit excellent angiogenesis and bone formation abilities. Therefore, the method of differentiating stem cells into osteoblasts of the present invention, or the osteoblasts obtained by the method, can be effectively used as a cell therapy or treatment method for bone diseases, thereby having industrial applicability.

Claims

1. A method for differentiating mesenchymal stem cells into osteoblasts, characterized in that: include: Step i), coating a breathable polymer film with poloxamer bubbles; Step ii), mesenchymal stem cells were cultured at a rate of 1×10 3 pieces / cm 2 to 1×10 5 pieces / cm 2 The density of is seeded in the bubbles of step i) above; Step iii), differentiating the mesenchymal stem cells from step ii) into osteoblasts in a differentiation medium; and Step iv), isolating and obtaining the osteoblasts differentiated in the above step iii).

2. The method for differentiating mesenchymal stem cells into osteoblasts according to claim 1, wherein The bubbles in step i) are prepared by adding poloxamer to the breathable polymer film and allowing it to move, thereby generating bubbles.

3. The method for differentiating mesenchymal stem cells into osteoblasts according to claim 1, wherein: The mesenchymal stem cells in step ii) are derived from at least one selected from the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicles, teeth, dental pulp and dermis.

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