JPWO2019189947A1 - Differentiation induction technology by actin polymerization inhibitor for the production of osteoblasts from human umbilical cord-derived mesenchymal stem cells - Google Patents

Abstract

The present invention relates to highly efficient differentiation of osteoblasts from stem cells capable of differentiating osteoblasts. The present invention provides an osteoblast differentiation promoter containing an actin polymerization inhibitor.

Description

The present invention provides a method for inducing osteoblast differentiation, which comprises culturing an osteoblast differentiation promoting agent containing an actin polymerization inhibitor, a stem cell having an osteoblast differentiation ability together with an osteodifferentiation inducer and an actin polymerization inhibitor. Etc.

Bone regeneration treatment has received a great deal of attention because it brings about a much earlier healing than natural recovery. The size of the regenerative medicine market for bones and joints is predicted to reach US$3.9 billion (BCC Research) in 2018 by the global total market size.

For example, periodontal disease has a high morbidity in Japan, and the number of patients with some symptoms of gingiva is approximately 94 million (2011 Dental Health Survey, Dental Health Division, Medical Policy Bureau, Ministry of Health, Labor and Welfare). It is said. As the periodontal disease progresses, the gums become inflamed and the alveolar bone is absorbed. Although local application of a protein preparation such as basic fibroblast growth factor (bFGF) has been applied as a treatment for bone resorption caused by periodontal disease, it is difficult to maintain the effect expression for a long time. The effect is limited when the absorption is widespread.

Regenerative treatment for periodontal disease is also performed, but current bone regeneration requires highly invasive surgery combined with autologous bone transplantation (around 10,000 patients each year, 2013 survey by social medical practice) Therefore, the burden on the patient is large. Therefore, there is a demand for the development of a new bone regeneration method that is minimally invasive and highly effective.

In recent years, a method using mesenchymal stem cells (MSCs) has been proposed as a novel bone regeneration method that is relatively less invasive and highly effective, but there are many problems to be overcome. MSCs are cell populations characterized by self-proliferation and pluripotency that can be extracted from mesenchymal tissues. Some MSCs are derived from bone marrow, umbilical cord, and fat, but they have different differentiation and proliferative characteristics depending on the tissue from which they are derived, and the most common source is bone marrow-derived stem cells (bone marrow MSCs). It can induce bone differentiation and also has an immunosuppressive action. However, bone marrow collection is still highly invasive and requires culturing to secure the required number of cells at the time of transplantation, and there are individual differences in the proliferation/differentiation capacity of the cultured cells, making it difficult to perform the culturing operation. There is such a problem.

Umbilical cord MSCs have no HLAII expression and can be transplanted as allogeneic (allo) transplantation, and because of the progress of banking, stable supply of cells is possible, which is highly useful. Furthermore, it has been reported that umbilical cord MSCs can be collected non-invasively, have high proliferative activity, and have the ability to differentiate into bone tissue. However, it takes longer time to induce differentiation into osteoblasts than MSCs isolated from other tissues, and even if differentiation is induced, Alkaline Phosphatase (ALP) activity is relatively low (Fig. 1), and the differentiation There are many unclear points about the mechanism.

MSC derived from fat does not have particularly high bone differentiation ability, contains many impurities, and has low quality. It is also highly invasive. MUSE cells and iPS/ES cells are also stem cells capable of differentiating osteoblasts, but the former have a small number of cells and have low proliferative properties, and the latter do not stably obtain homogeneous cells, and thus proliferate and differentiate. slow.

Furthermore, bone morphogenetic protein (BMP) is used in inducing osteoblast differentiation into stem cells having osteoblast differentiation ability, but BMP is expensive and has side effects, so the amount used is suppressed. Is required. When BMP is included in the coated gel, strong induction is observed even in a small amount, but the coated gel cannot be used in the human body and cannot be used for treatment. As described above, it has been difficult to differentiate osteoblasts for use in a novel bone regeneration method with low invasiveness and high efficiency from stem cells with high efficiency.

On the other hand, the cytoskeleton may show a dynamic structural change due to polymerization/depolymerization during cell differentiation (Non-patent document 1) or may be involved in mechanical signal transduction (Non-patent document 2). It has never been known or predicted that a polymerization inhibitor significantly promotes osteoblast differentiation.

Nobusue H, Onishi N, Shimizu T, et al. Regulation of MKL1 via actin cytoskeleton dynamics drives adipose differentiation. Nat. Commun. 2014; 5: 3368. J. Gao, et al. Cyclic stretch promotes osteogenesis-related gene expression in osteoblast-like cells thru a cofilin-associated mechanism, Mol. Med. Rep. 14, 218-224 (2016).

An object of the present invention is to differentiate osteoblasts from stem cells having osteoblast differentiation ability with high efficiency. For example, if it is possible to establish a treatment condition that enhances the differentiation induction efficiency of umbilical cord MSC to the same extent as that of bone marrow MSC, a large amount of osteoblasts can be produced, and thus inexpensive and safe cell therapy can be realized.

The present inventors have made an effort to develop a culture method that enables the production of umbilical cord-derived mesenchymal stem cells having a high osteoblast differentiation potential. As a result, it is possible to mass-produce osteoblasts as a transplant cell for bone regeneration from a stem cell having osteoblast differentiation ability by a new culture technique applying an actin polymerization inhibitor, and the present invention Completed

That is, the present invention is as follows.
[1] An osteoblast differentiation promoting agent containing an actin polymerization inhibitor.
[2] The agent according to [1], wherein the osteoblast differentiation is osteoblast differentiation from a stem cell having osteoblast differentiation ability.
[3] The agent according to [2], wherein the stem cells having osteoblast differentiation ability are mesenchymal stem cells.
[4] The agent according to [3], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.
[5] The agent according to any one of [1] to [4], wherein the actin polymerization inhibitor is latrunculin A and/or swinholide A.
[6] A method for inducing osteoblast differentiation, which comprises culturing a stem cell having an osteoblast differentiation ability together with an osteoinduction factor and an actin polymerization inhibitor.
[7] The method according to [6], wherein the stem cells having osteoblast differentiation ability are mesenchymal stem cells.
[8] The method according to [7], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.
[9] The method according to any one of [6] to [8], wherein the bone differentiation inducing factor is a bone morphogenetic protein.
[10] The method according to [9], wherein the bone morphogenetic protein is BMP-2.
[11] The method according to any one of [6] to [10], wherein the actin polymerization inhibitor is latrunculin A and/or swinholide A.
[12] The method according to any one of [6] to [11], which further comprises measuring the bone differentiation marker gene expression or the activity of the bone differentiation marker enzyme to confirm the differentiation status of the stem cells having osteoblast differentiation ability. the method of.
[13] The method according to [12], wherein the bone differentiation marker gene is the ALP gene and the bone differentiation marker enzyme is ALP.
[14] The method according to any one of [6] to [13], wherein the culture is performed for 5 days or more.
[15] At the same time as the start of culture (day 0), and 1 day, 2 days, 3 days, 4 days, or 5 days after the start of culture, the osteodifferentiation factor is contained in the medium. The method described in either.
[16] Simultaneously with the start of culture (0 days), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days after the start of culture. The method according to any one of [6] to [15], wherein the actin polymerization inhibitor is contained in the medium on the 13th, 14th or 15th day.
[17] The method according to any one of [6] to [16], which is a method for producing a bone treatment material.
[18] A bone treatment kit comprising the osteoblast differentiation promoting agent according to any one of [1] to [5] and a stem cell having osteoblast differentiation ability.

The present invention enables highly efficient osteoblast differentiation induction from stem cells, which has been difficult so far. In particular, when umbilical cord-derived mesenchymal stem cells are used as the stem cells, they can be collected non-invasively, and allogeneic transplantation can be performed because the stem cells do not have HLAII expression, so the burden on the patient to be treated can be reduced.

FIG. 1 is a graph showing the alkaline phosphatase (ALP) activity when osteoblast differentiation was induced in umbilical cord (UC)-derived and bone marrow (BM)-derived human mesenchymal stem cells and in an uninduced control. FIG. 2 is a graph showing the expression of alkaline phosphatase (ALP) mRNA when osteoblast differentiation was induced in umbilical cord (UC)-derived and bone marrow (BM)-derived human mesenchymal stem cells and in an uninduced control. As the umbilical cord (UC)-derived human mesenchymal stem cells, both those cultured on collagen gel (gel) and those not cultured (non-coat) were used. FIG. 3 is a diagram recording the migration state of mesenchymal stem cells that have been induced to differentiate by culturing without coating, on a collagen gel, and on a collagen coating, and non-induced control cells. FIG. 4 is a graph quantifying the migration distance based on the data of FIG. FIG. 5 shows changes in cytoskeleton (actin) of umbilical cord (UC)-derived and bone marrow (BM)-derived human mesenchymal stem cells during culture on collagen gel or without coating. FIG. 6 is a schematic diagram of the BMP-2-BMP receptor II (BMPRII)-cofilin transduction pathway. FIG. 7 is a graph showing the expression of BMPRII mRNA when osteoblast differentiation was induced in umbilical cord (UC)-derived human mesenchymal stem cells and in an uninduced control. Umbilical cord (UC)-derived human mesenchymal stem cells used were both those cultured on collagen gel (gel) and those not cultured (non-coating). FIG. 8 is a graph showing cofilin mRNA expression when osteoblast differentiation was induced in umbilical cord (UC)-derived human mesenchymal stem cells and in an uninduced control. Umbilical cord (UC)-derived human mesenchymal stem cells used were both those cultured on collagen gel (gel) and those not cultured (non-coating). FIG. 9: is a figure which shows the observation result of the intracellular localization of actin in culture on the displayed conditions. FIG. 10 shows ALPs of cells treated with the actin inhibitors Latrunculin A, Swinholide A, and non-differentiation-induced and inhibitor-free cells 3 days after induction of osteoblast differentiation. It is a figure which shows mRNA expression. FIG. 11 is a diagram showing actin kinetic changes in cells treated with actin inhibitors Latrunculin A, Swinholide A, and control cells 3 days after induction of osteoblast differentiation. Is. FIG. 12 shows actin in cells treated with the actin inhibitors Latrunculin A, Swinholide A after induction of osteoblast differentiation, as well as cells in non-differentiation, inhibitor-free and collagen gel culture cells. It is a figure which shows the dynamic change of. FIG. 13 shows expression of undifferentiated markers (Oct4, Nanog) of umbilical cord-derived human mesenchymal stem cells (gel) cultured on a collagen gel and umbilical cord-derived human mesenchymal stem cells (non-coating) not cultured. FIG. FIG. 14 is a Hematoxylin & Eosin (HE) stained image of a nude mouse in which osteoblasts treated with an inhibitor of umbilical cord MSC were transplanted to the crown together with β-TCP, and a control section specimen (4 weeks after transplantation).

Hereinafter, the present invention will be described in detail.

1. Osteoblast Differentiation Promoter The present invention provides an osteoblast differentiation promoter containing an actin polymerization inhibitor.

Spherical actin (G-actin) is a globular protein having a molecular weight of about 42 kD, which polymerizes in a spiral form to form filamentous actin (F-actin). In the present specification, an actin polymerization inhibitor refers to a substance that inhibits this polymerization.

As the actin polymerization inhibitor, latrunculin A, latrunculin B, latrunculin C, latrunculin C, latrunculin, etc., Swinholide A, Swinholide B, Swinholide C, Swinholide D, Swinholide E, Swinholid F , Swinholide G, Swinholid H, Swinholide I, Swinholide J, Swinholide K, etc. Low molecular weight compounds such as cytochalasins such as J, actin-binding proteins such as cofilin, profilin, villin, fragmin, actinkin, gelsolin, and depactin, and proteins that control the activity of actin-binding proteins (eg, the activity of cofilin LIM kinase (LIMK) and the like which regulate it, and preferably include latrunkulin A and swinholide A.

Latrunculins form a 1:1 complex with G actin (monomer) to inhibit actin polymerization. By removing latrunculin from the complex by washing, the actin polymerization capacity is restored. Swinholides bind to G-actin at a ratio of 1:1 to inhibit actin polymerization, and also have F-actin cleavage activity to rapidly disintegrate filamentous actin.

In one aspect, the present invention provides an osteoblast differentiation promoting agent, which comprises a vector encoding an actin polymerization inhibitor.

As the actin polymerization inhibitor encoded by the vector, cofilin, profilin, villin, fragmin, actinkin, gelsolin, actin-binding proteins such as depactin, and proteins that control the activity of actin-binding proteins (e.g., the activity of cofilin The LIM kinase (LIMK) etc. which control is mentioned.

In the case of a low molecular weight compound, an actin polymerization inhibitor may be introduced by allowing it to permeate (permeate) into cells, for example, by adding it to a medium during culturing, and act by contacting with actin in bone cells. .. The concentration of the actin polymerization inhibitor in the medium can be appropriately set within the range in which the effects of the present invention can be achieved. The substance is usually used at a concentration of 1 nM to 10 μM, preferably 10 nM to 800 nM, more preferably 10 nM to 500 nM.

The actin polymerization inhibitor can be used alone as the differentiation promoting agent of the present invention, but may further contain components such as a dissolution promoting agent, a stabilizer, a preservative, and an antioxidant. Examples of the dissolution accelerator include dimethyl sulfoxide, ethanol, methanol, N,N-dimethylformamide and the like.

In the case where the actin polymerization inhibitor is in the form of a low molecular weight compound or protein, for example, lipofection, fusion with a cell membrane-penetrating peptide (for example, HIV-derived TAT and polyarginine), and microinjection are used for culturing. It may be introduced into cells and act on intracellular actin.

On the other hand, when it is in the form of DNA, it can be introduced into cells by a method such as virus, plasmid, vector such as artificial chromosome, lipofection, liposome, microinjection, and act on intracellular actin. Viral vectors include retrovirus vectors and lentivirus vectors (above, Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1971-1920, 2007). ), adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector and the like. The artificial chromosome vector includes, for example, human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC) and the like. As a plasmid, a plasmid for mammalian cells can be used (Science, 322:949-953, 2008). The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that an actin polymerization inhibitor can be expressed, and further, if necessary, a drug resistance gene ( For example, a puromycin resistance gene etc.), a thymidine kinase gene, a selection marker sequence such as a diphtheria toxin gene, a green fluorescent protein (GFP), β-glucuronidase (GUS), a reporter gene sequence such as FLAG, etc. can be included.

In the case of RNA, for example, lipofection, microinjection, or the like may be used to act on cells, and RNA that incorporates 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation. Good (Warren L, (2010) Cell Stem Cell. 7:618-630).

The osteoblast differentiation promoting agent of the present invention can promote the differentiation of stem cells having osteoblast differentiation ability into osteoblasts.

Osteoblasts are cells that are present on the surface of bone tissue and have the function of differentiating into osteocytes and creating new bone. When the osteoblast differentiation promoting agent of the present invention is used for stem cells having osteoblast differentiation ability, the differentiated cells (differentiation-induced cells) have not only osteoblast differentiation ability but also osteoblast differentiation ability. It means any cell observed in the differentiation of a stem cell into a bone, and is, for example, slightly differentiated from an osteocyte, a preosteoblast, an osteoprogenitor cell, or a mesenchymal stem cell. Mentioned mesenchymal precursor cells and the like can also be mentioned.

The stem cells having osteoblast differentiation ability of which differentiation into osteoblasts is promoted by the osteoblast differentiation promoter of the present invention are preferably vertebrate-derived cells. Examples of the vertebrates include mammals and birds. Examples of mammals include, for example, mice, rats, hamsters, experimental animals such as rodents such as guinea pigs and rabbits, pigs, cattle, goats, horses, sheep, minced domestic animals, dogs, pets such as cats, humans, and the like. Examples include primates such as monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. Examples of birds include chickens, quails, ducks, geese, turkeys, ostriches, emu, ostriches, guinea fowls, pigeons and the like. The vertebrate is preferably a mammal, more preferably a rodent (such as mouse) or a primate (such as human).

Examples of the stem cells having osteoblast differentiation ability include mesenchymal stem (MS) cells, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, and induced pluripotency. Examples include sex stem (iPS) cells, cultured fibroblasts, and bone marrow stem cell-derived pluripotent cells (Muse cells).

Mesenchymal stem cells are somatic stem cells derived from mesenchyme and have the ability to differentiate into cells belonging to mesenchymal system such as bone cells, adipocytes and muscle cells. It has also been shown to have plasticity capable of differentiating into non-mesoderm tissues such as ectodermal-derived cells and endoderm-derived cells. Mesenchymal stem cells include, for example, umbilical cord-derived stem cells, bone marrow mesenchymal cells and adipose-derived stem cells. Mesenchymal stem cells are considered to be present in all tissues with mesenchymal tissues (eg, cord blood, bone marrow, adipose tissue, endometrium, dermis, skeletal muscle, periosteum, periodontal ligament, dental pulp and tooth germ) ing. Umbilical cord blood is blood contained in the umbilical cord (umbilical cord), which is a fetal tissue that connects the fetus and the mother. It was known that umbilical cord blood contains a large amount of umbilical cord blood-derived stem cells (hematopoietic stem cells), but umbilical cord and umbilical cord blood contain mesenchymal stem cells that are somatic stem cells other than hematopoietic stem cells. It has been revealed that (umbilical cord (blood)-derived mesenchymal stem cells) are present. On the other hand, bone marrow mesenchymal stem cells are contained in the bone marrow stromal cells, and the bone marrow stromal cells are a kind of cells that support the hematopoietic cells that are the main components in the bone marrow. Bone marrow mesenchymal stem cells can be easily collected by bone marrow puncture, and culture techniques have been established. Adipose tissue-derived mesenchymal stem cells have relatively fast proliferation and high cell activity.

In the living body, mesenchymal stem cells are present at low frequency in, for example, bone marrow, peripheral blood, umbilical cord (blood), and adipose tissue. Mesenchymal stem cells can be isolated or purified from these tissues by a known method. The term "isolated or purified" means artificially placed in a state different from a naturally occurring state, for example, an operation of removing components other than the intended component from the naturally occurring state. Means that

For example, to isolate mesenchymal stem cells from human umbilical cord, Romanov Y. et al. A. (Stem Cells. 2003; 21:105-10.) can be used to isolate mesenchymal stem cells. Also, for example, from human bone marrow, Haynesworth S. E. (Bone. 1992; 13: 81-8.) according to the method reported by Bone. From the obtained bone marrow cell fluid, Pittenger M. F. Mesenchymal stem cells can be isolated by removing other cells mixed in the bone marrow cell fluid using the method reported by et al. (Science. 1999; 284:143-7.). For example, when mesenchymal stem cells are isolated from human adipose tissue, Zuk P. et al. A. (Tissue Eng. 2001 Apr; 7: 211-28., Mol Biol Cell. 2002 Dec; 13(12): 4279-95.) can be used. Mesenchymal stem cells can be collected as described above, but the method is not limited to the above.

The mesenchymal stem cells can be immortalized cells. Immortal means having acquired infinite autonomous growth ability. Immortalization of mesenchymal cells can be performed by introducing various immortalizing genes. For example, introducing the Bmi1 gene and the TERT gene (BBRC, vol. 353, p. 60-66 (2007)) or introducing the TERT gene and the E6 and E7 genes of human papillomavirus (BBRC, vol. 295, p. .354-361 (2002)), it is possible to immortalize mesenchymal stem cells.

Embryonic stem cells can be obtained from a prescribed institution, or commercially available products can be purchased. For example, human embryonic stem cells KhES-1, KhES-2 and KhES-3 are available from Institute for Frontier Medical Sciences, Kyoto University. EB5 cells, both of which are mouse embryonic stem cells, are available from National Institute of Physical and Chemical Research, and D3 strain is available from ATCC. A nuclear transfer ES cell (ntES cell), which is one of ES cells, can be established from a cloned embryo produced by transplanting somatic cell nuclei into an ovum from which a cell line has been removed. EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Cell, 70:841-847, 1992).

The induced pluripotent stem cells are cells that have induced pluripotency by reprogramming somatic cells by a known method or the like. Specifically, somatic cells differentiated such as fibroblasts and peripheral blood mononuclear cells are treated with Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28. , Esrrb and the like, and cells that have been reprogrammed to induce pluripotency by the expression of any combination of genes selected from a group of reprogramming genes. Preferred combinations of reprogramming factors include (1) Oct3/4, Sox2, Klf4, and Myc (c-Myc or L-Myc), (2) Oct3/4, Sox2, Klf4, Lin28, and L-Myc (Stem). Cells, 2013; 31: 458-466).

Muse cells can be obtained from skin tissues such as bone marrow fluid and dermal connective tissue, and are also scattered in connective tissues of each organ. In addition, this cell is a cell having both properties of a pluripotent stem cell and a mesenchymal stem cell, and for example, “SSEA-3 (Stage-specific embryonic antigen-3)” and “Cell-surface marker”, which are cell surface markers of each cell, are used. Identified as double positive for "CD105". Therefore, Muse cells or a cell population containing Muse cells can be separated from living tissues using these antigen markers as indicators. Details of Muse cell isolation, identification, characteristics, and the like are disclosed in International Publication No. WO2011/007900. See also Wakao, S, et al. , Proc. Natl. Acad. Sci. USA, Vol. 108, p. As reported by 9875-9880 (2011), when the cells obtained by culturing mesenchymal cells such as bone marrow and skin are used as a population of Muse cells, all SSEA-3-positive cells are CD105-positive. It is known to be a cell. Therefore, in the present invention, when Muse cells are isolated from living mesenchymal tissues or cultured mesenchymal stem cells, MUSE cells can be purified and used simply by using SSEA-3 as an antigen marker.

The stem cells having osteoblast differentiation potential may be adherently cultured in an appropriate medium before using the osteoblast differentiation promoting agent of the present invention. Specifically, for example, by culturing a suspension of stem cells on a suitable extracellular matrix, preferably on an extracellular matrix immobilized on the surface of a carrier, the stem cells are grown without differentiation. Can be made. The solution for suspending the stem cells is not particularly limited, but a stem cell medium (culture solution) can be preferably used. As a medium for such stem cells, a medium known per se suitable for culture can be used depending on the type of stem cells used. The basal medium used is not particularly limited, but includes, for example, MEM medium, α-MEM medium, D-MEM medium, Eagle MEM medium, BME medium, IMDM medium, Medium 199 medium, RPMI1640 medium, BGJb medium, CMRL1066 medium, Glassgow. Examples thereof include MEM medium, Improved MEM Zinc Option medium, ham medium, Fischer's medium and mixed medium thereof. These media may contain serum (eg, fetal bovine serum, human serum, etc.). Alternatively, an alternative additive to serum (eg, Knockout Serum Replacement (KSR) (manufactured by Invitrogen) or the like) may be used. The concentration of serum is not particularly limited, but is usually in the range of 5 to 20 (v/v)%.

Furthermore, the above-mentioned medium may contain additives known per se. The additives are not particularly limited, but include, for example, growth factors (such as insulin), iron sources (such as transferrin), minerals (such as sodium selenate), sugars (such as glucose), organic acids (such as pyruvic acid, Lactic acid etc.), serum proteins (eg albumin etc.), amino acids (eg L-glutamine etc.), reducing agents (eg 2-mercaptoethanol etc.), vitamins (eg ascorbic acid, d-biotin etc.), antibiotics (eg streptomycin). , Penicillin, gentamicin, etc.), buffers (eg HEPES, etc.) and the like. When proliferating stem cells, a stem cell differentiation inhibitor (eg, LIF, Wnt, TGF-β, bFGF, etc.) is usually used. Each of these additives can be contained within a concentration range known per se.

Further, in the case of promoting the induction of the differentiation of stem cells having osteoblast differentiation ability into osteoblasts with the osteoblast differentiation promoting agent of the present invention, usually, in advance or with the osteoblast differentiation promoting agent of the present invention At the same time, a bone differentiation inducing factor is added to the medium. Such a bone differentiation inducing factor can be appropriately determined depending on the stem cells used. For example, when mesenchymal stem cells are used as stem cells to induce differentiation into osteoblasts, immunosuppressive agents such as dexamethasone, tacrolimus and cyclosporine; BMP-2, BMP-4, BMP-5, BMP-6. , Bone morphogenetic proteins such as BMP-7 and BMP-9; morphogenetic humoral factors such as TGF-β; hydrocortisone, ascorbic acid, β-glycerophosphate and the like can be used, but preferably bone morphogenetic proteins are used. And more preferably BMP-2 is used. These bone differentiation-inducing factors can be used alone or in combination of two or more. The concentration of the bone differentiation inducing factor to be added is not particularly limited and can be within the concentration range known per se. For example, the bone differentiation inducing factor is usually at a concentration of about 0.01 ng/ml to about 300 μg/ml, preferably about 0.1 ng/ml to about 500 ng/ml, more preferably about 1 ng/ml to about 300 ng/ml. Can be added to the medium.

The number (density) of stem cells having an osteoblast differentiation potential to be seeded can be appropriately set according to the type of stem cells used, the culture method, the culture conditions, the target for inducing differentiation of the stem cells, and the like. When performing cell proliferation, the number of cells is usually 10,000 to 500,000 cells/ml, preferably 10,000 to 100,000 cells/ml, more preferably 10,000 to 50,000 cells/ml. is there. When inducing differentiation of cells, the number of cells is usually 100,000 to 1,000,000 cells/ml, preferably 100,000 to 500,000 cells/ml, more preferably 100,000 to 250. 1,000 pieces/ml.

The culture condition of the stem cell having osteoblast differentiation ability is not particularly limited as long as it is a condition usually used in cell culture technology. For example, the culture temperature is usually in the range of 30 to 40°C, preferably about 37°C. It is illustrated. The CO ₂ concentration is usually in the range of 1 to 10%, preferably about 5%. Humidity is usually in the range of 70 to 100%, preferably 85 to 95%.

2. Method for Inducing Osteoblast Differentiation The present invention provides a method for inducing osteoblast differentiation, which comprises culturing stem cells capable of differentiating osteoblast with an osteoinduction factor and an actin polymerization inhibitor.

In the osteoblast differentiation inducing method of the present invention, “stem cells capable of differentiating osteoblasts”, “bone differentiation inducing factor”, and “actin polymerization inhibitor” are described in 1. above. What was described in the osteoblast differentiation promoting agent can be used.

The culture of the osteoblast-differentiating stem cells in the above method may be suspension culture, adherent culture, or a combination thereof. “Suspension culture” in the present invention refers to culturing while maintaining the state in which cells (or cell aggregates) are suspended and present in a culture solution. “Adhesive culture” refers to culture performed under the condition that cells (or cell aggregates) are adhered to a culture device or the like. In this case, the cell adhesion means that a strong cell-substratum junction can be formed between the cell or the cell aggregate and the culture device. More specifically, the suspension culture refers to culture under conditions that do not form a strong cell-substrate bond between cells or aggregates of cells and culture equipment, and adherent culture refers to cells or cells. Culture under the condition that a strong cell-matrix bond is formed between the aggregate and the culture equipment.

The incubator used when carrying out suspension culture is not particularly limited as long as it can carry out suspension culture, and can be appropriately determined by those skilled in the art. Examples of such an incubator include a flask, a tissue culture flask, a culture dish (dish), a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a micropore, a multiplate, a multiwell plate. , Chamber slides, petri dishes, tubes, trays, culture bags, Erlenmeyer flasks, spinner flasks or roller bottles. It is preferable that these incubators are non-cell-adhesive in order to enable suspension culture. As a cell-non-adhesive incubator, the surface of the incubator is artificially treated (for example, a superhydrophilic treatment such as MPC polymer, a low protein adsorption treatment, etc.) for the purpose of reducing the adhesion to cells. Things etc. can be used. You may spin-culture using a spinner flask, a roller bottle, etc. The culture surface of the incubator may have a flat bottom or may have irregularities.

The incubator used when performing the adherent culture is not particularly limited as long as it is capable of performing the adherent culture, and those skilled in the art appropriately select an incubator according to the culture scale, culture conditions and culture period. It is possible to As such an incubator, for example, flask, flask for tissue culture, culture dish (dish), tissue culture dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate, chamber slide, petri dish, Examples include tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks or roller bottles. These incubators are preferably cell-adhesive in order to enable adherent culture. As the cell-adhesive incubator, the surface of the incubator includes an incubator that is artificially treated for the purpose of improving the adhesiveness with cells, and specifically, a surface-treated incubator, or An example is an incubator whose inside is coated with a coating agent. Examples of the surface-treated incubator include culture vessels that have been surface-treated by positive charge treatment or the like. Examples of the coating agent include laminin [including laminin α5β1γ1 (hereinafter, laminin 511), laminin α1β1γ1 (hereinafter, laminin 111) and laminin fragments (laminin 511E8 and the like]], entactin, collagen, gelatin, vitronectin (Vitronectin), Examples include extracellular matrix such as Synthemax (Corning) and Matrigel, and polymers such as polylysine and polyornithine.

The medium used for culturing cells in the osteoblast differentiation inducing method of the present invention can be prepared by using a medium normally used for culturing animal cells as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, D-MEM medium, F- 12 medium, D-MEM/F12 medium, IMDM/F12 medium, ham medium, RPMI1640 medium, Fischer's medium, a mixed medium thereof, and the like, which can be used for culturing animal cells can be mentioned. These media may be serum-free media or serum (eg fetal calf serum, human serum etc.) media, but serum-free media are preferred.

By serum-free medium is meant a medium that does not contain unconditioned or unpurified serum. In the present invention, a medium in which a purified blood-derived component or animal tissue-derived component (eg, growth factor) is mixed is also included in the serum-free medium as long as it does not contain unadjusted or unpurified serum.

The serum-free medium may contain a serum replacement. Examples of the serum substitute include those appropriately containing albumin, transferrin, fatty acid, collagen precursor, trace element, 2-mercaptoethanol or 3'thiolglycerol, or equivalents thereof. Such a serum substitute can be prepared, for example, by the method described in WO98/30679. A commercially available product may be used as the serum substitute. As such a commercially available serum substitute, for example, Knockout™ Serum Replacement (manufactured by Life Technologies; current ThermoFisher: sometimes referred to as KSR hereinafter), Chemically-defined GF (Chemically-defined TF) purified (Chemically-defined TF), chemically-defined monolithic lenticulated (modified). Manufactured by Life Technologies), B27 (manufactured by Life Technologies), and N2 supplement (manufactured by Life Technologies).

In order to avoid the complexity of preparation, as the serum-free medium, commercially available KSR (manufactured by Life Technologies) is used in an appropriate amount (for example, from about 0.5% to about 30%, preferably from about 1%). About 20%) added serum-free medium (for example, a medium containing 10% KSR, 1x Chemically Defined Lipid Concentrate (CDLC) and 450 μM 1-monothioglycerol in a 1:1 mixture of F-12 medium and IMDM medium). May be used. In addition, as the KSR-equivalent product, the medium disclosed in JP-A-2001-508302 can be mentioned.

The culture in the osteoblast differentiation inducing method of the present invention is preferably performed under xeno-free conditions. “Zenofree” means conditions under which components derived from an organism different from the organism of the cell to be cultured are excluded.

The concentration of the osteoblast-differentiating stem cells to be seeded is, for example, about 1×10 ³ to about 1×10 ⁸ cells/ml, preferably about 3×10 ³ to about 5×10 ⁷ cells/ml, more preferably Is about 4×10 ³ to about 2×10 ⁶ cells/ml, more preferably about 4×10 ³ to about 1×10 ⁶ cells/ml, and even more preferably about 1×10 ⁴ to about 1×10 ^6. It can be cells/ml.

Culture conditions such as culture temperature and CO ₂ concentration can be appropriately set. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO ₂ concentration is, for example, about 1% to about 10%, preferably about 5%.

The number of culture days in the osteoblast differentiation inducing method of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but for example, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 or more. It is at least 15 days and at least 20 days. The culture period is preferably 10 days or longer or 15 days or longer, and 20 days or shorter.

In the osteoblast differentiation inducing method of the present invention, the bone differentiation inducing factor is not particularly limited as long as the stem cells having osteoblast differentiation ability are differentiated into osteoblasts, but is usually about 0.01 ng/ml to about 300 μg/ ml, preferably about 0.1 ng/ml to about 500 ng/ml, more preferably about 1 ng/ml to about 300 ng/ml can be added to the medium.

Further, the timing of adding the bone differentiation inducing factor to the medium is not particularly limited as long as the stem cells capable of differentiating osteoblasts are differentiated into osteoblasts. For example, at the same time as the start of culture (0 day), after the start of culture, 1 Days, 2 days, 3 days, 4 days or 5 days, preferably at the same time as the start of culture (0 days), 1 day, 2 days or 3 days after the start of culture, and more preferably start of culture It is the same time (0 day). The number of times of addition can be appropriately determined by those skilled in the art, but is usually once or more. In the method of the present invention, the time when the bone differentiation inducing factor is added to the medium can be the 0th day of culture, if necessary. The osteoblast-differentiating stem cells can be cultured (precultured) in an appropriate medium until the time point. The pre-culture period is, for example, 6 hours to 3 days, preferably 12 hours to 2 days, and more preferably 24 hours.

The time for containing the bone differentiation inducing factor in the medium is not particularly limited as long as the stem cells having osteoblast differentiation ability are differentiated into osteoblasts, but for example, 3 days or more, 5 days or more, 6 days or more, 7 days or more , 8 days or more, 9 days or more, 10 days or more, or 15 days or more, and 20 days or less. The number of days is preferably 10 days or more or 15 days or more and 20 days or less. The bone differentiation inducer may be allowed to act on cells at all times during culture.

In the osteoblast differentiation inducing method of the present invention, the actin polymerization inhibitor is not particularly limited as long as it promotes differentiation of stem cells having osteoblast differentiation ability into osteoblasts, but is usually 1 nM to 10 μM, preferably 10 nM. ˜800 nM, more preferably 10 nM to 500 nM.

The timing at which the actin polymerization inhibitor is added to the medium is not particularly limited as long as it promotes the differentiation of stem cells having osteoblast differentiation ability into osteoblasts. For example, the start of culture (0 day), the start of culture 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days, preferably Are 3 and 7 days after the start of culture. The number of times of addition can be appropriately determined by those skilled in the art, but is usually once or twice or more.

The time for containing the actin polymerization inhibitor in the medium is not particularly limited as long as it promotes the differentiation of the stem cells having osteoblast differentiation ability into osteoblasts, but for example, 1 hour to 20 hours, 2 hours to 15 hours. Can be 3 hours to 10 hours, 4 hours to 8 hours, 5 hours to 7 hours

Culturing of the actin polymerization inhibitor and the stem cells capable of differentiating osteoblasts may be carried out only once or may be carried out in plural times throughout the entire culture process.

When the culture of the stem cells having osteoblast differentiation ability and the bone differentiation inducing factor and/or the actin polymerization inhibitor is completed during the culture, for example, the bone differentiation inducing factor and/or the actin polymerization inhibitor is removed from the medium. To be removed. The removal is performed, for example, by replacing the original medium containing the bone differentiation inducing factor and the actin polymerization inhibitor with a medium not containing the bone differentiation inducing factor and/or the actin polymerization inhibitor. When performing the medium exchange operation, for example, an operation of adding a new medium without discarding the original medium (medium addition operation), about half of the original medium (about 30 to 90% of the volume of the original medium, for example 40 to About 60%) Discard and add about half amount of the new medium (about 30 to 90% of the volume of the original medium, for example, about 40 to 60%) (half volume medium replacement operation), about the entire amount of the original medium (the original amount) An operation (90% or more of the volume of the medium) is discarded and a new medium is added to the entire amount (90% or more of the volume of the original medium) (total medium exchange operation).

When adding a specific component (for example, a bone differentiation inducing factor or an actin polymerization inhibitor) at a certain time point, for example, after calculating the final concentration, discard about half of the original medium, You may perform the operation (half volume medium exchange operation) which adds about half the volume of the new medium containing a high concentration (specifically, 1.5 to 3.0 times the final concentration, for example, about twice the final concentration). ..

To maintain the concentration of a specific component contained in the original medium at a certain time point, for example, discard about half of the original medium, and half the volume of a new medium containing the same concentration of the specific component contained in the original medium. You may perform the addition operation to some extent.

When diluting the components contained in the original medium at a certain time to reduce the concentration, for example, the medium exchange operation is performed multiple times a day, preferably multiple times within 1 hour (for example, 2 to 3 times). Good. Further, at a certain point, when the components contained in the original medium are diluted to reduce the concentration, the cells or aggregates may be transferred to another culture container.

The tool used for the medium exchange operation is not particularly limited, and examples thereof include a pipetter, a micropipette (pipetteman), a multichannel micropipette (multichannel Pipetman), and a continuous pipetting machine. For example, when using a 96-well plate as a culture container, a multi-channel micropipette (multi-channel Pipetman) may be used.

As described above, by using the osteoblast differentiation inducing method of the present invention, it is possible to induce the differentiation of stem cells having osteoblast differentiation ability into osteoblasts. Confirmation that osteoblasts have been obtained by the method for inducing osteoblast differentiation of the present invention is performed by measuring the activity of a bone differentiation marker gene expression or a bone differentiation marker enzyme to determine the differentiation status of stem cells having osteoblast differentiation ability. Can be carried out by checking Therefore, the osteoblast differentiation inducing method of the present invention may further include measuring the bone differentiation marker gene expression or the activity of the bone differentiation marker enzyme to confirm the differentiation status of the stem cells having osteoblast differentiation ability. Examples of bone differentiation marker genes include ALP (alkaline phosphatase), osteocalcin (OC), osteopontin, Runx2 and the like, and preferably ALP.

Runx2 is an essential transcription factor in bone formation. Runx2 plays an essential role in the differentiation of mesenchymal stem cells into osteoblasts in the living body. Forced expression of Runx2 in mesenchymal stem cells increases osteoblast-specific genes such as OC (osteocalcin), BSP (Bone sialo-protein), ALP (alkaline phosphatase), and COL1A1.

ALP (alkaline phosphatase) is an early- to mid-phase differentiation marker for osteoblasts. It is abundant in the membrane surface of osteoblasts and matrix vesicles secreted by osteoblasts and is involved in the initiation of calcified matrix production.

Osteocalcin (OC) is considered to be osteoblast-specifically expressed and contributes to promotion of bone formation.

Furthermore, a method of detecting a calcified component produced by a bone-differentiated cell can also be used. The detection method is not particularly limited, and examples thereof include von Kossa staining and alizarin red staining.

Von Kossa staining is a method of detecting calcium phosphate, which is a calcification component, using silver nitrate. Specifically, cells fixed with paraffin or the like are reacted with a 1 to 5 wt% silver nitrate aqueous solution. After the reaction, the part where calcium phosphate is present is colored black by shining light. Bone differentiation can be evaluated by measuring the area of this colored portion.

Alizarin red staining is a method that utilizes that alizarin red S specifically binds to calcium ions and stains the calcium ion deposits. Specifically, when 0.01 to 5 wt% of alizarin red S solution is reacted with cells fixed with paraffin or the like, a color reaction of magenta to orange red is observed. Bone differentiation can be evaluated by measuring the area of this colored portion.

3. Bone therapeutic material The present invention provides a bone therapeutic material containing osteoblasts produced by the osteoblast differentiation inducing method of the present invention. Moreover, the osteoblast differentiation inducing method of the present invention may be a method for producing a bone therapeutic material.

The bone therapeutic material refers to an implant material containing osteoblasts, which is introduced into a living body for repair and regeneration of bone tissue. The transplant material includes a material in which bone tissue is partially or completely regenerated in vitro and transplanted into the same or another individual. The osteoblasts obtained in the present invention can be used for producing a transplant material. The osteoblast itself is also a transplant material. Therefore, osteoblasts can be transplanted to a patient as a cell preparation, and together with a base material (scaffold) made of an artificial material such as β-TCP (β-tricalcium phosphate), hydroxyapatite, and bioabsorbable ceramic. It can be transplanted into bone tissue in need of repair or regeneration, or can be cultured with a scaffold before transplantation. In these cases, the scaffold can be made to have various three-dimensional shapes depending on the purpose of implantation. The cells may be either autologous or allotransplanted for the administration subject, and in the case of allotransplantation, allogeneic, allogeneic or xenogeneic cells may be used. In particular, when osteoblasts differentiated from umbilical cord-derived mesenchymal stem cells are used in the bone therapeutic material of the present invention, umbilical cord-derived mesenchymal stem cells do not express HLAII, and therefore allogeneic transplantation is possible.

The diseases to be treated with the osteoblasts (bone treatment materials) obtained by the present invention include periodontal disease, alveolar bone resorption, bone tumors, bone defects associated with trauma and osteomyelitis, and bone tumors. After curettage of bone, bone fracture, osteoporosis, intractable oral wounds, jaw fracture, rheumatoid arthritis, sudden femoral head necrosis, osteoarthritis, lumbar deformable spondylosis, spinal canal stenosis, disc herniation , Spinal cord injury, spondylolisthesis, spondylolisthesis, scoliosis, cervical spondylotic myelopathy, posterior longitudinal ligament ossification, osteoarthritis of the knee, osteoarthritis of the knee, slipped femoral head, osteomalacia, Reconstruction of fracture site destroyed by complex fracture such as mandibular reconstruction, bone repair after surgery (stembone repair after heart surgery, etc.), repair of defects due to artificial ankle surgery, osteomyelitis, osteonecrosis Etc. In addition, if osteoblasts are transplanted, there is a possibility that the therapeutic effect can be enhanced by joint use with bone grafts, artificial bone grafts, artificial joints and implants. Further, the above diseases can be treated by culturing osteoblasts using a three-dimensional scaffold or the like to prepare various forms of bone tissue outside the body and transplanting the bone tissue. In addition to the above, various diseases related to the loss, deficiency or functional decline of osteoblasts are targeted.

The osteoblasts obtained by the present invention can also be used for cosmetic purposes, not limited to the treatment of diseases. For example, by implanting osteoblasts or bone tissue produced by the same into a site that is defective due to an accident, surgery, or the like, it is possible to produce a bone matrix and repair the defective site to make it inconspicuous.

The bone therapeutic material of the present invention may contain, in addition to the above osteoblasts, any carrier, for example, a pharmaceutically acceptable carrier, a stabilizer or a buffer solution component, other therapeutic agents or supplements and the like. Examples of the pharmaceutically acceptable carrier include, but are not limited to, diluents such as water and physiological saline.

The dose of the bone treatment material of the present invention, the number of administration, the administration period, etc., and the concentration of osteoblasts in the bone treatment material of the present invention are not particularly limited, depending on the physical condition of the administration subject, medical condition, weight, age, sex, etc. It can be adjusted appropriately, and other surgery and medication can be used in combination.

The bone treatment material of the present invention includes, for example, rodents such as mouse, rat, hamster, and guinea pig, Lagomorpha such as rabbit, pigs, cows, goats, horses, ungulates such as sheep, dogs, cats such as cats. It can be used for transplantation into primates such as eyes, humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans and chimpanzees. The bone treatment material of the present invention is preferably for transplantation into primates or rodents, more preferably for transplantation into humans.

The therapeutic material of the present invention is transplanted by a medical staff according to an appropriate transplantation method in accordance with the guidelines. For example, it can be transplanted by incising the mucosa-periosteum to expose the jawbone at the site to be transplanted and administering the therapeutic material of the present invention to the site.

When a therapeutically effective amount of the therapeutic material of the present invention is transplanted into a site in need of bone tissue repair, the transplanted cells are differentiated into bone cells, and the bone cells supplement the defect site, thereby repairing the bone tissue. And the therapeutic effect is achieved. As a result, it becomes possible to regenerate bone tissue with the therapeutic material of the present invention.

In the present specification, the “effective amount” means the amount of active ingredient (eg, osteoblast) that produces a desired effect. As used herein, "therapeutically effective amount" means the amount of active ingredient that, when administered to a subject, produces the desired therapeutic effect (eg, bone regeneration, etc.). The therapeutically effective amount may be administered once (transplanted) or may be administered in multiple doses (transplanted). The number of times the transplant is applied is determined according to the medical staff and guidelines according to the disease. When transplanting a plurality of times, the interval is not particularly limited, but a period of several days to several weeks may be set.

4. Bone regeneration treatment method The present invention comprises culturing a stem cell having an osteoblast differentiation ability together with a bone differentiation inducing factor and an actin polymerization inhibitor, and transplanting the obtained osteoblast into a bone defect site. Treatment methods are also provided.

The method can include isolating osteoblasts from the cultured cells. The term "isolated" of osteoblasts means that cells other than the desired osteoblasts have been subjected to an operation to remove the naturally existing state. Therefore, most preferably, "isolated osteoblasts" does not include cells other than osteoblasts produced from cells or tissues to be cultured and contained in the medium or medium. The purity of "isolated osteoblasts" (percentage of the number of osteoblasts in the total number of cells) is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 99% or more. , And more preferably 100%. In the present invention, when the purity of “isolated osteoblasts” is less than 100%, the cells other than osteoblasts present in “isolated osteoblasts” are osteocyte (osteocyte), It is preferable to use preosteoblasts, osteoprogenitor cells, mesenchymal progenitor cells that have been slightly differentiated from mesenchymal stem cells, or a combination thereof.

5. The present invention provides a bone treatment kit .
There is provided a bone treatment kit containing the osteoblast differentiation promoting agent of the present invention and a stem cell having osteoblast differentiation ability.

The above-mentioned kit for bone treatment uses, for example, the osteoblast differentiation promoting agent of the present invention for stem cells having osteoblast differentiation ability as described in the osteoblast differentiation inducing method of the present invention. Then, osteoblasts are produced, and further, a bone therapeutic material containing the osteoblasts is produced and transplanted to a subject having the above-mentioned disease, whereby the disease can be treated.

The kit of the present invention contains the osteoblast differentiation promoting agent of the present invention and stem cells having osteoblast differentiation ability in separate containers. The osteoblast-differentiating stem cells are preferably provided in a cryopreserved form in order to allow proliferation and induction of differentiation over multiple times. Cryopreservation is carried out by suspending the cells in a physiological solution such as a culture solution containing a cryoprotective agent such as 5 to 20% of dimethylsulfoxide, glycerin, ethylene glycol, propylene glycol, etc. By freezing at -80°C or -196°C. Alternatively, it may be cryopreserved according to the method described in JP5630979B2.

Further, the kit of the present invention includes a statement that the osteoblast differentiation promoting agent of the present invention and a stem cell having an osteoblast differentiation ability can be or should be used for bone treatment. You can leave.

The bone treatment kit preferably further contains a substance capable of detecting the expression of the bone differentiation marker gene. The bone differentiation marker is not particularly limited as long as it is a marker capable of confirming the bone differentiation of mesenchymal stem cells, and for example, the marker shown in the above differentiation induction method can be used. Preferably, BMP2, ALP and OCN bone differentiation markers are used.

The bone treatment kit may include any carrier, for example, a pharmaceutically acceptable carrier, a stabilizer or a buffer component, other therapeutic agents or supplements, and the like. Examples of the pharmaceutically acceptable carrier include, but are not limited to, diluents such as water and physiological saline.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1]
(Experimental conditions)
The experimental conditions in the gel culture are as follows. Porcine tendon-derived Type I collagen solution adjusted to 3.0 mg/ml, pH 3.0 0.5 ml of concentrated medium was added to 4 ml of Cellmatrix Type I-A and stirred, and 0.05 N sodium hydroxide solution containing sodium bicarbonate and HEPES was added. In addition, what was further stirred was used as a collagen mixed solution, which was spread thinly on a 60 mm dish (manufactured by FALCON) and allowed to stand at 37°C for 20 minutes. 5×10 ⁴ cell/ml of D-MEM (Low-glucose, 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml) added) cell suspension was seeded at 5 ml/dish and incubated for 24 hours. did.
For the inhibitor experiment, 5×10 ⁴ cell/ml D-MEM (Low-glucose, 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL) added) cell suspension was added at 5 ml/dish. Seed and incubate for 24 hours. After that, BMP-2 was added to a final concentration of 300 ng/ml (this day is designated as day 0 of culture), and on day 3 and day 7 of culture, 100 nM of Swinholide A in serum-free medium and latrunc Phosphorus A was added to 400 nM and allowed to act for 6 hours. After the treatment, the medium was returned to the medium containing BMP-2 again, cells were collected on the 10th day after the culture, and the expression analysis of the ALP gene was performed by RT-PCR.

Until now, stem cell manipulation techniques based on the extracellular environment design have mainly focused on the regulation of differentiation induction by the induction of exogenous signals by the addition of humoral factors such as BMP-2. However, there have been problems and problems for clinical application in terms of side effects such as the development of edema and local administration to a necessary site. Therefore, in the present invention, a culture method based on induction of endogenous signaling via cell behavior was considered as a novel differentiation control method.

Umbilical cord MSCs, like other cell types, are surrounded by extracellular matrix (ECM). It has been reported that the ECM not only provides a scaffold for cell adhesion, but also contributes to many physiological activities such as cell proliferation, differentiation, and migration via a receptor present on the cell membrane surface. Collagen is a typical molecule that constitutes an extracellular matrix, capable of binding to a cell adhesion molecule involved in cell adhesion and activating an intracellular signal. When umbilical cord MSCs were cultured using this collagen substrate, ALP activity and elevation were observed in both undifferentiated/osteoblast cells, and in particular, a remarkable elevation of ALP gene expression was observed in culture on collagen gel, which increased to the same level as bone marrow MSCs. (Figure 2). We have found that collagen gel culture is effective in a collagen comparison experiment (FIGS. 3-5).

When the “BMP-2-BMP receptor II (BMPRII)-cofilin” transduction pathway (FIG. 6) was analyzed for these control mechanisms, both BMPRII and cofilin showed increased mRNA expression (FIGS. 7 and 8). Since cofilin is a depolymerization/cleavage factor for actin filament (F-actin), it is known that F-actin is transferred to G-actin when cofilin is activated.

In order to realize a system for producing and supplying cell products, it is necessary to prepare transplanted cells without special treatment as much as possible and to handle the cells easily. The gel culture is not suitable for practical use because it requires labor such as collagenase treatment for cell collection. Therefore, it is important to design and optimize a new culturing method for umbilical cord MSC that induces differentiation promotion while securing the cell number in normal culturing.

From the observation result of the intracellular localization of actin in the gel culture, it was possible to confirm that G-actin, which was not expressed before the differentiation, showed a marked increase in the intracellular expression after the culture period (FIG. 9). .. From the obtained findings, it was suggested that umbilical cord MSC promote osteoblast differentiation by culturing on gel, but F-actin depolymerization is largely involved in the mechanism of action. Therefore, it was thought that osteoblast differentiation could be induced by treating umbilical cord MSCs with an actin polymerization inhibitor.

Therefore, we investigated the promotion of bone differentiation using two types of actin polymerization inhibitors. When treated with the actin inhibitors Latrunculin A and Swinholide A 3 days after the induction of osteoblast differentiation with BMP-2, an increase in the expression of ALP mRNA was observed in both addition groups. It was seen (Fig. 10). Regarding the change in actin kinetics, it was confirmed that the cells treated with the inhibitor had the same intracellular localization change as the morphology observed in the gel culture (FIG. 9). That is, regarding the change in actin kinetics, F-actin was disrupted in cells treated with an inhibitor, but F-actin was reconstituted when the culture was repeated thereafter, and expression of G-actin was also frequently seen (FIG. 11, 12), the change in intracellular localization similar to the morphology in gel culture (FIG. 9) was shown. The results of this experiment suggested that treatment of umbilical cord MSCs with an actin polymerization inhibitor promotes osteoblast differentiation.

On the other hand, it is difficult to stably obtain a homogeneous differentiated cell group in the process of inducing differentiation of various cells from stem cells. It is considered that the main cause is cell heterogeneity and cell behavior such as intercellular communication and induction in a multidirectional and stepwise differentiation induction process. Umbilical cord MSCs maintain high expression of undifferentiated markers such as Oct4 and Nanog even after induction of differentiation, but in collagen gel culture, expression of undifferentiated markers is significantly reduced after long-term culture (FIG. 13).

[Example 2]
(experimental method)
Umbilical cord MSCs were seeded in a 100 mm dish together with D-MEM (Low-glucose) containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL), and incubated for 24 hours. After that, BMP-2 was added to a final concentration of 300 ng/ml and 400 nM latrunculin A was allowed to act thereon to prepare osteoblasts (this day is designated as day 0 of culture). On the 10th day of culture, the cells were detached using a 0.025% trypsin solution to form a complex of 3×10 ⁶ cells and β-tricalcium phosphate (β-TCP), and then on a skull of a 10-week-old male nude mouse. Transplanted.

(Experimental result)
Umbilical cord MSCs were treated with inhibitors, and osteoblasts were transplanted together with β-TCP to nude mice under general anesthesia at the crown. A control group was transplanted with a complex of BMP-2 induced osteoblasts and β-TCP.

Four weeks after the transplantation, a section specimen was prepared and evaluated by Hematoxylin & Eosin (HE) staining. As a result, the absorption of the complex proceeded in the inhibitor-treated umbilical cord MSC transplantation group, and new bone formation was confirmed (FIG. 14).

Umbilical cord MSCs can be collected non-invasively and are usually treated as medical wastes, and therefore, they are easy to bank and can be stably supplied. Therefore, they have many advantages over bone regeneration using other adult tissue-derived MSCs.

This differentiation-inducing method makes it possible to mass-produce osteoblasts from human umbilical cord MSCs as a cell preparation for transplantation for bone regeneration without a genetic manipulation, by a new culture technique applying an actin polymerization inhibitor.

The present invention enables mass production of osteoblasts as a cell preparation for transplantation for bone regeneration. In particular, when umbilical cord-derived mesenchymal stem cells are used, the cells can be collected non-invasively, and are normally treated as medical waste, so banking is easy and stable supply is possible.

This application is based on a Japanese patent application filed on Mar. 30, 2018, Japanese Patent Application No. 2018-069695, the entire contents of which are incorporated herein.

Claims (18)

An osteoblast differentiation promoting agent containing an actin polymerization inhibitor. The agent according to claim 1, wherein the osteoblast differentiation is osteoblast differentiation from a stem cell having an osteoblast differentiation ability. The agent according to claim 2, wherein the stem cell having an osteoblast differentiation ability is a mesenchymal stem cell. The agent according to claim 3, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. The agent according to any one of claims 1 to 4, wherein the actin polymerization inhibitor is latrunculin A and/or swinholide A. A method for inducing osteoblast differentiation, which comprises culturing a stem cell having osteoblast differentiation ability together with an osteoinduction factor and an actin polymerization inhibitor. The method according to claim 6, wherein the stem cell having an osteoblast differentiation ability is a mesenchymal stem cell. The method according to claim 7, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. The method according to any one of claims 6 to 8, wherein the bone differentiation-inducing factor is a bone morphogenetic protein. The method of claim 9, wherein the bone morphogenetic protein is BMP-2. The method according to any one of claims 6 to 10, wherein the actin polymerization inhibitor is latrunculin A and/or swinholide A. The method according to any one of claims 6 to 11, which further comprises measuring the bone differentiation marker gene expression or the activity of a bone differentiation marker enzyme to confirm the differentiation status of stem cells having osteoblast differentiation potential. The method according to claim 12, wherein the bone differentiation marker gene is the ALP gene and the bone differentiation marker enzyme is ALP. The method according to any one of claims 6 to 13, wherein the culture is performed for 5 days or more. The osteodifferentiation inducing factor is contained in the medium at the same time (0 days) as the start of culture or 1 day, 2 days, 3 days, 4 days or 5 days after the start of culture, according to any one of claims 6 to 14. The described method. Simultaneous with the start of culture (0 days), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days after the start of culture The method according to any one of claims 6 to 15, wherein an actin polymerization inhibitor is contained in the medium on the 14th or 15th day. The method according to any one of claims 6 to 16, which is a method for producing a bone treatment material. A bone treatment kit comprising the osteoblast differentiation promoting agent according to any one of claims 1 to 5 and a stem cell having osteoblast differentiation ability.