WO2025178117A1 - Composition for use in suppressing differentiation into osteoclasts - Google Patents

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

The present disclosure relates to a composition for use in inhibiting osteoclast differentiation.

Attempts are being made to develop cell preparations containing cells that promote tissue regeneration, such as mesenchymal stem cells, as therapeutic agents for regenerative medicine. Furthermore, it is believed that these cells promote tissue regeneration by releasing physiologically active proteins, such as growth factors.

Furthermore, in regenerative medicine in the dental field, platelet-rich plasma is used in bone augmentation treatments and other procedures. However, because platelet-rich plasma is prepared by separating and centrifuging isolated human blood, the platelet count is not standardized, and its efficacy is unknown.

The present disclosure therefore aims to provide a composition that can indirectly induce bone formation, for example, by inhibiting differentiation into osteoclasts and/or promoting differentiation into osteoblasts.

To achieve the above objective, the composition disclosed herein for use in inhibiting osteoclast differentiation contains proliferating megakaryocytes and/or mature megakaryocytes.

The compositions disclosed herein for use in suppressing the inhibition of bone formation by osteoclasts include the compositions disclosed herein for use in suppressing differentiation into osteoclasts.

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.

The kit for use in bone formation of the present disclosure includes a composition for use in inhibiting differentiation into osteoclasts of the present disclosure and/or a composition for use in inhibiting inhibition of bone formation by osteoclasts of the present disclosure, and a scaffold material for bone formation.

The kit for use in inhibiting bone destruction of the present disclosure includes a composition for use in inhibiting differentiation into osteoclasts of the present disclosure and/or a composition for use in inhibiting inhibition of bone formation by osteoclasts of the present disclosure, and a scaffold material for bone formation.

The composition disclosed herein for use in promoting differentiation into osteoblasts contains mature megakaryocytes.

The composition disclosed herein for use in promoting bone formation by osteoblasts includes the composition disclosed herein for use in promoting differentiation into osteoblasts.

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.

The kit for use in bone formation of the present disclosure includes the composition for use in promoting differentiation into osteoblasts of the present disclosure and/or the composition for use in promoting bone formation by osteoblasts of the present disclosure, and a scaffold material for bone formation.

The disclosed method for inhibiting osteoclast differentiation uses the disclosed composition for inhibiting osteoclast differentiation.

The disclosed method for suppressing the inhibition of bone formation by osteoclasts uses the disclosed composition for use in suppressing the inhibition of bone formation by osteoclasts.

The disclosed method for promoting differentiation into osteoblasts uses the disclosed composition for promoting differentiation into osteoblasts.

The disclosed method for promoting bone formation by osteoblasts uses the disclosed composition for promoting bone formation by osteoblasts.

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

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 number and ALP activity. FIG. 9 is a graph showing cell number and ALP activity. FIG. 10 is a graph showing cell number 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.

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

As used herein, "osteoclast precursor cells" refer 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, which is expressed by osteoblasts. The osteoclast precursor cells can be identified, for example, by markers. Examples of such markers include RANK and C-FMS (CD115).

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

In this specification, "osteogenesis" can mean the increase in existing bone tissue, i.e., "bone augmentation" (bone formation), the formation of new bone, i.e., "neosteogenesis," or the restoration of lost (defective) bone or parts thereof, i.e., "bone regeneration."

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. Cells with equivalent functions refer to cells that have the ability to produce platelets. Megakaryocytes can be identified, for example, by markers. Examples of such markers include the megakaryocyte markers described below.

As used herein, "proliferative" means that the target cells or cell population are in the proliferation phase. The "proliferative" can be evaluated using 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.

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. 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.

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.

As used herein, "oncogene" refers to a gene capable of inducing canceration of cells in the body, and examples 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).

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

As used herein, "apoptosis-inhibiting gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples 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).

As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., a composition, a base material such as a scaffold material, instructions, etc.) are provided in two or more compartments. The kit is suitable for providing compositions that, for stability reasons, are not provided in a mixed state, but are preferably mixed immediately before use. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., a composition, a base material such as a scaffold material, etc.), or instructions or instructions describing how to process the components. When the kit is used as a treatment kit in the present specification, the kit may also include instructions or instructions describing how to use the composition, scaffold material, etc. for treatment.

As used herein, "instructions" or "instructions" refer to written 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 the regulatory authority 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 that regulatory authority. The instructions may be a package insert, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, email, etc.).

As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and is used to particularly include 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.

　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.

The present disclosure will now be described in detail using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

<Composition for use in inhibiting differentiation into osteoclasts>
In one aspect, the present disclosure provides a composition capable of suppressing osteoclast differentiation. The composition for use in suppressing 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 suppress osteoclast differentiation, i.e., suppress 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.

As a result of extensive research, the inventors discovered that megakaryocytes in the proliferative phase, i.e., proliferative megakaryocytes and/or mature megakaryocytes, may suppress the activity of osteoclasts. Further research led the inventors to discover that the proliferative megakaryocytes and/or mature megakaryocytes suppress the differentiation of osteoclast precursors into osteoclasts, thereby suppressing bone destruction by osteoclasts and promoting bone formation, leading to the establishment of the present disclosure. The inventors have also discovered 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 proliferative megakaryocytes and/or mature megakaryocytes. This presumption does not in any way limit the present disclosure.

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 also be referred to as a composition for use in suppressing, reducing, lowering, and/or inhibiting differentiation or differentiation induction into osteoclasts, or as 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 referred to as a composition for suppressing the inhibition of the induction, promotion, acceleration (induction promotion), enhancement, potentiation, and/or strengthening of bone formation due to the inhibition of bone destruction by osteoclasts, for example.

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 sets, and specifically ranges from 16 to 32 sets.

The origin of the megakaryocytes is not particularly limited, and examples include humans and non-human animals. Examples of 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 the subject to which the first composition of the present disclosure is administered.

In the present disclosure, the megakaryocytes can be identified by cell surface markers. When the megakaryocytes are of human origin, 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 of human origin, 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.

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 that have the ability to differentiate into megakaryocytes.

When the megakaryocytes are isolated from a living organism, the megakaryocytes are present in bone marrow, for example, and can therefore be isolated from bone marrow. In this case, the megakaryocytes may contain other cells derived from the living organism.

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.

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 proliferative properties can be imparted, 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 can be induced, for example, by imparting the proliferative properties. When proliferating, the megakaryocytes 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.

Examples of megakaryocyte proliferation-inducing factors include thrombopoietin (TPO), stem cell factor (SCF), IL-6, IL-11, IL-3, etc.

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, polycomb genes, C-KIT, C-MPL, etc. The megakaryocyte proliferation-inducing gene is preferably used in combination with the apoptosis-inhibiting gene, since it can suppress cell death of the resulting proliferative megakaryocytes, for example.

The proliferative megakaryocytes are, for example, megakaryocytes in which expression of the oncogene, the polycomb gene, and/or the apoptosis-inhibiting gene is enhanced, preferably megakaryocytes in which expression of the oncogene and/or the polycomb gene and the apoptosis-inhibiting gene is enhanced, and more preferably megakaryocytes in which expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene is enhanced. The phrase "enhanced gene expression" means, for example, that expression of the target gene is higher compared to non-proliferative megakaryocytes, such as megakaryocytes isolated from a living body before multinucleation. The gene expression can be evaluated, for example, based on 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 the non-proliferative megakaryocytes (reference (1)).

When the proliferative megakaryocytes are proliferative megakaryocytes induced from precursor cells, the proliferative megakaryocytes are preferably immortalized megakaryocytes. Compared to megakaryocytes induced by other megakaryocyte induction methods, the immortalized megakaryocytes have a high degree of homogeneity in the differentiation stage of the cells, and are therefore suitable for use as the active ingredient of 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 precursor cells, as described below.

The immortalized megakaryocytes are preferably megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes. The term "exogenous" refers to genes introduced into the cell from outside the cell. The exogenous genes may be present on the chromosomes of the cell, or in the nucleus or cytoplasm. The exogenous genes can be detected, for example, by measuring the number of genes. If the genes are autosomal, one gene is present on each autosome, meaning that two genes exist in one cell. Therefore, if the exogenous genes are not present, two genes will be detected in one cell. On the other hand, if the exogenous genes are present, three or more genes will be detected in one cell. In this case, the exogenous genes can be detected using, for example, PCR using primers, probes, or a combination thereof. If the exogenous genes have a tag sequence or a selection marker, the exogenous genes 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.

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 proliferation has ceased. The mature megakaryocytes may be, for example, those obtained by multinucleating the proliferative megakaryocytes after proliferation has ceased to form multinucleated megakaryocytes, and then differentiating them into the mature megakaryocytes. Furthermore, the proliferative megakaryocytes acquire the ability to produce platelets 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, and more preferably megakaryocytes in which the expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene is suppressed. The phrase "suppressed gene expression" means, for example, that the expression of a target gene is lower than that of the proliferative megakaryocytes. The gene expression can be evaluated, for example, by quantitative PCR. In the mature megakaryocytes, the expression level of the target gene is, for example, 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, relative to the expression level of the target gene in the proliferative megakaryocytes (100%).

The number of chromosome sets (nuclear phase: N) possessed by the multinucleated megakaryocytes may be more than two 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).

The mature megakaryocytes are preferably derived from the immortalized megakaryocytes.

The physiological activities of the first composition of the present disclosure include, for example, the activity of promoting the proliferation of mesenchymal stem cells and the 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.

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.

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. In this case, the "proliferation-promoting activity" may also be referred to as, for example, suppression of a decrease in proliferation activity. As a specific example, the proliferation activity of 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.

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 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 Remodelling and Associated Disorders. Front Bioeng Biotechnol. 2018 Oct 11;6:134. doi: 10.3389/fbioe.2018.00134. PMID: 30364287; PMCID: PMC6193121.

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

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.

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.

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

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 osteoclasts, or 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 subsequently improved. The seeding density of proliferating megakaryocytes and/or mature megakaryocytes in the maintenance medium is, for example, 5 x ¹⁰ to 5 x ¹⁰ cells/cm, or 5 ^{x 10 to} 5 x ¹⁰ cells/cm ^. The seeding density of the proliferating megakaryocytes and/or mature megakaryocytes in the differentiation medium is, for example, 5×10 ³ to 5×10 ⁷ cells/cm ² , or 5×10 ⁴ to 5×10 ⁶ cells/cm ² .

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. Specifically, 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, and specific examples include 1×10 ⁵ to 1×10 ⁶ cells. When administered to humans, the daily dosage of the first composition is not particularly limited and can be appropriately set depending on the intended use, and specific examples include 1×10 ⁵ to 1×10 ⁶ cells. The number of administrations per day is, for example, 1 to 5 times, 1 to 3 times, or 1 or 2 times.

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.

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. Examples of the dosage form include liquid and solid forms.

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

The manufacturing method for the first composition of the present disclosure (hereinafter also referred to as the "manufacturing 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 proliferative megakaryocytes. Furthermore, the manufacturing 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 inducing proliferative megakaryocytes by imparting proliferative properties to the obtained megakaryocytes. Furthermore, the manufacturing method of the present disclosure may include, for example, a multinucleation step of inducing multinucleated megakaryocytes by multinucleating the proliferative megakaryocytes prior to the mature megakaryocyte induction step.

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, that is, 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

In the proliferative megakaryocyte induction step, for example, the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcibly expressed. In this case, the forcible expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be carried out simultaneously or separately. Specifically, in the megakaryocyte induction step, the forcible expression of the oncogene and the polycomb gene may be followed by the release of the forcible expression and then the apoptosis-inhibiting gene; the forcible expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be carried out; or the forcible expression of the oncogene and the polycomb gene may be followed by the 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 from the oncogene, the polycomb gene, and/or the apoptosis-inhibiting gene, can also be referred to as growth factors for the proliferative megakaryocytes.

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-inhibiting gene, such as the BCL-XL gene, in the undifferentiated cells, since this can improve the efficiency of gene introduction.

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 known methods. Specific examples of methods 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.

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 canceling all of the forced expression. By canceling the forced expression, the proliferating megakaryocytes can be induced to become mature megakaryocytes, allowing platelets to be produced.

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

The isolation of megakaryocytes can be carried out, for example, with reference to Reference 7 below. Next, the induction step can be carried out, 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 carried out, 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 carried out, for example, by introducing the proliferation-inducing gene into the megakaryocytes, and specifically, can be carried out 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.

The medium is not particularly limited, and examples thereof include known media suitable for inducing megakaryocytes and media equivalent thereto. Specific examples of the medium include a basal medium used for culturing animal cells. 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, RPMI1640 medium, Fischer's medium, and Neurobasal® Medium (manufactured by Thermo Fisher Scientific), as well as mixtures 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. As a specific example, when the megakaryocytes are derived from a human, the serum and plasma are preferably also derived from a human.

The medium may contain, for example, other components. The other components are not particularly limited, and examples include 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, etc. The other components may be, for example, one type, or two or more types. The 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 aromatic hydrocarbon receptor (AhR) inhibitors. The medium is preferably IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The medium may further contain 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).

The first composition of the present disclosure can be used, for example, as described below, as a composition for use in inhibiting bone destruction by osteoclasts, a composition for use in inhibiting the inhibition of bone formation by osteoclasts, and a composition for use in treating diseases caused by osteoclasts. The first composition of the present disclosure can, for example, inhibit differentiation into osteoclasts, thereby inhibiting bone destruction by osteoclasts, and therefore can be suitably used in 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 in question.

<Composition for inhibiting bone destruction by osteoclasts>
In another aspect, the present disclosure provides a composition capable of suppressing bone destruction by osteoclasts. The composition for use in suppressing bone destruction 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 by osteoclasts. The second composition of the present disclosure can, for example, suppress bone destruction by osteoclasts, and therefore indirectly suppress the inhibition of bone formation by 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 by osteoclasts.

The subject (subject) and conditions of use (administration conditions) of the second composition of the present disclosure can be referenced from the description of the first composition of the present disclosure.

The inhibitory effect on bone destruction by osteoclasts 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 coexisted with the test substance, compared to the absence of the test substance (control group). Specifically, the inhibitory effect on bone destruction by osteoclasts can be evaluated using a pit formation assay, etc. The method for inducing the differentiation of the osteoclast precursor cells into osteoclasts can be carried out by referring to Reference 4 mentioned above. In the evaluation, for example, if 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.

<Composition for treating diseases caused by osteoclasts>
In another aspect, the present disclosure provides a composition that can be used to treat diseases caused by osteoclasts. The composition for use in treating diseases caused by osteoclasts 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 diseases caused by osteoclasts, for example, because it can inhibit bone destruction caused by the osteoclasts.

The administration target (subject) and use conditions (administration conditions) of the third composition of the present disclosure can be referenced from the explanation of the first composition of the present disclosure.

<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.

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 described as a material to which, for example, bone cells 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.

Since the scaffold material is placed in the body together with the composition, it is preferable that it exhibits biocompatibility and/or biodegradability. The term "biocompatibility" means, for example, that it has affinity with tissues and/or organs in the body and does not cause foreign body reactions or rejection reactions. The term "biodegradable" means, for example, that its constituent components are decomposable in the body.

The components of the scaffold material include, for example, 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 of the components may be used alone, or multiple components may be used in combination.

The scaffold material can be prepared, for example, by crosslinking or compounding the components of the scaffold material.

Examples of the scaffolding material include Neobone (hydroxyapatite, manufactured by Aimdic MMT), APACERAM (registered trademark, hydroxyapatite, manufactured by HOYA Corporation), Bonarc (collagen/calcium octaphosphate, manufactured by Toyobo Co., Ltd.), Ospherion (β-TCP, manufactured by Olympus Terumo Biomaterials Corporation), Cytotrans Granule (carbonate apatite, manufactured by GC Corporation), and Refit (collagen/apatite, manufactured by HOYA Corporation).

In the first kit of the present disclosure, the composition and the scaffold material may exist independently or together. 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 together, the composition may be, for example, adsorbed onto the surface of the scaffold material or may be contained within the scaffold material.

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, in the first kit of the present disclosure, if the composition and the scaffold material are separate, the kit can be used by impregnating the scaffold material with the composition and then transplanting the scaffold material. Furthermore, in the first kit of the present disclosure, if the composition and the scaffold material are separate, the kit can be used by implanting the scaffold material and then introducing the composition of the present disclosure into the implantation site. Furthermore, in the first kit of the present disclosure, if the composition and the scaffold material are integrated, the kit can be used by implanting the scaffold material.

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.

<Bone destruction suppression and formation kit>
In another aspect, the present disclosure provides a kit for use in inhibiting bone destruction. The kit for use in inhibiting bone destruction of the present disclosure (hereinafter also referred to as a "second kit") includes a 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 inhibit differentiation into osteoclasts, thereby inhibiting bone destruction. The bone destruction can also be referred to as bone destruction caused by osteoclasts, for example.

<Composition for use in promoting differentiation into osteoblasts>
In another aspect, the present disclosure provides a composition for use in promoting osteoblast differentiation. The composition for use in promoting osteoblast differentiation of the present disclosure (hereinafter also referred to as the "fourth composition") 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 osteoblast differentiation, 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.

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.

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 identical except for the addition of the fourth composition of the present disclosure. 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 in accordance with Example 1(6) described below.

<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.

The administration target (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.

The promotion of bone formation by osteoblasts can be evaluated, for example, by determining whether bone mass at the defect site increases when a test article is placed in the presence of 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 placed in the presence of 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 placed in the presence of the test article.

<Composition for treating diseases caused by osteoblasts>
In another aspect, the present disclosure provides a composition usable for treating diseases caused by osteoblasts. The composition for use in treating diseases caused by osteoblasts (hereinafter also referred to as the "sixth composition") of the present disclosure 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 diseases caused by osteoblasts, for example, because it can promote bone formation by the osteoblasts. The descriptions of the first composition and the fifth composition can be used to describe the sixth composition of the present disclosure.

The administration target (subject) and use conditions (administration conditions) of the sixth composition of the present disclosure can be referenced from the explanation of the first composition of the present disclosure.

<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 the "second kit") includes the fourth composition of the present disclosure and/or the fifth composition of the present disclosure (hereinafter also referred to collectively 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 second kit of the present disclosure can be incorporated by reference into the descriptions of the first composition, the fourth composition, the fifth composition, and the first kit of the present disclosure.

<Method for inhibiting differentiation into osteoclasts>
In another aspect, the present disclosure provides a method for inhibiting osteoclast differentiation. The osteoclast differentiation inhibiting method 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 osteoclast differentiation, particularly the differentiation of osteoclast precursor cells into osteoclasts.

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 may, for example, include 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.

<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.

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 may, for example, include 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.

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

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 may be determined, for example, from the description of the recipient, administration conditions, and dosage form of the first composition of the present disclosure.

<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.

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 description of the recipient, administration conditions, and dosage form of the first composition of the present disclosure.

<Method for treating diseases caused by osteoclasts>
In another aspect, the present disclosure provides a method for treating a disease caused by osteoclasts. The method for treating a disease 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.

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.

<Method for treating diseases caused by osteoblasts>
In another aspect, the present disclosure provides a method for treating a disease caused by osteoblasts. The method for treating a disease 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.

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.

<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 osteoclast-mediated bone destruction. 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 producing a composition for use in inhibiting osteoclast-mediated bone destruction. The present disclosure 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 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 relates to the use of mature megakaryocytes for the manufacture of a composition for use in promoting differentiation into osteoblasts. Also, the present disclosure relates to the use of mature megakaryocytes for the manufacture of a composition for use in promoting bone formation by osteoblasts. The present disclosure relates to the use of mature megakaryocytes for the manufacture of a composition for use in treating diseases caused by osteoblasts.

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

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

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

(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 iPS cell maintenance and the first 7 days of HPC _induction were cultured at 37°C, 5% _O , and 5% CO , and the subsequent culture conditions were ₃₇ °C, 20% _O , and 5% CO (unless otherwise specified).
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, vo.13, pages 2817-2830

(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

(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.

(1-4) Megakaryocytic Cell Line Growth Culture The resulting MKCL 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). Culture conditions were 37°C, 5% _CO , and the shaking speed was set at 100 rpm using a shaker with a rotation diameter of 19 mm. Cells cultured under these conditions were collected as growth-phase MK (Tet-on).

FBS (Hyclone #SH30071.03, fetal bovine serum) 15%
Glutamax (GIBCO #3505-061) 2mmol/l
ITS-G (Gibco #41400-045) 100x dilution
MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157) 450 μmol/l
Ascorbic acid (NIPRO #49871900040329) 50μg/ml
SCF (Wako Pure Chemical #193-15513) 50ng/ml
TPO (Fujifilm Wako Pure Chemical #207-17581) 50ng/ml
Doxycycline (DOX) (clontech #631311) 1μg/ml

(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 cells/ml. After two days of culture, the immortalized megakaryocyte cell line was collected as mature MKs (mature megakaryocytes, Tet-off Day 2).
(Platelet-producing medium)
Human AB serum 10% (AccessBiologicals)
Glutamax (GIBCO #3505-061) 2mmol/L
ITS-G (Gibco #41400-045) 100x dilution
MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157) 450 μmol/l
Ascorbic acid (NIPRO #49871900040329) 50μg/ml
SCF (Wako Pure Chemical #193-15513) 50ng/ml
TPO (Fujifilm Wako Pure Chemical #207-17581) 50ng/ml
Aromatic hydrocarbon receptor (AhR) inhibitor GNF351 (Calbiochem #182707) 500nmol/l
ROCK inhibitor Y-39983 (Medchemexpress #MCH-HY-13300-10) 500nmol/l
Enoxaparin (Sanofi) 1 U/mL

(3) Examination of the proliferation of bone marrow-derived mesenchymal stem cells. We investigated whether immortalized megakaryocytes contribute to the proliferation of bone marrow-derived mesenchymal stem cells. Specifically, bone marrow-derived mesenchymal stem cells were first seeded into a 6-well plate at 3.0 × 10 ⁵ cells/well and cultured for 4 hours. The culture medium used was DMEM containing 10% FBS. After the culture, megakaryocytes obtained in Examples (1-6) or (2) were added to the plate at 1.7 × 10 ⁵ cells/well for co-culture. The co-culture medium used was a 2% FBS-containing mix (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 (hereinafter the same). Cell counts were measured on days 0, 5, and 10, based on the start of the co-culture. Furthermore, 1 ml of medium was added on day 5, based on the start of the co-culture. The cell counts were 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.

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

(4) Examination of Gene Expression in Coculture of Megakaryocytes and Bone Marrow-Derived Mesenchymal Stem Cells We investigated whether coculture of 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, bone marrow-derived mesenchymal stem cells were seeded into 6-well plates at 3.0 × ¹⁰ cells/well and cultured for 4 days. The culture medium used was DMEM containing 10% FBS. After the culture, megakaryocytes obtained in Example 1 (1-4) or Example 1 (2) were added to the coculture medium at 1.7 × ¹⁰ cells/well (Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, Tet-off Day 2 group). The coculture medium used was 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, or 14, based on the start of the culture of bone marrow-derived mesenchymal stem cells. 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, as described below, and a qPCR kit (Brilliant III Ultra-Fast SYBR QPCR MasterMix, Agilent, Cat. No.: 600882). The PCR reaction was performed by heating 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.

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'

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

Figure 3 is a graph showing the OPG/RANKL ratio. In Figure 3, the horizontal axis indicates the type of sample, and the vertical axis indicates 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 the differentiation of osteoclast precursor cells by increasing the gene expression level of OPG and decreasing the gene expression level of RANKL in bone marrow-derived mesenchymal stem cells.

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.

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) indicates the gene expression level of GDF15, and (B) indicates the gene expression level of BMP4. As shown in Figure 5(A), compared to the start of co-culture, the expression level 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 level 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.

Figure 6 is a graph showing the expression level of each gene in megakaryocytes co-cultured 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 shows the type of sample, and the vertical axis shows the relative expression level of each gene. In Figure 6(C), the horizontal axis shows the type of sample, and the vertical axis shows the OPG/RANKL ratio. As shown in Figure 6(A), compared to the start of co-culture, the expression level 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 on both Day 9 and Day 14 compared to the Tet-on [Dox(-)] group and the Tet-off Day 2 group. These results suggest that the immortalized megakaryocytes are able to suppress the differentiation of osteoclast precursor cells, thereby promoting the induction of osteoblast differentiation.

(5) Study on Promotion of Osteoblast Differentiation in Co-Culture of Immortalized Megakaryocytes and Bone Marrow-Derived Mesenchymal Stem Cells. We investigated whether co-culture of immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, bone marrow-derived mesenchymal stem cells were seeded into 6-well plates at 3.0 × ¹⁰ cells/well and cultured for 4 days. The culture medium used was DMEM containing 10% FBS. After the culture, megakaryocytes obtained in Example 1(1-4) or Example 1(2) were added to the plates at 1.7 × ¹⁰ cells/well for co-culture (Tet-on [Dox(+)] group, Tet-on [Dox(-)] group, Tet-off Day 2 group). The co-culture medium used was a 2% FBS-containing mixed medium (DMEM:IMDM = 1:1). ALP activity, an indicator of osteoblast differentiation, was measured on days 0, 9, and 14, based on the start date of the culture of bone marrow-derived mesenchymal stem cells. The ALP activity was examined by measuring the absorbance at 405 nm using p-nitrophenyl phosphate (SIGMAFAST™ p-nitrophenyl phosphate tablets, SIGMA, Cat. No. N1891). The ALP activity value per 1 ng of total protein was then calculated. These results are shown in Figure 7.

Figure 7 is a graph showing ALP activity. In Figure 7, the horizontal axis indicates the type of sample, and the vertical axis indicates 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 to the control group. These results suggest that the immortalized megakaryocytes promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

(6) Examination of Promotion of Osteoblast Differentiation in Co-Culture of Megakaryocytes and Bone Marrow-Derived Mesenchymal Stem Cells We investigated whether co-culture of immortalized megakaryocytes and bone marrow-derived mesenchymal stem cells contributes to promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Specifically, bone marrow-derived mesenchymal stem cells were seeded into 24-well plates at 1.0 × ¹⁰ cells/well and cultured for 3 days. The culture medium used was DMEM containing 10% FBS. After the culture, osteoblast differentiation-inducing medium (DGA) and/or megakaryocytes (Tet-on [Dox(+)], Tet-off Day 2) obtained in Example 1 (1-4) or Example 1 (2) above were added to the plates at 1.7 × ¹⁰ cells/well, and co-culture was continued. The 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). Samples containing only DGA were designated the DGA group; samples containing only Tet-on [Dox(+)] were designated the Tet-on group; samples containing only Tet-off Day 2 were designated the Tet-off group; samples containing both DGA and Tet-on [Dox(+)] were designated the Tet-on + DGA group; and samples containing both DGA and Tet-off Day 2 were 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.

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

Figure 9 is a graph showing cell number and ALP activity. In Figure 9, (A) to (C) show the results when Tet-on [Dox(+)] megakaryocytes were used in co-culture (Tet-on group), and (D) to (F) show the results when DGA and Tet-on [Dox(+)] were used in co-culture (Tet-on + DGA group). In Figures 9 (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 9 (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 ALP activity (absorbance OD405). In Figures 9 (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 ALP activity per cell (ALP activity/cell). As shown in Figure 9, compared to the control and DGA groups, the Tet-on group showed an increase in cell number, while the Tet-on + DGA group showed a suppressed increase in cell number. Furthermore, compared to the control group, the Tet-on group showed a suppressed ALP activity, while the Tet-on + DGA group showed an increase in ALP activity.

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 in co-culture (Tet-off group), and (D) to (F) show the results when DGA and Tet-off Day 2 were used in 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 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 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, compared to the control group, ALP activity was increased in the DGA group, Tet-off group, and Tet-off + DGA group. Furthermore, on Day 9, ALP activity was increased in the Tet-off + DGA group compared to the Tet-off group.

(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.

Figure 11 is a graph showing the expression level of each gene. In Figure 11, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression level of each gene. In Figure 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 Figure 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 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, DGA group, and Tet-on group. As shown in Figure 11(B), on Day 9 and Day 14, the Tet-off + DGA group showed increased Runx2 gene expression levels compared to the control group, DGA group, and Tet-off group.

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

(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, samples were first prepared using 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 centrifugation, the megakaryocytes were suspended in physiological saline to prepare a preparation solution. After the preparation, an amount of the preparation solution equivalent to 1.0 × ¹⁰ cells was added to an artificial bone material (octacalcium phosphate (OCP)/Collagen (Col), Bonarc®). 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). The negative control group was transplanted 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 by μCT. The results are shown in Figure 13.

Figure 13 is a photograph showing bone formation four 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 top row is a photograph showing the results of a sagittal image of the defect site in the example, and the bottom 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 in 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 to promote bone formation, it was confirmed that bone formation occurred throughout the entire defect site, and bone formation was promoted throughout the entire 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 immortalized megakaryocytes suppress the activity of osteoclasts present in the bone tissue in the peripheral area, resulting in 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.

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

We investigated whether mature megakaryocytes contribute to bone formation and bone augmentation in a skull defect in vivo . Specifically, samples were first 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 centrifugation, the megakaryocytes were suspended in physiological saline to prepare a preparation solution. After the preparation, an amount of the preparation solution equivalent to 4.0 × ¹⁰ cells was added to an artificial bone material (octacalcium phosphate (OCP)/Collagen (Col), Bonarc®). 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 the area of the skull other than the defect site (Example 2). The negative control group was implanted in the same manner, except that the artificial bone material was impregnated with PBS. Four weeks after implantation, the artificial bone material, including the surrounding skull, was removed, and the resulting excised area was subjected to μCT imaging. Histological observation of the excised area was also performed using HE staining. Furthermore, the bone mass of the newly formed bone from the implanted material was evaluated. For the evaluation, bone mass BV ( ^cm3 ) was measured using 3D bone analysis software (TRI/3D-BON; manufactured by Ratoc System Engineering). These results are shown in Figures 14 and 15.

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 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 form new bone in the skull defect site.

Next, Figure 15 is a graph showing bone volume four weeks after transplantation. In Figure 15, the horizontal axis indicates the type of experimental group, and the vertical axis indicates bone volume (cm ³ ). As shown in Figure 15, compared to the negative control group, Example 2 showed an increase in bone volume. 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.

The present disclosure has been described above with reference to embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various modifications that would be understandable to a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

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.

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

As described above, the present disclosure provides a composition that can indirectly induce bone formation, for example, by inhibiting differentiation into osteoclasts and/or promoting differentiation into osteoblasts. For this reason, the present disclosure is extremely useful in the fields of medicine, regenerative medicine, and the like.

Claims (40)

A composition for use in inhibiting differentiation into osteoclasts, comprising proliferating megakaryocytes and/or mature megakaryocytes. 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. The composition described in claim 1 or 2, wherein the proliferative megakaryocytes are megakaryocytes in which expression of oncogenes, polycomb genes, and/or apoptosis-suppressing genes is enhanced. The composition described in any one of claims 1 to 3, wherein the proliferative megakaryocytes are immortalized megakaryocytes. The composition described in any one of claims 1 to 4, wherein the mature megakaryocytes are megakaryocytes in which expression of oncogenes, polycomb genes, and/or apoptosis-inhibiting genes is suppressed. The composition described in any one of claims 1 to 5, wherein the mature megakaryocytes are derived from the proliferative megakaryocytes. The composition described in any one of claims 1 to 6, wherein the mature megakaryocytes are derived from immortalized megakaryocytes. The oncogene is the MYC gene.
The composition according to any one of claims 2 to 7, wherein the polycomb gene is a BMI gene and/or the apoptosis-inhibiting gene is a BCL-XL gene. The composition according to claim 1 , wherein the proliferative megakaryocytes and/or the mature megakaryocytes are megakaryocytes induced in vitro . The composition described in any one of claims 1 to 9, wherein the proliferative megakaryocytes and/or the mature megakaryocytes are derived from pluripotent cells. A composition described in any one of claims 1 to 10, which inhibits differentiation of osteoclast precursor cells into osteoclasts. 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. A composition for use in treating a disease caused by osteoclasts, comprising the composition for use in inhibiting differentiation into osteoclasts described in any one of claims 1 to 11 and/or the composition for use in inhibiting inhibition of bone formation by osteoclasts described in claim 12. A kit for use in bone formation, comprising a composition for use in inhibiting osteoclast differentiation described in any one of claims 1 to 11 and/or a composition for use in inhibiting osteoclast-mediated bone formation inhibition described in claim 12, and a scaffold material for bone formation. A kit for use in inhibiting bone destruction, comprising a composition for use in inhibiting osteoclast differentiation described in any one of claims 1 to 11 and/or a composition for use in inhibiting osteoclast-mediated bone formation inhibition described in claim 12, and a scaffold material for bone formation. A composition for use in promoting differentiation of mature megakaryocytes into osteoblasts. The composition of claim 16, wherein the mature megakaryocytes are megakaryocytes containing an exogenous oncogene, polycomb gene, and/or apoptosis-inhibiting gene. The composition described in claim 16 or 17, wherein the mature megakaryocytes are megakaryocytes in which the expression of oncogenes, polycomb genes, and/or apoptosis-inhibiting genes is suppressed. The composition described in any one of claims 16 to 18, wherein the mature megakaryocytes are derived from proliferative megakaryocytes. 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. The composition described in claim 19 or 20, wherein the proliferative megakaryocytes are derived from immortalized megakaryocytes. The oncogene is the MYC gene.
The composition of any one of claims 17 to 21, wherein the polycomb gene is a BMI gene and/or the apoptosis-inhibiting gene is a BCL-XL gene. 23. The composition of any one of claims 16 to 22, wherein the mature megakaryocytes are in vitro induced megakaryocytes. The composition described in any one of claims 16 to 23, wherein the mature megakaryocytes are derived from pluripotent cells. A composition described in any one of claims 16 to 24, which promotes differentiation of mesenchymal stem cells into osteoblasts. A composition for use in promoting bone formation by osteoblasts, comprising the composition for use in promoting differentiation into osteoblasts described in any one of claims 16 to 25. A composition for use in treating a disease caused by osteoblasts, comprising the composition for use in promoting differentiation into osteoblasts described in any one of claims 16 to 25 and/or the composition for use in promoting bone formation by osteoblasts described in claim 26. 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. A method for inhibiting osteoclast differentiation, comprising using a composition for inhibiting osteoclast differentiation described in any one of claims 1 to 15. The suppression method described in claim 29, comprising an administration step of administering to a subject a composition for use in inhibiting differentiation into osteoclasts. The method of suppression according to claim 29 or 30, which is used in vitro or in vivo . A method for suppressing osteoclast-mediated bone formation inhibition, comprising using the composition for use in suppressing osteoclast-mediated bone formation inhibition described in claim 12. The suppression method described in claim 32, comprising an administration 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 . A method for promoting differentiation into osteoblasts, comprising using a composition for use in promoting differentiation into osteoblasts described in any one of claims 16 to 25. The method for promoting differentiation into osteoblasts described in claim 35, comprising an administration step of administering to a subject a composition for use in promoting differentiation into osteoblasts. 37. The method of claim 35 or 36, which is used in vitro or in vivo . A method for promoting bone formation by osteoblasts, using the composition for promoting bone formation by osteoblasts described in claim 26. The promotion method described in claim 38, comprising an administration step of administering to a subject a composition for use in promoting bone formation by osteoblasts. 40. The method of claim 38 or 39, which is used in vitro or in vivo .