Bone-forming composition

Abstract

The purpose of the present invention is to provide a bone-forming composition comprising enlarged megakaryocytes and / or extracellular secretions derived from enlarged megakaryocytes.　The bone-forming composition according to the present disclosure comprises enlarged megakaryocytes and / or extracellular secretions derived from enlarged megakaryocytes.

Description

osteogenic composition

[0001] The present disclosure relates to osteogenic compositions.

[0002] Extracellular secretions such as secretomes and extracellular vesicles are known to be involved in intercellular / interorgan signal transduction and tissue functional regeneration (Patent Document 1, Non-Patent Documents 1 and 2). Applications of these extracellular secretions to regenerative medicine, etc., are being explored.

[0003] Furthermore, in regenerative medicine in the dental field, platelet-rich plasma is used in bone augmentation treatments, etc. However, the therapeutic application of platelet-rich plasma requires preparation just before use, which requires steps from blood collection to production during surgery, making the process complicated. Furthermore, because platelet-rich plasma is produced by separation and centrifugal concentration from isolated human blood, the platelet count is not standardized, and its efficacy is often unclear, presenting a hurdle to its use (Non-Patent Document 3).

[0004] International Publication No. 2010 / 079086

[0005] Beer, Lucian et al. “Peripheral blood mononuclear cell secretome for tissue repair.” Apoptosis : an international journal on programmed cell death vol. 21,12 (2016): 1336-1353. doi:10.1007 / s10495-016-1292-8Gonzalez-Gonzalez, Alberto et al. “Mesenchymal stem cells secretome: The cornerstone of cell-free regenerative medicine.” World journal of stem cells vol. 12,12 (2020): 1529-1552. doi:10.4252 / wjsc.v12.i12.1529Fernandes, G. & Yang, S. “Application of platelet-rich plasma with stem cells in bone and periodontal tissue engineering.” Bone Res., 4 (2016): 16036. doi:10.1038 / boneres.2016.36. eCollection 2016

[0006] Therefore, an object of the present disclosure is to provide a novel composition having osteogenic activity derived from megakaryocyte cells.

[0007] To achieve the above object, the osteogenic composition of the present disclosure (hereinafter also referred to as "composition") contains hypertrophic megakaryocyte cells and / or extracellular secretions derived from hypertrophic megakaryocyte cells.

[0008] The osteogenic kit (hereinafter also referred to as "kit") of the present disclosure includes an osteogenic composition and an osteogenic scaffold material, and the osteogenic composition is the osteogenic composition of the present disclosure.

[0009] According to the present disclosure, a novel composition having osteogenic activity derived from megakaryocyte cells can be provided, and the composition can be provided as an off-the-shelf product that does not require blood sampling or preparation.

[0010] Figure 1 is a photograph showing bone formation 4 weeks after transplantation in Example 1. Figure 2 is a graph showing bone mass 4 weeks after transplantation in Example 1. Figure 3 is a photograph showing bone formation 4 weeks after transplantation in Example 1. Figure 4 is a graph showing bone mass 4 weeks after transplantation in Example 1. Figure 5 is a graph showing cell number in Example 1.

[0011] <Osteogenic Composition> As described above, the osteogenic composition of the present disclosure contains hypertrophic megakaryocyte cells and / or extracellular secretions derived from hypertrophic megakaryocyte cells.

[0012] In the present disclosure, "osteogenesis" may mean the increase in existing bone, i.e., "bone augmentation" (bone creation), the formation of new bone, i.e., "neosteogenesis," or the restoration of lost (defective) bone or a portion thereof, i.e., "bone regeneration." Therefore, the osteogenic composition of the present disclosure can also be referred to as a bone augmentation (bone creation) composition, a new bone formation composition, and / or a bone regeneration composition.

[0013] In the present disclosure, "secretome" refers to a group of molecules secreted by a cell. Examples of the group of molecules include proteins, extracellular vesicles, etc. Examples of the proteins include growth factors, cytokines, chemokines, adhesion factors, proteases, etc.

[0014] In the present disclosure, "extracellular vesicles" (EVs) refer to particles secreted by cells and surrounded by a lipid bilayer membrane. The extracellular vesicles contain, for example, nucleic acid molecules, growth factors, proteins, etc. Examples of the nucleic acid molecules include DNA, mRNA, miRNA, etc. Examples of the extracellular vesicles include exosomes, microvesicles, etc.

[0015] In the present disclosure, "extracellular secretions" refers to substances secreted by cells, such as the secretome and extracellular vesicles.

[0016] In the present disclosure, "megakaryocyte cells" (megakaryocytes) are the largest cells present in the bone marrow and the like in vivo, and refer to cells capable of releasing platelets, cells that release platelets, and cells with equivalent functions. The cells with equivalent functions refer to cells capable of producing platelets. In the present disclosure, megakaryocytes may be megakaryocytes before multinucleation (polyploidization), i.e., immature megakaryocytes or megakaryocytes in the proliferative phase, megakaryocytes after multinucleation (multinucleated megakaryocytes), or megakaryocytes after hypertrophy (hypertrophic megakaryocytes). The multinucleated megakaryocytes can also be referred to as megakaryocytes that have undergone multinucleation but have not yet undergone hypertrophy. Specific examples of megakaryocytes include promegakaryocytes, megakaryoblasts, promegakaryocytes, and mature megakaryocytes. The number of chromosome sets (nuclear phase: N) possessed by the megakaryocytes after multinucleation may be more than 2 sets, and specific examples include 4 to 128 sets (4 to 128N), 4 to 64 sets (4N to 64N), 4 to 32 sets (4N to 32N), 8 to 128 sets (8 to 128N), 8 to 64 sets (8 to 64N), 8 to 32 sets (8N to 32N), 16 to 128 sets (16 to 128N), 16 to 64 sets (16 to 64N), or 16 to 32 sets (16 to 32N).

[0017] The origin of the megakaryocytes is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animals include primates such as monkeys, gorillas, chimpanzees, and marmosets, as well as mice, rats, dogs, cats, rabbits, sheep, horses, and guinea pigs.

[0018] In the present disclosure, the megakaryocytes can be identified by cell surface markers. When the megakaryocytes are derived from humans, the cell surface markers include CD41a, CD42a, and CD42b. That is, the megakaryocytes are cells that are positive for CD41a, CD42a, and CD42b. When the megakaryocytes are derived from humans, the cell surface marker may be, for example, at least one selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c.

[0019] The pre-multinucleated megakaryocytes and the multinucleated megakaryocytes can be identified, for example, by the number of chromosome sets. As a specific example, megakaryocytes of a subject having a chromosome set number (N) of less than 8N can be evaluated as pre-multinucleated megakaryocytes. On the other hand, megakaryocytes of a subject having a chromosome set number (N) of 8N or more, preferably 16N or more, can be evaluated as multinucleated megakaryocytes.

[0020] When the subject has a plurality of megakaryocytes, i.e., when the subject has a population (cell population) of megakaryocytes, if the proportion of cells with a chromosome set number (N) of 8N or more in the subject's megakaryocyte cell population is less than 10%, the subject's megakaryocyte cell population can be evaluated as a population of megakaryocytes before multinucleation. Furthermore, if the proportion of cells with a chromosome set number (N) of 8N or more in the subject's megakaryocyte cell population is 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more, the subject's megakaryocyte cell population can be evaluated as a population containing multinucleated megakaryocytes. The subject's megakaryocyte cell population can be, for example, 1 × 10 ５ ~1 x 10 ６ Each cell is an individual cell.

[0021] In the present disclosure, the term "hypertrophic megakaryocyte" refers to a megakaryocyte in which the cytoplasm of the multinucleated megakaryocyte has been enlarged. The multinucleated megakaryocyte contains, for example, α-granules. The number of chromosome sets (nuclear phase: N) possessed by the multinucleated megakaryocyte is the same as the number of chromosome sets possessed by the multinucleated megakaryocyte before hypertrophy. Therefore, the number of chromosome sets (nuclear phase: N) possessed by the enlarged megakaryocytes may be more than two sets, and specific examples include 4 to 128 sets (4N to 128N), 4 to 64 sets (4N to 64N), 4 to 32 sets (4N to 32N), 8 to 128 sets (8N to 128N), 8 to 64 sets (8 to 64N), 8 to 32 sets (8N to 32N), 16 to 128 sets (16N to 128N), 16 to 64 sets (16 to 64N), or 16 to 32 sets (16 to 32N).

[0022] In the present disclosure, the hypertrophic megakaryocytes can be identified, for example, by a marker. When the hypertrophic megakaryocytes are derived from human-derived immortalized megakaryocytes, the marker can be Ki-67 and / or MYC. In the hypertrophic megakaryocytes, for example, the expression level of the marker is reduced to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.1% or less, or 0.01% or less compared to the multinucleated megakaryocytes. Therefore, when the target megakaryocyte cells contain multiple megakaryocytes, i.e., when the target megakaryocyte cells are a population (cell population) of megakaryocytes, the population of megakaryocyte cells can be evaluated as a population containing hypertrophic megakaryocytes when the expression of the marker in the target megakaryocyte cell population is reduced to 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.1% or less, or 0.01% or less compared to the multinucleated megakaryocytes. ５ ~1 x 10 ６ The number of cells is 1. One type of marker may be used, or multiple types of markers may be used. When multiple types of markers are used, the combination of markers is, for example, Ki-67 and / or MYC.

[0023] The hypertrophic megakaryocytes can also be distinguished by, for example, cell size. Among the megakaryocytes, the multinucleated megakaryocytes are relatively larger in cell size than, for example, immature megakaryocytes or megakaryocytes in the proliferative phase. Furthermore, the hypertrophic megakaryocytes are relatively larger in cell size than, for example, the multinucleated megakaryocytes. For example, the immature megakaryocytes or the multinucleated megakaryocytes have a cell size of 10 μm or less. The multinucleated megakaryocytes have a cell size of, for example, more than 10 μm and not more than 60 μm. The hypertrophic megakaryocytes have a cell size of, for example, more than 50 μm and not more than 100 μm. The cell size is preferably measured by, for example, measuring the major axis. The measurement is preferably performed under an optical microscope with an objective lens magnification of 10 to 20 times.

[0024] In the present disclosure, hypertrophic megakaryocytes may be composed solely of hypertrophic megakaryocytes or may contain other cells. In the latter case, the hypertrophic megakaryocytes may also be referred to as a cell population containing hypertrophic megakaryocytes. The hypertrophic megakaryocytes may be, for example, hypertrophic megakaryocytes of 8N or more, hypertrophic megakaryocytes of 16N or more, or hypertrophic megakaryocytes of 32N or more. The hypertrophic megakaryocytes include, for example, the hypertrophic megakaryocytes of 8N or more, the hypertrophic megakaryocytes of 16N or more, and / or the hypertrophic megakaryocytes of 32N or more.

[0025] When the hypertrophic megakaryocytes include hypertrophic megakaryocytes with a nuclear phase of 8N or more, the lower limit of the proportion (cell number) of hypertrophic megakaryocytes of 8N or more in the cell population is, for example, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more. In the cell population, the upper limit of the proportion (cell number) of 8N or greater enlarged megakaryocytes is, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, or 46% or less. The proportion (cell number) of 8N or more enlarged megakaryocytes is, for example, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%, 40 to 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 15 to 85%, 15 to 80%, 15 to 75%, 15 to 70%, 15 to 65%, 16 to 60%, 17 to 59%, 18 to 58%, 19 to 57%, 20 to 56%, 21 to 55%, 22 to 54%, 23 to 54%, 24 to 53%, 25 to 51%, 26 to 50%, 27 to 49%, 28 to 48%, 29 to 47%, or 30 to 46%. The proportion (cell number) can be measured, for example, by thoroughly suspending a target cell population, taking a portion of the resulting cell suspension, and measuring the set number of chromosomes (nuclear phase) (the same applies hereinafter).

[0026] When the hypertrophic megakaryocytes include those with a nuclear phase of 16N or greater, the lower limit of the proportion (cell number) of 16N or greater hypertrophic megakaryocytes in the cell population is, for example, 15% or greater, 16% or greater, 17% or greater, 18% or greater, 19% or greater, 20% or greater, 21% or greater, 22% or greater, 23% or greater, 24% or greater, or 25% or greater. The upper limit of the proportion (cell number) of 16N or greater hypertrophic megakaryocytes in the cell population is, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, or 43% or less. The proportion (cell number) of enlarged megakaryocytes of 16N or more is, for example, 15 to 90%, 15 to 85%, 15 to 80%, 15 to 75%, 15 to 70%, 15 to 65%, 15 to 60%, 16 to 55%, 17 to 54%, 18 to 53%, 19 to 52%, 20 to 90%, 20 to 70%, 20 to 51%, 21 to 50%, 22 to 49%, 23 to 48%, 24 to 47%, 25 to 46%, 25 to 45%, 25 to 44%, or 25 to 43%.

[0027] When the hypertrophic megakaryocytes include those with a nuclear phase of 32N or greater, the lower limit of the proportion (cell number) of hypertrophic megakaryocytes with a nuclear phase of 32N or greater in the cell population is, for example, 5% or greater, 6% or greater, 7% or greater, 8% or greater, 9% or greater, 10% or greater, 11% or greater, 12% or greater, 13% or greater, 14% or greater, or 15% or greater. The upper limit of the proportion (cell number) of hypertrophic megakaryocytes with a nuclear phase of 32N or greater in the cell population is, for example, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, or 30% or less. The proportion (cell number) of enlarged megakaryocytes of 32N or more is, for example, 5 to 50%, 5 to 49%, 5 to 48%, 5 to 47%, 5 to 46%, 5 to 45%, 5 to 44%, 5 to 43%, 5 to 42%, 5 to 41%, 5 to 40%, 6 to 39%, 7 to 38%, 8 to 37%, 9 to 36%, 10 to 35%, 11 to 34%, 12 to 33%, 13 to 32%, 14 to 31%, 15 to 31%, or 15 to 30%.

[0028] The hypertrophic megakaryocytes are, for example, cells derived from pluripotent cells, preferably cells induced from pluripotent cells in vitro. The hypertrophic megakaryocytes are preferably derived from immortalized megakaryocytes. The immortalized megakaryocytes can be prepared, for example, by introducing an oncogene, a polycomb gene, and / or an apoptosis inhibitor gene, as described below. Therefore, the pre-hypertrophic megakaryocytes (the multinucleated megakaryocytes) and the hypertrophic megakaryocytes contain, for example, an exogenous oncogene, a polycomb gene, and / or an apoptosis inhibitor gene. The oncogene is preferably the c-MYC gene. The polycomb gene is preferably the BMI1 gene. The apoptosis inhibitor gene is preferably the BCL-XL gene. The pre-hypertrophic megakaryocytes and the hypertrophic megakaryocytes express, for example, an exogenous oncogene, a polycomb gene, and / or an apoptosis inhibitor gene.

[0029] The osteogenic composition of the present disclosure may contain factors such as growth factors. Examples of the growth factors include basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB). Examples of the growth factors include transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF). The content of a factor in the composition can be defined, for example, as the concentration or the content in a composition prepared from a predetermined amount of cells.

[0030] The content of the factor in the composition is, for example, 1 × 10 ６ The evaluation can be based on a composition prepared using enlarged megakaryocytes. ６ When converted into a composition prepared from enlarged megakaryocytic cells, the content of the bFGF ranges, for example, 1,000 to 100,000 pg, 2,000 to 80,000 pg, or 3,000 to 71,000 pg. The content of the PDGF-BB ranges, for example, 1,000 to 50,000 pg, 2,000 to 40,000 pg, or 3,000 to 30,000 pg. The content of the TGF-β ranges, for example, 20 to 3,000 pg, 30 to 2,000 pg, or 35 to 1,500 ng. The content of the VEGF ranges, for example, 50 to 7,000 pg, 65 to 5,000 pg, or 80 to 4,500 pg.

[0031] The hypertrophic megakaryocytic cells may be produced by a production method including, for example, a multinucleation step of multinucleating pre-multinucleated megakaryocytic cells or their precursor cells to induce multinucleated megakaryocytic cells, and a hypertrophy step of maturing the multinucleated megakaryocytes to induce hypertrophic megakaryocytic cells. Furthermore, the multinucleation step may involve inhibiting the activity of at least one selected from the group consisting of aryl hydrocarbon receptor (AhR), Rho-associated kinase (ROCK), dual specificity tyrosine phosphorylation-regulated kinase (DYRK), and myosin 2, and multinucleating the pre-multinucleated megakaryocytic cells in the presence of harmine to induce multinucleated megakaryocytic cells. The present disclosure provides a method for inducing hypertrophic megakaryocytes using the multinucleated megakaryocytes induced in the multinucleation step, thereby improving the secretion of secretomes and extracellular vesicles from the hypertrophic megakaryocytes. This allows the present disclosure to, for example, increase the content of secretome, extracellular vesicles, and / or factors in the culture medium. The factor promoting the proliferation of pre-multinucleated megakaryocytes, described below, may be present in the culture medium or within the pre-multinucleated megakaryocytes. The AhR activity inhibitor (AhR inhibitor), the ROCK activity inhibitor (ROCK inhibitor), the DYRK activity inhibitor (DYRK inhibitor), the myosin 2 activity inhibitor (myosin 2 inhibitor), and / or harmine are, for example, contained in the culture medium for pre-multinucleated megakaryocytes, described below. When the factor promoting the proliferation of pre-multinucleated megakaryocytes, the AhR activity inhibitor, the ROCK activity inhibitor, the DYRK activity inhibitor, and / or the myosin 2 activity inhibitor are proteins or nucleic acid molecules, they may be introduced into the pre-multinucleated megakaryocytes by lipofection, electroporation, or the like in the multinucleation step.

[0032] In the multinucleation step, the activity of any one or two or more of AhR, ROCK, DYRK, and myosin 2 may be inhibited. In the latter case, the combination of targets whose activities are inhibited may be any combination, and, because of their high ability to promote multinucleation, the combination of AhR and myosin 2, the combination of AhR, ROCK, and myosin 2, the combination of AhR, DYRK, and myosin 2, or the combination of AhR, ROCK, DYRK, and myosin 2 is preferred. Therefore, the multinucleation process is preferably carried out by inhibiting the activities of AhR and myosin 2 and in the presence of harmine, by inhibiting the activities of AhR, ROCK, and myosin 2 and in the presence of harmine, by inhibiting the activities of AhR, DYRK, and myosin 2 and in the presence of harmine, or by inhibiting the activities of AhR, ROCK, DYRK, and myosin 2 and in the presence of harmine.

[0033] The AhR refers to an aryl hydrocarbon receptor. The AhR is a transcription factor belonging to the Per / ARNT / SIM (PAS) family. For example, AhR is inactive when no ligand is bound, and translocates into the nucleus when an aromatic hydrocarbon compound binds as a ligand. After translocation into the nucleus, the AhR forms a heterodimer with, for example, ARNT (Ahr Nuclear Translocator), binds to a xenobiotic responsive element (XRE, also known as DRE), and activates transcription.

[0034] In the multinucleation step, the suppression of AhR activity means that the activity of AhR is reduced compared to that in the absence of the AhR activity inhibitor. The AhR activity can be measured, for example, using AhR nuclear translocation as an index. The AhR activity can be measured, for example, using a commercially available AhR protein activity measurement kit, a specific example of which is a nuclear receptor assay kit (manufactured by PURACYP). In the present disclosure, the suppression of activity can also be referred to, for example, as inhibition of activity, reduction of activity, suppression of activation, prevention of activation, etc.

[0035] The AhR inhibitor is not particularly limited, and examples thereof include AhR antagonists, expression-inhibiting nucleic acid molecules capable of suppressing the expression of AhR, and the like. The AhR antagonist is not particularly limited, and examples thereof include 4-(2-(2-(Benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (SR1), α-naphthoflavone (CAS 604-59-1), 1,4-dihydrocyanthraquinone, 1,5-dihydrocyanthraquinone, 1,8-dihydrocyanthraquinone, galangin (CAS 548-83-4), resveratrol, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), N-(2-(3H-Indol-3-yl)ethyl)-9-isopropyl-2-(5-methyl-3-pyridyl)-7H-purin-6-amine (GNF351), 2-(29- Examples of the AhR antagonist include (amino-39-methoxyphenyl)-oxanaphthalen-4-one (PD98059), (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (TSU-16), 6,2',4'-trimethoxyflavone (TMF), and 3',4'-dimethoxyflavone (DMF). The AhR antagonist may be, for example, a compound described as an AhR antagonist in International Publication No. 2012 / 015914. Examples of the AhR expression-inhibiting nucleic acid molecule include expression-inhibiting nucleic acid molecules such as siRNA and miRNA that target the mRNA encoding AhR. The AhR expression-inhibiting nucleic acid molecule can be appropriately designed, for example, based on the base sequence of AhR mRNA registered in a database. A specific example of human AhR mRNA is a polynucleotide consisting of the nucleotide sequence registered in Genbank under accession number NM_001621. The AhR activity inhibitors may be used singly or in combination.

[0036] In the multinucleation step, the concentration of the AhR inhibitor is not particularly limited and can be appropriately determined depending on the type of compound and its effective concentration. The concentration of the AhR inhibitor can be, for example, the following: SR1: 200 nmol / L or more and less than 1000 mmol / L CH-223191: 0.2 μmol / L or more and less than 4 μmol / L GNF351: 20 nmol / L or more and less than 300 nmol / L, 20 nmol / L or more and less than 1000 nmol / L TMF: 2.5 μmol / L or more and less than 40 μmol / L DMF: 2.5 μmol / L or more and less than 40 μmol / L

[0037] The above-mentioned ROCK means Rho-associated coiled-coil forming kinase (ROCK).

[0038] In the multinucleation step, inhibition of ROCK activity means, for example, that ROCK activity is reduced compared to that in the absence of the ROCK activity inhibitor. The ROCK activity can be measured, for example, using phosphorylation of a substrate by ROCK as an indicator. The ROCK activity can be measured, for example, using a commercially available ROCK protein activity measurement kit, a specific example of which is a ROCK activity measurement kit (96-Well ROCK Activity Assay Kit, manufactured by Cell Biolabs). In the present disclosure, inhibition of activity can also be referred to, for example, as inhibition of activity, reduction of activity, suppression of activation, prevention of activation, etc.

[0039] Examples of the ROCK inhibitor include a ROCK antagonist, an expression-inhibiting nucleic acid molecule capable of suppressing the expression of ROCK, and the like.The ROCK inhibitor includes, for example, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), 4-[(1R)-1-aminoethyl]-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide (Y-39983), fasudil hydrochloride (Fasudil (HA1077)), 4-fluoro-5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]-isoquinoline (Ripasudil), 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide (SLx-2119), N-[(3-Hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI-1447), 6-Chloro-N4-[3,5-difluoro-4-[(3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl]-2,4-pyrimidinediamine (TC-S 7001, Azaindole 1), N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide (SR-3677), Staurosporine, (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine (H-1152), rac-(2R)-2-(dimethylamino)-N-(1-oxo-1,2-dihydroisoquinolin-6-yl)-2-(thiophen-3-yl)acetamide (AR-12286), N-(1-{[4-(methylsulfanyl)phenyl]methyl}piperidin-3-yl)-1H-indazol-5-amine (INS-117548), and the like.Examples of the ROCK expression-inhibiting nucleic acid molecule include expression-inhibiting nucleic acid molecules such as siRNA and miRNA that target the mRNA encoding ROCK. The ROCK expression-inhibiting nucleic acid molecule can be appropriately designed, for example, based on the nucleotide sequence of ROCK mRNA registered in a database. Specific examples of human ROCK1 mRNA include a polynucleotide consisting of the nucleotide sequence registered in GenBank under accession number NM_005406. Furthermore, examples of human ROCK2 mRNA include a polynucleotide consisting of the nucleotide sequence registered in GenBank under accession number NM_004850. The ROCK activity-inhibiting substances may be used singly or in combination.

[0040] In the polynucleation step, the concentration of the ROCK inhibitor is not particularly limited and can be appropriately determined depending on the type of compound and its effective concentration. When the ROCK activity inhibitor is Y-39983, the concentration of the ROCK activity inhibitor is, for example, 1 × 10 －8 ~1 x 10 －３ mol / l, preferably 1×10 －７ ~1 x 10 －５ mol / l.

[0041] DYRK stands for Dual Specificity Tyrosine Phosphorylation Regulated Kinase.

[0042] In the inhibition of DYRK activity, examples of the target DYRK include DYRK1A, DYRK1B, DYRK2, etc., and preferably DYRK1A. As an example, the case of inhibiting DYRK1A activity will be described below.

[0043] In the multinucleation step, the inhibition of DYRK1A activity means that the activity of DYRK1A is reduced compared to that in the absence of the DYRK1A activity inhibitor. The DYRK1A activity can be measured, for example, using a measurement system that uses a DYRK1A protein and a substrate, and using phosphorylation of the substrate as an indicator. For the former, for example, a commercially available DYRK1A protein activity measurement kit can be used, and specific examples include the DYRK1A Kinase Enzyme System (Promega, Cat. No. VA7423, VA7424). For the latter, see, for example, Reference 1 below. In the present disclosure, the inhibition of activity can also be referred to, for example, as inhibition of activity, reduction of activity, inhibition of activation, prevention of activation, etc. Reference 1: Isao Kii et al., "Selective inhibition of the kinase DYRK1A by targeting its folding process," Nat. Commun., 2016, vol. 7, article number 11391

[0044] The substance that inhibits DYRK1A activity (DYRK1A inhibitor) is not particularly limited and includes, for example, a compound, protein, nucleic acid molecule, etc. that inhibits the activity or expression of DYRK1A. The compound that inhibits DYRK1A activity can be, for example, a known compound that inhibits DYRK1A activity. Specific examples of the DYRK1A activity-inhibiting compound (DYRK1A antagonist) include harmine (harmine, 7-Methoxy-1-methyl-9H-pyrido[3,4-b]indole, Cas No. 442-51-3), INDY ((1Z)-1-(3-Ethyl-5-hydroxy-2(3H)-benzothiazolylidene)-2-propanone, Pubchem No.: 69538603), GSK626616 ((5Z)-2-[(2,6-Dichlorophenyl)amino]-5-(6-quinoxalinylmethylene)-4(5H)-thiazolone, Cas No. 1025821-33-3), and TBB (casein kinase 2 inhibitor 1, 4,5,6,7-tetrabromobenzotriazole, Cas No. 17374-26-4), AZ191 (N-[2-Methoxy-4-(4-methyl-1-piperazinyl)phenyl]-4-(1-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-pyrimidinamine, Cas No. 1594092-37-1), Mrik-IN-1 (TC-S 7004, N-[2-Chloro-5-[[[(3-chlorophenyl)methyl]amino]carbonyl]phenyl]-7,8-dihydro-2-methoxy-7-oxopyrido[2,3-d]pyrimidine-6-carboxamide, Cas No. 1386979-55-0), EHT5372 (methyl 9-((2,4-dichlorophenyl)amino)thiazolo[5,4-f]quinazoline-2-carbimidate, Cas No.1425945-60-3)、GNF4877((R)-1-(3-(3-Amino-6-(2-fluoro-5-isopropoxyphenyl)pyrazine-2-carboxamido)pyridin-4-yl)piperidine-3-carboxylic acid、Cas No. 2041073-22-5)、FINDY((5Z)-5-[[4-Methoxy-3-[2-(trimethylsilyl)ethynyl]phenyl]methylene]-2-thioxo-4-thiazolidinone 、Cas No. 1507367-37-4)、ML351(5-(Methylamino)-2-(1-naphthalenyl)-4-oxazolecarbonitrile 、Cas No. 847163-28-4)、CLK-IN-T3(N-(6-(3,5-di-tert-butylphenyl)imidazo[1,2-a]pyridin-2-yl)-4-(2-methyl-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl)benzamide、Cas No. 2109805-56-1)、casein kinase 2 inhibitor IX.IQA(5-Oxo-5,6-dihydroindolo[1,2-a]quinazoline-7-acetic acid 、Cas No. 391670-48-7)、protein kinase inhibitor 1((E)-5-((2-Oxo-6'-(piperazin-1-yl)-1,2-dihydro-[3,3'-bipyridin]-5-yl)methylene)thiazolidine-2,4-dione hydrochloride、Cas No. 1365986-44-2)、Leucettine L41((5Z)-5-(1,3-benzodioxol-5-ylmethylene)-3,5-dihydro-2-(phenylamino)-4H-imidazol-4-one、Cas No.1112978-84-3). The DYRK1A activity-inhibiting compound may, for example, be free of harmine. As described above, the multinucleation step is performed in the presence of harmine. For this reason, it is preferable to use a compound other than harmine in addition to harmine as the DYRK1A activity-inhibiting compound, and a specific example is AZ191. The DYRK1A activity-inhibiting protein may, for example, be a dominant-negative (DN) form of DYRK1A protein. A specific example of a human DYRK1A DN protein is a mutant protein in which lysine (K) at position 188 is substituted with arginine (R). The DYRK1A expression-inhibiting nucleic acid molecule may, for example, be an expression-inhibiting nucleic acid molecule such as siRNA or miRNA that targets the mRNA encoding DYRK1A. The DYRK1A expression-inhibiting nucleic acid molecule may be appropriately designed, for example, based on the base sequence of DYRK1A mRNA registered in a database. Specific examples of human DYRK1A mRNA include polynucleotides consisting of the nucleotide sequences registered in GenBank under accession numbers NM_001396, NM_101395, NM_130436, NM_130437, NM_130438, NM_001347721, NM_001347722, and NM_001347723. The DYRK1A activity inhibitors may be used singly or in combination.

[0045] The concentration of the DYRK1A activity inhibitor is not particularly limited and can be appropriately set depending on, for example, the type and effective concentration of the DYRK1A activity inhibitor. When the DYRK1A activity inhibitor is Harmine, the concentration of the DYRK1A activity inhibitor is, for example, 1 × 10 －8 ~1 x 10 －３ mol / l, preferably 1×10 －６ ~1 x 10 －４ When the DYRK1A activity inhibitor is AZ191, the concentration of the DYRK1A activity inhibitor is, for example, 1 × 10 －９ ~1 x 10 －３mol / l, preferably 1×10 －７ ~1 x 10 －５ mol / l.

[0046] Myosin 2 is a protein that has ATPase activity and moves on actin, and refers to a complex composed of two heavy chains and two light chains attached to each heavy chain (four in total).

[0047] In the multinucleation step, the inhibition of myosin 2 activity means that the ATPase activity of myosin 2 is reduced compared to that in the absence of the myosin 2 activity inhibitor. The myosin 2 activity can be measured, for example, using phosphate released from myosin 2 as an indicator. The myosin 2 activity can be measured, for example, using a commercially available myosin 2 protein activity measurement kit. In the present disclosure, the inhibition of activity can also be referred to, for example, as inhibition of activity, reduction of activity, inhibition of activation, prevention of activation, etc.

[0048] Examples of the myosin 2 inhibitor include a myosin 2 antagonist, an expression-inhibiting nucleic acid molecule capable of inhibiting the expression of myosin 2, a myosin light chain kinase (MLCK) inhibitor, etc. Examples of the myosin 2 inhibitor include 3a-hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one (Blebbistatin), CK1122534, Omecamtiv mecarbil (methyl 4-[[2-fluoro-3-[(6-methylpyridin-3-yl)carbamoylamino]phenyl]methyl]piperazine-1-carboxylate), Ammosamides A&B, CTK2018448, and Manassantin. Examples of the MLCK inhibitor include ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine) and ML9 (1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine). Examples of the myosin 2 expression-inhibiting nucleic acid molecule include expression-inhibiting nucleic acid molecules such as siRNA and miRNA that target mRNA encoding myosin 2 or myosin light chain kinase. The myosin 2 expression-inhibiting nucleic acid molecule can be appropriately designed based on, for example, the base sequence of the heavy chain or light chain constituting myosin 2 or the mRNA of myosin light chain kinase registered in a database.Specific examples of mRNA of the heavy chain that constitutes human myosin 2 include polynucleotides consisting of the nucleotide sequences registered in GenBank under the accession numbers NM_005963, NM_001100112, NM_017534, NM_002470, NM_017533, NM_002147, NM_002471, NM_000257, NM_002472, NM_002473, NM_001256012, NM_002474, NM_001382347, NM_003802, NM_001145809, and NM_014981. Examples of mRNA for the light chain that constitutes human myosin 2 include polynucleotides consisting of the nucleotide sequences registered in GenBank under accession numbers NM_079420, NM_000432, NM_000258, NM_002476, NM_001363650, NM_021019, NM_021223, NM_006097, NM_138403, and NM_001324458. Examples of human myosin light chain kinase mRNA include polynucleotides consisting of the nucleotide sequences registered in GenBank under accession numbers NM_001321309, NM_053025, NM_053026, NM_053027, NM_053028, NM_053031, NM_053032, NM_033118, NM_053032, and NM_18249. The myosin 2 activity inhibitors may be used singly or in combination.

[0049] In the multinucleation step, the concentration of the myosin 2 inhibitor is not particularly limited and can be appropriately determined depending on the type of compound and its effective concentration. When the myosin 2 activity inhibitor is Blebbistatin, the concentration of the myosin 2 activity inhibitor is, for example, 1 × 10 －8 ~1 x 10 －３ mol / l, preferably 1×10 －６ ~1 x 10 －４ mol / l.

[0050] In the polynuclearization step, the concentration of the harmine is, for example, 1×10 －8 ~1 x 10 －３mol / l, preferably 1×10 －６ ~1 x 10 －４ mol / l.

[0051] In the multinucleation step, the inhibition of the activity of AhR, ROCK, DYRK, and / or myosin 2 and the coexistence of harmine may be carried out simultaneously or at different times, but are preferably carried out simultaneously. That is, the induction of the multinucleated megakaryocytes from the pre-multinucleated megakaryocytes is preferably carried out under conditions in which the activity of at least one selected from the group consisting of AhR, ROCK, DYRK, and myosin 2 is inhibited and the cells are coexistent with harmine.

[0052] When the megakaryocytes before multinucleation are immortalized megakaryocytes, the multinucleation step can be carried out, for example, as follows. Specifically, as described above, the proteins expressed by the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene function as factors that promote the proliferation of megakaryocytes before multinucleation in the immortalized megakaryocytes. Therefore, when the immortalized megakaryocytes are used, multinucleated megakaryocytes can be induced by, for example, maintaining the forced expression of the oncogene, the polycomb gene, and / or the apoptosis-inhibiting gene, inhibiting the activity of at least one selected from the group consisting of AhR, ROCK, DYRK, and myosin 2, and culturing the immortalized megakaryocytes in the presence of harmine. In this case, the multinucleation step can be carried out by, during the culture of the immortalized megakaryocytes, replacing the medium containing the immortalized megakaryocytes with a medium containing harmine and at least one selected from the group consisting of the AhR inhibitor, the ROCK inhibitor, the DYRK inhibitor, and the myosin 2 inhibitor, or by adding harmine and at least one selected from the group consisting of the AhR inhibitor, the ROCK inhibitor, the DYRK inhibitor, and the myosin 2 inhibitor to the medium containing the immortalized megakaryocytes. The addition or replacement may be carried out, for example, once or two or more times.

[0053] The medium used in the multinucleation step is not particularly limited and may include, for example, known media suitable for producing megakaryocytes and media equivalent thereto. Specifically, the medium may be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Neurobasal® Medium (manufactured by Thermo Fisher Scientific), as well as a mixture of these media. The medium may contain, for example, serum or plasma, or may be serum-free without serum or plasma, although serum-free media are preferred. Using a serum-free medium in the multinucleation step can improve the degree of multinucleation of the pre-multinucleated megakaryocytes into multinucleated megakaryocytes, for example. Examples of the serum-free medium include StemSpan ACF (manufactured by StemCell Technologies, Cat. No.: 9855) and Stemline 2 (manufactured by Sigma-Aldrich, Cat. No.: S0192). The serum and plasma are preferably derived from the same source as the megakaryocytes. Specifically, when the megakaryocytes are derived from humans, the serum and plasma are preferably derived from humans. By using the megakaryocytes and serum from the same source, for example, the multinucleation of the megakaryocytes can be further promoted in the multinucleation step.

[0054] The medium may contain, for example, other components. The other components are not particularly limited and include, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like. The other components may be, for example, one type or two or more types. Cytokines are substances that promote the differentiation of hematopoietic cells, and specific examples include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, ADAM inhibitors, FLT inhibitors, WNT inhibitors, and the like. The medium is preferably an IMDM medium containing, for example, serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The medium may further contain, for example, SCF or heparin. The concentrations of the other components are not particularly limited. The TPO concentration is, for example, about 10 ng / ml to about 200 ng / ml, or about 50 ng / ml to about 100 ng / ml. The SCF concentration is, for example, about 10 ng / ml to about 200 ng / ml, or about 50 ng / ml. The heparin concentration is, for example, about 10 U / ml to about 100 U / ml, or about 25 U / ml. The medium may further contain, for example, a phorbol ester (e.g., phorbol-12-myristate-13-acetate; PMA).

[0055] In the multinucleation step, the cell density of the megakaryocytes before multinucleation at the start is not particularly limited. The lower limit of the cell density is, for example, 1 × 10 ５ cells / ml, 2 x 10 ５ cells / ml, 3 x 10 ５ cells / ml, 4 x 10 ５ The upper limit of the cell density is not particularly limited, and may be, for example, 4 × 10 ５ cells / ml, 6 x 10 ５cells / ml, 8x10 ５ cells / ml, 1 x 10 ７ cells / ml, 5 x 10 ７ cells / ml, 1 x 10 ８ The cell density range is, for example, 1 x 10 ５ cells / ml ~ 1 x 10 ８ cells / ml, 1 x 10 ５ cells / ml~5×10 ７ cells / ml, 1 x 10 ５ cells / ml ~ 1 x 10 ７ cells / ml, 1 x 10 ５ cells / ml~8×10 ５ cells / ml, 1 x 10 ５ cells / ml~5×10 ５ cells / ml, 2 x 10 ５ cells / ml~8×10 ５ cells / ml, 3 x 10 ５ cells / ml~6×10 ５ cells / ml, 4 x 10 ５ cells / ml~6×10 ５ cells / ml, and can promote multinucleation or improve platelet production, so preferably 1 x 10 ５ cells / ml~8×10 ５ cells / ml, or 1 x 10 ５ cells / ml~5×10 ５ The cell density can be calculated, for example, by dividing the number of megakaryocytes before multinucleation by the volume of the medium in which the megakaryocytes before multinucleation are suspended.

[0056] The culture conditions in the multinucleation step are not particularly limited, and ordinary culture conditions for megakaryocytes can be used. Specific examples of the culture temperature include about 35 to about 42°C, about 36 to about 40°C, and about 37 to about 39°C. ２ The concentration is, for example, about 5 to about 15%. ２ The concentration is, for example, about 15 to about 25%, about 20%.

[0057] The culture period for the polynucleation step can be set, for example, as the period required for the pre-polynuclear megakaryocytes to polynucleate. The culture period for the polynucleation step may be appropriately set depending on the proportion of polynucleated megakaryocytes among the megakaryocytes being cultured. The culture period for the polynucleation step is preferably set, for example, to a period during which the proportion of 8N or larger polynucleated megakaryocytes among the megakaryocytes being cultured is 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more. Specific examples of the culture period for the polynucleation step include 0 to 20 days and 1 to 15 days. The proportion of multinucleated megakaryocytes among the megakaryocytes can be calculated, for example, by sampling a portion of the megakaryocytes during culture.

[0058] In the multinucleation step, the culture of megakaryocytes before multinucleation may be carried out, for example, on feeder cells or without feeder cells. The megakaryocytes before multinucleation can be cultured, for example, in suspension culture, and therefore can be cultured without feeder cells. The feeder cells refer to cells that are co-cultured with target cells (target cells) to be proliferated or differentiated in order to create an environment necessary for the culture of the target cells. The feeder cells may be any cells that can be distinguished from the target cells, and may be cells of the same species as the target cells or cells of a different species. The feeder cells may be cells that have been treated to prevent proliferation, for example, with antibiotics, anticancer drugs, gamma-ray irradiation, etc.

[0059] The polynucleated megakaryocytes after the polynucleation step may be composed solely of polynucleated megakaryocytes, or may contain other cells. In the latter case, the polynucleated megakaryocytes after the polynucleation step can also be referred to as a cell population containing polynucleated megakaryocytes. The polynucleated megakaryocytes may be, for example, 8N or larger polynucleated megakaryocytes, 16N or larger polynucleated megakaryocytes, or 32N or larger polynucleated megakaryocytes. The polynucleated megakaryocytes after the polynucleation step include, for example, the 8N or larger polynucleated megakaryocytes, the 16N or larger polynucleated megakaryocytes, and / or the 32N or larger polynucleated megakaryocytes.

[0060] When the multinucleated megakaryocytes include multinucleated megakaryocytes with a nuclear phase of 8N or more, the lower limit of the proportion (cell number) of multinucleated megakaryocytes of 8N or more in the cell population is, for example, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more. In the cell population, the upper limit of the proportion (cell number) of 8N or more multinucleated megakaryocytes is, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, or 46% or less. The proportion (cell number) of 8N or more multinucleated megakaryocytes is, for example, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%, 40 to 90%, 45 to 90%, 50 to 90%, 55 to 90%, 60 to 90%, 15 to 85%, 15 to 80%, 15 to 75%, 15 to 70%, 15 to 65%, 16 to 60%, 17 to 59%, 18 to 58%, 19 to 57%, 20 to 56%, 21 to 55%, 22 to 54%, 23 to 54%, 24 to 53%, 25 to 51%, 26 to 50%, 27 to 49%, 28 to 48%, 29 to 47%, or 30 to 46%.

[0061] When the multinucleated megakaryocytes include multinucleated megakaryocytes with a nuclear phase of 16N or greater, the lower limit of the proportion (cell number) of the multinucleated megakaryocytes of 16N or greater in the cell population is, for example, 15% or greater, 16% or greater, 17% or greater, 18% or greater, 19% or greater, 20% or greater, 21% or greater, 22% or greater, 23% or greater, 24% or greater, or 25% or greater. The upper limit of the proportion (cell number) of the multinucleated megakaryocytes of 16N or greater in the cell population is, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, or 43% or less. The proportion (cell number) of 16N or more multinucleated megakaryocytes is, for example, 15 to 90%, 15 to 85%, 15 to 80%, 15 to 75%, 15 to 70%, 15 to 65%, 15 to 60%, 16 to 55%, 17 to 54%, 18 to 53%, 19 to 52%, 20 to 90%, 20 to 70%, 20 to 51%, 21 to 50%, 22 to 49%, 23 to 48%, 24 to 47%, 25 to 46%, 25 to 45%, 25 to 44%, or 25 to 43%.

[0062] When the multinucleated megakaryocytes include multinucleated megakaryocytes with a nuclear phase of 32N or greater, the lower limit of the proportion (cell number) of multinucleated megakaryocytes with a nuclear phase of 32N or greater in the cell population is, for example, 5% or greater, 6% or greater, 7% or greater, 8% or greater, 9% or greater, 10% or greater, 11% or greater, 12% or greater, 13% or greater, 14% or greater, or 15% or greater. The upper limit of the proportion (cell number) of multinucleated megakaryocytes with a nuclear phase of 32N or greater in the cell population is, for example, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, or 30% or less. The proportion (cell number) of 32N or more multinucleated megakaryocytes is, for example, 5 to 50%, 5 to 49%, 5 to 48%, 5 to 47%, 5 to 46%, 5 to 45%, 5 to 44%, 5 to 43%, 5 to 42%, 5 to 41%, 5 to 40%, 6 to 39%, 7 to 38%, 8 to 37%, 9 to 36%, 10 to 35%, 11 to 34%, 12 to 33%, 13 to 32%, 14 to 31%, 15 to 31%, or 15 to 30%.

[0063] The pre-multinucleated megakaryocytes can be induced, for example, from cells less differentiated than megakaryocytes (also referred to as "pre-multinucleated megakaryocyte precursor cells"). Therefore, the production method may include, for example, a megakaryocyte induction step of inducing pre-multinucleated megakaryocytes from cells less differentiated than megakaryocytes prior to the multinucleation step. The medium, culture conditions, etc. used in the megakaryocyte induction step can be the same as those described above for the multinucleation step.

[0064] The "cells less differentiated than megakaryocytes" refer to cells capable of differentiating into megakaryocytes. Specific examples of the cells less differentiated than megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor cells (MEPs), and megakaryocyte progenitor cells. The cells less differentiated than megakaryocytes may be isolated from bone marrow, umbilical cord blood, peripheral blood, or the like, or may be derived from pluripotent cells or multipotent stem cells, such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), nuclear transfer ES cells (ntES cells), germline stem cells, somatic stem cells, and embryonic tumor cells.

[0065] The method for inducing megakaryocytes is not particularly limited and can be carried out using known induction methods. Specific examples of the megakaryocyte induction method include those described in International Publication Nos. 2011 / 034073 and 2012 / 157586. Specifically, in the megakaryocyte induction step, for example, an oncogene and a polycomb gene may be forced to express in cells less differentiated than megakaryocytes. This allows the megakaryocyte induction step to obtain immortalized megakaryocytes that proliferate indefinitely, corresponding to megakaryocytes before multinucleation. Furthermore, for example, by canceling the forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced into multinucleated megakaryocytes, which can then be induced into hypertrophic megakaryocytes. Therefore, the immortalized megakaryocytes correspond to megakaryocytes before multinucleation. Furthermore, in the megakaryocyte induction step, for example, an apoptosis-inhibiting gene may be forced to express in the megakaryocyte progenitor cells. As a result, the immortalized megakaryocytes can be obtained in the megakaryocyte induction step. Furthermore, in the hypertrophy step described below, for example, the forced expression of the immortalized megakaryocytes is cancelled, whereby the immortalized megakaryocytes are matured and induced into the hypertrophied megakaryocytes.

[0066] In the megakaryocyte induction step, for example, the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcedly expressed. In this case, the forced expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be performed simultaneously or separately. Specifically, in the megakaryocyte induction step, the oncogene and the polycomb gene may be forcedly expressed, and then the forced expression may be released, followed by the forced expression of the apoptosis-inhibiting gene; the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcedly expressed; or the oncogene and the polycomb gene may be forcedly expressed, followed by the expression of the apoptosis-inhibiting gene. This allows the megakaryocyte induction step to obtain the immortalized megakaryocytes. Furthermore, in the hypertrophy step described below, for example, the forced expression of the immortalized megakaryocytes may be released, thereby maturing the immortalized megakaryocytes and induced into the hypertrophied megakaryocytes.

[0067] The megakaryocyte induction step preferably includes, for example, a first expression step of forcibly expressing an oncogene and a polycomb gene in cells less differentiated than megakaryocytes, since this can improve the efficiency of gene introduction; a second expression step of forcibly expressing an apoptosis-inhibiting gene such as the BCL-XL gene in the undifferentiated cells; and a release step of releasing all of the forced expression. As described above, by releasing the forced expression, for example, multinucleated megakaryocytes are induced from the immortalized megakaryocytes, and then the hypertrophied megakaryocytes are induced. Therefore, the release step can also be referred to as the hypertrophy step described below.

[0068] 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 2 listed below, or by methods equivalent thereto. 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 2: 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

[0069] In the present disclosure, "oncogene" means a gene capable of inducing canceration of cells in a living body, and examples thereof include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as c-kit (CD117), PDGFR (platelet growth factor receptor), and Abl (Abelson murine leukemia viral oncogene homolog).

[0070] In the present disclosure, "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 3 to 5 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)), Mel18 (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 3: 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 4: 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 5: 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

[0071] In the present disclosure, "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples thereof include BCL2 (B-cell lymphoma 2), BCL-XL (B-cell lymphoma-extra large), Survivin (Baculoviral IAP Repeat Containing 5), and MCL1 (BCL2 Family Apoptosis Regulator).

[0072] The production method may include a proliferation step of proliferating the pre-multinucleated megakaryocytes in the presence of a factor that promotes the proliferation of the pre-multinucleated megakaryocytes. In this case, the proliferation step involves proliferating the pre-multinucleated megakaryocytes without inhibiting the activity of AhR, ROCK, DYRK, and myosin 2 and in the absence of harmine. This allows the pre-multinucleated megakaryocytes to be proliferated while maintaining their pre-multinucleated state. Specifically, in the proliferation step, pre-multinucleated megakaryocytes are cultured in the presence of a factor that promotes the proliferation of the pre-multinucleated megakaryocytes, thereby proliferating the pre-multinucleated megakaryocytes. The factor that promotes the proliferation of the pre-multinucleated megakaryocytes may be present in the medium or may be present within the pre-multinucleated megakaryocytes. Furthermore, in the proliferation step, by not adding to the medium harmine or a substance that inhibits the activity of AhR, ROCK, DYRK, and myosin 2, proliferation can be carried out in the absence of harmine without suppressing the activities of AhR, ROCK, DYRK, and myosin 2. The medium, culture conditions, etc. used in the proliferation step can be, for example, those described above for the multinucleation step.

[0073] When the production method includes the proliferation step, the multinucleation step is carried out after the proliferation step. Examples of growth factors for megakaryocytes before multinucleation include stem cell factor (SCF) and thrombopoietin (TPO).

[0074] The concentration of the growth factor for the megakaryocytes before multinucleation is not particularly limited and can be appropriately set depending on, for example, the type and effective concentration of the growth factor. When the growth factor is SCF or TPO, the concentration of the growth factor is, for example, 0.1 to 1000 ng / ml, or 1 to 200 ng / ml.

[0075] The culture period for the proliferation step can be, for example, the period until a desired number of pre-multinucleated megakaryocytes is obtained. Specifically, the culture period for the proliferation step is, for example, one day or more, and specific examples are 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 5 days, 2 to 5 days, or 2 to 4 days.

[0076] When immortalized megakaryocytes are used as the pre-multinucleated megakaryocytes, the immortalized megakaryocytes proliferate while maintaining their pre-multinucleated state if the oncogene and the polycomb gene, or the oncogene, the polycomb gene, and the apoptosis-inhibiting gene, are expressed. Therefore, when immortalized megakaryocytes are used, the first expression step and / or the second expression step can be referred to as a proliferation step. Furthermore, 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 factors that promote the proliferation of the pre-multinucleated megakaryocytes. When immortalized megakaryocytes are used as the pre-multinucleated megakaryocytes, the same explanation as for the megakaryocyte induction step for the immortalized megakaryocytes can be applied to the proliferation step.

[0077] In the hypertrophy step, the multinucleated megakaryocytes are matured to induce hypertrophic megakaryocytic cells. Specifically, the hypertrophy step can be carried out, for example, by culturing the multinucleated megakaryocytes in the presence of a medium. The multinucleated megakaryocytes can be cultured, for example, on feeder cells or without feeder cells. When the multinucleated megakaryocytes are derived from immortalized megakaryocytes, the hypertrophy step can be carried out, for example, by suppressing the expression of the oncogene and the polycomb gene, or the oncogene, the polycomb gene, and the apoptosis-inhibiting gene in the multinucleated megakaryocytes.

[0078] In the hypertrophy step, the period for inducing the hypertrophied megakaryocytes from the multinucleated megakaryocytes is not particularly limited and may be, for example, 1 to 10 days, 1 to 5 days, or 2 to 3 days. In the hypertrophy step, for example, by relatively extending the induction period, the contents of secretome, extracellular vesicles, and / or factors in the resulting culture medium can be increased.

[0079] In the hypertrophy step, for example, the activity of AhR, ROCK, DYRK, and myosin 2 may be inhibited, and the pre-multinucleated megakaryocytes may be proliferated in the presence of harmine. In this case, the hypertrophy step can be performed by adding harmine and a substance that inhibits the activity of AhR, ROCK, DYRK, and myosin 2 to the medium, thereby inhibiting the activity of AhR, ROCK, DYRK, and myosin 2, and performing hypertrophy in the presence of harmine. In this case, the hypertrophy step can be performed, for example, without inhibiting the activity of AhR, ROCK, DYRK, and myosin 2, and in the absence of harmine, after the multinucleation step. The medium, culture conditions, etc. used in the enlargement step can be the same as those described above for the multinucleation step.

[0080] In the hypertrophy step, the cell density of the multinucleated megakaryocytes at the start of induction of the hypertrophied megakaryocytes is not particularly limited.５ cells / ml, 2 x 10 ５ cells / ml, 3 x 10 ５ cells / ml, 4 x 10 ５ The upper limit of the cell density is not particularly limited, and may be, for example, 4 × 10 ５ cells / ml, 6 x 10 ５ cells / ml, 8x10 ５ cells / ml, 1 x 10 ６ cells / ml, 1 x 10 ７ The cell density range is, for example, 1 x 10 ５ cells / ml~8×10 ５ cells / ml, 1 x 10 ５ cells / ml ~ 1 x 10 ６ cells / ml, 1 x 10 ５ cells / ml ~ 1 x 10 ７ cells / ml, 2 x 10 ５ cells / ml~8×10 ５ cells / ml, 3 x 10 ５ cells / ml~6×10 ５ cells / ml, 4 x 10 ５ cells / ml~6×10 ５ The cell density can be calculated, for example, by dividing the number of the polynucleated megakaryocytes by the volume of the medium in which the polynucleated megakaryocytes are suspended.

[0081] In this way, the present disclosure allows hypertrophic megakaryocytes to be induced from the megakaryocytes. For example, by culturing the hypertrophic megakaryocytes in the medium, the secretome and / or the extracellular vesicles are secreted into the medium. Therefore, the medium (culture medium) during the hypertrophy process, the culture medium after the hypertrophy process, or the culture medium of the hypertrophic megakaryocytes cultured after the hypertrophy process (hereinafter also referred to as "culture medium of multinucleated megakaryocytes") contains, for example, the secretome and / or the extracellular vesicles. The osteogenic composition of the present disclosure may contain, for example, the culture medium of the hypertrophic megakaryocyte cells as the secretome and / or the extracellular vesicles.

[0082] During or after the enlargement step, the present disclosure may include a stimulation step of stimulating the enlarged megakaryocytes to increase the amount of secretome and / or extracellular vesicles released into the culture medium. Examples of the stimulation step include optical stimulation such as X-ray irradiation, physical stimulation such as preparation with glass beads, and chemical stimulation using collagen, ADP, TRAP6, etc.

[0083] The method may include a concentration step of concentrating the culture medium after the enlargement step. The concentration may be performed using a concentration member, or by centrifugation or the like. Examples of the concentration member include a membrane filter, an ultrafiltration membrane, and a hollow fiber membrane. The pore size of the concentration member is, for example, a pore size that can capture the secretome and / or extracellular vesicles. Specific examples of the pore size are 50 to 100 nm. The concentration step is preferably performed after the stimulation step.

[0084] The osteogenic composition of the present disclosure may comprise, for example, a concentrate of the culture medium after the concentration step. The concentrate may be, for example, a liquid or a solid.

[0085] The present disclosure may include, for example, an isolation step of isolating the extracellular vesicles from the culture medium after the enlargement step. The isolation may be performed using, for example, ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, affinity purification, co-precipitation, or the like, or a combination of these methods.

[0086] The osteogenic composition of the present disclosure may comprise, for example, the isolated extracellular vesicles after the isolation step, where "isolated" means identified, separated, or removed from a natural (natural) state.

[0087] The osteogenic composition of the present disclosure may have, for example, cell proliferation-promoting activity. When the osteogenic composition of the present disclosure has cell proliferation-promoting activity, the osteogenic composition of the present disclosure may also be referred to, for example, as a "composition for promoting cell proliferation." In the cell proliferation-promoting activity, the cells are not particularly limited, and examples thereof include mesenchymal stem cells. The mesenchymal stem cells are cells that have the ability to self-renew and the ability to differentiate into bone, cartilage, and adipocytes. The mesenchymal stem cells can be identified by cell surface markers. The mesenchymal stem cells are, for example, CD73, CD90, and CD105 positive, and CD14, CD34, and CD45 negative. The osteogenic composition of the present disclosure may be used in combination with mesenchymal stem cells, for example.

[0088] The cell proliferation-promoting activity may be, for example, an improvement in the proliferation ability of cells compared to a control group that is otherwise identical except for the addition of the osteogenic composition of the present disclosure; for example, the proliferation ability of 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 the mesenchymal stem cells decreases with each passage. Since the osteogenic composition of the present disclosure can suppress a decrease in the proliferation activity of the mesenchymal stem cells, it can be said that the osteogenic composition of the present disclosure exhibits proliferation-promoting activity. The proliferation-promoting activity of the cells can be measured, for example, under culture conditions in which the target cells proliferate. The culture conditions can be appropriately set, for example, depending on the type of the cells.

[0089] The activity of promoting differentiation into osteoblasts may be, for example, an improvement in the ability to differentiate into osteoblasts compared to a control group that is identical except for not adding the osteogenic composition of the present disclosure. The improvement 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. For example, Japanese Patent Application Laid-Open No. 2012-120529 can be referred to for the differentiation into osteoblasts.

[0090] The osteogenic compositions of the present disclosure may be used, for example, in vitro or in vivo.

[0091] When the osteogenic 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.

[0092] When the osteogenic 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, such as a mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, or guinea pig.

[0093] The conditions for use (administration conditions) of the osteogenic composition of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on the type of subject to be administered, for example.

[0094] When the osteogenic composition of the present disclosure is used in vitro, it can be used by, for example, adding it to a culture medium for target progenitor cells. For example, the osteogenic composition of the present disclosure may be added to a maintenance medium used to maintain the progenitor cells.

[0095] When the osteogenic 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.

[0096] The administration form of the osteogenic composition of the present disclosure is not particularly limited. When the osteogenic composition of the present disclosure is administered in vivo, it may be administered orally or 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.

[0097] The dosage form of the osteogenic composition of the present disclosure is not particularly limited and can be appropriately determined depending on, for example, the administration form, such as a liquid or solid form.

[0098] The osteogenic 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.

[0099] The osteogenic composition of the present disclosure may contain, for example, a lysate of the hypertrophied megakaryocytes. The lysate may be, for example, a lysate, and may be prepared by physically or chemically disrupting the hypertrophied megakaryocytes. The lysate may be, for example, a liquid or solid.

[0100] The osteogenic composition of the present disclosure is presumed to be suitable for use in treating, for example, jaw bone defects caused by jaw bone tumors, cysts, osteonecrosis, trauma, or bone system diseases (ectodermal dysplasia, cleft lip and palate, etc.); alveolar bone defects such as alveolar bone defects caused by periodontal disease, peri-implantitis or associated alveolar bone defects, or alveolar bone defects caused by caries or trauma; osteoporosis such as primary osteoporosis (postmenopausal osteoporosis, senile osteoporosis, etc.) and secondary osteoporosis (osteogenesis imperfecta, etc.); intractable fractures caused by hyperthyroidism, Cushing's syndrome, hypogonadism), trauma, etc.; rib fixation due to open chest surgery; bone defects due to craniotomy; and systemic bone defects due to tumor resection or cyst removal.

[0101] <Osteogenesis Kit> The osteogenic kit of the present disclosure includes an osteogenic composition and an osteogenic scaffold material, and the osteogenic composition is the osteogenic composition of the present disclosure. The kit of the present disclosure can easily induce bone formation by combining the osteogenic composition of the present disclosure with the scaffold material. The kit of the present disclosure can be applied to the same description of the osteogenic composition of the present disclosure.

[0102] In the kit of the present disclosure, the scaffold material may be, for example, a scaffold material used for bone or cartilage regeneration. The scaffold material may also be a material to which, for example, osteocytes or osteoblasts, or their precursor cells, can attach, proliferate, and / or differentiate. The scaffold material is, for example, a structure (construct) having a three-dimensional hollow or porous structure, and specific examples include a porous body, a porous support, a porous three-dimensional structure (construct), a gel (hydrogel), a sponge structure, etc.

[0103] The scaffold material is preferably biocompatible and / or biodegradable, for example, since it is placed in a living body together with the composition of the present disclosure. The "biocompatible" means, for example, that it has affinity with tissues and / or organs in a living body and does not cause foreign body reactions, rejection reactions, etc. The "biodegradable" means, for example, that its constituent components can be decomposed in a living body.

[0104] Examples of the components of the scaffold material include proteins such as collagen, gelatin, albumin, keratin, fibrin, and fibroin; polysaccharides such as agarose, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, and alginic acid; polyglycolic acid (PGA), polylactic acid (PLA), copolymers of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polyethylene glycol (PEG), polycaprolactone, polybutylene succinate, calcium phosphate (e.g., β-tricalcium phosphate), calcium carbonate, hydroxyapatite, polyether ketone, and polyether ether ketone. One type of the components may be used alone, or multiple types may be used in combination.

[0105] The scaffold material can be prepared, for example, by crosslinking or conjugating the components of the scaffold material.

[0106] Examples of the scaffolding material include Neobone (made of hydroxyapatite, manufactured by Aimdic MMT), APACERAM (registered trademark, made of hydroxyapatite, manufactured by HOYA Corporation), Bonarc (made of collagen / calcium octaphosphate, manufactured by Toyobo Co., Ltd.), Ospherion (made of βTCP, manufactured by Olympus Terumo Biomaterials Corporation), Cytotrans Granule (made of carbonated apatite, manufactured by GC Corporation), and Refit (made of collagen / apatite, manufactured by HOYA Corporation).

[0107] In the kit of the present disclosure, the osteogenic composition and the scaffold material may exist independently or integrally. In the latter case, the kit of the present disclosure can also be referred to as, for example, an osteogenic scaffold material. When the osteogenic composition and the scaffold material are integrally formed, the osteogenic composition may be, for example, adsorbed onto the surface of the scaffold material or contained within the scaffold material.

[0108] The kit of the present disclosure may contain, for example, a physiologically active substance in addition to the osteogenic composition. The physiologically active substance is, for example, a substance that promotes bone formation or the differentiation of osteoblasts or osteocytes. Examples of the physiologically active substance include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), growth differentiation factor-5 (GDF5), erythropoietin (EPO), transforming growth factor (TGF), and bone morphogenetic protein (BMP). One type of physiologically active substance may be used alone, or multiple types may be used in combination.

[0109] The kit of the present disclosure can be used, for example, by transplanting (embedding or embedding) the osteogenic composition and scaffold material into the desired location in the subject where bone formation is to be induced. Specifically, when the composition and the scaffold material are separate in the kit of the present disclosure, the kit can be used by impregnating the osteogenic composition into the scaffold material and then implanting the scaffold material. Furthermore, when the osteogenic composition and the scaffold material are separate in the kit of the present disclosure, the scaffold material may be implanted and then the osteogenic composition may be introduced into the implantation site. Furthermore, when the osteogenic composition and the scaffold material are integrated in the kit of the present disclosure, the kit can be used by implanting the scaffold material.

[0110] <Bone formation method> The bone formation method of the present disclosure uses the bone formation composition of the present disclosure. According to the bone formation method of the present disclosure, for example, bone formation can be induced. The bone formation method of the present disclosure can be applied to the explanations of the bone formation composition and bone formation kit of the present disclosure.

[0111] The bone formation method of the present disclosure includes, for example, an administration step of administering the bone forming composition of the present disclosure to a recipient. When bone formation is performed in vivo using the bone formation method of the present disclosure, the administration site of the bone forming composition of the present disclosure can be the desired site for bone formation. In the bone formation method of the present disclosure, the recipient and administration conditions can be the same as those described for the bone forming composition.

[0112] In the bone formation method of the present disclosure, the bone formation kit of the present disclosure may be used as the bone formation composition of the present disclosure.

[0113] <Use> The present disclosure relates to use of an osteogenic composition comprising hypertrophic megakaryocyte cells and / or an extracellular secretion product derived from hypertrophic megakaryocyte cells for use in bone formation. The present disclosure relates to use of an osteogenic composition comprising hypertrophic megakaryocyte cells and / or an extracellular secretion product derived from hypertrophic megakaryocyte cells for producing an osteogenic composition. For example, the above-described descriptions of the osteogenic composition, osteogenic kit, and osteogenic method of the present disclosure can be used in conjunction with the present disclosure.

[0114] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the aspects described in the examples.

[0115] Example 1 The osteogenic composition of the present disclosure was produced, and it was confirmed that the osteogenic composition of the present disclosure exhibited osteogenic effects.

[0116] (1) Preparation of Immortalized Megakaryocytes Immortalized megakaryocytes were prepared by the following procedure. Immortalized megakaryocytes were used as megakaryocytes before multinucleation. The immortalized megakaryocytes were prepared by the following procedure.

[0117] (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 6 below. Specifically, human iPS cell colonies maintained on laminin 511-E8 (Nippi) were cultured overnight in the presence of 50 ng / ml activin A (WAKO) and 3 μM CHIR99021 (WAKO), and then cocultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / mL VEGF (R&D SYSTEMS) for 14 days to generate hematopoietic progenitor cells (HPCs). The culture conditions for iPS cell maintenance and the first 7 days of HPC induction were 37°C, 5% O. ２ , 5% CO ２ and then at 37°C, 20% O ２ , 5% CO ２ Reference 6: Takayama N. et al., "Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells", J. Exp. Med., 2010, vol.13, pages 2817-2830

[0118] (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. The vectors were 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 2, supra, and Reference 7, 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 7: 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

[0119] (1-3) Induction of Megakaryocytic Cell Lines by Introduction of Three Genes into Hematopoietic Progenitor Cells The HPCs prepared in Example 1 (1-1) above were infected with the three lentiviruses prepared in Example 1 (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) antibody, 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, equivalent to megakaryocytic cells prior to multinucleation) and used for further studies.

[0120] (1-4) Growth and culture of megakaryocytic cell lines The resulting MKCLs were grown in 10 cm dishes (10 ml / dish) or 125 ml shake flasks. IMDM (Sigma Aldrich #I3390) was used as the base medium, to which the following components were added (concentrations are final concentrations in the growth medium). The culture conditions were 37°C, 5% CO ２ The shaking speed was set at 100 rpm using a 19 mm diameter shaker. The following ingredients were used: 15% FBS (Hyclone #SH30071.03, fetal bovine serum); 2 mmol / L Glutamax (GIBCO #3505-061); 2 mmol / L ITS-G (Gibco #41400-045) diluted 100 times; 450 μmol / L MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157); 50 μg / mL ascorbic acid (NIPRO #49871900040329); 50 ng / mL SCF (Wako Pure Chemical #193-15513); 200 ng / mL TPO-like substance; 1 μg / mL doxycycline (DOX) (clontech #631311).

[0121] (2) Induction of Multinucleation of Immortalized Megakaryocytes Next, multinucleation of immortalized megakaryocytes was induced by culturing in a medium supplemented with an AhR inhibitor, a ROCK inhibitor, a myosin 2 inhibitor, and harmine in addition to DOX. Specifically, the immortalized megakaryocyte cell line obtained in Example 1(1) was cultured in the following multinucleation medium at a concentration of 3.0 × 10 ５ The cells were suspended at 25 ml / flask in 125 ml Erlenmeyer flasks and cultured for 3 days with shaking at 100 rpm under the conditions of 37°C and 5% CO. ２ It was decided.

[0122] (Polynucleation medium) 10% human AB serum (Access Biologicals) Glutamax (GIBCO #3505-061) 2mmol / L ITS-G (Gibco #41400-045) 100x diluted MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157) 450μmol / L ascorbic acid (NIPRO #49871900040329) 50μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO mimetic 200ng / ml Doxycycline (clontech #631311) 1μg / ml aryl hydrocarbon receptor (AhR) inhibitor GNF351 (Calbiochem #182707) 500nmol / l ROCK inhibitor Y-39983 (Medchemexpress) #MCH-HY-13300-10) 500nmol / l Harmine (Sigma Aldrich #286044-1G) 5μmol / l Myosin 2 inhibitor (-)-Blebbistatin (Medchem Express #HY-13441) 10μmol / l

[0123] (3) Induction of Hypertrophic Megakaryocytes Next, the polynucleated megakaryocytes were further cultured to induce hypertrophic megakaryocytes from the polynucleated megakaryocytes. Specifically, the polynucleated megakaryocytes obtained in Example 1(2) were cultured in the following hypertrophy medium at a concentration of 1.0 × 10 5The cells were suspended at 25 ml / flask in 125 ml Erlenmeyer flasks and cultured for 2 or 3 days with shaking at 100 rpm under the conditions of 37°C and 5% CO ２ Next, the culture medium containing the hypertrophied megakaryocytes was centrifuged at 300 × g for 5 minutes, and the precipitate was suspended in 30 ml of a washing preservative solution (2.5% human serum albumin, 20% ACD-A solution, bicanate infusion (pH 7.3)). After the suspension, the cells were irradiated with X-rays at 25 Gy to inhibit the proliferation of the hypertrophied megakaryocytes. After the physical stimulation, the hypertrophied megakaryocytes were stored at 4°C. (Hypertrophy medium) 10% human AB serum (Access Biologicals) Glutamax (GIBCO #3505-061) 2mmol / L ITS-G (Gibco #41400-045) 100x diluted MTG (monothioglycerol, Fujifilm Wako Pure Chemical #195-157) 450μmol / L Ascorbic acid (NIPRO #49871900040329) 50μg / mL SCF (Wako Pure Chemical #193-15513) 50ng / mL TPO mimetic 200ng / mL Aryl 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

[0124] (4) In Vivo Study of Bone Formation and Bone Augmentation at Skull Defect Sites We investigated whether the hypertrophied megakaryocytes of the present disclosure contribute to bone formation and bone augmentation at skull defect sites in vivo. Specifically, first, samples were prepared using the hypertrophied megakaryocytes (cultured for 2 days) obtained in Example 1(3). The cell suspension containing the hypertrophied megakaryocytes was centrifuged at 300 × g for 5 minutes. After the centrifugation, the hypertrophied megakaryocytes were suspended in physiological saline to prepare a preparation solution. After the preparation, 1.0 × 10 ６A cell-equivalent preparation solution was added to an artificial bone material (octacalcium phosphate (OCP) / Collagen (Col), Bonarc®). Next, a 4 mm diameter 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 outside the defect site (Example). For the negative control group, transplantation was performed in the same manner, except that the artificial bone material was impregnated with PBS. For the reference example, transplantation was performed in the same manner, except that the artificial bone material was impregnated with a composition (25 mg / ml iMDF) prepared from a platelet-removed megakaryocyte culture. The platelet-removed megakaryocyte culture was prepared using the same number of megakaryocytes, following the method described in International Publication No. 2022 / 092169. Four weeks after the transplantation, the artificial bone material, including the surrounding skull, was removed, and the excised area was subjected to μCT imaging. The bone mass of the newly formed bone from the artificial bone material was also evaluated. In the evaluation, 3D bone analysis software (TRI / 3D-BON; manufactured by Ratoc System Engineering) was used to measure the bone mass (BV (cm 3 The results are shown in Figures 1 and 2.

[0125] Figure 1 is a photograph showing bone formation 4 weeks after transplantation. From left to right in Figure 1, photographs show the results of the negative control group, Reference Example, and Example. In Figure 1, the upper row is a photograph showing the results of the right side of the skull defect site, and the lower row is a photograph showing the results of the left side of the skull defect site. As shown in Figure 1, compared to the negative control group, new bone was formed at the skull defect site in the Reference Example and Example.

[0126] Figure 2 is a graph showing bone mass four weeks after transplantation. In Figure 2, the vertical axis represents bone mass (BV (cm ３)), and the horizontal axis indicates the type of experimental group. As shown in Figure 2, bone mass increased in the Reference Example and Examples compared to the negative control group. Furthermore, bone mass increased in the Examples compared to the Reference Example. These results demonstrate that the composition containing hypertrophied megakaryocytes of the present disclosure can induce and promote bone formation.

[0127] (5) Preparation of Secretome Derived from Hypertrophic Megakaryocytes Hypertrophic megakaryocytes were used to prepare secretome derived from hypertrophic megakaryocytes. ５ Citric acid (ACD-A) was added to 300 ml of culture medium containing 1000 cells / ml of hypertrophied megakaryocytes (cultured for 2 days or 3 days), and the mixture was centrifuged for 20 minutes at 2000 x g. After the centrifugation, the hypertrophied megakaryocytes were collected and suspended in citrate / Ringer's solution. After the suspension, the mixture was centrifuged for 20 minutes at 2000 x g. After the centrifugation, the hypertrophied megakaryocytes were collected and suspended in 15-20 ml of DMEM, and the resulting mixture was centrifuged at 1-2 x 10 ６ The concentration was adjusted to 1000 cells / ml. Subsequently, the hypertrophied megakaryocytes were physically stimulated using 60 Gy X-ray irradiation and glass beads (Cell-Aid, manufactured by JMS Co., Ltd.). After the physical stimulation, the cells were centrifuged for 20 minutes at 2000 × g. After the centrifugation, filtration was performed. A 0.8 μm filter and a 0.22 μm filter were used for the filtration. After the filtration, the culture medium was ultraconcentrated at 100 kJ / ml to obtain a secretome preparation derived from hypertrophied megakaryocytes. Hereinafter, secretomes derived from hypertrophied megakaryocytes cultured for 2 days are also referred to as Day 2 secretomes, and secretomes derived from hypertrophied megakaryocytes cultured for 3 days are also referred to as Day 3 secretomes.

[0128] (6) In Vivo Study of Bone Formation and Bone Augmentation at Skull Defect Sites by Secretomes Derived from Hypertrophied Megakaryocytes We investigated whether the secretomes derived from hypertrophied megakaryocytes of the present disclosure contribute to bone formation and bone augmentation at skull defect sites in vivo. Specifically, instead of the hypertrophied megakaryocytes, 20 μl of Day 2 secretomes or Day 3 secretomes (1.6 × 10 ５ ~1.8 x 10 ５ The artificial bone material was transplanted into wild-type rats in the same manner as in Example 1 (4), except that the artificial bone material was impregnated with PBS. 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 μCT images were taken of the extracted area. The bone mass of the newly formed bone from the artificial bone material was also evaluated. In the evaluation, 3D bone analysis software (TRI / 3D-BON; manufactured by Ratoc System Engineering) was used to measure bone mass (BV (cm 3 The results are shown in Figures 3 and 4.

[0129] Figure 3 is a photograph showing bone formation 4 weeks after transplantation. In Figure 3, the upper row shows the negative control group, the middle row shows the group using Day 2 secretome, and the lower row shows the group using Day 3 secretome. As shown in Figure 3, compared to the negative control group, new bone was formed at the skull defect site in the groups using Day 2 secretome and Day 3 secretome.

[0130] Figure 4 is a graph showing bone mass 4 weeks after implantation. In Figure 4, the vertical axis represents bone mass (BV (cm ３ )), and the horizontal axis indicates the type of experimental group. As shown in Figure 4, bone mass increased in the group using Day 2 secretome and the group using Day 3 secretome, compared to the negative control group. These results demonstrate that the composition of the present disclosure containing secretome derived from hypertrophied megakaryocytes can induce and promote bone formation.

[0131] (7) Effect of secretome derived from hypertrophied megakaryocytes on bone marrow mesenchymal stem cells The effect of secretome derived from hypertrophied megakaryocytes of the present disclosure on bone marrow mesenchymal stem cells was investigated. ４ Bone marrow mesenchymal stem cells (MSCs) were seeded onto a 24-well plate and cultured until 80% confluent. After the culture, Day 3 secretome or osteoblast differentiation induction medium (DGA) prepared in Example 1(5) was added. 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).

[0132] The cell count was measured on days 0, 1, 5, and 10, based on the start of the culture. 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 results are shown in Figure 5.

[0133] Figure 5 is a graph showing cell counts. In Figure 5, the horizontal axis represents the number of days since the start of culture of bone marrow mesenchymal stem cells, and the vertical axis represents cell counts. As shown in Figure 5, when DGA was added, the increase in bone marrow mesenchymal stem cell counts was suppressed and remained stable, whereas when Day 3 secretome was added, the bone marrow mesenchymal stem cell counts increased. Furthermore, it was found that the bone marrow mesenchymal stem cell count increased in a concentration-dependent manner of Day 3 secretome. These results demonstrate that the secretome derived from hypertrophied megakaryocytes of the present disclosure promotes the proliferation of bone marrow mesenchymal stem cells.

[0134] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0135] This application claims priority based on Japanese Patent Application No. 2024-019962, filed February 14, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0136] <Appendix> Some or all of the above embodiments and examples can be described as in the appendix below, but are not limited to the following. <Osteogenic Composition> (Appendix 1) An osteogenic composition comprising hypertrophic megakaryocytes and / or extracellular secretions derived from hypertrophic megakaryocytes. (Appendix 2) The osteogenic composition according to appendix 1, wherein the hypertrophic megakaryocytes contain α granules. (Appendix 3) The osteogenic composition according to appendix 1 or 2, wherein the hypertrophic megakaryocytes have an expression level of Ki-67 and / or MYC that is 50% or less of the expression level of Ki-67 and / or MYC in multinucleated megakaryocytes. (Appendix 4) The osteogenic composition according to any one of Appendices 1 to 3, wherein the hypertrophic megakaryocytes are a cell population containing the hypertrophic megakaryocytes, the hypertrophic megakaryocytes include hypertrophic megakaryocytes with a nuclear phase of 16N or more, and the proportion (cell number) of the hypertrophic megakaryocytes of 16N or more in the cell population containing the hypertrophic megakaryocytes is 20% or more. (Appendix 5) The osteogenic composition according to Appendices 4, wherein the proportion (cell number) of the hypertrophic megakaryocytes of 16N or more in the cell population is 20 to 90%. (Appendix 6) The osteogenic composition according to Appendices 5, wherein the proportion (cell number) of the hypertrophic megakaryocytes of 16N or more in the cell population is 20 to 70%. (Appendix 7) The osteogenic composition according to any one of Appendices 1 to 6, wherein the hypertrophic megakaryocytes are a cell population containing the hypertrophic megakaryocytes, the hypertrophic megakaryocytes comprising hypertrophic megakaryocytes having a nuclear phase of 32N or greater, and the proportion (cell number) of the hypertrophic megakaryocytes of 32N or greater in the cell population is 5 to 50%. (Appendix 8) The osteogenic composition according to Appendices 7, wherein the hypertrophic megakaryocytes comprise hypertrophic megakaryocytes having a nuclear phase of 32N or greater, and the proportion (cell number) of the hypertrophic megakaryocytes of 32N or greater in the cell population is 5 to 40%.(Appendix 9) The osteogenic composition according to any one of Appendices 1 to 8, wherein the hypertrophic megakaryocytes are produced by a production method comprising: a multinucleation step of multinucleating pre-multinucleated megakaryocytes or their precursor cells to induce multinucleated megakaryocytes; and a hypertrophy step of maturing the multinucleated megakaryocytes to induce hypertrophic megakaryocytes, wherein in the multinucleation step, the activity of at least one selected from the group consisting of aryl hydrocarbon receptor (AhR), Rho-associated kinase (ROCK), dual specificity tyrosine phosphorylation-regulated kinase (DYRK), and myosin 2 is inhibited, and the pre-multinucleated megakaryocytes are multinucleated in the presence of harmine to induce multinucleated megakaryocytes. (Appendix 10) The osteogenic composition according to Appendix 9, wherein in the multinucleation step, the pre-multinucleated megakaryocyte cells or their precursor cells are cultured to multinucleate in the presence of an AhR inhibitor, a myosin 2 inhibitor, and harmine, thereby inducing multinucleated megakaryocytes. (Appendix 11) The osteogenic composition according to Appendix 9 or 10, wherein the production method includes a proliferation step of proliferating the pre-multinucleated megakaryocyte cells, and in the proliferation step, the pre-multinucleated megakaryocyte cells are proliferated without suppressing the activities of AhR, ROCK, DYRK, and myosin 2, and in the absence of harmine. (Appendix 12) The osteogenic composition according to Appendix 11, wherein the multinucleation step is carried out after the proliferation step. (Appendix 13) The osteogenic composition according to any one of Appendices 1 to 12, comprising a culture medium of the hypertrophic megakaryocytic cells, wherein the culture medium contains the hypertrophic megakaryocytic cells and / or extracellular secretions derived from the hypertrophic megakaryocytic cells. (Appendix 14) The osteogenic composition according to Appendices 13, comprising a concentrate of the culture medium. (Appendix 15) The osteogenic composition according to any one of Appendices 1 to 14, comprising isolated extracellular vesicles. (Appendix 16) The osteogenic composition according to any one of Appendices 1 to 15, wherein the hypertrophic megakaryocytic cells are hypertrophic megakaryocytic cells induced in vitro. (Appendix 17) The osteogenic composition according to any one of Appendices 1 to 16, wherein the hypertrophic megakaryocytic cells are derived from immortalized megakaryocytes. (Appendix 18) The osteogenic composition according to any one of Appendices 1 to 17, wherein the hypertrophic megakaryocytic cells contain an exogenous oncogene, polycomb gene, and / or apoptosis inhibitor gene.(Appendix 19) The osteogenic composition according to any one of Appendices 1 to 18, which has cell proliferation-promoting activity. (Appendix 20) The osteogenic composition according to Appendices 19, wherein the cells are mesenchymal stem cells. (Appendix 21) The osteogenic composition according to Appendices 20, wherein the mesenchymal stem cells are precursor cells of osteoblasts. <Osteogenic Kit> (Appendix 22) An osteogenic kit comprising an osteogenic composition and an osteogenic scaffold material, wherein the osteogenic composition is the osteogenic composition according to any one of Appendices 1 to 21. (Appendix 23) The osteogenic kit according to Appendices 22, wherein the scaffold material is biocompatible and / or biodegradable. (Appendix 24) The kit according to Appendices 22 or 23, wherein the scaffold material is at least one selected from the group consisting of collagen, gelatin, albumin, keratin, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, polyglycolic acid, polylactic acid, copolymers of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polycaprolactone, polybutylene succinate, calcium phosphate, hydroxyapatite, polyether ketone, and polyether ether ketone. (Appendix 25) The kit according to any of Appendices 22 to 24, wherein the scaffold material has a porous structure. (Appendix 26) The kit according to any of Appendices 22 to 25, further comprising a physiologically active substance. (Appendix 27) The kit according to any one of Appendices 22 to 26, comprising at least one selected from the group consisting of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), growth differentiation factor-5 (GDF5), erythropoietin (EPO), transforming growth factor (TGF), and bone morphogenetic protein. <Bone formation method> (Appendix 28) A bone formation method using the bone forming composition according to any one of Appendices 1 to 21. (Appendix 29) The bone formation method according to Appendices 28, comprising an administration step of administering the bone forming composition to a subject. (Appendix 30) The bone formation method according to Appendices 28 or 29, using the bone forming composition in vitro or in vivo.<Use> (Appendix 31) Use of the osteogenic composition according to any one of Appendices 1 to 21 for bone formation.

[0137] As described above, the present disclosure provides an osteogenic composition containing secretomes and / or extracellular vesicles derived from hypertrophic megakaryocytes. Therefore, the present disclosure is extremely useful in, for example, the fields of regenerative medicine and cosmetics.

Claims

1. An osteogenic composition comprising hypertrophic megakaryocyte cells and / or extracellular secretions derived from hypertrophic megakaryocyte cells.

2. The osteogenic composition of claim 1, wherein the hypertrophic megakaryocyte cells contain alpha granules.

3. The osteogenic composition according to claim 1 or 2, wherein the hypertrophic megakaryocytes have an expression level of Ki-67 and / or MYC of 50% or less of the expression level of Ki-67 and / or MYC in multinucleated megakaryocytes.

4. The bone-forming composition according to any one of claims 1 to 3, wherein the hypertrophic megakaryocytes are a cell population containing the hypertrophic megakaryocytes, the hypertrophic megakaryocytes include hypertrophic megakaryocytes having a nuclear phase of 16N or more, and the proportion (cell number) of the hypertrophic megakaryocytes having a nuclear phase of 16N or more in the cell population containing the hypertrophic megakaryocytes is 20% or more.

5. The bone-forming composition according to any one of claims 1 to 4, wherein the hypertrophic megakaryocytes are a cell population containing the hypertrophic megakaryocytes, the hypertrophic megakaryocytes include hypertrophic megakaryocytes with a nuclear phase of 32N or more, and the proportion (cell number) of hypertrophic megakaryocytes with a nuclear phase of 32N or more in the cell population is 5 to 50%.

6. The osteogenic composition according to any one of claims 1 to 5, wherein the hypertrophic megakaryocytic cells are produced by a production method comprising: a multinucleation step of multinucleating pre-multinucleated megakaryocytic cells or their precursor cells to induce multinucleated megakaryocytic cells; and a hypertrophy step of maturing the multinucleated megakaryocytes to induce hypertrophic megakaryocytic cells, wherein in the multinucleation step, the activity of at least one selected from the group consisting of aryl hydrocarbon receptor (AhR), Rho-associated kinase (ROCK), dual specificity tyrosine phosphorylation-regulated kinase (DYRK), and myosin 2 is inhibited, and the pre-multinucleated megakaryocytic cells are multinucleated in the presence of harmine to induce multinucleated megakaryocytic cells.

7. The osteogenic composition described in claim 6, wherein in the multinucleation process, the pre-multinucleated megakaryocyte cells or their precursor cells are cultured in the presence of an AhR inhibitor, a myosin 2 inhibitor and harmine to multinucleate the cells and induce multinucleated megakaryocytes.

8. The osteogenic composition according to claim 6 or 7, wherein the production method includes a proliferation step of proliferating the pre-multinucleated megakaryocyte cells, and in the proliferation step, the pre-multinucleated megakaryocyte cells are proliferated without suppressing the activity of AhR, ROCK, DYRK, and myosin 2, and in the absence of harmine.

9. The osteogenic composition according to claim 8, wherein the multinucleation step is carried out after the proliferation step.

10. An osteogenic composition according to any one of claims 1 to 9, comprising a culture medium for the hypertrophic megakaryocyte cells, the culture medium containing the hypertrophic megakaryocyte cells and / or extracellular secretions derived from the hypertrophic megakaryocyte cells.

11. The osteogenic composition of claim 10, comprising a concentrate of said culture medium.

12. An osteogenic composition according to any one of claims 1 to 11, comprising isolated extracellular vesicles.

13. The osteogenic composition according to any one of claims 1 to 12, wherein the hypertrophic megakaryocyte cells are in vitro induced hypertrophic megakaryocyte cells.

14. The osteogenic composition of any one of claims 1 to 13, wherein the hypertrophic megakaryocyte cells are derived from immortalized megakaryocytes.

15. The osteogenic composition of any one of claims 1 to 14, wherein the hypertrophic megakaryocytic cells contain an exogenous oncogene, polycomb gene, and / or apoptosis-inhibiting gene.

16. An osteogenic composition according to any one of claims 1 to 15, having cell proliferation promoting activity.

17. The osteogenic composition of claim 16, wherein the cells are mesenchymal stem cells.

18. An osteogenic kit comprising an osteogenic composition and an osteogenic scaffold material, wherein the osteogenic composition is the osteogenic composition described in any one of claims 1 to 17.

19. The osteogenic kit according to claim 18, wherein the scaffold material is biocompatible and / or biodegradable.

20. The kit of claim 18 or 19, wherein the scaffold material is composed of at least one selected from the group consisting of collagen, gelatin, albumin, keratin, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, polyglycolic acid, polylactic acid, copolymers of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polycaprolactone, polybutylene succinate, calcium phosphate, hydroxyapatite, polyether ketone, and polyether ether ketone.

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