Composition for use in suppressing differentiation into osteoclasts

Abstract

To provide a composition capable of indirectly inducing osteogenesis by suppressing differentiation into osteoclasts and / or promoting differentiation into osteoblasts. A composition for use in suppressing differentiation into osteoclasts and / or promoting differentiation into osteoblasts according to the present disclosure includes proliferative megakaryocytes and / or mature megakaryocytes.

Description

Composition for use in inhibiting osteoclast differentiation

[0001] The present disclosure relates to compositions for use in inhibiting osteoclast differentiation.

[0002] Attempts have been made to develop cell preparations containing cells that promote tissue regeneration, such as mesenchymal stem cells, as therapeutic agents for regenerative medicine. Furthermore, the cells that promote tissue regeneration are thought to promote tissue regeneration by releasing physiologically active proteins, such as growth factors.

[0003] In addition, in regenerative medicine in the dental field, platelet-rich plasma is used in bone augmentation treatments, etc. However, because platelet-rich plasma is prepared by separation and centrifugal concentration from isolated human blood, the platelet count is not standardized, and its efficacy is unknown.

[0004] Therefore, an object of the present disclosure is to provide a composition that can indirectly induce bone formation, for example, by suppressing differentiation into osteoclasts and / or promoting differentiation into osteoblasts.

[0005] To achieve the above object, the composition of the present disclosure for use in inhibiting differentiation into osteoclasts contains proliferating megakaryocytes and / or mature megakaryocytes.

[0006] The composition of the present disclosure for use in suppressing inhibition of bone formation by osteoclasts includes the composition of the present disclosure for use in suppressing differentiation into osteoclasts.

[0007] The compositions disclosed herein for use in treating diseases caused by osteoclasts include compositions disclosed herein for use in inhibiting differentiation into osteoclasts and / or compositions disclosed herein for use in inhibiting inhibition of bone formation by osteoclasts.

[0008] A kit for use in bone formation according to the present disclosure includes a composition for use in inhibiting differentiation into osteoclasts according to the present disclosure and / or a composition for use in inhibiting inhibition of bone formation by osteoclasts according to the present disclosure, and an osteogenic scaffold material.

[0009] A kit for use in inhibiting bone destruction according to the present disclosure includes a composition for use in inhibiting differentiation into osteoclasts according to the present disclosure and / or a composition for use in inhibiting inhibition of bone formation by osteoclasts according to the present disclosure, and an osteogenic scaffold material.

[0010] The composition of the present disclosure for use in promoting differentiation into osteoblasts comprises mature megakaryocytes.

[0011] The composition of the present disclosure for use in promoting bone formation by osteoblasts includes the composition of the present disclosure for use in promoting differentiation into osteoblasts.

[0012] The compositions disclosed herein for use in treating diseases caused by osteoblasts include compositions disclosed herein for use in promoting differentiation into osteoblasts and / or compositions disclosed herein for use in promoting bone formation by osteoblasts.

[0013] A kit for use in bone formation according to the present disclosure includes a composition for use in promoting differentiation into osteoblasts according to the present disclosure and / or a composition for use in promoting bone formation by osteoblasts according to the present disclosure, and an osteogenic scaffold material.

[0014] The method for inhibiting differentiation into osteoclasts of the present disclosure uses the composition for use in inhibiting differentiation into osteoclasts of the present disclosure.

[0015] The method of the present disclosure for suppressing the inhibition of bone formation by osteoclasts uses the composition of the present disclosure for use in suppressing the inhibition of bone formation by osteoclasts.

[0016] The method for promoting differentiation into osteoblasts of the present disclosure uses the composition for use in promoting differentiation into osteoblasts of the present disclosure.

[0017] The disclosed method for promoting bone formation by osteoblasts uses the disclosed composition for use in promoting bone formation by osteoblasts.

[0018] According to the present disclosure, bone formation can be induced indirectly, for example, by inhibiting differentiation into osteoclasts and / or promoting differentiation into osteoblasts.

[0019] FIG. 1 is a graph showing cell counts. FIG. 2 is a graph showing the expression level of each gene in bone marrow-derived mesenchymal stem cells. FIG. 3 is a graph showing the OPG / RANKL ratio. FIG. 4 is a graph showing the expression level of each gene in megakaryocytes co-cultured with bone marrow-derived mesenchymal stem cells. FIG. 5 is a graph showing the expression level of each gene in megakaryocytes co-cultured with bone marrow-derived mesenchymal stem cells. FIG. 6 is a graph showing the expression level of each gene in megakaryocytes co-cultured with bone marrow-derived mesenchymal stem cells. FIG. 7 is a graph showing ALP activity. FIG. 8 is a graph showing cell counts and ALP activity. FIG. 9 is a graph showing cell counts and ALP activity. FIG. 10 is a graph showing cell counts and ALP activity. FIG. 11 is a graph showing the expression level of each gene. FIG. 12 is a graph showing the expression level of each gene. FIG. 13 is a photograph showing bone formation 4 weeks after transplantation. FIG. 14 is a photograph showing bone formation 4 weeks after transplantation. FIG. 15 is a graph showing bone mass four weeks after transplantation.

[0020] <Definition> As used herein, "osteoclast" refers to a multinucleated cell having an acidophilic cytoplasm and capable of resorbing bone tissue. The osteoclast can be identified, for example, by a marker. Examples of the marker include tartrate-resistant acid phosphatase (TRAP), cathepsin K, and calcitonin receptor (CALCR).

[0021] As used herein, "osteoclast precursor cells" refers to cells that have the ability to differentiate into osteoclasts and differentiate into multinucleated osteoclasts upon stimulation with macrophage colony-stimulating factor (M-CSF) and RANKL expressed by osteoblasts. The osteoclast precursor cells can be identified, for example, by markers. Examples of the markers include RANK and C-FMS (CD115).

[0022] As used herein, "bone destruction" refers to the reduction of bone tissue caused by osteoclasts. The bone destruction can also be referred to as bone resorption, for example.

[0023] In this specification, "osteogenesis" may mean an increase in existing bone tissue, i.e., "bone augmentation" (bone formation), or the formation of new bone, i.e., "neosteogenesis," or the restoration of lost (defective) bone or a portion thereof, i.e., "bone regeneration."

[0024] As used herein, "megakaryocytes" are the largest cells present in the bone marrow in vivo, and refer to cells that release platelets and cells with equivalent functions. The cells with equivalent functions refer to cells that have the ability to produce platelets. The megakaryocytes can be identified, for example, by a marker. Examples of the marker include the megakaryocyte markers described below.

[0025] As used herein, "proliferative" means that a target cell or cell population is in a proliferation phase. The "proliferative" can be evaluated using, for example, proliferation markers such as Ki67 and cellular DNA content as indicators. The "proliferative" can be evaluated as having proliferative properties when, for example, the positive rate of the proliferation marker is 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0026] As used herein, "positive (+)" means that a higher signal is detected by an analytical method such as flow cytometry that utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. Also, as used herein, "negative (-)" means that a signal that is equal to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0027] As used herein, the term "cell population" refers to a collection of cells that includes a desired cell and is composed of one or more cells. In the cell population, the proportion of the desired cells among all cells (also referred to as "purity") can be quantified, for example, as the proportion of cells expressing one or more markers expressed by the desired cells. The purity is, for example, the proportion among live cells. The purity can be measured by methods such as flow cytometry, immunohistochemistry, and in situ hybridization. The purity of the desired cells in the cell population is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The cell population can also be referred to as, for example, a cell preparation.

[0028] As used herein, the term "oncogene" refers to a gene capable of inducing canceration of cells in vivo, and examples thereof include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as C-KIT (CD117), PDGFR (platelet growth factor receptor), and ABL (Abelson murine leukemia viral oncogene homolog).

[0029] As used herein, "Polycomb genes" refer to genes known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to prevent cellular senescence (see References 1 to 3 below). Specific examples of the polycomb genes include BMI1 (Polycomb complex protein BMI-1, polycomb group RING finger protein 4 (PCGF4), RING finger protein 51 (RNF51)), MELL8 (Polycomb group RING finger protein 2), RING (Ring Finger Protein) 1a / b, PHC (Polyhomeotic Homolog) 1 / 2 / 3, CBX (Chromobox) 2 / 4 / 6 / 7 / 8, EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit), EED (Embryonic Ectoderm Development), SUZ12 (SUZ12 Polycomb Repressive Complex 2 Subunit), HADC (Histone deacetylases), and DNMT (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b. Reference 1: Hideyuki Oguro et al., "Control of stem cell aging by polycomb group protein complexes," Regenerative Medicine, 2007, Vol. 6, No. 4, pp. 26-32 Reference 2: Jesus Gil et.al, "Regulation of the INK4b-ARF-INK4a tumor suppressor locus: all for one or one for all," Nature Reviews Molecular Cell Biology, 2007, vol. 7, pages 667-677 Reference 3: Soo-Hyun Kim et.al., "Absence of p16 INK4a and truncation of ARF tumor suppressors in chickens”, PNAS, 2003, vol.100, No.1, pages 211-216

[0030] As used herein, the term "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples thereof include BCL2 (B-cell lymphoma 2), BCL-XL (B-cell lymphoma-extra large), BIRC5 (Baculoviral IAP Repeat Containing 5), and MCL1 (BCL2 Family Apoptosis Regulator).

[0031] As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., a composition, a substrate such as a scaffold material, instructions, etc.) are provided separately in two or more compartments. The kit can be suitably used to provide a composition that is not provided in a mixed state, but is preferably mixed immediately before use, for reasons of stability, etc. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., a composition, a substrate such as a scaffold material, etc.), or instructions or instructions describing how to treat the components. As used herein, when the kit is used as a treatment kit, the kit may include instructions or instructions describing how to use the composition, scaffold material, etc. for treatment.

[0032] As used herein, "instructions" or "instructions" refer to instructions to a physician or other user on how to use the present disclosure. The instructions may, for example, include instructions on how to use the composition or kit of the present disclosure. The instructions may be prepared in accordance with a format specified by a regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, the European Medicines Agency (EMA) in Europe, etc.), and may clearly state that they have been approved by the regulatory agency. The instructions may be a package insert, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, an electronic medium (e.g., a website provided on the Internet, email, etc.).

[0033] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and particularly includes humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0034] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a patient who develops a target disease, or treatment of an animal model of the target disease.

[0035] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0036] <Composition for Use in Inhibiting Osteoclast Differentiation> In one aspect, the present disclosure provides a composition capable of inhibiting osteoclast differentiation. The composition for use in inhibiting osteoclast differentiation of the present disclosure (hereinafter also referred to as the "first composition") contains proliferating megakaryocytes and / or mature megakaryocytes. The first composition of the present disclosure can inhibit osteoclast differentiation, i.e., inhibit the differentiation of osteoclast precursor cells into osteoclasts. Therefore, the first composition of the present disclosure can indirectly induce bone formation, for example, by suppressing or inhibiting bone destruction. Therefore, the first composition of the present disclosure is expected to be suitable for use in, for example, diseases caused by osteoclasts, such as bone diseases caused by osteoclast-mediated bone destruction, such as osteoporosis, bone destruction in rheumatoid arthritis, and osteopetrosis.

[0037] As a result of extensive research, the present inventors have discovered that proliferating megakaryocytes, i.e., proliferating megakaryocytes and / or mature megakaryocytes, may suppress the activity of osteoclasts. Further research by the present inventors has revealed that the proliferating megakaryocytes and / or mature megakaryocytes suppress the differentiation of osteoclast precursors into osteoclasts, thereby suppressing bone destruction by osteoclasts and promoting bone formation, thereby establishing the present disclosure. The present inventors have also found that the use of an extract of megakaryocytes after platelet production can promote the induction of osteoblast differentiation from mesenchymal stem cells, thereby promoting bone formation by osteoblasts. However, the extract has not been observed to suppress the differentiation of osteoclast precursors into osteoclasts. Therefore, it is presumed that the inhibitory function of osteoclast precursors into osteoclasts is mediated by a mechanism mediated by contact with the proliferating megakaryocytes and / or mature megakaryocytes. This presumption does not limit the present disclosure in any way.

[0038] The first composition of the present disclosure is presumed to indirectly induce bone formation by suppressing, reducing, lowering, and / or inhibiting differentiation or differentiation induction into osteoclasts. Therefore, the first composition of the present disclosure can be said to be a composition for use in suppressing, reducing, lowering, and / or inhibiting differentiation or differentiation induction into osteoclasts, or a composition for suppressing, reducing, lowering, and / or inhibiting bone destruction by osteoclasts. Furthermore, the first composition of the present disclosure can indirectly induce bone formation by suppressing bone destruction by osteoclasts. Therefore, the first composition of the present disclosure can also be said to be a composition for suppressing the inhibition of induction, promotion, acceleration (induction promotion), enhancement, potentiation, and / or strengthening of bone formation due to the inhibition of bone destruction by osteoclasts, for example.

[0039] In the present disclosure, megakaryocytes may be megakaryocytes before multinucleation (polyploidization), i.e., immature megakaryocytes or megakaryocytes in the proliferative phase (proliferative megakaryocytes), or megakaryocytes after multinucleation (polynucleated megakaryocytes), or megakaryocytes in the mature phase that release platelets (mature megakaryocytes). Specific examples of megakaryocytes include promegakaryocytes, megakaryoblasts, promegakaryocytes, and mature megakaryocytes. The number of chromosome sets possessed by the megakaryocytes after multinucleation may be more than two, specifically 16 to 32 sets.

[0040] The origin of the megakaryocytes is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animals include primates such as monkeys, gorillas, chimpanzees, and marmosets, as well as mice, rats, dogs, cats, rabbits, sheep, horses, and guinea pigs. The origin of the megakaryocytes may be the same as or different from that of the subject to which the first composition of the present disclosure is administered.

[0041] In the present disclosure, the megakaryocytes can be identified by cell surface markers. When the megakaryocytes are derived from humans, the cell surface markers include CD41a, CD42a, and CD42b. That is, the megakaryocytes are cells that are positive for CD41a, CD42a, and CD42b. When the megakaryocytes are derived from humans, the cell surface marker may be, for example, at least one selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c.

[0042] The megakaryocytes may be megakaryocytes isolated from a living body, or may be megakaryocytes induced from cells less differentiated than megakaryocytes, such as pluripotent cells (hereinafter also referred to as "progenitor cells"). The "cells less differentiated than megakaryocytes" refer to cells capable of differentiating into megakaryocytes.

[0043] When the megakaryocytes are isolated from a living body, the megakaryocytes can be isolated from bone marrow, for example, since they are present in bone marrow. In this case, the megakaryocytes may contain other cells derived from the living body.

[0044] When the megakaryocytes are megakaryocytes induced from progenitor cells, the megakaryocytes can be induced in vitro as described below. In this case, the megakaryocytes may contain the progenitor cells. Examples of the progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor cells (MEPs), and megakaryocyte progenitor cells. The progenitor cells may be isolated from bone marrow, umbilical cord blood, peripheral blood, or the like, or may be induced from pluripotent cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), nuclear transfer ES cells (ntES cells), germ stem cells, somatic stem cells, and embryonic tumor cells.

[0045] The proliferative megakaryocytes can be prepared, for example, by imparting proliferative properties to megakaryocytes isolated from a living organism or megakaryocytes induced from the precursor cells. The imparting of proliferative properties may be achieved, for example, by contacting the megakaryocytes with a substance that induces cell proliferation (a proliferation-inducing factor), by inducing the expression of an endogenous proliferation-inducing gene in the megakaryocytes, or by introducing an exogenous proliferation-inducing gene into the megakaryocytes and inducing its expression. When inducing megakaryocytes from the precursor cells, the proliferative megakaryocytes may be induced, for example, by imparting the proliferative properties. For example, when the megakaryocytes are proliferating, they do not produce platelets. Therefore, the proliferative megakaryocytes are, for example, megakaryocytes that have substantially no platelet-producing ability or do not have platelets. The proliferative megakaryocytes have a platelet production capacity per cell of 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the mature megakaryocytes, for example.

[0046] Examples of the megakaryocyte proliferation-inducing factors include thrombopoietin (TPO), stem cell factor (SCF), IL-6, IL-11, and IL-3.

[0047] The megakaryocyte proliferation-inducing gene is not particularly limited as long as it is a gene known to confer proliferation to the megakaryocytes, and specific examples include the oncogenes, the polycomb genes, C-KIT, C-MPL, etc. The megakaryocyte proliferation-inducing gene is preferably used in combination with the apoptosis-inhibiting gene, for example, because it can suppress cell death of the obtained proliferative megakaryocytes.

[0048] The proliferative megakaryocytes are, for example, megakaryocytes in which the expression of the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene is enhanced, preferably megakaryocytes in which the expression of the oncogene and / or the polycomb gene and the apoptosis-inhibiting gene is enhanced, and more preferably megakaryocytes in which the expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene is enhanced. The "enhanced gene expression" refers to, for example, higher expression of a target gene compared to non-proliferative megakaryocytes, such as megakaryocytes isolated from a living body before multinucleation. The gene expression can be evaluated, for example, by quantitative PCR. The proliferative megakaryocytes preferably have an expression level of the target gene that is 1.1 to 70 times, 1.1 to 50 times, or 1.1 to 10 times higher than the expression level of the target gene in non-proliferative megakaryocytes (Reference (1)).

[0049] When the proliferative megakaryocytes are proliferative megakaryocytes induced from progenitor cells, the proliferative megakaryocytes are preferably immortalized megakaryocytes. The immortalized megakaryocytes have a higher degree of homogeneity in the differentiation stage of cells compared to megakaryocytes induced by other megakaryocyte induction methods, and are therefore suitable for use as an active ingredient in the first composition of the present disclosure. The immortalized megakaryocytes are, for example, megakaryocytes induced by introducing an oncogene and a polycomb gene, or an oncogene, a polycomb gene, and an apoptosis-inhibiting gene, into the progenitor cells, as described below.

[0050] The immortalized megakaryocytes are preferably megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes. The term "exogenous" refers to a gene introduced into a cell from outside the cell. The exogenous gene may be present on the chromosome of the cell, or in the nucleus or cytoplasm. The exogenous gene can be detected, for example, by measuring the number of genes. When the gene is an autosomal gene, one copy of the gene is present on each autosome, resulting in two genes present in a single cell. Therefore, if the exogenous gene is absent, two copies of the gene are detected in a single cell. On the other hand, if the exogenous gene is present, three or more copies of the gene are detected in a single cell. In this case, the exogenous gene can be detected using, for example, PCR using primers, a probe, or a combination thereof. When the exogenous gene has a tag sequence or a selection marker, the exogenous gene may be detected by detecting the tag sequence or the selection marker. Furthermore, the exogenous gene can be detected, for example, using an antibody or the like against the protein translated from the gene.

[0051] The mature megakaryocytes may be, for example, megakaryocytes induced from the proliferative megakaryocytes, i.e., derived from the proliferative megakaryocytes. The proliferative megakaryocytes mature and differentiate into mature megakaryocytes, for example, after cessation of proliferation. The mature megakaryocytes may be, for example, proliferative megakaryocytes that have been multinucleated to form multinucleated megakaryocytes after cessation of proliferation, and then differentiated into the mature megakaryocytes. Furthermore, the proliferative megakaryocytes acquire platelet production ability by differentiating into the mature megakaryocytes. The induction can be achieved, for example, by suppressing the expression of the proliferation-inducing gene in the proliferative megakaryocytes. Therefore, the mature megakaryocytes are, for example, megakaryocytes in which the expression of the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene is suppressed, preferably megakaryocytes in which the expression of the oncogene and / or the polycomb gene and the apoptosis-inhibiting gene is suppressed, more preferably megakaryocytes in which the expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene is suppressed. The term "suppressed gene expression" means, for example, that the expression of a target gene is lower compared to the proliferative megakaryocytes. The gene expression can be evaluated, for example, by quantitative PCR. For example, the expression level of the target gene in the mature megakaryocytes is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, or 0.01% or less, based on the expression level of the target gene in the proliferative megakaryocytes (100%).

[0052] The number of chromosome sets (nuclear phase: N) possessed by the multinucleated megakaryocytes may be more than 2 sets, and specific examples include 4 to 64 sets (4N to 64N), 4 to 32 sets (4N to 32N), 8 to 64 sets (8 to 64N), 16 to 32 sets (16 to 32N), or 16 to 64 sets (16 to 64N).

[0053] The mature megakaryocytes are preferably derived from the immortalized megakaryocytes.

[0054] The physiological activity of the first composition of the present disclosure includes, for example, an activity of promoting the proliferation of mesenchymal stem cells and an activity of inhibiting the differentiation of osteoclast precursor cells into osteoclasts. The first composition of the present disclosure may have, for example, one activity or multiple activities.

[0055] The mesenchymal stem cells are cells that have the ability to self-renew and differentiate into bone, cartilage, and adipocytes. The mesenchymal stem cells can be identified by cell surface markers. For example, the mesenchymal stem cells are CD73, CD90, and CD105 positive, and CD14, CD34, and CD45 negative.

[0056] The mesenchymal stem cell proliferation-promoting activity may be, for example, an improvement in the proliferation ability of mesenchymal stem cells compared to a control group that is otherwise identical except for the addition of the first composition of the present disclosure. For example, the proliferation ability of mesenchymal stem cells may be decreased from the start of the treatment. In this case, the "proliferation-promoting activity" may also be referred to as, for example, suppression of a decrease in proliferation activity. Specifically, the proliferation activity of the mesenchymal stem cells decreases with each passage. Since the first composition of the present disclosure can suppress a decrease in the proliferation activity of the mesenchymal stem cells, it can be said that the first composition of the present disclosure exhibits proliferation-promoting activity. The mesenchymal stem cell proliferation-promoting activity can be measured, for example, under culture conditions in which the target mesenchymal stem cells proliferate. The culture conditions can be set, for example, to normal culture conditions for the mesenchymal stem cells.

[0057] The inhibitory activity against osteoclast differentiation may be, for example, a reduction in the ability to differentiate into osteoclasts compared to a control group that is otherwise identical except that the first composition of the present disclosure is not added. The inhibition of differentiation ability may mean, for example, a slowing of the rate of differentiation into osteoclasts or a reduction in the proportion of cells that differentiate into osteoclasts. For the differentiation into osteoclasts, see, for example, Reference 4 below. Reference 4: Owen R, Reilly GC. In vitro Models of Bone Remodeling and Associated Disorders. Front Bioeng Biotechnol. 2018 Oct 11;6:134. doi: 10.3389 / fbioe.2018.00134. PMID: 30364287; PMCID: PMC6193121.

[0058] The first composition of the present disclosure may be used in vitro or in vivo.

[0059] When the first composition of the present disclosure is used in vitro, the subject to be administered may be, for example, a cell, tissue, organ, etc., and the cells may be, for example, a cell collected from a living body, a cultured cell, etc.

[0060] When the first composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a human or a non-human animal other than a human, such as a mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, or guinea pig.

[0061] The conditions for use (administration conditions) of the first composition of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on, for example, the type of subject to be administered.

[0062] When the first composition of the present disclosure is used in vitro, it can be used, for example, by adding it to a culture medium for target osteoclast precursor cells. The first composition of the present disclosure may be added, for example, to a maintenance medium used to maintain the precursor cells, or to a medium used to differentiate the osteoclast precursor cells into the osteoclasts, or may be added to both the maintenance medium and the differentiation medium. According to the first composition of the present disclosure, for example, by maintaining the osteoclast precursor cells in a maintenance medium containing the first composition, it is expected that the maintenance rate of the osteoclast precursor cells can be improved thereafter. The seeding density of proliferating megakaryocytes and / or mature megakaryocytes in the maintenance medium can be, for example, 5 x 10 ３ ~5 x 10 ７ cells / cm ２ , or 5 x 10 ４ ~5 x 10 ６ cells / cm ２ The seeding density of the proliferative megakaryocytes and / or mature megakaryocytes in the differentiation medium is, for example, 5 × 10 ３ ~5 x 10 ７ cells / cm ２ , or 5 x 10 ４ ~5 x 10 ６ cells / cm ２ is.

[0063] When the first composition of the present disclosure is used in vivo, the dosage can be appropriately determined depending on, for example, the type, symptoms, age, and administration method of the subject. As a specific example, when administered to mice, the daily dosage of the first composition is not particularly limited and can be appropriately set depending on the intended use. For example, 1 x 10 ５ ~1 x 10 ６ When administered to humans, the daily dose of the first composition is not particularly limited and can be appropriately determined depending on the intended use. ５ ~1 x 10 ６ The number of times per day is, for example, 1 to 5 times, 1 to 3 times, or 1 or 2 times.

[0064] The administration form of the first composition of the present disclosure is not particularly limited. When the first composition of the present disclosure is administered in vivo, it may be administered parenterally. Examples of parenteral administration include intravenous injection (intravenous administration), intramuscular injection (intramuscular administration), transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, and topical administration.

[0065] The dosage form of the first composition of the present disclosure is not particularly limited and can be determined appropriately depending on, for example, the administration form. The dosage form may be, for example, a liquid or solid form.

[0066] The first composition of the present disclosure may contain, for example, an additive, if necessary. The additive is preferably a pharmaceutically acceptable additive or a pharmaceutically acceptable carrier.

[0067] The method for producing the first composition of the present disclosure (hereinafter also referred to as the "production method") may include a proliferative megakaryocyte induction step of inducing the proliferative megakaryocytes from cells less differentiated than megakaryocytes and / or a mature megakaryocyte induction step of inducing mature megakaryocytes from the proliferative megakaryocytes. Furthermore, the production method of the present disclosure may include, for example, a megakaryocyte induction step of inducing megakaryocytes from cells less differentiated than megakaryocytes, or an isolation step of isolating megakaryocytes and an induction step of imparting proliferative properties to the obtained megakaryocytes to induce proliferative megakaryocytes. Furthermore, the production method of the present disclosure may include, for example, a multinucleation step of multinucleating the proliferative megakaryocytes to induce multinucleated megakaryocytes prior to the mature megakaryocyte induction step.

[0068] In the megakaryocyte or proliferative megakaryocyte induction step, the method for inducing megakaryocytes or proliferative megakaryocytes is not particularly limited and can be performed using known induction methods. Specific examples of the megakaryocyte induction method include immortalized megakaryocyte induction methods such as those described in International Publication No. 2011 / 034073 (U.S. Patent Application Publication No. 2012 / 0238023), International Publication No. 2012 / 157586 (U.S. Patent Application Publication No. 2014 / 0127815), and International Publication No. 2014 / 123242 (U.S. Patent Application Publication No. 2016 / 0002599); and the megakaryocyte induction method described in Reference 5 below, which are incorporated herein by reference. Specific examples of the proliferative megakaryocyte induction step include forcibly expressing the oncogene and the polycomb gene in cells less differentiated than megakaryocytes. As a result, in the megakaryocyte induction step, for example, immortalized megakaryocytes that proliferate indefinitely, i.e., proliferative megakaryocytes, can be obtained. Reference 5: Ann-Kathrin Borger et al., "Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions," Mol. Med., 2016, vol. 22, pages 274-288

[0069] In the proliferative megakaryocyte induction step, for example, the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcedly expressed. In this case, the forced expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be performed simultaneously or separately. Specifically, in the megakaryocyte induction step, the oncogene and the polycomb gene may be forcedly expressed, followed by deactivating the forced expression and then forcibly expressing the apoptosis-inhibiting gene; the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcedly expressed; or the oncogene and the polycomb gene may be forcedly expressed, followed by further expression of the apoptosis-inhibiting gene. In this way, the immortalized megakaryocytes can be obtained in the megakaryocyte induction step. The proteins encoded by the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene, i.e., the proteins expressed by the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene, can also be referred to as growth factors for the proliferative megakaryocytes.

[0070] The proliferative megakaryocyte induction step preferably includes a first expression step of forcibly expressing an oncogene and a polycomb gene in cells less differentiated than megakaryocytes, and a second expression step of forcibly expressing an apoptosis-suppressing gene such as the BCL-XL gene in the undifferentiated cells, since this can improve the efficiency of introducing each gene, for example.

[0071] As described above, the multinucleation step can be carried out, for example, prior to the mature megakaryocyte induction step. The multinucleation step is not particularly limited and can be carried out by a known method. Specific examples of the method for multinucleating proliferative megakaryocytes include those disclosed in International Publication No. 2022 / 265117 (U.S. Patent Application Publication No. 2023 / 0399617), which are incorporated herein by reference.

[0072] The mature megakaryocyte induction step may involve, for example, canceling the forced expression of the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene, or may involve canceling all of the forced expression. By canceling the forced expression, the proliferating megakaryocytes can be induced to mature megakaryocytes, allowing platelets to be produced.

[0073] The forced expression and release of forced expression of each gene can be carried out by known methods, such as those described in International Publication Nos. 2011 / 034073, 2012 / 157586, and 2014 / 123242, or Reference 6 listed below, or by methods equivalent thereto, which are incorporated herein by reference. Specifically, the forced expression and release of forced expression of each gene can be carried out using, for example, a drug-responsive gene expression induction system. Examples of such gene expression induction systems include the Tet-on (registered trademark) system and the Tet-off (registered trademark) system. When the Tet-on system is used, for example, the forced expression step involves culturing in the presence of a drug that induces gene expression, such as tetracycline or doxycycline, and the release of forced expression involves culturing in the absence of the drug. Reference 6: Nakamura S et al, “Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells.”, Cell Stem Cell, 2014, vol.14, No.4, pages 535-548

[0074] The isolation of megakaryocytes can be performed with reference to, for example, Reference 7 below. Next, the induction step can be performed, for example, by contacting the megakaryocytes with the proliferation-inducing factor and / or a substance that induces expression of the endogenous proliferation-inducing gene. The contact can be performed, for example, by adding the proliferation-inducing factor and / or a substance that induces expression of the endogenous proliferation-inducing gene to a medium for the megakaryocytes while culturing the megakaryocytes. The culture conditions can be similar to the culture conditions used for megakaryocyte induction. Furthermore, the induction step can be performed, for example, by introducing the proliferation-inducing gene into the megakaryocytes. Specifically, the induction step can be performed in the same manner as the forced expression of a gene in the proliferative megakaryocyte induction step. Reference 7: Mott K and Schulze H, "Isolation, In Vitro Differentiation, and Culture of Murine Megakaryocytes From Fetal Liver and Adult Bone Marrow," Current Protocols, 3(5), e783.

[0075] The medium is not particularly limited and may be, for example, a known medium suitable for inducing megakaryocytes or a medium similar thereto. Specifically, the medium may be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Neurobasal® Medium (manufactured by Thermo Fisher Scientific), as well as a mixture thereof. The medium may contain, for example, serum or plasma, or may be a serum-free medium that does not contain these. The serum and plasma are preferably derived from the same source as the megakaryocytes. Specifically, when the megakaryocytes are derived from humans, the serum and plasma are preferably derived from humans.

[0076] The medium may contain, for example, other components. The other components are not particularly limited and include, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like. The other components may be, for example, one type or two or more types. Cytokines are substances that promote the differentiation of hematopoietic cells, and specific examples include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, ADAM inhibitors, FLT inhibitors, WNT inhibitors, ROCK inhibitors, and aryl hydrocarbon receptor (AhR) inhibitors. The medium is preferably an IMDM medium containing, for example, serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The medium may further contain, for example, SCF or heparin. The concentrations of the other components are not particularly limited. The TPO concentration is, for example, about 10 ng / ml to about 200 ng / ml, or about 50 ng / ml to about 100 ng / ml. The SCF concentration is, for example, about 10 ng / ml to about 200 ng / ml, or about 50 ng / ml. The heparin concentration is, for example, about 10 U / ml to about 100 U / ml, or about 25 U / ml. The medium may further contain, for example, a phorbol ester (e.g., phorbol-12-myristate-13-acetate; PMA).

[0077] The first composition of the present disclosure can be used, for example, as a composition for suppressing bone destruction caused by osteoclasts, a composition for suppressing the inhibition of bone formation by osteoclasts, and a composition for treating diseases caused by osteoclasts, as described below.The first composition of the present disclosure can, for example, suppress differentiation into osteoclasts, thereby suppressing bone destruction caused by osteoclasts, and therefore can be suitably used for treating diseases caused by osteoclasts.Examples of diseases caused by osteoclasts include osteoporosis, bone destruction in rheumatoid arthritis, osteopetrosis, etc. Furthermore, the first composition of the present disclosure is presumed to be suitable for use in treating, for example, jaw bone defects due to jaw bone tumors, cysts, osteonecrosis, trauma, bone system diseases (ectodermal dysplasia, cleft lip and palate, etc.); alveolar bone defects such as alveolar bone defects due to periodontal disease or associated alveolar bone defects, caries, or trauma; osteoporosis such as primary osteoporosis (postmenopausal osteoporosis, senile osteoporosis, etc.) and secondary osteoporosis (osteogenesis imperfecta, etc.); intractable fractures due to hyperthyroidism, Cushing's syndrome, hypogonadism), trauma, etc.; rib fixation due to thoracotomy; bone defects due to craniotomy; systemic bone defects due to tumor resection or cyst removal; etc. Therefore, the first composition of the present disclosure is expected to be suitable for use in, for example, patients with these diseases or subjects suspected of having these diseases, particularly patients with or suspected of having an increased number of osteoclasts, or patients with or suspected of having accelerated differentiation into osteoclasts. The suspected patient is, for example, a subject who has been diagnosed by a doctor as possibly having the condition.

[0078] <Composition for Suppressing Bone Destruction Caused by Osteoclasts> In another aspect, the present disclosure provides a composition capable of suppressing bone destruction caused by osteoclasts. The composition for use in suppressing bone destruction caused by osteoclasts of the present disclosure (hereinafter also referred to as the "second composition") comprises the first composition of the present disclosure, i.e., the proliferating megakaryocytes and / or the mature megakaryocytes. The second composition of the present disclosure can suppress differentiation into osteoclasts, particularly the differentiation of osteoclast precursor cells into osteoclasts, thereby suppressing bone destruction caused by the osteoclasts. The second composition of the present disclosure can, for example, suppress bone destruction caused by osteoclasts, and therefore indirectly suppress the inhibition of bone formation caused by the osteoclasts. Therefore, the second composition of the present disclosure can also be referred to as, for example, the composition of the present disclosure used for suppressing the inhibition of bone formation caused by osteoclasts.

[0079] The administration subject (subject) and use conditions (administration conditions) of the second composition of the present disclosure can be determined from the explanation of the first composition of the present disclosure.

[0080] The inhibitory effect on osteoclast-mediated bone destruction can be evaluated, for example, by determining whether the differentiation of the osteoclast precursor cells into osteoclasts can be inhibited when the osteoclast precursor cells are coexisting with a test substance, compared to the absence of the test substance (control group). Specifically, the inhibitory effect on osteoclast-mediated bone destruction can be evaluated using a pit formation assay or the like. The method for inducing the differentiation of osteoclast precursor cells into osteoclasts can be carried out with reference to Reference 4 mentioned above. In the evaluation, for example, when the proportion of cells differentiated into osteoclasts in the group coexisting with the test substance is 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, based on the proportion of cells differentiated into osteoclasts in the group in the absence of the test substance (100%), the test substance can be evaluated as having an inhibitory effect on bone destruction in osteoclasts.

[0081] <Composition for treating osteoclast-induced diseases> In another aspect, the present disclosure provides a composition that can be used to treat osteoclast-induced diseases. The composition for use in treating osteoclast-induced diseases of the present disclosure (hereinafter also referred to as the "third composition") comprises the first composition of the present disclosure, i.e., the proliferating megakaryocytes and / or the mature megakaryocytes. The third composition of the present disclosure can inhibit differentiation into osteoclasts, particularly the differentiation of osteoclast precursor cells into osteoclasts, thereby inhibiting bone destruction caused by the osteoclasts. Therefore, the third composition of the present disclosure is expected to be suitable for use in treating osteoclast-induced diseases, for example, because it can inhibit bone destruction caused by the osteoclasts.

[0082] The administration subject (subject) and use conditions (administration conditions) of the third composition of the present disclosure can be determined from the explanation of the first composition of the present disclosure.

[0083] <Bone Formation Kit> In another aspect, the present disclosure provides a kit for use in osteogenesis. The kit for use in osteogenesis of the present disclosure (hereinafter also referred to as a "first kit") includes the first composition of the present disclosure and / or the second composition of the present disclosure (hereinafter also referred to collectively as a "composition"), and an osteogenic scaffold material. By combining the composition of the present disclosure with the scaffold material, the first kit of the present disclosure can, for example, inhibit bone destruction by osteoclasts, thereby indirectly inducing osteogenesis.

[0084] In the first kit of the present disclosure, the scaffold material can be, for example, a scaffold material used for bone or cartilage regeneration. The scaffold material can also be a material to which, for example, osteocytes or osteoblasts, or their precursor cells, can attach, proliferate, and / or differentiate. The scaffold material is, for example, a structure (construct) having a three-dimensional hollow or porous structure, and specific examples include a porous body, a porous support, a porous three-dimensional structure (construct), a gel (hydrogel), a sponge structure, etc.

[0085] The scaffold material is preferably biocompatible and / or biodegradable, for example, since it is placed in a living body together with the composition. The "biocompatible" means, for example, that it has affinity with tissues and / or organs in a living body and does not cause foreign body reactions or rejection reactions. The "biodegradable" means, for example, that its constituent components can be decomposed in a living body.

[0086] Examples of components of the scaffold material include proteins such as collagen, gelatin, albumin, keratin, fibrin, and fibroin; polysaccharides such as agarose, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, and alginic acid; polyglycolic acid (PGA), polylactic acid (PLA), copolymers of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polyethylene glycol (PEG), polycaprolactone, polybutylene succinate, calcium phosphate (e.g., β-tricalcium phosphate), calcium carbonate, hydroxyapatite, polyether ketone, and polyether ether ketone. One type of the components may be used alone, or multiple types may be used in combination.

[0087] The scaffold material can be prepared, for example, by crosslinking or conjugating the components of the scaffold material.

[0088] Examples of the scaffolding material include Neobone (made of hydroxyapatite, manufactured by Aimdic MMT), APACERAM (registered trademark, made of hydroxyapatite, manufactured by HOYA Corporation), Bonarc (made of collagen / calcium octaphosphate, manufactured by Toyobo Co., Ltd.), Ospherion (made of βTCP, manufactured by Olympus Terumo Biomaterials Corporation), Cytotrans Granule (made of carbonated apatite, manufactured by GC Corporation), and Refit (made of collagen / apatite, manufactured by HOYA Corporation).

[0089] In the first kit of the present disclosure, the composition and the scaffold material may exist independently or integrally. In the latter case, the kit of the present disclosure can also be referred to as, for example, an osteogenic scaffold material. When the composition and the scaffold material are integrally formed, the composition may be, for example, adsorbed onto the surface of the scaffold material or may be contained within the scaffold material.

[0090] The first kit of the present disclosure can be used, for example, by transplanting (embedding, embedding) the composition and scaffold material into the desired location in the subject where bone formation is to be induced. Specifically, when the composition and the scaffold material are separate in the first kit of the present disclosure, the kit can be used by impregnating the scaffold material with the composition and then transplanting the scaffold material. Furthermore, when the composition and the scaffold material are separate in the first kit of the present disclosure, the kit can be used by implanting the scaffold material and then introducing the composition of the present disclosure into the implantation site. Furthermore, when the composition and the scaffold material are integrated in the first kit of the present disclosure, the kit can be used by implanting the scaffold material.

[0091] In the first kit of the present disclosure, the content of the composition can be set, for example, in accordance with the dosage of the composition of the present disclosure.

[0092] <Kit for suppressing bone destruction> In another aspect, the present disclosure provides a kit for use in suppressing bone destruction. The kit for use in suppressing bone destruction of the present disclosure (hereinafter also referred to as the "second kit") includes the composition of the present disclosure and an osteogenic scaffold material. By combining the composition of the present disclosure with the scaffold material, the second kit of the present disclosure can suppress the differentiation into osteoclasts, thereby suppressing bone destruction. The bone destruction can also be referred to as, for example, bone destruction caused by the osteoclasts.

[0093] <Composition for Use in Promoting Differentiation into Osteoblasts> In another aspect, the present disclosure provides a composition for use in promoting differentiation into osteoblasts. The composition for use in promoting differentiation into osteoblasts (hereinafter also referred to as the "fourth composition") of the present disclosure contains mature megakaryocytes. The fourth composition of the present disclosure can promote the differentiation of progenitor cells into osteoblasts, i.e., promote the differentiation of mesenchymal stem cells into osteoblasts. Therefore, the fourth composition of the present disclosure can induce bone formation, for example, by increasing the number of osteoblasts. A decrease in osteoblasts disrupts the balance between the functions of osteoblasts and osteoclasts, and osteoclast activity exceeds that of osteoblasts, resulting in osteoclast-related bone diseases. Therefore, by promoting differentiation into osteoblasts, the fourth composition is expected to be suitable for use in bone diseases such as osteoporosis, bone destruction in rheumatoid arthritis, and osteopetrosis. The description of the first composition can be used to describe the fourth composition of the present disclosure.

[0094] The fourth composition of the present disclosure is presumed to induce bone formation by promoting, increasing, increasing, and / or encouraging differentiation or differentiation induction into osteoblasts. Therefore, the fourth composition of the present disclosure can also be said to be a composition for use in promoting, increasing, increasing, and / or encouraging differentiation or differentiation induction into osteoblasts.

[0095] The physiological activity of the fourth composition of the present disclosure may include, for example, the activity of promoting differentiation of precursor cells into osteoblasts. The activity of promoting differentiation into osteoblasts may be, for example, an increase in the ability to differentiate into osteoblasts compared to a control group that is otherwise identical except that the fourth composition of the present disclosure is not added. The increase in differentiation ability may mean, for example, either an increase in the rate of differentiation into osteoblasts or an increase in the proportion of cells that differentiate into osteoblasts. The activity of promoting differentiation into osteoblasts can be evaluated, for example, by measuring the ALP activity per cell according to Example 1(6) described below.

[0096] <Composition for Promoting Bone Formation by Osteoblasts> In another aspect, the present disclosure provides a composition capable of promoting bone formation by osteoblasts. The composition for use in promoting bone formation by osteoblasts of the present disclosure (hereinafter also referred to as the "fifth composition") comprises the fourth composition of the present disclosure, i.e., the mature megakaryocytes. The fifth composition of the present disclosure can promote differentiation of progenitor cells into osteoblasts, particularly differentiation of mesenchymal stem cells into osteoblasts, thereby promoting bone formation by the osteoblasts. The descriptions of the first composition and the fourth composition can be used to explain the fifth composition of the present disclosure.

[0097] The administration subject (subject) and use conditions (administration conditions) of the fifth composition of the present disclosure can be referenced from the explanation of the first composition of the present disclosure.

[0098] The promotion of bone formation by osteoblasts can be evaluated, for example, by determining whether bone mass at the defect site is increased when a test article is coexisted with a partially defective bone compared to the absence of the test article (control group). Specifically, in the evaluation, the test article can be evaluated as having the effect of promoting bone formation by osteoblasts if the rate of increase in bone mass when a partially defective bone is coexisted with the test article is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the rate of increase in bone mass in a control group in which the partially defective bone is not coexisted with the test article.

[0099] <Composition for treating osteoblast-induced diseases> In another aspect, the present disclosure provides a composition usable for treating osteoblast-induced diseases. The composition for use in treating osteoblast-induced diseases of the present disclosure (hereinafter also referred to as the "sixth composition") comprises the fourth composition of the present disclosure, i.e., the mature megakaryocytes. The sixth composition of the present disclosure can promote differentiation of progenitor cells into osteoblasts, particularly differentiation of mesenchymal stem cells into osteoblasts, thereby promoting bone formation by the osteoblasts. Therefore, the sixth composition of the present disclosure is expected to be suitable for treating osteoblast-induced diseases, for example, because it can promote bone formation by the osteoblasts. The explanation of the sixth composition of the present disclosure can be incorporated into the explanations of the first composition and the fifth composition.

[0100] The administration subject (subject) and use conditions (administration conditions) of the sixth composition of the present disclosure can be determined from the explanation of the first composition of the present disclosure.

[0101] <Bone Formation Kit> In another aspect, the present disclosure provides a kit for use in bone formation. The kit for use in bone formation of the present disclosure (hereinafter also referred to as the "second kit") includes the fourth composition of the present disclosure and / or the fifth composition of the present disclosure (hereinafter collectively referred to as the "composition") and an osteogenic scaffold material. The second kit of the present disclosure can induce bone formation by, for example, osteoblasts by combining the composition of the present disclosure with the scaffold material. The description of the first composition, the fourth composition, the fifth composition, and the first kit of the present disclosure can be used to explain the second kit of the present disclosure.

[0102] <Method for inhibiting differentiation into osteoclasts> In another aspect, the present disclosure provides a method for inhibiting differentiation into osteoclasts. The method for inhibiting differentiation into osteoclasts of the present disclosure (hereinafter also referred to as the "differentiation-inhibiting method") uses the first composition of the present disclosure, i.e., the proliferating megakaryocytes and / or the mature megakaryocytes. The differentiation-inhibiting method of the present disclosure can inhibit differentiation into osteoclasts, particularly the differentiation of osteoclast precursor cells into osteoclasts.

[0103] The differentiation-inhibiting method of the present disclosure may be performed, for example, in vitro or in vivo. The differentiation-inhibiting method of the present disclosure includes, for example, an administration step of administering the differentiation-inhibiting composition of the present disclosure to a recipient. The recipient and administration conditions of the first composition of the present disclosure can be determined, for example, from the description of the recipient and administration conditions of the first composition of the present disclosure.

[0104] <Method for Promoting Differentiation into Osteoblasts> In another aspect, the present disclosure provides a method capable of promoting differentiation into osteoblasts. The method for promoting differentiation into osteoblasts (hereinafter also referred to as the "differentiation promotion method") of the present disclosure uses the fourth composition of the present disclosure, i.e., the mature megakaryocytes. The differentiation promotion method of the present disclosure can promote differentiation into osteoblasts, particularly the differentiation of mesenchymal stem cells into osteoblasts.

[0105] The differentiation-promoting method of the present disclosure may be performed, for example, in vitro or in vivo. The differentiation-promoting method of the present disclosure includes, for example, an administration step of administering the differentiation-promoting composition of the present disclosure to a recipient. The recipient and administration conditions of the fourth composition of the present disclosure can be determined, for example, from the description of the recipient and administration conditions of the first composition of the present disclosure.

[0106] <Method for inhibiting bone destruction caused by osteoclasts> In another aspect, the present disclosure provides a method for inhibiting bone destruction caused by osteoclasts. The method for inhibiting bone destruction caused by osteoclasts of the present disclosure (hereinafter also referred to as the "bone destruction inhibiting method") uses the second composition of the present disclosure, i.e., the proliferating megakaryocytes and / or the mature megakaryocytes. According to the method for inhibiting bone destruction of the present disclosure, differentiation into osteoclasts can be inhibited, thereby inhibiting bone destruction caused by osteoclasts. The method for inhibiting bone destruction of the present disclosure can, for example, inhibit bone destruction caused by osteoclasts, and therefore indirectly inhibit the inhibition of bone formation by osteoclasts. Therefore, the method for inhibiting bone destruction of the present disclosure can also be referred to as, for example, the method for inhibiting inhibition of bone formation caused by osteoclasts of the present disclosure.

[0107] The method for inhibiting bone destruction of the present disclosure may be carried out, for example, in vitro or in vivo. The method for inhibiting bone destruction of the present disclosure may, for example, include an administration step of administering the second composition of the present disclosure to a recipient. The recipient, administration conditions, and dosage form of the second composition of the present disclosure can be determined, for example, from the description of the recipient, administration conditions, and dosage form of the first composition of the present disclosure.

[0108] <Method for promoting bone formation by osteoblasts> In another aspect, the present disclosure provides a method for promoting bone formation by osteoblasts. The method for promoting bone formation by osteoblasts of the present disclosure (hereinafter also referred to as the "osteogenesis promoting method") uses the fifth composition of the present disclosure, i.e., the mature megakaryocytes. According to the method for promoting bone formation of the present disclosure, differentiation into osteoblasts can be promoted, thereby promoting bone formation by osteoblasts.

[0109] The method for promoting bone formation of the present disclosure may be carried out, for example, in vitro or in vivo. The method for promoting bone formation of the present disclosure includes, for example, an administration step of administering the fifth composition of the present disclosure to a recipient. The recipient, administration conditions, and dosage form of the fifth composition of the present disclosure can be determined, for example, from the descriptions of the recipient, administration conditions, and dosage form of the first composition of the present disclosure.

[0110] <Method for treating diseases caused by osteoclasts> In another aspect, the present disclosure provides a method for treating diseases caused by osteoclasts. The method for treating diseases caused by osteoclasts of the present disclosure (hereinafter also referred to as the "first treatment method") uses the third composition of the present disclosure, i.e., the proliferating megakaryocytes and / or the mature megakaryocytes. According to the first treatment method of the present disclosure, differentiation into osteoclasts, particularly differentiation from osteoclast precursor cells to osteoclasts, can be suppressed, thereby suppressing bone destruction caused by the osteoclasts. Therefore, the first treatment method of the present disclosure is expected to be suitable for treating diseases caused by osteoclasts, for example, because it can suppress bone destruction caused by the osteoclasts.

[0111] The first treatment method of the present disclosure may be performed, for example, in vitro or in vivo. The first treatment method of the present disclosure may, for example, include an administration step of administering the third composition of the present disclosure to a subject. The subject, administration conditions, and dosage form of the third composition of the present disclosure may be determined, for example, from the description of the subject, administration conditions, and dosage form of the first composition of the present disclosure.

[0112] <Method for treating diseases caused by osteoblasts> In another aspect, the present disclosure provides a method for treating diseases caused by osteoblasts. The method for treating diseases caused by osteoblasts of the present disclosure (hereinafter also referred to as the "second treatment method") uses the sixth composition of the present disclosure, i.e., the mature megakaryocytes. According to the second treatment method of the present disclosure, differentiation into osteoblasts, particularly differentiation from stem cells into osteoblasts, can be promoted, thereby promoting bone formation by the osteoblasts. Therefore, the second treatment method of the present disclosure is expected to be suitable for use in treating diseases caused by osteoblasts, for example, because it can promote bone formation by the osteoblasts.

[0113] The second treatment method of the present disclosure may be performed, for example, in vitro or in vivo. The second treatment method of the present disclosure may, for example, include an administration step of administering the sixth composition of the present disclosure to a subject. The subject, administration conditions, and dosage form of the sixth composition of the present disclosure may be determined, for example, from the description of the subject, administration conditions, and dosage form of the first composition of the present disclosure.

[0114] <Use> The present disclosure relates to a first composition of the present disclosure or use thereof for use in inhibiting osteoclast differentiation. The present disclosure relates to a second composition of the present disclosure or use thereof for use in inhibiting bone destruction by osteoclasts. The present disclosure relates to a second composition of the present disclosure or use thereof for use in inhibiting osteoclast-mediated bone formation inhibition. The present disclosure relates to a third composition of the present disclosure or use thereof for use in treating diseases caused by osteoclasts. The present disclosure relates to a fourth composition of the present disclosure or use thereof for use in promoting osteoblast differentiation. The present disclosure relates to a fifth composition of the present disclosure or use thereof for use in promoting osteoblast-mediated bone formation. The present disclosure relates to a sixth composition of the present disclosure or use thereof for use in treating diseases caused by osteoblasts. The present disclosure relates to the use of proliferating megakaryocytes and / or mature megakaryocytes for producing a composition for use in inhibiting osteoclast differentiation. The present disclosure also relates to the use of proliferating megakaryocytes and / or mature megakaryocytes for the manufacture of a composition for use in suppressing bone destruction by osteoclasts. The present disclosure also relates to the use of proliferating megakaryocytes and / or mature megakaryocytes for the manufacture of a composition for use in suppressing the inhibition of bone formation by osteoclasts. The present disclosure also relates to the use of proliferating megakaryocytes and / or mature megakaryocytes for the manufacture of a composition for use in treating diseases caused by osteoclasts. The present disclosure also relates to the use of mature megakaryocytes for the manufacture of a composition for use in promoting differentiation into osteoblasts. The present disclosure also relates to the use of mature megakaryocytes for the manufacture of a composition for use in promoting bone formation by osteoblasts. The present disclosure also relates to the use of mature megakaryocytes for the manufacture of a composition for use in treating diseases caused by osteoblasts.

[0115] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the aspects described in the examples.

[0116] Example 1 It was confirmed that immortalized megakaryocytes contribute to promoting differentiation of preosteoblasts into osteoblasts.

[0117] (1) Preparation of Immortalized Megakaryocytes Immortalized megakaryocytes were prepared by the following procedure.

[0118] (1-1) Preparation of Hematopoietic Progenitor Cells from iPS Cells Human iPS cells were cultured to differentiate into blood cells according to the method described in Reference 8 below. Specifically, human ES / iPS cell colonies maintained on laminin 511-E8 (Nippi) were cultured overnight in the presence of 50 ng / ml activin A (WAKO) and 3 μM CHIR99021 (WAKO), and then cocultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / ml VEGF (R&D SYSTEMS) for 14 days to generate hematopoietic progenitor cells (HPCs). The culture conditions for iPS cell maintenance and the first 7 days of HPC induction were 37°C, 5% O. ２ , 5% CO ２ and then at 37°C, 20% O ２ , 5% CO ２ (Unless otherwise specified, the same conditions apply hereinafter.) Reference 8: Takayama N. et al., "Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells", J. Exp. Med., 2010, vol.13, pages 2817-2830

[0119] (1-2) Gene Delivery System The gene delivery system utilized a lentiviral vector system. The lentiviral vector used was the Tetracycline-regulated Tet-on® gene expression induction system vector. The vectors were constructed as follows: c-MYC, BMI1, or BCL-XL vectors were introduced under the control of the TRE promoter. Each vector was constructed as follows: EN-TRE-c-Myc-Ubc-rtTA-KR, EN-TRE-BMI1-Ubc-rtTA-KR, and EN-TRE-BCL-XL-Ubc-rtTA-KR, respectively. Viruses containing the c-MYC, BMI1, and BCL-XL vectors were then prepared by transfecting the lentiviral vectors into 293T cells (see Reference 6, supra, and Reference 9, supra). The resulting viruses were then used to infect target cells, allowing the c-MYC, BMI1, and BCL-XL genes to be introduced into the genomic sequence of the target cells. Furthermore, when the c-MYC, BMI1, and BCL-XL genes are stably introduced into the genomic sequence of the resulting cells, the cells can be forced to express the c-MYC, BMI1, and BCL-XL genes by adding doxycycline (clontech#631311) to the culture medium. Reference 9: Yamaguchi et al., "Development of an All-in-One Inducible Lentiviral Vector for Gene Specific Analysis of Reprogramming." PLoS ONE, 2012, vol.7 (7) e41007

[0120] (1-3) Induction of megakaryocytic cell lines by introduction of three genes into hematopoietic progenitor cells. The HPCs prepared in (1-1) above were infected with the three lentiviruses prepared in (1-2) above to induce megakaryocytic progenitor cells. Approximately 25 days after infection, cells showing sufficient proliferation capacity were immunostained using anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibodies, and then analyzed using a FACS Verse™. Cells with CD41a and CD42b positivity rates of 50% or higher were designated as immortalized megakaryocytic cell lines (MKCL) and used for further studies.

[0121] (1-4) Growth and culture of megakaryocytic cell lines The resulting MKCLs were grown in 10 cm dishes (10 ml / dish) or 125 ml shake flasks. IMDM (Sigma Aldrich #I3390) was used as the base medium, to which the following components were added (concentrations are final concentrations in the growth medium). The culture conditions were 37°C, 5% CO ２ The shaking speed was set to 100 rpm using a shaker with a rotation diameter of 19 mm. Cells cultured under these conditions were collected as proliferative MK (Tet-on). FBS (Hyclone #SH30071.03, fetal bovine serum) 15%. Glutamax (GIBCO #3505-061) 2 mmol / L. ITS-G (Gibco #41400-045) 100x diluted. MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157) 450 μmol / L. Ascorbic acid (NIPRO #49871900040329) 50 μg / mL. SCF (Wako Pure Chemical #193-15513) 50 ng / mL. TPO (Fujifilm Wako Pure Chemical #207-17581) 50 ng / mL. Doxycycline (DOX) (clontech #631311) 1 μg / mL.

[0122] (2) Production of megakaryocyte cultures. Forced expression was reversed by culturing in a DOX-free medium. Specifically, the immortalized megakaryocyte cell line obtained by the method in (1) above was washed twice with PBS(-) and suspended in the platelet production medium described below. The cell seeding density was 1.0 × 10 5 After 2 days of culture, the immortalized megakaryocytic cell line was collected as mature MKs (mature megakaryocytes, Tet-off Day 2). Platelet-producing medium: 10% human AB serum (Access Biologicals), Glutamax (GIBCO #3505-061), 2 mmol / L ITS-G (Gibco #41400-045), 100x dilution, MTG (monothioglycerol, Fujifilm Wako Pure Chemical Industries, Ltd. #195-157), 450 μmol / L ascorbic acid (NIPRO #49871900040329), 50 μg / ml SCF (Wako Pure Chemical Industries, Ltd. #193-15513), 50 ng / ml TPO (Fujifilm Wako Pure Chemical Industries, Ltd. #207-17581), 50 ng / ml aryl hydrocarbon receptor (AhR) inhibitor GNF351 (Calbiochem #182707), 500 nmol / l ROCK inhibitor Y-39983 (Medchemexpress). #MCH-HY-13300-10) 500nmol / l Enoxaparin (Sanofi) 1U / mL

[0123] (3) Examination of proliferation of bone marrow-derived mesenchymal stem cells We examined whether immortalized megakaryocytes contribute to the proliferation of bone marrow-derived mesenchymal stem cells. Specifically, first, bone marrow-derived mesenchymal stem cells were cultured at 3.0 × 10 ５ The cells were seeded in a 6-well plate at 1.7 x 10 cells / well and cultured for 4 hours. In the culture, DMEM containing 10% FBS was used as the medium. After the culture, 1.7 x 10 megakaryocytes obtained in Example (1-6) or Example (2) were added to the plate. ５The cells were added to the medium at a ratio of 1 cell / well and co-cultured. The co-culture was performed using a 2% FBS-containing mixed medium (DMEM:IMDM = 1:1). The co-culture was performed in the presence (Tet-on [Dox(+)]) or absence (Tet-on [Dox(-)]) of doxycycline. When the immortalized megakaryocytes were cultured in the presence of doxycycline, they exhibited proliferation and became proliferative megakaryocytes (the same applies below). Cell counts were measured on days 0, 5, and 10, based on the start of the culture. On day 5, based on the start of the co-culture, 1 ml of medium was added. The cell count was calculated using a cell counting kit (WST8, Cell Counting Kit-8, Dojindo, Cat. No. 347-07621) according to the attached protocol. These results are shown in Figure 1.

[0124] Figure 1 is a graph showing cell counts. In Figure 1, the horizontal axis represents the number of days since the start of co-culture of bone marrow mesenchymal stem cells and megakaryocytes, and the vertical axis represents cell counts. As shown in Figure 1, it was found that bone marrow mesenchymal stem cells proliferated in the following order of megakaryocyte types: Tet-on [Dox(+)], Tet-on [Dox(-)], and Tet-off Day 2.

[0125] (4) Examination of various gene expression in co-culture of megakaryocytes and bone marrow-derived mesenchymal stem cells We examined whether co-culture of the immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to promoting the expression of genes involved in the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, first, bone marrow-derived mesenchymal stem cells were cultured at 3.0 × 10 ５ The cells were seeded in a 6-well plate at 1.7 × 10 cells / well and cultured for 4 days. In the culture, DMEM containing 10% FBS was used as the medium. After the culture, 1.7 × 10 megakaryocytes obtained in Example 1 (1-4) or Example 1 (2) were added to the plate. ５The cells were added at a cell / well ratio and co-cultured (Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group). The co-culture was performed using a 2% FBS-containing mixed medium (DMEM:IMDM = 1:1). The co-culture was performed in the presence (Tet-on [Dox(+)]) or absence (Tet-on [Dox(-)]) of doxycycline. Gene expression analysis was performed on days 0, 4 (the start of co-culture), 9, and 14, based on the start of the bone marrow-derived mesenchymal stem cell culture. After the culture, total RNA was recovered and purified from the cells using an RNA extraction kit (TRI Reagent, Cosmo Bio, Cat. No.: TR118). RNA was extracted according to the protocol provided with the kit. cDNA was synthesized from the purified RNA using a reverse transcription kit (Revertra Ace, Toyobo). The reverse transcription reaction was performed according to the protocol provided with the kit. Next, quantitative PCR was performed using the resulting cDNA and primer sets for the Runx2 gene, SP7 gene, OPG gene, RANKL gene, b-FGF gene, PDGF-bb gene, GDF15 gene, BMP4 gene, or GAPDH gene listed below, and a qPCR kit (Brilliant III Ultra-Fast SYBR QPCR MasterMix, Agilent, Cat. No.: 600882). The PCR reaction was heated at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, after which the resulting PCR product was dissociated. The qPCR was performed using a qPCR device (Aria Mx Real-time PCR system, Agilent, Cat. No.: G8830A). A control was performed in the same manner, except that the megakaryocytes were not added. From the obtained measurement data, the expression level of each gene was calculated by the ΔΔCt method, corrected by the expression level of the GAPDH gene, and further, the relative expression level was calculated by setting the expression level of the control at 1. These results are shown in Figures 2 to 6.

[0126] Primer set for Runx2 gene Forward primer (SEQ ID NO: 1) 5'-gcagttcccaagcatttcat-3' Reverse primer (SEQ ID NO: 2) 5'-cactctggctttgggaagag-3' Primer set for SP7 gene Forward primer (SEQ ID NO: 3) 5'-taatgggctcctttcacctg-3' Reverse primer (SEQ ID NO: 4) 5'-cactgggcagacagtcagaa-3' Primer set for OPG gene Forward primer (SEQ ID NO: 5) 5'-ggcaacacagctcacaagaa-3' Reverse primer (SEQ ID NO: 6) 5'-ctgggtttgcatgcctttat-3' Primer set for RANKL gene Forward primer (SEQ ID NO: 7) 5'-agagcgcagatggatcctaa-3' Reverse primer (SEQ ID NO: 8) 5'-ttccttttgcacagctcctt-3' Primer set for b-FGF gene Forward primer (SEQ ID NO: 9) 5'-agagcgaccctcacatcaag-3' Reverse primer (SEQ ID NO: 10) 5'-actgcccagttcgtttcagt-3' Primer set for PDGF-bb gene Forward primer (SEQ ID NO: 11) 5'-gggttgagggtacagggact-3' Reverse primer (SEQ ID NO: 12) 5'-tcgagtggtcactcagcatc-3' Primer set for GDF15 gene Forward primer (SEQ ID NO: 13) 5'-ctccagattccgagagttgc-3' Reverse primer (SEQ ID NO: 14) 5'-agagatacgcaggtgcaggt-3' Primer set for BMP4 gene Forward primer (SEQ ID NO: 15) 5'-tgatacctgagacggggaag-3' Reverse primer (SEQ ID NO: 16) 5'-ccagactgaagccggtaaag-3' Primer set for GAPDH gene Forward primer (SEQ ID NO: 17) 5'-gagtcaacggatttggtcgt-3'Reverse primer (SEQ ID NO: 18) 5'-ttgattttggagggatctcg-3'

[0127] Figure 2 is a graph showing the expression levels of each gene in bone marrow-derived mesenchymal stem cells. In Figure 2, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression levels of each gene. In Figure 2, (A) shows the gene expression level of Runx2, (B) shows the gene expression level of SP7, (C) shows the expression level of OPG, and (D) shows the gene expression level of RANKL. As shown in Figure 2(A), compared to the control group, the Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group showed increased gene expression of Runx2, a master transcription factor for bone development. As shown in Figure 2(B), compared to the control group, the Tet-on [Dox(+)] group and Tet-off Day 2 group co-cultured with the composition of the present disclosure showed increased gene expression of SP7, a transcription factor required for differentiation of preosteoblasts into osteoblasts. As shown in Figure 2(C), the gene expression level of OPG, a decoy receptor for the osteoclast differentiation factor RANKL, was increased in the Tet-on [Dox(-)] group compared to the control group. As shown in Figure 2(D), the gene expression level of RANKL, an osteoclast differentiation factor, was decreased in the Tet-on [Dox(+)] group and the Tet-off Day 2 group compared to the control group. These results demonstrated that the immortalized megakaryocytes can enhance the expression of genes involved in osteoblast differentiation. Furthermore, it was demonstrated that megakaryocytes from the Tet-on [Dox(+)] and Tet-off Day 2 groups of the present disclosure can suppress the expression of RANKL in bone marrow-derived mesenchymal stem cells, from which osteoblasts are derived.

[0128] Figure 3 is a graph showing the OPG / RANKL ratio. In Figure 3, the horizontal axis represents the type of sample, and the vertical axis represents the OPG / RANKL ratio. As shown in Figure 3, the OPG / RANKL ratio was found to be elevated in the Tet-on [Dox(+)] group compared to the control group. These results suggest that the Tet-on [Dox(+)] megakaryocytes of the present disclosure can indirectly suppress osteoclast precursor cell differentiation by increasing the gene expression level of OPG and decreasing the gene expression level of RANKL in bone marrow-derived mesenchymal stem cells.

[0129] Figure 4 is a graph showing the expression levels of each gene in megakaryocytes co-cultured with bone marrow-derived mesenchymal stem cells. In Figure 4, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression levels of each gene. In Figure 4, (A) indicates the gene expression level of b-FGF, and (B) indicates the gene expression level of PDGF-bb. As shown in Figure 4(A), compared to the start of co-culture, the expression level of bFGF, a gene that promotes the proliferation of pre-osteoblasts, increased in the Tet-on [Dox(-)] group on Day 14. As shown in Figure 4(B), compared to the start of co-culture, the expression level of PDGF-bb, a bone metabolism coupling factor, increased in the Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group on Days 9 and 14.

[0130] Figure 5 is a graph showing the expression levels of each gene in megakaryocytes co-cultured with bone marrow-derived mesenchymal stem cells. In Figure 5, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression levels of each gene. In Figure 5, (A) shows the gene expression level of GDF15, and (B) shows the gene expression level of BMP4. As shown in Figure 5(A), compared to the start of co-culture, the expression levels of GDF15, which is involved in bone formation, increased in the Tet-on [Dox(-)] group on Day 9 and Day 14 and the Tet-off Day 2 group. As shown in Figure 5(B), the expression levels of BMP4, which induces differentiation into osteoblasts, increased in the Tet-on [Dox(-)] group on Day 14 and the Tet-off Day 2 group on Day 9 and Day 14.

[0131] Figure 6 is a graph showing the expression levels of each gene in megakaryocytes cocultured with bone marrow-derived mesenchymal stem cells. In Figure 6, (A) shows the gene expression level of OPG, (B) shows the gene expression level of RANKL, and (C) shows the OPG / RANKL ratio. In Figures 6(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression level of each gene. In Figure 6(C), the horizontal axis indicates the type of sample, and the vertical axis indicates the OPG / RANKL ratio. As shown in Figure 6(A), compared to the start of coculture, the expression levels of OPG, a decoy receptor for the osteoclast differentiation factor RANKL, increased in the Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group on Days 9 and 14. As shown in Figure 6(C), the OPG / RANKL ratio was higher in the Tet-on [Dox(+)] group than in the Tet-on [Dox(-)] group and the Tet-off Day 2 group on both Day 9 and Day 14. These results suggest that the immortalized megakaryocytes can suppress the differentiation of osteoclast precursor cells, thereby promoting the induction of osteoblast differentiation.

[0132] (5) Examination of promotion of differentiation into osteoblasts in co-culture of immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells We examined whether co-culture of the immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to promotion of differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, first, bone marrow-derived mesenchymal stem cells were cultured at 3.0 × 10 ５ The cells were seeded in a 6-well plate at 1.7 × 10 cells / well and cultured for 4 days. In the culture, DMEM containing 10% FBS was used as the medium. After the culture, 1.7 × 10 megakaryocytes obtained in Example 1 (1-4) or Example 1 (2) were added to the plate. ５The cells were added to the co-culture medium at a ratio of 1 cell / well (Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group). A 2% FBS-containing mixed medium (DMEM:IMDM = 1:1) was used for the co-culture. ALP activity, an indicator of osteoblast differentiation, was measured on days 0, 9, and 14, based on the start date of the bone marrow-derived mesenchymal stem cell culture. The ALP activity was assessed by measuring the absorbance at 405 nm using p-nitrophenyl phosphate (SIGMAFAST™ p-nitrophenyl phosphate tablets, SIGMA, Cat. No. N1891). The ALP activity per 1 ng of total protein was calculated. These results are shown in Figure 7.

[0133] Figure 7 is a graph showing ALP activity. In Figure 7, the horizontal axis represents the type of sample, and the vertical axis represents ALP activity (absorbance OD405). As shown in Figure 7, ALP activity increased in the Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, and Tet-off Day 2 group compared with the control group. These results suggest that the immortalized megakaryocytes promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0134] (6) Examination of promotion of differentiation into osteoblasts in co-culture of megakaryocytes and bone marrow-derived mesenchymal stem cells We examined whether co-culture of immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to promotion of differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, first, bone marrow-derived mesenchymal stem cells were cultured at 1.0 × 10 ５ The cells were seeded in a 24-well plate at 100 cells / well and cultured for 3 days. The medium used for the culture was DMEM containing 10% FBS. After the culture, 1.7 × 10 ５The cells were added to the culture medium at a density of 100 cells / well and co-cultured. DGA was prepared by adding 10% FBS, 50 mg / ml ascorbic acid, 10 mmol / L β-glycerophosphate, and 100 nmol / L dexamethasone to DMEM (low glucose). The sample containing only DGA was designated the DGA group; the sample containing only Tet-on [Dox(+)] was designated the Tet-on group; the sample containing only Tet-off Day 2 was designated the Tet-off group; the sample containing both DGA and Tet-on [Dox(+)] was designated the Tet-on + DGA group; and the sample containing both DGA and Tet-off Day 2 was designated the Tet-off + DGA group. Cell counts were measured on days 0, 1, 3, 9, and 14, based on the start date of bone marrow-derived mesenchymal stem cell culture. Additionally, ALP activity, an indicator of osteoblast differentiation, was measured on days 0, 1, 3, 9, and 14, based on the start date of the culture of bone marrow-derived mesenchymal stem cells. The cell count was calculated using a cell counting kit (WST8, Cell Counting Kit-8, Dojindo, Cat. No. 347-07621) according to the attached protocol. The ALP activity was determined by measuring the absorbance at 405 nm using p-nitrophenyl phosphate (SIGMAFAST™ p-nitrophenyl phosphate tablets, SIGMA, Cat. No. N1891). The ALP activity per cell was then calculated. A control was performed in the same manner, except that neither DGA nor megakaryocytes were added. These results are shown in Figures 8 to 10.

[0135] Figure 8 is a graph showing cell number and ALP activity. In Figure 8, (A) to (C) show the results when megakaryocytes were co-cultured with Tet-on [Dox(+)] (Tet-on group), and (D) to (F) show the results when megakaryocytes were co-cultured with Tet-off Day 2 (Tet-off group). In Figures 8(A) and (D), the horizontal axis indicates the sample type and the number of days from the start of culture, and the vertical axis indicates the cell number. In Figures 8(B) and (E), the horizontal axis indicates the number of days from the start of culture and the sample type, and the vertical axis indicates ALP activity (absorbance OD405). In Figures 8(C) and (F), the horizontal axis indicates the sample type and the number of days from the start of culture, and the vertical axis indicates ALP activity per cell (ALP activity / cell). As shown in Figure 8, the Tet-on and Tet-off groups showed increased cell numbers compared to the control and DGA groups. Furthermore, ALP activity increased in the DGA and Tet-off groups compared to the control group, whereas ALP activity was suppressed in the Tet-on group. These results suggest that megakaryocytes from the Tet-on [Dox(+)] group inhibited the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts, while megakaryocytes from the Tet-off Day 2 group promoted the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts.

[0136] Figure 9 is a graph showing cell number and ALP activity. In Figure 9, (A) to (C) show the results when megakaryocytes with Tet-on [Dox(+)] were co-cultured (Tet-on group), and (D) to (F) show the results when DGA and Tet-on [Dox(+)] were co-cultured (Tet-on + DGA group). In Figures 9(A) and (D), the horizontal axis indicates the sample type and the number of days from the start of culture, and the vertical axis indicates the cell number. In Figures 9(B) and (E), the horizontal axis indicates the sample type and the number of days from the start of culture, and the vertical axis indicates the ALP activity (absorbance OD405). In Figures 9(C) and (F), the horizontal axis indicates the sample type and the number of days from the start of culture, and the vertical axis indicates the ALP activity per cell (ALP activity / cell). As shown in Figure 9, compared with the control and DGA groups, the Tet-on group showed an increase in cell number, whereas the Tet-on + DGA group showed a suppressed increase in cell number. Furthermore, compared with the control group, the Tet-on group showed a suppression of ALP activity, whereas the Tet-on + DGA group showed an increase in ALP activity.

[0137] Figure 10 is a graph showing cell number and ALP activity. In Figure 10, (A) to (C) show the results when megakaryocytes on Tet-off Day 2 were used for co-culture (Tet-off group), and (D) to (F) show the results when DGA and Tet-off Day 2 were used for co-culture (Tet-off + DGA group). In Figures 10(A) and (D), the horizontal axis shows the sample type and the number of days from the start of culture, and the vertical axis shows the cell number. In Figures 10(B) and (E), the horizontal axis shows the sample type and the number of days from the start of culture, and the vertical axis shows the ALP activity (absorbance OD405). In Figures 10(C) and (F), the horizontal axis shows the sample type and the number of days from the start of culture, and the vertical axis shows the ALP activity per cell (ALP activity / cell). As shown in Figure 10, an increase in cells was observed in the Tet-off group compared to the control group and DGA group, whereas no increase in cell number was observed in the Tet-off + DGA group. Furthermore, ALP activity was increased in the DGA group, Tet-off group, and Tet-off + DGA group compared to the control group. Furthermore, on Day 9, ALP activity was increased in the Tet-off + DGA group compared to the Tet-off group.

[0138] (7) Examination of Gene Expression in Coculture of Megakaryocytes and Bone Marrow-Derived Mesenchymal Stem Cells We investigated whether coculture of the immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to the promotion of expression of genes involved in the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, the same procedures as in Example 1(4) were used, except that DGA was added to the coculture, Tet-on [Dox(+)] (Tet-on group) or Tet-off Day 2 (Tet-off group) was used for megakaryocytes, and gene expression analysis was performed on days 1, 3, 9, or 14, relative to the start of bone marrow-derived mesenchymal stem cell culture. These results are shown in Figures 11 and 12.

[0139] FIG. 11 is a graph showing the expression level of each gene. In FIG. 11, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression level of each gene. In FIG. 11, (A) and (B) indicate the gene expression level of Runx2, and (C) and (D) indicate the gene expression level of SP7. As shown in FIGS. 11(A) and (C), on Day 9, the gene expression levels of Runx2 and SP7 were increased in the Tet-on + DGA group compared to the control group and the Tet-on group. Furthermore, on Day 14, the gene expression levels of Runx2 and SP7 were increased in the Tet-on + DGA group compared to the control group, the DGA group, and the Tet-on group. As shown in FIG. 11(B), on Day 9 and Day 14, the expression level of Runx2 gene was increased in the Tet-off+DGA group compared to the control group, DGA group, and Tet-off group.

[0140] FIG. 12 is a graph showing the expression level of each gene. In FIG. 12, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression level of each gene. In FIG. 12, (A) and (B) indicate the gene expression level of OPG, and (C) and (D) indicate the gene expression level of RANKL. As shown in FIG. 12(A), on Day 9 and Day 14, the gene expression level of OPG was increased in the Tet-on + DGA group compared to the control group, DGA group, and Tet-on group. As shown in FIG. 12(B), on Day 9 and Day 14, the gene expression level of OPG was increased in the Tet-off + DGA group compared to the control group and Tet-off group. Furthermore, on Day 9, the gene expression level of OPG was increased in the Tet-off + DGA group compared to the DGA group.

[0141] (8) In Vivo Study of Bone Formation and Bone Augmentation at Skull Defect Sites We investigated whether the immortalized megakaryocytes contribute to bone formation and bone augmentation at skull defect sites in vivo. Specifically, first, a sample was prepared using the megakaryocytes (Tet-on [Dox(+)]) obtained in Example 1 (1-4). The cell suspension containing the megakaryocytes was centrifuged at 300 × g for 5 minutes. After the centrifugation, the megakaryocytes were suspended in physiological saline to prepare a preparation solution. After the preparation, 1.0 × 10 ６ A cell-equivalent preparation solution was added to an artificial bone material (octacalcium phosphate (OCP) / Collagen (Col), Bonarc (registered trademark)). Next, a 4 mm defect was created in the skull of a nude rat (11 weeks old) under general anesthesia. The artificial bone material was transplanted into the defect site and onto the skull other than the defect site (Example). For the negative control group, transplantation was carried out in the same manner, except that the artificial bone material was impregnated with PBS. Four weeks after the transplantation, the artificial bone material, including the surrounding skull, was extracted, and the extracted area was subjected to μCT imaging. These results are shown in Figure 13.

[0142] Figure 13 shows photographs showing bone formation 4 weeks after transplantation. In Figure 13, (A) is a photograph showing the results of the negative control group, and (B) is a photograph showing the results of the example group. In Figure 13(B), the upper row is a photograph showing the results of a sagittal image of the defect site in the example, and the lower row is a photograph showing the results of a skull image of the defect site in the example. As shown in Figure 13, immortalized megakaryocytes were able to form new bone at the skull defect site. Furthermore, as shown in Figure 13, based on the thickness of the newly formed bone, new bone formation occurred not in the center of the defect site but on the periphery of the defect site and progressed toward the center. Although not shown in the figure, when the megakaryocyte extract was used as a composition for promoting bone formation, it was confirmed that bone formation occurred throughout the defect site, and bone formation was promoted throughout the defect site by promoting osteoblast differentiation. On the other hand, since bone formation occurs from the peripheral side of the defect, it was presumed that the activity of osteoclasts present in the bone tissue at the peripheral area is suppressed, resulting in the dominance of bone formation by osteoblasts. Therefore, it was thought that the immortalized megakaryocytes indirectly promote bone formation by osteoblasts by suppressing the differentiation of the osteoclast precursor cells into osteoclasts. It was also presumed that the immortalized megakaryocytes contribute to the proliferation of osteoblasts.

[0143] Example 2 It was confirmed that mature megakaryocytes induce bone formation and bone augmentation in a cranial defect site in vivo.

[0144] We investigated whether mature megakaryocytes contribute to bone formation and bone augmentation in a cranial defect site in vivo. Specifically, first, samples were prepared using mature MKs (Tet-off Day 2) obtained in Example 1(2). The cell suspension containing megakaryocytes was centrifuged at 300 × g for 5 minutes. After the centrifugation, the megakaryocytes were suspended in physiological saline to prepare a preparation solution. After the preparation, 4.0 × 10 ５A cell-equivalent preparation solution was added to an artificial bone material (octacalcium phosphate (OCP) / Collagen (Col), Bonarc (registered trademark)). Next, a 4 mm defect was created in the skull of a nude rat (11 weeks old) under general anesthesia. The artificial bone material was transplanted into the defect site and onto the skull other than the defect site (Example 2). For the negative control group, transplantation was carried out in the same manner, except that the artificial bone material was impregnated with PBS. Four weeks after the transplantation, the artificial bone material including the surrounding skull was extracted, and the extracted area was photographed with μCT. In addition, the extracted area was subjected to histological observation using HE staining. Furthermore, the bone mass of the newly formed bone from the transplant material was evaluated. In the evaluation, bone mass BV (cm) was measured using 3D bone analysis software (TRI / 3D-BON; manufactured by Ratoc System Engineering). 3 The results are shown in Figures 14 and 15.

[0145] Figure 14 is a set of photographs showing bone formation four weeks after transplantation. (A) is a photograph showing the results of the negative control group, and (B) is a photograph showing the results of the Example 2 group. In Figure 14, the left column shows the results of skull surface images of the defect site, and the right column shows the results of HE staining. As shown in Figure 14, mature megakaryocytes were able to newly form bone at the defect site of the skull.

[0146] Next, Figure 15 is a graph showing bone volume 4 weeks after transplantation. In Figure 15, the horizontal axis indicates the type of experimental group, and the vertical axis indicates bone volume (cm ３ 15 , compared to the negative control group, Example 2 showed an increase in bone mass. From the above, it was found that the mature megakaryocytes of the present disclosure can newly form bone at the defect site of the skull.

[0147] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0148] This application claims priority based on Japanese Patent Application No. 2024-024967, filed February 21, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0149] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendices. <Compositions for use in inhibiting osteoclast differentiation> (Appendix 1) A composition for use in inhibiting osteoclast differentiation, comprising proliferating megakaryocytes and / or mature megakaryocytes. (Appendix 2) The composition according to Appendix 1, wherein the proliferating megakaryocytes and / or mature megakaryocytes are megakaryocytes containing an exogenous oncogene, polycomb gene, and / or apoptosis inhibitor gene. (Appendix 3) The composition according to Appendix 1 or 2, wherein the proliferating megakaryocytes are megakaryocytes in which expression of an oncogene, polycomb gene, and / or apoptosis inhibitor gene has been enhanced. (Appendix 4) The composition according to any of Appendices 1 to 3, wherein the proliferating megakaryocytes are immortalized megakaryocytes. (Appendix 5) The composition according to any of Appendices 1 to 4, wherein the mature megakaryocytes are megakaryocytes in which expression of an oncogene, polycomb gene, and / or apoptosis inhibitor gene has been inhibited. (Appendix 6) The composition according to any one of Appendices 1 to 5, wherein the mature megakaryocytes are derived from the proliferative megakaryocytes. (Appendix 7) The composition according to any one of Appendices 1 to 6, wherein the mature megakaryocytes are derived from immortalized megakaryocytes. (Appendix 8) The composition according to any one of Appendices 2 to 7, wherein the oncogene is the MYC gene, the polycomb gene is the BMI gene, and / or the apoptosis-inhibiting gene is the BCL-XL gene. (Appendix 9) The composition according to any one of Appendices 1 to 8, wherein the proliferative megakaryocytes and / or the mature megakaryocytes are megakaryocytes induced in vitro. (Appendix 10) The composition according to any one of Appendices 1 to 9, wherein the proliferative megakaryocytes and / or the mature megakaryocytes are derived from pluripotent cells. (Appendix 11) The composition according to any one of Appendices 1 to 10, which inhibits differentiation of osteoclast precursor cells into osteoclasts. <Composition for use in suppressing inhibition of bone formation by osteoclasts> (Appendix 12) A composition for use in suppressing inhibition of bone formation by osteoclasts, comprising the composition for use in suppressing differentiation into osteoclasts described in any one of Appendices 1 to 11.<Composition for use in treating diseases caused by osteoclasts> (Appendix 13) A composition for use in treating diseases caused by osteoclasts, comprising the composition for use in inhibiting osteoclast differentiation according to any one of Appendices 1 to 11 and / or the composition for use in inhibiting osteoclast-mediated bone formation inhibition according to Appendix 12. <Kit for use in bone formation> (Appendix 14) A kit for use in bone formation, comprising the composition for use in inhibiting osteoclast differentiation according to any one of Appendices 1 to 11 and / or the composition for use in inhibiting osteoclast-mediated bone formation inhibition according to Appendix 12, and a scaffold for bone formation. <Kit for use in inhibiting bone destruction> (Appendix 15) A kit for use in inhibiting bone destruction, comprising the composition for use in inhibiting osteoclast differentiation according to any one of Appendices 1 to 11 and / or the composition for use in inhibiting osteoclast-mediated bone formation inhibition according to Appendix 12, and a scaffold for bone formation. <Composition for use in promoting differentiation into osteoblasts> (Appendix 16) A composition for use in promoting differentiation into osteoblasts, comprising mature megakaryocytes. (Appendix 17) The composition according to Appendix 16, wherein the mature megakaryocytes are megakaryocytes containing an exogenous oncogene, polycomb gene, and / or apoptosis inhibitor gene. (Appendix 18) The composition according to Appendix 16 or 17, wherein the mature megakaryocytes are megakaryocytes in which expression of an oncogene, polycomb gene, and / or apoptosis inhibitor gene is suppressed. (Appendix 19) The composition according to any of Appendixes 16 to 18, wherein the mature megakaryocytes are derived from proliferative megakaryocytes. (Appendix 20) The composition according to Appendix 19, wherein the proliferative megakaryocytes are megakaryocytes in which expression of an oncogene, polycomb gene, and / or apoptosis inhibitor gene is enhanced. (Appendix 21) The composition according to Appendix 19 or 20, wherein the proliferative megakaryocytes are derived from immortalized megakaryocytes. (Appendix 22) The composition according to any one of Appendices 17 to 21, wherein the oncogene is the MYC gene, the polycomb gene is the BMI gene, and / or the apoptosis-inhibiting gene is the BCL-XL gene. (Appendix 23) The composition according to any one of Appendices 16 to 22, wherein the mature megakaryocytes are megakaryocytes induced in vitro.(Appendix 24) The composition according to any one of Appendices 16 to 23, wherein the mature megakaryocytes are derived from pluripotent cells. (Appendix 25) The composition according to any one of Appendices 16 to 24, which promotes differentiation of mesenchymal stem cells into osteoblasts. <Composition for use in promoting bone formation by osteoblasts> (Appendix 26) A composition for use in promoting bone formation by osteoblasts, comprising the composition for use in promoting differentiation into osteoblasts according to any one of Appendices 16 to 25. <Composition for use in treating diseases caused by osteoblasts> (Appendix 27) A composition for use in treating diseases caused by osteoblasts, comprising the composition for use in promoting differentiation into osteoblasts according to any one of Appendices 16 to 25 and / or the composition for use in promoting bone formation by osteoblasts according to Appendix 26. <Kit for use in bone formation> (Appendix 28) A kit for use in bone formation, comprising the composition for use in promoting osteoblast differentiation according to any one of Appendices 16 to 25 and / or the composition for use in promoting bone formation by osteoblasts according to Appendices 26, and a scaffold for bone formation. <Method for inhibiting osteoclast differentiation> (Appendix 29) A method for inhibiting osteoclast differentiation, using the composition for use in inhibiting osteoclast differentiation according to any one of Appendices 1 to 15. (Appendix 30) The inhibiting method according to Appendices 29, comprising an administration step of administering the composition for use in inhibiting osteoclast differentiation to a subject. (Appendix 31) The inhibiting method according to Appendices 29 or 30, used in vitro or in vivo. <Method for inhibiting osteoclast-mediated bone formation inhibition> (Appendix 32) A method for inhibiting osteoclast-mediated bone formation inhibition, using the composition for use in inhibiting osteoclast-mediated bone formation inhibition according to Appendices 12. (Appendix 33) The suppression method according to Appendix 32, comprising an administration step of administering to a subject a composition for use in suppressing the inhibition of bone formation by osteoclasts. (Appendix 34) The suppression method according to Appendix 32 or 33, which is used in vitro or in vivo. <Method for promoting differentiation into osteoblasts> (Appendix 35) A method for promoting differentiation into osteoblasts, using a composition for use in promoting differentiation into osteoblasts according to any of Appendices 16 to 25. (Appendix 36) The promotion method according to Appendix 35, comprising an administration step of administering to a subject the composition for use in promoting differentiation into osteoblasts.(Appendix 37) The method for promoting bone formation according to Appendix 35 or 36, which is used in vitro or in vivo. <Method for promoting bone formation> (Appendix 38) A method for promoting bone formation by osteoblasts, using the composition for use in promoting bone formation by osteoblasts according to Appendix 26. (Appendix 39) The method for promoting bone formation according to Appendix 38, comprising an administration step of administering to a subject the composition for use in promoting bone formation by osteoblasts. (Appendix 40) The method for promoting bone formation according to Appendix 38 or 39, which is used in vitro or in vivo. <Method for treating diseases caused by osteoblasts> (Appendix 41) A method for treating diseases caused by osteoblasts, using the composition for use in promoting differentiation into osteoblasts according to any of Appendixes 16 to 25, the composition for use in promoting bone formation by osteoblasts according to Appendix 26, and / or the composition for use in treating diseases caused by osteoblasts according to Appendix 27. (Appendix 42) A method of treatment according to Appendix 41, comprising an administration step of administering the composition to a subject. (Appendix 43) A method of treatment according to Appendix 41 or 42, used in vitro or in vivo. <Use> (Appendix 44) A composition for use in promoting differentiation into osteoblasts according to any of Appendixes 16 to 25, for use in promoting differentiation into osteoblasts. (Appendix 45) A composition for use in promoting differentiation into osteoblasts according to any of Appendixes 16 to 25, for use in promoting bone formation by osteoblasts. (Appendix 46) A composition for use in promoting differentiation into osteoblasts according to any of Appendixes 16 to 25, for use in a method of treating a disease caused by osteoblasts.

[0150] As described above, the present disclosure provides a composition that can indirectly induce osteogenesis by, for example, inhibiting differentiation into osteoclasts and / or promoting differentiation into osteoblasts, and is therefore extremely useful in the fields of medicine, regenerative medicine, and the like.

Claims

1. A composition for use in inhibiting differentiation of proliferating megakaryocytes and / or mature megakaryocytes into osteoclasts.

2. The composition according to claim 1, wherein the proliferating megakaryocytes and / or the mature megakaryocytes are megakaryocytes containing an exogenous oncogene, polycomb gene, and / or apoptosis-inhibiting gene.

3. The composition according to claim 1 or 2, wherein the proliferative megakaryocytes are megakaryocytes in which expression of oncogenes, polycomb genes, and / or apoptosis-suppressing genes is enhanced.

4. The composition according to any one of claims 1 to 3, wherein the proliferative megakaryocytes are immortalized megakaryocytes.

5. The composition according to any one of claims 1 to 4, wherein the mature megakaryocytes are megakaryocytes in which the expression of oncogenes, polycomb genes, and / or apoptosis-suppressing genes is suppressed.

6. The composition of any one of claims 1 to 5, wherein the mature megakaryocytes are derived from the proliferative megakaryocytes.

7. The composition of any one of claims 1 to 6, wherein the mature megakaryocytes are derived from immortalized megakaryocytes.

8. The composition of any one of claims 2 to 7, wherein the oncogene is the MYC gene, the polycomb gene is the BMI gene, and / or the apoptosis-inhibiting gene is the BCL-XL gene.

9. The composition according to any one of claims 1 to 8, wherein the proliferative megakaryocytes and / or the mature megakaryocytes are megakaryocytes induced in vitro.

10. The composition of any one of claims 1 to 9, wherein the proliferating megakaryocytes and / or the mature megakaryocytes are derived from pluripotent cells.

11. A composition described in any one of claims 1 to 10, which inhibits the differentiation of osteoclast precursor cells into osteoclasts.

12. A composition for use in suppressing inhibition of bone formation by osteoclasts, comprising the composition for use in suppressing differentiation into osteoclasts described in any one of claims 1 to 11.

13. A composition for use in treating a disease caused by osteoclasts, comprising a composition for use in inhibiting differentiation into osteoclasts as described in any one of claims 1 to 11 and / or a composition for use in inhibiting inhibition of bone formation by osteoclasts as described in claim 12.

14. A kit for use in bone formation, comprising a composition for use in inhibiting differentiation into osteoclasts as described in any one of claims 1 to 11 and / or a composition for use in inhibiting inhibition of bone formation by osteoclasts as described in claim 12, and a scaffold material for bone formation.

15. A kit for use in inhibiting bone destruction, comprising a composition for use in inhibiting differentiation into osteoclasts as described in any one of claims 1 to 11 and / or a composition for use in inhibiting inhibition of bone formation by osteoclasts as described in claim 12, and a scaffold material for bone formation.

16. A composition for use in promoting differentiation of mature megakaryocytes into osteoblasts.

17. The composition according to claim 16, wherein the mature megakaryocytes are megakaryocytes containing an exogenous oncogene, polycomb gene, and / or apoptosis-inhibiting gene.

18. The composition according to claim 16 or 17, wherein the mature megakaryocytes are megakaryocytes in which the expression of oncogenes, polycomb genes, and / or apoptosis-suppressing genes is suppressed.

19. The composition of any one of claims 16 to 18, wherein the mature megakaryocytes are derived from proliferative megakaryocytes.

20. The composition of claim 19, wherein the proliferative megakaryocytes are megakaryocytes in which expression of oncogenes, polycomb genes, and / or apoptosis-inhibiting genes is enhanced.

21. The composition of claim 19 or 20, wherein the proliferative megakaryocytes are derived from immortalized megakaryocytes.

22. The composition of any one of claims 17 to 21, wherein the oncogene is the MYC gene, the polycomb gene is the BMI gene, and / or the apoptosis-inhibiting gene is the BCL-XL gene.

23. The composition of any one of claims 16 to 22, wherein the mature megakaryocytes are megakaryocytes induced in vitro.

24. The composition of any one of claims 16 to 23, wherein the mature megakaryocytes are derived from pluripotent cells.

25. A composition described in any one of claims 16 to 24, which promotes differentiation of mesenchymal stem cells into osteoblasts.

26. A composition for use in promoting bone formation by osteoblasts, comprising the composition for use in promoting differentiation into osteoblasts according to any one of claims 16 to 25.

27. A composition for use in treating a disease caused by osteoblasts, comprising a composition for use in promoting differentiation into osteoblasts as described in any one of claims 16 to 25 and / or a composition for use in promoting bone formation by osteoblasts as described in claim 26.

28. A kit for use in bone formation, comprising a composition for use in promoting differentiation into osteoblasts according to any one of claims 16 to 25 and / or a composition for use in promoting bone formation by osteoblasts according to claim 26, and a scaffold material for bone formation.

29. A method for inhibiting differentiation into osteoclasts, comprising using a composition for use in inhibiting differentiation into osteoclasts described in any one of claims 1 to 15.

30. The method for suppression according to claim 29, comprising the step of administering to a subject a composition for use in inhibiting differentiation into osteoclasts.

31. The method of suppression according to claim 29 or 30, which is used in vitro or in vivo.

32. A method for suppressing the inhibition of bone formation by osteoclasts, comprising using the composition for use in suppressing the inhibition of bone formation by osteoclasts described in claim 12.

33. The method for suppression according to claim 32, comprising the step of administering to a subject a composition for use in suppressing the inhibition of bone formation by osteoclasts.

34. The method of suppression according to claim 32 or 33, which is used in vitro or in vivo.

35. A method for promoting differentiation into osteoblasts, comprising using a composition for use in promoting differentiation into osteoblasts as defined in any one of claims 16 to 25.

36. The method for promoting differentiation into osteoblasts according to claim 35, comprising the step of administering to a subject a composition for use in promoting differentiation into osteoblasts.

37. The promotion method according to claim 35 or 36, which is used in vitro or in vivo.

38. A method for promoting bone formation by osteoblasts, comprising using the composition for use in promoting bone formation by osteoblasts described in claim 26.

39. The method of promoting bone formation according to claim 38, comprising the step of administering to a subject a composition for use in promoting bone formation by osteoblasts.

40. The promotion method according to claim 38 or 39, which is used in vitro or in vivo.

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