Method for producing osteoblast-containing cell mass

Abstract

The purpose of the present invention is to three-dimensionally culture osteoblasts produced with a direct conversion technique, and to provide a three-dimensional bone-like tissue composed of the osteoblasts and bone matrix. The present invention relates to, for example, a method for producing an osteoblast-containing three-dimensional cell mass, comprising: a first step for performing direct conversion from somatic cells of mammals into osteoblasts; a second step for subjecting cells having undergone the first step to high-density culture, and forming a cell sheet containing the osteoblasts; and a third step for obtaining an osteoblast-containing three-dimensional cell mass from the cell sheet obtained in the second step.

Description

Method for producing osteoblast-containing cell aggregates

[0001] The present invention relates to a method for producing a cell mass containing osteoblasts, particularly a cell mass containing osteoblasts obtained by a direct conversion technique. The present invention also relates to a method for preparing a three-dimensional bone-like tissue and an inducer thereof.

[0002] Regenerative medicine is attracting attention as a new technology for treating dysfunctional or dysfunctional tissues and organs by utilizing in vivo cells to regenerate lost functions, potentially bringing about new medical treatments for disorders and diseases for which there were no previous treatments. For example, the prevalence of osteoporosis is increasing with aging, and fractures in elderly people can lead to prolonged bed rest. Transplanting osteoblasts into bone defects in these diseases is expected to promote healing of traumatic fractures, intractable fractures, and pseudofractures. Furthermore, transplanting osteoblasts into the lesions for the purpose of repairing bone defects associated with bone resorption due to various inflammations, such as osteomyelitis, periodontal disease, and dental caries, as well as bone defects following bone tumor ablation, may promote bone formation and improve functional and morphological outcomes. Diseases and conditions for which osteoblast transplantation therapy is useful include osteoporosis (a disease in which a decrease in bone density increases the risk of fracture; osteoblast transplantation promotes bone formation), osteonecrosis (diseases such as femoral head necrosis in which blood flow is cut off and bone dies; osteoblast transplantation helps regenerate bone at the necrotic site), fracture repair failure (when a fracture does not heal normally, osteoblast transplantation promotes bone fusion), spinal fusion (surgery to restore spinal stability; osteoblast transplantation enhances bone formation at the fusion site), and congenital osteogenesis imperfecta (a genetic disease in which bones are very fragile and break easily; osteoblast transplantation improves bone strength).

[0003] Periodontal disease, also known as the fourth lifestyle-related disease, is an inflammatory disease caused by bacterial infection that dissolves the gums around the teeth and the bones that support them. It has a high prevalence and is a cause of various systemic diseases. As periodontal disease progresses, alveolar bone resorption occurs, making bone tissue regeneration extremely difficult and often leading to tooth loss. Similarly, in inflammatory diseases at the roots of teeth, such as dental caries, inflammation-induced bone resorption progresses at the roots of the teeth, reducing the tooth's scaffolding in the jawbone and leading to tooth loss. Osteoblast transplantation therapy is considered as a bone augmentation treatment to create the tooth scaffolding (jawbone augmentation) necessary for subsequent implant treatment.

[0004] If osteoblasts can be efficiently transplanted into the localized bone resorption site for diseases and conditions for which osteoblast transplantation therapy is effective, it may lead to bone regeneration therapy. Furthermore, if osteoblast transplantation is combined with bone transplantation or artificial bone transplantation, the therapeutic effect may be enhanced.

[0005] In regenerative medicine, the means of supplying the cells used (e.g., invasiveness to humans during cell collection, cell recovery methods after culture, formulation methods, and the form of cell products intended for human administration) are one of the major challenges for practical application. For example, bone marrow-derived mesenchymal stem cells and bone marrow cells containing bone marrow-derived mesenchymal stem cells have been used as osteoblasts for transplantation. However, bone marrow cell collection is highly invasive to patients and has problems such as the inability to supply sufficient numbers of bone marrow cells. On the other hand, the use of human embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) eliminates the need to collect bone marrow cells from patients and potentially provides a sufficient number of osteoblasts. For example, Non-Patent Document 1 describes a method for inducing differentiation of induced pluripotent stem cells (iPS cells) derived from cynomolgus monkeys into chondrocytes. Furthermore, ES cells, which have the same pluripotency, pose ethical issues. If even a small number of undifferentiated cells are present in these ES cells and iPS cells during the multi-step manufacturing process that induces differentiation into osteoblasts, these undifferentiated cells are expected to become cancerous, and if even a small number of undifferentiated cells are mixed into osteoblasts induced from ES cells or iPS cells, there is a risk that they will become tumorigenic in the body after transplantation.

[0006] Furthermore, methods for inducing differentiation of mesenchymal stem cells or mesenchymal stromal cells (MSCs) into cell clusters containing osteoblasts have been reported (Non-Patent Document 2, Patent Document 5, and Patent Document 6). Patent Document 5 discloses an implant and a method for producing the same, in which MSCs are cultured three-dimensionally in a growth medium containing a factor that causes MSCs to produce collagen, to form cell clusters, and the cell clusters are then frozen. Patent Document 6 discloses a method for producing three-dimensional bone-like tissue consisting of a mineral-deposited bone matrix and encapsulated osteocytes by culturing three-dimensionally cultured MSC clusters embedded in an artificial gel using an osteoblast medium. However, the use of MSCs has several unresolved issues, including (1) the significant invasiveness to patients, such as the harvesting of bone marrow or adipose tissue, (2) the difficulty of culturing them while maintaining pluripotency, (3) variations in bone differentiation potential between patient cell lots, and (4) the carriers (artificial scaffolding materials) used for MSC transplantation are artificial foreign bodies similar to bone fillers, and they have metabolic and absorption issues, undesirable inflammatory responses, and incompatibility with material quality and safety standards. In addition, cell aggregates induced from MSCs cannot be induced to differentiate into mature osteoblasts, and remain as cell groups (such as precursor osteoblasts or immature osteoblasts) that retain the differentiation potential of MSCs while they are still in the process of differentiating into osteoblasts.

[0007] Techniques for direct conversion of somatic cells such as fibroblasts into osteoblasts are known (Non-Patent Document 3, Patent Documents 1 to 4). Patent Document 1 discloses a method for preparing osteoblasts by introducing reprogramming-related genes or expression products, including the OCT4 gene, into somatic cells and culturing them in a differentiation-inducing medium that causes differentiation into osteoblasts. Patent Document 2 discloses a method for preparing osteoblasts by culturing differentiated mammalian somatic cells, including fibroblasts, in a medium in the presence of at least one compound selected from the group consisting of (1) a statin compound, (2) a casein kinase 1 inhibitor, (3) a cAMP inducer, and (4) a histone methyltransferase inhibitor. Patent Document 3 discloses a method for converting somatic cells into osteoblasts by introducing a gene group, including the OCT9 gene or expression products, into somatic cells. Patent Document 4 discloses a method for preparing various cells by direct conversion from somatic cells and an inducer for converting them into osteoblasts. It is difficult to recover osteoblasts in a state that is useful for cell therapy (efficiently recovering viable osteoblasts and recovering mature osteoblasts in an active state). Taking this into consideration, a method for producing active mature osteoblasts by converting them into a cylindrical artificial scaffold (Non-Patent Document 4) has been reported. However, when using an artificial scaffold material, an artificial object whose effects on the body (especially safety) are unknown is introduced into the body, so safety for humans must be ensured. Therefore, selecting an artificial material as a scaffold material for osteoblasts produced by direct conversion technology poses challenges for practical application of cell therapy, such as verifying the safety of the artificial scaffold material and the manufacturing costs.

[0008] Patent No. 6516672 Patent No. 6705115 Patent No. 6941868 Patent No. 7475723 Patent No. 7105487 WO2020 / 226043

[0009] Abe K et al, Nature Communications 14: 804, 2023Kittaka M et al., International Society for Cellular Therapy; Cytotherapy, 2015, 17, 860-873Yamamoto K et al, Scientific Reports 8: 8463, 2018Nakai K et al., Front Bioeng Biotechnol. 2021 Sep 3;9:713932

[0010] The present invention aims to provide a three-dimensional bone-like tissue consisting of osteoblasts and bone matrix by three-dimensionally culturing osteoblasts produced by direct conversion technology. It also aims to provide a method for producing osteoblasts that can recover osteoblasts in sufficient numbers to enable transplantation and in a state useful as a cell therapy. It also aims to provide a technology for repairing extensive fractures and bone destruction by producing three-dimensional bone-like tissue using the osteoblast-containing cell masses obtained by direct conversion technology.

[0011] In light of the above-mentioned problems, the present inventors have used a method they have developed to directly convert somatic cells such as fibroblasts into osteoblasts by direct conversion. By converting fibroblasts into osteoblasts in three steps, they have succeeded in obtaining cell masses containing osteoblasts, using extracellular matrix proteins (including bone matrix) produced by the directly converted osteoblasts as a scaffold. Furthermore, they have surprisingly discovered that the osteoblasts form a "cell-extracellular matrix structure" with the extracellular matrix proteins they themselves produce, resulting in cell masses containing an extracellular microenvironment that allows infiltration of oxygen and nutrients from culture medium components and allows osteoblasts to properly function. They have successfully produced three-dimensional bone-like tissue using the cell masses, thereby completing the present invention. Specifically, the present invention provides the following:

[0012] [1] A method for producing a three-dimensional cell mass containing osteoblasts, comprising the step of obtaining a three-dimensional cell mass containing osteoblasts from a cell sheet obtained by high-density culturing of cells to form a cell sheet. [1A] A method for producing a three-dimensional cell mass containing osteoblasts (referred to as a three-dimensional bone-like tissue or 3D-DCob), comprising the step of obtaining a three-dimensional cell mass containing osteoblasts from a cell sheet obtained by high-density culturing of a cell population containing osteoblasts to form a cell sheet containing osteoblasts. [2] The method described in [1], comprising: a first step of culturing mammalian somatic cells under conditions that convert the somatic cells into osteoblasts while increasing the number of somatic cells; a second step of culturing the cells that have undergone the first step at high density under conditions that convert the somatic cells into osteoblasts to form a cell sheet; and a third step of obtaining a three-dimensional cell mass containing osteoblasts from the cell sheet obtained in the second step. [2A] The method of [1A], comprising: a first step of performing direct conversion from mammalian somatic cells to osteoblasts under culture conditions that convert the somatic cells into osteoblasts while expanding them; a second step of high-density culturing the cells that have undergone the first step to form a cell sheet containing osteoblasts; and a third step of obtaining a three-dimensional cell mass containing osteoblasts from the cell sheet obtained in the second step. [3] The method of [2] or [2A], further comprising, before the first step (step 0), a step of culturing the somatic cells under conditions that do not induce direct conversion. [4] The culture in the first step is performed at a seeding density of 1,000 to 32,000 cells / cm. ２ [5] The method according to [2], [2A] or [3], wherein the high-density culture or the high-density culture in the second step is carried out at a seeding density of 20,000 to 400,000 cells / cm. ２The method according to any one of [1] to [4] and [2A], wherein the period of the 0th step is 1 to 7 days. [6] The method according to [3], wherein the period of the 1st step is 1 to 20 days. [7] The method according to any one of [2] to [6] and [2A], wherein the period of the 1st step is 1 to 20 days. [8] The method according to any one of [2] to [7] and [2A], wherein the period of the 2nd step is 1 to 20 days. [9] The method according to any one of [2] to [8] and [2A], wherein the period of the 3rd step is 3 days or more, preferably 3 to 100 days.

[10] The method according to any one of [3] to [9], wherein the period of the series of steps 0 to 3 is 6 to 147 days.

[11] The method according to any one of [3] to

[10] , wherein the period of the series of steps 0 to 3 is 19 to 75 days.

[12] The method according to any one of [2] to

[11] and [2A], wherein the direct conversion is achieved by the addition of a compound.

[13] The method of

[12] , wherein the compound is at least one selected from the group consisting of (1) a TGF-β pathway inhibitor, (2) a statin compound, (3) a casein kinase 1 inhibitor, (4) a cAMP inducer, (5) a histone methyltransferase inhibitor, and (6) a histone deacetylase inhibitor (HDAC inhibitor).

[14] The method of

[12] or

[13] , wherein the compound is a TGF-β pathway inhibitor.

[15] The method of

[14] , wherein the TGF-β pathway inhibitor is ALK5 inhibitor II.

[16] The method of [2] or [2A], wherein the direct conversion is achieved by gene transfer.

[17] The method of

[16] , wherein the gene is at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc (M), L-Myc (L), GLIS family, Klf family, Lin-28, and Sox2.

[18] The method described in

[16] , wherein the genes are a combination of at least one bone-related gene selected from the group consisting of Runx2 (R), Osterix (O), and Dlx5 (D) and at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc (M), L-Myc (L), GLIS family, Klf family, Lin-28, and Sox2.

[19] The method according to any one of [2] to

[18] and [2A], wherein the mammalian somatic cells are fibroblasts.

[20] The method according to any one of [2] to

[19] and [2A], wherein the medium contains at least one of the following components in at least one of the first, second, and third steps (excluding the compound used in direct conversion): (1) a TGF-β pathway inhibitor, (2) a statin compound, (3) a casein kinase 1 inhibitor, (4) a cAMP inducer, (5) a histone methyltransferase inhibitor, and (6) a histone deacetylase inhibitor (HDAC inhibitor).

[21] The method according to

[20] , wherein the medium contains a histone deacetylase inhibitor in the second step.

[22] The method according to any one of [1] to

[21] and [2A], wherein the three-dimensional cell mass expresses OCN (osteocalcin).

[0013] Three-dimensional bone-like tissue can be created by the method of the present invention, which involves the preparation of a cell sheet by high-density culturing of osteoblasts, followed by suspension culture of the three-dimensional cell clusters. The method of the present invention allows for the production of a sufficient number of osteoblasts in a state useful as a cell therapy (including transplant materials). The resulting osteoblasts can form three-dimensional bone-like tissue. Since the bone-like tissue does not contain cells in the process of differentiation, such as undifferentiated cells, but contains many mature osteoblasts, efficient bone formation and bone regeneration can be achieved using the bone-like tissue. According to the present invention, an effective treatment for the above-mentioned diseases can be provided by producing three-dimensional bone-like tissue containing osteoblasts and transplanting it into a bone defect site. According to the present invention, differentiated somatic cells such as fibroblasts can be cultured in a three-step process combining a direct conversion osteoblast induction method and three-dimensional cell cluster culture to obtain three-dimensional bone-like tissue containing directly converted osteoblasts and bone matrix proteins produced by the osteoblasts, without the use of artificial materials. The osteoblasts used in the method of the present invention may be obtained by a method other than the direct conversion method. Even in such cases, three-dimensional bone-like tissue can be produced by producing a cell sheet through high-density culture and then by suspension culture of the three-dimensional cell aggregates.

[0014] This figure shows a scheme illustrating the steps of the method of the present invention for producing three-dimensional bone-like tissue (3D-DCob) by combining osteoblast induction by direct conversion (DC-OBs induction) and clump culture. HE staining was performed on cell clumps obtained from human fibroblasts through the three-stage culture steps of the present invention. Furthermore, to observe the level of bone matrix protein production, immunostaining was performed for OCN, a protein specifically produced by osteoblasts, and COL1, a type of bone matrix protein. This figure shows the results of HE staining and COL1 / OCN immunostaining. This figure shows a graph (left) showing the results of ELISA measurement of the amount of OCN protein contained in the cell lysate of cell clumps obtained from human fibroblasts through the three-stage culture steps of the present invention, and a graph (right) showing the results of quantification of ALP activity. Cell clumps cultured in osteoblast medium or DC medium were used. Graphs showing the results of measuring the OCN protein amount (top graph) and ALP activity (bottom graph) for cell clumps obtained from human fibroblasts through the four steps (step 0, step 1, step 2, and step 3) of the present invention. The cell clumps were prepared by varying the culture period in step 3 to 15 days (day 15), 16 days (day 16), 25 days (day 25), and 36 days (day 36). Graphs showing the results of measuring the OCN protein amount (top graph) and ALP activity (bottom graph) for cell clumps obtained from human fibroblasts through the four steps (step 0, step 1, step 2, and step 3) of the present invention. The cell clumps were prepared by varying the culture period in step 3 to 22 days (day 22), 29 days (day 29), and 46 days (day 46). HE staining was performed on cell clusters obtained from human fibroblasts through the three-stage culture steps of the present invention. To observe the level of bone matrix protein production, OCN, a protein produced specifically by osteoblasts, and COL1, a type of bone matrix protein, were observed by immunostaining. Figure 1 shows the results of HE staining and COL1 / OCN immunostaining. HE staining was performed on cell clusters obtained from human fibroblasts through the three-stage culture steps of the present invention. To observe the level of bone matrix protein production, OCN, a protein produced specifically by osteoblasts, and COL1, a type of bone matrix protein, were observed by immunostaining.

[0033] Figure 1 shows the results of HE staining and COL1 / OCN immunostaining. HE staining was performed on cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention. Samples were prepared from the obtained cell clumps, and real-time RT-PCR was performed to quantify the mRNA expression levels of osteoblast-specific markers OCN and ALP genes.

[0034] Figure 1 shows the results of HE staining and ALP / OCN mRNA expression (qPCR).

[0035] Figure 1 shows the results of HE staining on cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention (days 5 and 15 of the third step).

[0036] Figure 1 shows the results of TUNEL staining on cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention.

[0037] Figure 1 shows the results of measuring the viable cell count on cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention (days 2 and 15 of the third step). This graph shows the results of measuring the differentiation stage of osteoblasts by qPCR using cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention. The mRNA expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and RUNX2 were quantified as indicators. This graph shows the results of measuring the differentiation stage of osteoblasts by qPCR using cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention. The mRNA expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen (hCOLI) were quantified as indicators. This graph shows the results of drug efficacy evaluation using a pathological animal model. Cell clumps obtained from human fibroblasts by the three-stage culture steps of the present invention were transplanted into a nude rat large calvarial defect model animal. Drug efficacy evaluation was performed by performing μCT tomography using an X-ray CT device and reconstructing 3D images. Fig. 1 shows the results of Alizarin staining of osteoblasts induced from human fibroblasts, and Fig. 2 shows the results of Alizarin staining of osteoblasts induced from human fibroblasts, and the results of quantifying the mRNA expression level of the osteoblast-specific marker OCN by real-time RT-PCR.

[0015] The present invention relates to a method for producing a three-dimensional bone-like tissue (osteoblast-containing cell mass) containing directly converted osteoblasts and bone matrix proteins produced by the osteoblasts (hereinafter also referred to as the method of the present invention), which is characterized by comprising three culture steps. The three steps are: a first step (first step) of promoting the conversion of somatic cells (e.g., fibroblasts) into osteoblasts by two-dimensional plate culture; a second step (second step) of promoting the production of extracellular matrix (ECM) by cells partially containing directly converted osteoblasts by two-dimensional high-density culture, and preparing for three-dimensionalization in the subsequent third step using the autologous ECM as a scaffold; and a third step (third step) of promoting the formation of cell masses containing directly converted osteoblasts to increase the efficiency of osteoblast conversion. In one embodiment of the present invention, a method for producing a three-dimensional bone-like tissue is provided, comprising the steps of: (A) seeding fibroblasts and culturing them in an osteoblast medium in the presence of a compound (e.g., a TGF-β pathway inhibitor) to promote cell proliferation and direct conversion to osteoblasts (corresponding to step 1); (B) seeding cells containing some of the directly converted osteoblasts obtained in the previous step (A) at high density to enhance ECM production required for three-dimensional cell cluster formation (corresponding to step 2); and (C) culturing the cell / ECM complexes from the previous step (B) in suspension to promote the formation of three-dimensional cell clusters through self-aggregation, thereby promoting conversion (corresponding to step 3) (Figure 1). The three-dimensional bone-like tissue obtained by the present invention does not contain artificial materials and contains directly converted osteoblasts and bone matrix proteins produced by those osteoblasts. Thus, one embodiment of the three-dimensional bone-like tissue (3D-DCob) obtained by the present invention is a bone-like tissue characterized by being obtained via direct conversion (DC). The above is a case where a technique for direct conversion of somatic cells into osteoblasts using a compound is used, but a technique for direct conversion of somatic cells into osteoblasts by gene transfer can also be implemented in the same way.In this case, a gene required for direct conversion to osteoblasts (e.g., those described in Patent Documents 1 and 3) is introduced before the first step, and then the first step is performed. In this case, DC induction stimulation (addition of a compound) may be omitted in steps 1 to 3, but a compound may be added depending on the combination of genes to be introduced. Furthermore, the osteoblasts used in the method of the present invention may be obtained by methods other than direct conversion (e.g., a method in which osteoblasts are obtained by culturing MSCs in a medium containing osteoblast-inducing factors). Even in such cases, three-dimensional bone-like tissue (containing osteoblasts and bone matrix proteins produced by osteoblasts) can be produced by producing a cell sheet in high-density culture and subsequent suspension culture of the three-dimensional cell mass. Each step is described in detail below.

[0016] (Production Method of the Present Invention) 1. First Step (Plate Culture) In the production method of the present invention, the first step is a process of carrying out direct conversion from mammalian somatic cells to osteoblasts. The first step begins with seeding mammalian somatic cells at a cell concentration (cell density) that results in confluence before proceeding to the second step.

[0017] <Culture medium for osteoblasts> The direct conversion under culture conditions in the first step is carried out in a culture medium, particularly in a culture medium suitable for maintaining or inducing osteoblasts. The culture medium for maintaining or inducing osteoblasts is not particularly limited, but an osteoblast culture medium is preferred. As the osteoblast culture medium, there are many commercially available media as osteoblast maintenance media or osteoblast induction media, and any of them may be used. Furthermore, when preparing a customized osteoblast medium, a factor used for the maintenance culture of osteoblasts is added to the basal medium, and examples of such a factor (osteoblast culture factor) include one or more components selected from the group consisting of ascorbic acid (for example, a concentration of about 0.1 to 1000 μg / ml, preferably about 1 to 100 μg / ml); β-glycerophosphate (for example, a concentration of about 0.1 to 1000 mM, preferably about 1 to 100 mM); and glucocorticoids such as dexamethasone and hydrocortisone (about 1 nM to 10 mM, preferably about 10 to 1000 nM). Specific examples of osteoblast media include basal synthetic media (DMEM, IMDM, etc.) containing 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone (all at final concentrations) supplemented with serum or serum substitutes (FBS or UltraGro, recombinant albumin, etc.), media commercially available for the maintenance and proliferation of fibroblasts and MSCs, and media customized from basal synthetic media to which the osteoblast culture factors have been added. However, the media are not limited to these. Examples of basal media include DMEM, IMDM, and modified media thereof supplemented with serum, such as FBS (approximately 0.1 to 20%, preferably approximately 1 to 10%), and serum substitutes, such as UltraGro. ＴＭExamples of such basal media include those supplemented with recombinant HSA (human serum albumin: about 0.1 to 20%, preferably about 1 to 5%) (human platelet-derived components), and those supplemented with recombinant HSA (human serum albumin: about 0.1 to 20%, preferably about 1 to 5%). Non-essential amino acids (NEAA; about 1 μM to 1000 μM, preferably about 10 μM to 100 μM) may be added to these basal media (e.g., DMEM medium). Alternatively, commercially available media for the maintenance and proliferation of fibroblasts and MSCs (e.g., Prime-XV MSC XSFM MDF1) may be used. ＴＭ (Prime-MDF)) can also be used. However, the medium is not limited to these, and known serum-free media can also be used as the basal medium. In addition to the above-mentioned media, commercially available osteoblast media can also be used as the osteoblast medium. For example, a serum-free, xenogeneic animal protein-free bone induction medium (MSCgo Osteogenic XF ＴＭ , manufactured by BLG), MesenCult Osteogenic Differentiation kit (manufactured by STEMCELL Technologies), serum-free bone induction medium STK3 (manufactured by Two Cells), etc. However, the medium is not limited to these.

[0018] <Mammalian somatic cells (source cells)> "Mammalian somatic cells" are not particularly limited as long as they are derived from a mammal. Somatic cells refer to cells that have differentiated into a specific target cell, excluding cells that have the ability to differentiate into other cells (i.e., pluripotency), such as germ cells and MSCs. Examples of somatic cell types include fibroblasts (fibroblasts include gingival fibroblasts, pulmonary fibroblasts, myofibroblasts, and liver fibroblasts), epithelial cells such as skin epidermal cells, oral mucosal epithelial cells, respiratory tract mucosal epithelial cells, and intestinal mucosal epithelial cells, epidermal cells, gingival cells such as gingival epithelial cells, dental pulp cells, white adipocytes, subcutaneous fat, visceral fat, muscle, and blood cells such as peripheral blood mononuclear cells. Preferred examples include fibroblasts, gingival cells, oral mucosal epithelial cells, dental pulp cells, adipocytes, epidermal keratinocytes, and blood cells. Fibroblasts are particularly preferred. Other examples include somatic cells produced by inducing differentiation, dedifferentiation, or reprogramming from somatic stem cells or germline cells, such as MSCs, neural stem cells (NCs), hepatic stem cells, intestinal stem cells, skin stem cells, hair follicle stem cells, and melanocyte stem cells. Other examples include cells or cultured cells induced to differentiate, dedifferentiate, or reprogram from various somatic cells, and somatic cells induced by inducing differentiation, dedifferentiation, or reprogramming from cultured cells. Examples of mammals include humans, dogs, cats, monkeys, mice, rats, hamsters, rabbits, cows, horses, and pigs. Somatic cells are particularly preferably derived from humans. The age of the individual from which the somatic cells are derived is not limited, and they may be adults, children, or fetuses. In this specification, the term "somatic cells" may also encompass fetal-derived cells, as well as cells derived from the placenta, amnion, umbilical cord, etc.

[0019] <Prepared Osteoblasts> Using the production method of the present invention, preosteoblasts, immature osteoblasts, mature osteoblasts, osteocytes, etc. can be prepared by direct conversion, and these are collectively referred to as osteoblasts. The fact that osteoblasts have been obtained can be confirmed by measuring the mRNA of the ALP (alkaline phosphatase) gene, osteocalcin (OCN) gene, osteopontin gene, and Runx2 gene by real-time PCR, staining with Alizarin Red S (production of calcified (mineralized) bone matrix), etc. Runx2 is a transcription factor essential for bone formation. Runx2 plays an essential role in the differentiation of MSCs into osteoblasts in vivo. Forced expression of Runx2 in MSCs increases osteoblast-specific genes such as OCN (osteocalcin), BSP (bone sialoprotein), ALP (alkaline phosphatase), and COL1A1. Runx2 KO mice completely lack membranous or endochondral ossification due to the loss of mature osteoblasts. However, MSCs derived from these mice retain the ability to induce adipocytes and chondrocytes. ALP (alkaline phosphatase) is a marker for early to mid-stage osteoblast differentiation. It is abundant on the membrane surface of osteoblasts and in matrix vesicles secreted by osteoblasts, and is involved in the initiation of mineralized matrix production. Osteocalcin (OCN) is expressed specifically in osteoblasts and is thought to contribute to the promotion of bone formation. Staining with Alizarin Red S and von Kossa staining can detect the production of mineralized bone matrix, that is, calcium deposition, which is one of the important factors in bone formation.

[0020] Mature osteoblasts are characterized by the expression of OCN and ALP.

[0021] <Direct Conversion Method into Osteoblasts> Direct conversion of mammalian somatic cells into osteoblasts may be achieved by either the addition of a compound or by gene transfer. When a compound is added, the somatic cells are cultured in the above-mentioned osteoblast medium to which the compound has been added. When gene transfer is achieved, a vector or modified mRNA expressing the gene is introduced into the somatic cells, and then the cells are cultured in the osteoblast medium. For convenience, a medium in which a compound for direct conversion has been added to the osteoblast medium may be referred to as a direct conversion medium (DC medium). When gene transfer is achieved, a vector or modified mRNA expressing the gene is introduced into the somatic cells, and then the cells are cultured in the osteoblast medium; however, it is not precluded to additionally add a compound for direct conversion (i.e., culture in DC medium). In direct conversion using compounds, (1) TGF-β pathway inhibitors, (2) statin compounds, (3) casein kinase 1 inhibitors, (4) cAMP inducers, (5) histone methyltransferase inhibitors, and (6) histone deacetylase inhibitors (HDAC inhibitors) may be used alone or in combination.

[0022] (1) TGF-β Pathway Inhibitors Examples of "TGF-β pathway inhibitors" include TGF-β / SMAD pathway inhibitors, TGF-β / Erk pathway inhibitors, TGF-β / JNK pathway inhibitors, TGF-β / p38 pathway inhibitors, and TGF-β / RhoA pathway inhibitors. That is, the TGF-β pathway inhibitors used in the present invention are inhibitors that suppress one or more of TGF-β receptor family molecules, their ligands, cytokines of the TGF-β superfamily, and molecules that constitute the TGF-β / SMAD pathway, TGF-β / Erk pathway, TGF-β / JNK pathway, TGF-β / p38 pathway, and TGF-β / RhoA pathway downstream of TGF-β receptor family molecules. "TGF-β pathway inhibitors" are not limited to low molecular weight compounds that are inhibitors in the narrow sense, but also include cytokine-neutralizing antibodies, receptor antagonists, soluble receptors, antibodies that have the activity of binding to pathway proteins and inhibiting their action, aptamers, peptides, mutant proteins and peptides that act as dominant negatives, and their analogs, as well as siRNA, shRNA, and microRNA that suppress the expression of pathway proteins. Preferred examples of TGF-β pathway inhibitors include inhibitors that suppress one or more of ALK5 or its ligand, cytokines of the TGF-β family, and molecules downstream of ALK5 that constitute the TGF-β / SMAD pathway, TGF-β / Erk pathway, TGF-β / JNK pathway, TGF-β / p38 pathway, or TGF-β / RhoA pathway. More preferred examples include D4476, SB431542, LY2157299, SD208, and ALK5 inhibitor II. Examples of TGF-β pathway inhibitors that can be preferably used include those described or listed in WO 2017 / 069222 A1. Direct conversion to osteoblasts is primarily carried out by adding a TGF-β pathway inhibitor to the culture medium, but is not limited thereto as long as the effects of the present invention are not impaired.The concentration of the TGF-β pathway inhibitor in the medium can be appropriately determined by those skilled in the art depending on the compound used, but is usually about 0.01 μM to 100 μM, particularly about 0.1 μM to 30 μM.

[0023] (2) Statin Compounds Statin compounds broadly encompass HMG-CoA reductase inhibitors and are not particularly limited, but examples include simvastatin, atorvastatin, lovastatin, fluvastatin, pravastatin, cerivastatin, pitavastatin, rosuvastatin, dihydrocompactin, compactin, bervastatin, carvastatin, crilvastatin, dalvastatin, glenvastatin, fluindostatin, velostatin, mevastatin, rivastatin, sirivastatin, and CI-981. Statin compounds developed in the future can also be used in the present invention. Preferred statin compounds include those described or listed in WO 2015 / 0123777 A1. Direct conversion to osteoblasts is typically carried out by adding a statin compound to the culture medium, but is not limited thereto as long as the effects of the present invention are not impaired. The concentration of the statin compound in the medium can be appropriately determined by those skilled in the art depending on the compound used, but is usually about 100 pM to 10 μM, preferably about 500 pM to 5 μM, more preferably about 1 nM to 1 μM, and even more preferably about 10 to 100 nM.

[0024] (3) Casein Kinase 1 Inhibitors Suitable examples of casein kinase 1 inhibitors include compounds such as D4476, IC261, CK1-7, A3, SB-431542, DRB, hymenialdisine, matairesinol, 5-iodotuberdicine, meridianin, and SB-203580 (including compounds that specifically inhibit casein kinase 1). Other examples include compounds having casein kinase 1 inhibitory activity such as fasudil, hydroxyfasudil, fenretinide, PKZ-ζ peptide pseudosubstrate, dimethylsphingosine, CVS-3989, AG1024, 648450, K252a, C3 transferase, 553502, LY333531, ruboxistaurin, Go-6976, IWR-1-endo (IWR1e), and IWP-2. As the casein kinase 1 inhibitor, those described or listed in WO2015 / 0123777A1 can be preferably used. Direct conversion to osteoblasts is mainly carried out in a mode in which a casein kinase 1 inhibitor is contained in the medium, but is not limited to this as long as the effects of the present invention are not impaired. The concentration of the casein kinase 1 inhibitor in the medium can be appropriately determined by those skilled in the art depending on the compound used, but is usually about 0.01 to 100 μM, preferably about 0.1 to 50 μM, and more preferably about 1 to 10 μM.

[0025] (4) cAMP Inducers cAMP inducers (which may also be referred to as adenylate cyclase activators) broadly encompass compounds that increase intracellular cAMP (cyclic AMP) levels by activating adenylate cyclase, including, for example, forskolin (FRK) and isoproterenol. Preferred cAMP inducers include those described or listed in WO 2015 / 0123777 A1. Direct conversion to osteoblasts is typically carried out by adding a cAMP inducer to the medium, but is not limited thereto as long as the effects of the present invention are not impaired. The concentration of the cAMP inducer in the medium can be appropriately determined by those skilled in the art depending on the compound used; however, it is typically about 0.01 to 100 μM, preferably about 0.1 to 50 μM, and more preferably about 1 to 10 μM.

[0026] (5) Histone Methyltransferase Inhibitors Examples of histone methyltransferase inhibitors include DZNep (3-deazaneplanocin A) and BIX-01294. Histone methyltransferase inhibitors described or listed in WO 2015 / 0123777 A1 can be preferably used. Direct conversion to osteoblasts is typically carried out by adding a histone methyltransferase inhibitor to the medium, but is not limited thereto as long as the effects of the present invention are not impaired. The concentration of the histone methyltransferase inhibitor in the medium can be appropriately determined by those skilled in the art depending on the compound used, but is typically about 100 pM to 50 μM, preferably about 1 nM to 10 μM, more preferably about 5 nM to 1 μM, and even more preferably about 10 to 100 nM.

[0027] (6) Histone deacetylase inhibitors (HDAC inhibitors) HDAC inhibitors are compounds that inhibit the activity of histone deacetylases. HDACs are enzymes that remove acetyl groups from histones and other proteins, thereby reducing chromatin structure and suppressing gene expression. By inhibiting the function of these enzymes, HDAC inhibitors keep chromatin open and promote gene expression. Examples of HDAC inhibitors include VPA (valproic acid), hydroxamic acid-based vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), panobinostat (LBH589), benzamide-based entinostat (MS-275), tacedinaline (CI994), mocetinostat (MGCD0103), trichostatin A (TSA), sodium butyrate (NaB), etc. Direct conversion to osteoblasts is mainly carried out in an embodiment in which an HDAC inhibitor is contained in the medium, but is not limited thereto as long as the effects of the present invention are not impaired. The concentration of the HDAC inhibitor in the medium can be appropriately determined by those skilled in the art depending on the compound used, but is generally about 1 μM to 100 mM, preferably about 10 μM to 50 mM, more preferably about 100 μM to 10 mM, and even more preferably about 500 μM to 5 mM.

[0028] A compound without direct conversion activity can be added to the DC medium as desired. The DC medium used in the examples contains vitamin D (for example, calcitriol, but any substance with similar activity can be used; calcitriol is active vitamin D3) along with the DC compound (a compound with direct conversion activity). However, vitamin D does not have direct conversion activity, and therefore it is not necessarily required to add vitamin D to the DC medium.

[0029] In direct conversion by gene transfer, at least one reprogramming-related gene, or at least one bone-related gene or its expression product, and at least one reprogramming-related gene or its expression product are introduced into somatic cells. Here, "expression product" includes mRNA or protein of genes such as bone-related genes and reprogramming-related genes. The bone-related gene is a gene introduced to enable reprogrammed osteoblasts to function as osteoblasts, and specifically, at least one gene can be selected from the group consisting of Runx2 (hereinafter sometimes abbreviated as "R"), Osterix (hereinafter sometimes abbreviated as "O"), and Dlx5 (hereinafter sometimes abbreviated as "D"). Preferably, at least one of Oxterix and Dlx5 is included. It is preferable to introduce one or more of these genes or their expression products into somatic cells, if necessary, in combination with other bone-related genes or their expression products. Reprogramming-related genes are genes that are introduced into somatic cells to convert them into osteoblasts, and include Oct4, Oct1A, Oct6, c-Myc (hereinafter sometimes abbreviated as "M"), L-myc (hereinafter sometimes abbreviated as "L"), N-myc, the Klf family (KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16, KLF17), Lin-28, Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, and Sox18, and one or more of these genes are introduced into somatic cells. Somatic cells can be induced to become osteoblasts simply by introducing a reprogramming-related gene. Preferred combinations of bone-related genes and reprogramming-related genes to be introduced into somatic cells, as well as preferred methods and timing for direct conversion, and other conditions can be appropriately selected / designed in accordance with the description in WO2015 / 159982A1.

[0030] Direct conversion by gene transfer involves the introduction of at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc(M), L-Myc(L), GLIS family, Klf family, Lin-28, and Sox2, or the introduction of a combination of at least one bone-related gene selected from the group consisting of Runx2(R), Osterix(O), and Dlx5(D) and at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc(M), L-Myc(L), GLIS family, Klf family, Lin-28, and Sox2.

[0031] In the present invention, the first step is characterized by culturing somatic cells in a DC medium. In the first step, the cell concentration (cell density) is not particularly limited as long as the cells are confluent before proceeding to the second step. However, in many cases, a relatively small number of cells are seeded in a culture medium (medium) and converted while being grown. In another embodiment, in order to shorten the production schedule, the cells may be seeded in advance at a seeding density of 50% or more. For example, in the first step, the seeding density is 1,000 to 32,000 cells / cm. ２ , preferably 2,000 to 16,000 cells / cm ２ , more preferably 4,000 to 8,000 cells / cm ２ In this specification, the culture under conditions that allow the cells to become confluent before proceeding to the second step can be referred to as a low-density culture, whereas the cell culture after confluence can be referred to as a high-density culture.

[0032] The culture of somatic cells in the first step is not particularly limited as long as direct conversion to osteoblasts is achieved, and the culture should enhance epigenic changes in somatic cells. The culture can be carried out under the same conditions as for culturing normal animal cells, for example, under conditions of 95% humidity and CO ２Although culture at a concentration of 5-10% (v / v) is exemplified, the present invention is not limited to such conditions. Culture can be performed, for example, at 30-37°C, but temperatures outside this range may also be used as long as the desired effect is achieved. In the first step, the culture period varies depending on the cells before conversion and the direct conversion method used, and is set appropriately accordingly. For example, in the case of direct conversion using a TGF-β inhibitor, the culture period is 1-20 days, preferably 1-10 days, and more preferably 2-10 days. When a statin compound is added, the culture period is 1-10 days, preferably 2-10 days, and more preferably 2-7 days. In the case of direct conversion using a statin compound alone, the culture period is 1-20 days, preferably 1-10 days, and more preferably 2-7 days. In the case of conversion using an HDAC inhibitor, the culture period is 1-20 days, preferably 1-10 days, and more preferably 2-7 days. In the case of conversion using an HDAC inhibitor, the culture period is 1-20 days, preferably 2-15 days, and more preferably 2-10 days. In the first step, the cells are transferred to the second step within 0-5 days after reaching confluence.

[0033] The culture vessel used for cell culture is not particularly limited as long as it is capable of culturing the target cells, but examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. However, when starting culture with a relatively small number of cells, dishes, Petri dishes, tissue culture dishes, multi-dishes, etc. are preferred. Culture vessels may be cell-adhesive, non-cell-adhesive, or low-cell-adhesive, and are selected appropriately depending on the purpose. Cell-adhesive culture vessels may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), to improve cell adhesion to the surface of the culture vessel. The cell-supporting substrate may be any material intended for cell adhesion. Furthermore, when starting culture with a relatively small number of cells, culturing in osteoblast medium or DC medium may result in detachment of adhesive cells, such as fibroblasts. To prevent cell detachment, a positive charge (-NH ２ It is recommended to use a product that has been processed so that negative charges (such as -COOH groups) or negative charges (such as -COOH groups) or both are exposed, or a product that has been coated with any cell-supporting substrate such as an extracellular matrix (ECM) such as collagen or fibronectin. This method is not limited to the first step and may also be used in other steps.

[0034] After the first step, the spindle-shaped cells proliferate and reach subconfluence, becoming slightly cuboidal. These cells produce abundant matrix proteins such as COL1, and ALP, an osteoblast marker gene, also begins to increase.

[0035] In the present invention, a step of culturing somatic cells under conditions that do not induce direct conversion may be performed before performing the first step, and this is preferable. "Conditions that do not induce direct conversion" refers to conditions in which various compounds and various genes (expression vectors) for direct conversion are not present, specifically, culturing in the osteoblast medium alone. In the present invention, this step may be referred to as step 0. Step 0 also refers to a step of increasing the number of cells until they reach a state suitable for step 1. This step 0 is a preliminary process for stably adhering and growing human-derived primary somatic cells (fibroblasts) before seeding them on a culture dish and culturing them directly in osteoblast medium. Because primary somatic cells (fibroblasts) have different properties, step 0 may be necessary, but is not limited to this. In the present invention, step 0 allows for a smaller seeding number because the somatic cells will be expanded in the subsequent first step. For example, a seeding density of 1,000 to 12,000 cells / cm is used. ２ , preferably 2,000 to 10,000 cells / cm ２ , more preferably 3,000 to 6,000 cells / cm ２ Sow seeds in.

[0036] The somatic cell culture in step 0 can be carried out under the same culture conditions as for culturing normal animal cells. For example, humidity 95%, CO ２ Although culture at a concentration of 5-10% (v / v) is exemplified, the present invention is not limited to such conditions. Culture can be performed, for example, at 30-37°C, but temperatures outside this range may also be used as long as the desired effect is achieved. The culture period is not particularly limited as long as it allows the somatic cells to be in a state sufficient for performing the first step. Typically, it is at least 1 day, 1-7 days, 1-5 days, 2-5 days, or 3-4 days. In another embodiment, the culture period for step 0 is at least 1 day, approximately 1 day to 2 weeks, or approximately 1 day to 1 week. The medium used is the same as that used to culture ordinary animal cells. When producing cell therapy drugs, it is preferable to use a medium that meets material eligibility requirements, taking human safety into consideration.

[0037] The incubator used for cell culture is not particularly limited, and the same incubator as that used in the first step can be used.

[0038] 2. Second Step (Two-Dimensional High-Density Culture) In the production method of the present invention, the second step is a step of high-density culture of the cells obtained in the first step to form cell clusters. In one embodiment, the cells obtained in the first step include osteoblasts. In this second step, the cells expanded after the first step are seeded at high density. The medium used for seeding can be either the medium used in step 0 or step 1, or a medium for osteoblasts or a DC medium. Although DC medium is not particularly required, DC medium may be used from the time of seeding. After culturing, the medium is replaced with DC medium and culture is continued anywhere from the day of seeding to three days later. The second step is a process of enhancing ECM production and promoting conversion to osteoblasts.

[0039] In the present invention, the second step is characterized by high-density culture of the cells that have undergone the first step in a DC medium. "High-density culture" refers to a state in which cells are cultured at a high density. For example, this includes cell culture after reaching confluence. For example, this refers to a culture environment designed to achieve a very high cell density, and generally refers to culture at a density at which contact inhibition occurs or at a density at which cells reach their limit. Typically, the seeding density is 20,000 to 400,000 cells / cm. ２ , preferably 30,000 to 200,000 cells / cm ２ , more preferably 50,000 to 120,000 cells / cm ２ The cells are seeded in DC medium at 100°C.

[0040] The DC medium can be the same as that used in the first step. The various compounds for direct conversion contained in the DC medium may be the same as or different from those used in the first step, but for convenience, it is preferable that they are the same. The somatic cells in the second step can be cultured under the same conditions as those used for culturing ordinary animal cells. For example, 95% humidity, CO ２Culture at a concentration of 5 to 10% (v / v) is exemplified, but the present invention is not limited to such conditions. Culture can be carried out, for example, at 30 to 37°C, but temperatures outside this range may also be used as long as the desired effect is achieved. Approximately three days after the start of culture, a sheet-like cell population (hereinafter referred to as a cell sheet) rich in ECM proteins is formed. The culture period is the period until the cell sheet becomes capable of being released from the culture vessel, and is usually 1 to 20 days, preferably 3.5 to 18 days, and particularly preferably 4 to 7 days. In another embodiment, the culture period is 1 to 15 days, preferably 3.5 to 10 days, and particularly preferably 4 to 6 days.

[0041] The incubator used for cell culture is not particularly limited, and the same incubator as that used in the first step can be used. However, from the viewpoint of facilitating high-density culture, an incubator with a small bottom area, such as a microwell plate, is preferred.

[0042] In the second step, high-density culture is initiated immediately after seeding to transition the cell state from the cell proliferation phase to the ECM production phase. Shortly after seeding, the cells settle on the culture dish and begin to produce ECM proteins from the proliferated cells. Subsequently, a cell sheet rich in ECM proteins is formed. To transition from the second step to the third step, the cell sheet is detached to allow it to self-aggregate. Specifically, the cell sheet is attached in a two-dimensional sheet form due to the tension of its adhesion to the culture dish. However, once the periphery of the cell sheet is detached from the culture dish, the cell sheet's self-tension causes it to contract and form a clump. This property can be utilized to separate the edge of the cell sheet, whose periphery is attached to the peripheral wall of the culture vessel, from the culture vessel. For example, the cell sheet can be separated from the culture vessel by inserting a sterilized thin rod (such as a tip for a 100-microliter or 200-microliter micropipette) into the inner wall of the culture vessel to which the edge of the cell sheet is attached and moving the rod around the inner wall of the culture vessel. This causes the cell sheet to float.

[0043] 3. Third Step (Three-Dimensional Culture, Step for Forming Osteoblast-Containing Cell Aggregates) In the manufacturing method of the present invention, the third step is a step in which the cell sheet obtained in the second step is subsequently cultured in DC medium to form osteoblast-containing cell aggregates. In the second step, the cell aggregates are obtained in the form of a cell sheet. In this step, the cell sheet is separated from the culture vessel, and the suspended cell sheet is further cultured to obtain osteoblast-containing cell aggregates as three-dimensional bone-like tissue. In the third step, the cell sheet obtained in the second step is cultured in suspension, whereby the cell sheet wraps around itself through its self-aggregation action, forming a cell aggregate rich in ECM proteins produced by the cells themselves. In particular, when somatic cells are converted into osteoblasts by culturing in DC medium, three-dimensional bone-like tissue consisting of osteoblasts and bone matrix proteins (COL1 / OCN) produced by the osteoblasts is obtained. The cell sheet (one) obtained in the second step is transferred to a culture vessel and cultured in suspension in DC medium. The incubator can be the same as that used in the first and second steps. For example, the size of the incubator for culturing one three-dimensional cell cluster is 2 cm2. ２ A low-adhesion 24-well plate or a slightly smaller 48-well plate is preferred. Furthermore, within a few days of the start of the third step, the cell sheet detached in the second step will have progressed to self-aggregation, and once three-dimensional cell aggregates have been formed using the extracellular matrix proteins produced by the cell sheet itself as a scaffold, it is possible to culture multiple or many three-dimensional cell aggregates together in a single large well at the appropriate time. Furthermore, to facilitate the suspension culture of a single cell sheet and the recovery of the resulting three-dimensional bone-like tissue, a low-adhesion culture plate is preferred. Using a cylindrical culture vessel allows for the production of granular cell aggregates (note that a culture vessel with a base area of ​​0.9 to 1.9 cm2 is required for one cell sheet). ２(When a cylindrical vessel such as this is used, granular cell clumps with a major axis of approximately 1.0 to 4.0 mm are obtained.) The culture vessel is preferably non- or low-adhesive to cells. Treatment methods for rendering the culture vessel non- or low-adhesive to cells include methods commonly practiced in the art. Examples include polyethylene glycol (PEG) coating, poly-2-hydroxyethyl methacrylate (Poly-HEMA) coating, and hydrophobic coating. Such treated culture vessels are also commercially available. Furthermore, in the third step, the cells are wrapped around each other by the self-aggregation of the cell sheet, resulting in a cell clump rich in ECM proteins produced by the cells themselves, i.e., a three-dimensional bone-like tissue. This three-dimensional bone-like tissue uses the autologous ECM as a scaffold, creating an appropriate arrangement and space within the cell clump for osteoblast survival. As a result, it has been confirmed that the cell population within this cell clump can be maintained as viable cells even during long-term culture, as long as the medium is changed. Generally, when using a standard medium instead of a medium for direct conversion (DC medium), the three-dimensional cell aggregates that form spheroids are tightly packed cell aggregates formed by cell-cell contact, and it is known that nutrients (oxygen and medium components) do not reach the center of the cell aggregate, causing central necrosis. However, using this production method, i.e., cell aggregates containing osteoblasts cultured in DC medium, are formed by ECM proteins that are permeable to nutrients, allowing the construction of larger, three-dimensional tissues than cell spheroids. In the actual production method (see Examples below), even when starting with the same somatic cells and going through the same first, second, and third steps, the internal structure of the 3D cell mass in the third step differed depending on whether or not directly converted osteoblasts were present when the culture medium was used for production using osteoblast medium alone or DC medium. In the 3D cell mass obtained by culturing using osteoblast medium alone, the number of dead cells increased as the culture progressed, but it was confirmed that a high viable cell rate was maintained for a long period of time as long as the culture medium was changed using the 3D cell mass obtained in DC medium.This is thought to be due to the fact that the directly converted osteoblasts are contained in the three-dimensional cell mass, and in the second and third steps, the bone tissue-related ECM proteins specifically produced by the directly converted osteoblasts serve as a scaffold to form a three-dimensional cell mass suitable for bone-like tissue.

[0044] The DC medium can be the same as that used in the first and second steps. The various compounds for direct conversion contained in the DC medium may be the same as or different from those used in the first and second steps. The somatic cells in the third step can be cultured under the same culture conditions as those used for culturing normal animal cells. For example, 95% humidity, CO ２ Culture at a concentration of 5 to 10% (v / v) is exemplified, but the present invention is not limited to such conditions. Culture can be carried out, for example, at 30 to 37°C, but may be carried out at temperatures outside this range as long as the desired effect is achieved. The culture period is usually 3 days or more. Since culture for a considerable period is possible as long as the medium is changed, the upper limit of the culture period can theoretically be any number of days, but is preferably within 100 days. More preferably, it is 3 to 100 days, 3 to 80 days, 10 to 70 days, 10 to 60 days, or 10 to 50 days. In another embodiment, the culture period is 3 to 30 days, preferably 10 to 25 days, and more preferably 12 to 18 days.

[0045] The three-dimensional cell mass containing directly converted osteoblasts obtained in the third step is a milky-white, spherical cell mass (various shapes, including ellipsoidal spheres) with a diameter of approximately 1.5 to 3 mm. Its viscoelastic properties allow it to be easily collected with tweezers or a medicine spoon, enabling it to be directly transplanted into bone defects. This cell mass is constructed from OCN-producing osteoblasts and the bone-related ECM proteins (COL1, OCN, etc.) produced by these osteoblasts. Furthermore, due to the abundant presence of osteoblasts, it possesses high ALPase activity.

[0046] In the production method of the present invention, the period for the series of steps 1 to 3 is 5 to 140 days, preferably 5 to 115 days, more preferably 12 to 105 days, even more preferably 15.5 to 95 days, and particularly preferably 16 to 70 days. When step 0 is carried out, the period for the series of steps in the production method of the present invention is 6 to 147 days, preferably 6 to 120 days, more preferably 13 to 110 days, even more preferably 17.5 to 100 days, and particularly preferably 19 to 75 days. In another embodiment, the period for the series of steps 1 to 3 is 5 to 65 days, preferably 14.5 to 45 days, and more preferably 18 to 31 days. When step 0 is carried out, the period for the series is 6 to 70 days, preferably 16.5 to 54 days, and more preferably 21 to 35 days.

[0047] 4. Conversion-Promoting Effect In the present invention, when direct conversion from mammalian somatic cells to osteoblasts is carried out, it is also possible, and this is a preferred embodiment, to promote direct conversion to osteoblasts by using other compounds in addition to the various compounds or genes (expression vectors) used for direct conversion. Examples of such compounds include epigenetic control compounds such as (1) TGF-β pathway inhibitors, (2) statin compounds, (3) casein kinase 1 inhibitors, (4) cAMP inducers, (5) histone methyltransferase inhibitors, and (6) histone deacetylase inhibitors (HDAC inhibitors) (also referred to as the "combined compounds" of the present invention). The (1) TGF-β pathway inhibitors, (2) statin compounds, (3) casein kinase 1 inhibitors, (4) cAMP inducers, (5) histone methyltransferase inhibitors, and (6) histone deacetylase inhibitors (HDAC inhibitors) used here include the same compounds as those described in "1. First Step (Two-Dimensional Culture)." However, because these compounds are expected to have a combined effect with the compounds used in the direct conversion of osteoblasts, compounds that are the same as those used in the direct conversion of osteoblasts are excluded.

[0048] The use of the combined compounds promotes osteoblast conversion. The inventors investigated various timings for their addition. Results showed that the compounds were effective in all three steps, but their effect was most potentiated by adding them from the second step of two-dimensional culture. Addition from the second step is preferable, and addition during the third step also promotes conversion. Furthermore, osteoblasts can be directly induced from human fibroblasts (direct conversion), and then the addition of these combined compounds can enhance osteoblast function, resulting in the formation of stronger bone-like tissue.

[0049] Examples are shown below, but the present invention is not limited to these examples. Example 11-1. HE staining and COL1 / OCN immunostaining (Figure 2) Using human normal gingival fibroblasts as an example of fibroblasts, 0.36 x 10e4 cells / cm ２ The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 10 days in osteoblast medium (basal medium (Prime MDF; Fujifilm Wako Pure Chemical Industries, Ltd., 552-37463) supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or osteoblast medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)) and 5 nM calcitriol (DC medium). The proliferated cells were then cultured at a density of 2 x 10e5 cells / 1.9 cm dish. ２The cells were seeded at a concentration of 1000 kJ / well (24-well plate) and cultured for 5 days in the three media mentioned above in the second step. The cell sheets were then suspended and transferred to low-adhesion 24-well plates. The third step involved suspension culture in the three media mentioned above for 15 days. The medium was replaced approximately every 3 to 4 days throughout all steps. The basal medium may be any medium that does not contain the osteogenic differentiation factor DAG. It has been confirmed that synthetic basal media such as DMEM and IMDM, as well as growth and maintenance media for MSCs and fibroblasts, can also be used. The DC medium was supplemented with 5 nM calcitriol (unless otherwise specified). However, it has been confirmed that direct conversion to osteoblasts can also be achieved in DC medium without calcitriol, so supplementation of the DC medium with vitamin D is not necessarily required. The obtained samples (cell clumps) were washed with PBS, fixed with 4% paraformaldehyde, and embedded in paraffin. Serial 8-μm sections were prepared and stained with HE. To observe the level of bone matrix protein production, immunohistochemistry was performed for OCN, a protein specifically produced by osteoblasts, and COL1, a type of bone matrix protein. The results are shown in Figure 2A.

[0050] HE staining revealed that cell clumps prepared in 3. "DC medium" were significantly larger in size than cell clumps prepared in 1. "basal medium only" or 2. "osteoblast medium only" (a three-dimensional structure with a ratio of approximately 2.5 times the sum of the major and minor axes). These three cells were cultured from the same source and cultured to the same cell number in the second step. Because there was little cell proliferation due to high-density culture in the second step, the cell numbers at the time of the third step were almost the same in 1., 2., and 3. Considering this, the difference in size between the three-dimensional cell clumps (3. is large, while 1. and 2. are small) was very large, and this difference in size was thought to be due to differences in internal structure. Furthermore, immunostaining revealed that cell clumps prepared in 1. "basal medium only" or 2. "osteoblast medium only" did not express the osteoblast-specific protein OCN, whereas 3. In addition to COL1, abundant OCN was observed in the cell clumps prepared in "DC medium." More specifically, COL1 (green) expression was observed in samples 1 and 2, but higher levels of COL1 were observed in sample 3. OCN (red) expression was minimal in samples 1 and 2, but strong in sample 3. Approximately half of the OCN-expressing cells also expressed COL1, as observed in the merged image (far left photo: COL1 / OCN / DAPI), demonstrating the presence of mature osteoblasts. The resulting samples (cell clumps) were washed with PBS, immersed in RIPA buffer (Nacalai Tesque 16488-34), and disrupted by ultrasonic vibration to obtain a cell lysate. The amount of OCN protein contained in this cell lysate was measured by ELISA (Human Osteocalcin Simple Step ELISA Kit, ab270202), and the ALPase enzyme activity of this cell lysate was similarly quantified using LabAssay ALP (FUJIFILM 297-93501). The results are shown in Figure 2B. The vertical axis indicates the amount of OCN protein expression or ALPase activity.

[0051] ELISA analysis of OCN protein showed that cell clumps prepared in osteoblast medium did not detect OCN protein, an osteoblast-specific extracellular matrix protein, whereas cell clumps cultured in DC medium produced abundant OCN protein. Similar to this OCN expression pattern, cell clumps cultured in DC medium exhibited significantly higher ALPase activity than cell clumps prepared in osteoblast medium alone. This ALPase activity was approximately two-fold higher in cell clumps prepared in DC medium (cell clumps containing directly converted osteoblasts) than in cell clumps prepared in osteoblast medium (cell clumps without direct conversion). This indicates that by performing steps 1 to 3 in DC medium supplemented with a DC compound, three-dimensional cell clumps consisting of directly converted osteoblasts and bone matrix proteins were obtained. That is, the sample (three-dimensional cell mass) obtained by culturing the third step for 15 days using the above method was confirmed to be a three-dimensional bone-like tissue (3D-DCob) rich in mature osteoblasts based on the results of histological staining and histological immunostaining (Figure 2A) and the results of OCN protein amount and ALP activity (Figure 2B).

[0052] Next, to confirm the appropriate culture period for the third step, the following experiment was carried out. Using human normal gingival fibroblasts, 0.36 x 10e4 cells / cm ２ The cells were seeded at a density of 2.73 x 10e5 cells / T-75 flask, and the 0th step of culturing in basal medium was performed for 3 days, and the 1st step of culturing in DC medium was performed for 3 days. Then, the cells were collected and cultured at a density of 1 x 10e5 cells / 0.95 cm. ２The cells were seeded at a density of 1000 kJ / well (48-well plate) and cultured in DC medium for 6 to 9 days in the second step. The cell sheets were then suspended and transferred to low-adhesion 48-well plates. The third step involved suspension culture in DC medium. Note that the medium was changed approximately every 3 to 5 days throughout all steps. In Experiment 1, the OCN protein levels and ALP activity of the 3D bone-like tissue were measured on days 15, 16, 25, and 36 after the start of the third step of culture. The measured values ​​were converted to a concentration dissolved in 300 microliters per 3D bone-like tissue, and the average values ​​at each time point are shown in a graph (Figure 2C). To confirm longer culture periods, Experiment 2 was conducted, in which similar measurements were performed on days 22, 29, and 46 after the start of the third step of culture. The results are shown in a graph (Figure 2D). In the third step of Experiments 1 and 2, to confirm that the majority of cells in the 3D bone-like tissue were viable at the time of sampling for measuring OCN protein levels and ALP activity, the 3D bone-like tissue was sampled, the cells were dispersed with collagenase, and the total cell number and viable cell number were measured. As a result, the total cell number was nearly maintained over all of the above culture days, and the viability was maintained at 80% to 90% or more, confirming that cells were normally maintained as viable cells throughout the culture. Based on these findings, when cultured using this method, 3D bone-like tissue (3D-DCob), which is rich in osteoblasts directly converted from fibroblasts and bone matrix proteins, maintained a high viability and maintained OCN and ALP expression for at least 15 to 46 days of culture in the third step. This suggests that the properties of 3D bone-like tissue can be maintained as long as the cells remain viable after medium changes.

[0053] 1-2. HE staining and COL1 / OCN immunostaining (Figure 3) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.72 x 10e4 cells / cm. ２The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 10 days in osteoblast medium (basal medium (Prime MDF; Fujifilm Wako Pure Chemical Industries, Ltd., 552-37463) supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in osteoblast medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)). The proliferated cells were then cultured at a density of 2 x 10e5 cells / 1.9 cm dish. ２ The cells were seeded at a concentration of 1000 kJ / well (24-well plate) and cultured for 5 days in the two media mentioned above (step 2). The cell sheets were then suspended and transferred to low-adhesion 24-well plates. The third step involved suspension culture for 10 days in either the two media mentioned above or in DC medium (basal medium: DMEM supplemented with 5% UltraGro, osteoblast medium supplemented with the bone differentiation inducer DAG, and DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)). Throughout all steps, the medium was changed approximately once every 3 days. The obtained samples (cell clumps) were washed with PBS, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sections of 8 μm were prepared and stained with HE. In addition, to observe the level of production of bone matrix proteins, we performed immunostaining for OCN, a protein produced specifically by osteoblasts, and COL1, a type of bone matrix protein. The results are shown in Figure 3.

[0054] Similar to the results of Example 1-1 above, in 1. osteoblast medium alone, the resulting cell clumps were small and no OCN expression was observed. On the other hand, in 2. DC medium, i.e., osteoblast medium cultured with the addition of DC compound, the cell clumps obtained were large and strongly expressed OCN protein. Furthermore, when the third step was performed as 3. osteoblast medium cultured with the addition of DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)), osteoblast clumps with significant OCN protein expression were obtained, similar to 2. More specifically, COL1 (green) expression was observed in 1., but more COL1 was expressed in 2. and 3. OCN (red) was hardly expressed in 1., but was expressed in 2. and 3. It was strongly expressed in OCN-expressing cells, and it was observed from the merged image (leftmost photograph: COL1 / OCN / DAPI) that about half of the OCN-expressing cells also expressed COL1, making it clear that they contained mature osteoblasts, confirming the same results as in Example 1-1.

[0055] Furthermore, even when the basal medium that forms the base of the osteoblast medium was changed in the third culture step, three-dimensional cell aggregates containing the desired directly converted osteoblasts were produced without any problems. Although not shown in the data, the same thing was confirmed not only in the third step but also in the first and second steps.

[0056] 1-3. HE staining and COL1 / OCN immunostaining (Figure 4) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36 x 10e4 cells / cm. ２ The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 8 days in osteoblast medium (basal medium (DMEM supplemented with 100 μM NEAA and 5% serum substitute UltraGro) supplemented with bone differentiation inducer DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in DC medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)). The proliferated cells were then cultured at a density of 2 x 10e4 cells / 1.9 cm dish. ２The cells were seeded at a density of 1000 x g (24-well plate) and cultured for 17 days in the two media mentioned above in the second step. The cell sheets were then suspended and transferred to low-adhesion 24-well plates, and the third step involved continuing suspension culture for 15 days in the two media mentioned above. The medium was changed approximately every three days throughout all steps. The resulting samples (cell clumps) were washed with PBS, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sections of 8 μm were prepared and stained with HE. Furthermore, OCN, a protein specifically produced by osteoblasts, and COL1, a bone matrix protein, were observed by immunostaining. The results are shown in Figure 4.

[0057] HE staining revealed that cell clumps prepared in 1. osteoblast medium alone were small, with diameters of less than 500 μm, whereas cell clumps prepared in 1. DC medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)) (2. DC medium (DMEM)) were large, with major axes of approximately 1.2 mm. Furthermore, immunostaining revealed that cell clumps prepared in DMEM and 5% Ulgragro-based osteoblast medium did not express the osteoblast-specific protein OCN, whereas cell clumps prepared in DMEM and 5% Ulgragro-based osteoblast medium supplemented with DC compound exhibited abundant OCN in addition to COL1. More specifically, expression of COL1 (green) was observed in 1. and 2. OCN (red) was observed in 1. 1. There was almost no expression in 1., but strong expression in 2., and most of the OCN-expressing cells also expressed COL1, as observed from the merged image (COL1 / OCN / DAPI), demonstrating that they had differentiated into mature osteoblasts. This indicates that even in DMEM and 5% Ulgragro-based osteoblast medium, the three-step culture process consisting of steps 1 to 3 using the DC compound of the present invention allowed for the production of three-dimensional bone-like tissue consisting of osteoblasts directly converted from fibroblasts and bone matrix proteins.

[0058] 1-4. HE staining and ALP / OCN mRNA expression (qPCR) (Figure 5) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.72 x 10e4 cells / cm. ２ The cells were seeded at a density of 4 x 10e5 cells / 10 cm dish and cultured for 7 days in osteoblast medium (basal medium DMEM / 10% FBS supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) in the first step. Then, in the second step, the proliferated cells were cultured at a density of 2 x 10e5 cells / 1.9 cm. ２ The cells were seeded at a concentration of 1000 μg / ml (24-well plate) and cultured for 5 days in osteoblast medium (basal medium Prime MDF supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in DC medium supplemented with a TGF-β pathway inhibitor (Alk5 inhibitor II) (4 μM). Then, in the third step, the cell sheet was suspended and transferred to a low-adhesion 24-well plate and cultured in osteoblast medium (known osteoblast medium MSCgo Osteogenic XF). ＴＭ , or MSCgo Osteogenic XF ＴＭ The cells were then cultured in suspension for 10 days in DC medium containing 4 μM Alk5 inhibitor II. The medium was changed approximately once every three days throughout the entire process.

[0059] The obtained samples (cell clumps) were washed with PBS, fixed with 4% paraformaldehyde, and embedded in paraffin. Serial 8 μm sections were prepared and stained with HE. The results are shown in Figure 5 (bottom left). The obtained samples (cell clumps) were washed with PBS, and total RNA was recovered from the cell clumps using RNAiso. cDNA was prepared using Rever Tra Ace qPCR RT Master Mix. SYBR Green Realtime PCR Master Mix, specific primers, and cDNA were mixed, and real-time RT-PCR was performed using StepOne systems to quantify the mRNA expression levels of osteoblast-specific markers OCN and ALP genes. The results are shown in Figure 5 (bottom right).

[0060] The mRNA expression of cell clumps prepared by culturing without adding DC compounds (in osteoblast medium alone) was evaluated as a control. The vertical axis represents the relative mRNA value of each gene, with the mRNA level of the control cells set at 1. It was confirmed that the cell clumps prepared by adding DC compounds to the osteoblast medium above highly expressed the osteoblast markers ALP and OCN.

[0061] Example 22-1. HE staining (FIG. 6) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36×10e4 cells / cm ２ The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 10 days in osteoblast medium (basal medium (Prime MDF) supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in osteoblast medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)). The proliferated cells were then cultured at a density of 2 x 10e5 cells / 1.9 cm dish. ２The cells were seeded into 24-well plates at a concentration of 100 μM each, and cultured for 5 days in either the osteoblast medium alone or DC medium (step 2). The cell sheets were then suspended and transferred to low-adhesion 24-well plates. The third step of suspension culture was performed in either the osteoblast medium alone (referred to as "1. Osteoblast Medium Only") or DC medium (referred to as "DC Medium AP") for 15 days. The DC medium culture was compared between a DC compound containing only 8 μM Alk5 inhibitor II and a medium containing 8 μM TGF-β pathway inhibitor (Alk5 inhibitor II) and 100 nM statin compound (Pravastatin). The former was designated "2. DC Medium A," and the latter "3. DC Medium AP." The medium was replaced approximately every 3 days throughout all steps. After 5 and 15 days of suspension culture in the third step, the samples (cell clumps) were washed with PBS, fixed with 4% paraformaldehyde, and embedded in paraffin. Serial sections of 8 μm were prepared and stained with HE. The results are shown in Figure 6.

[0062] In the third step, HE staining after 5 days of suspension culture showed that samples prepared in osteoblast medium were spherical cell clumps with tightly aggregated cells, whereas samples prepared in DC medium A or DC medium AP, which contained the DC compound in osteoblast medium, showed a sheet structure that folded and contracted to form a three-dimensional sphere, with the cells not densely packed within the internal structure, maintaining a certain amount of space between cells. Furthermore, high-magnification images revealed that in the "2. DC medium A" group or the "3. DC medium AP" group, cells existed as a scaffold using an extracellular matrix stained with eosin. Furthermore, after 15 days of suspension culture, the cell clumps in the "1. Osteoblast medium only" group prepared in osteoblast medium shrank and became smaller, with a high density of cells with aggregated nuclei present within. This suggests the possibility of central necrosis, in which nutrients do not reach the center due to cell aggregation caused by cell-cell adhesion structures. On the other hand, the size of the cell aggregates prepared in the "2. DC Medium A" group or the "3. DC Medium AP" group was clearly larger, and it was observed that the cells (converted osteoblasts) and the abundant extracellular matrix proteins produced by the osteoblasts formed a three-dimensional structure. High-magnification images showed that the osteoblasts existed using the extracellular matrix as a scaffold, and that the surrounding area was also filled with extracellular matrix. Thus, the cell aggregates prepared in the medium supplemented with DC compounds formed bone-like tissue primarily composed of directly converted osteoblasts and the extracellular matrix produced by osteoblasts. This suggests that large tissue-like structures with a diameter of 1 mm or more could be constructed, allowing for easy infiltration of nutrient-containing medium and avoiding central necrosis.

[0063] 2-2. TdT-mediated dUTP nick end labeling (TUNEL) staining (Figure 7) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36 x 10e4 cells / cm. ２The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 10 days in osteoblast medium (basal medium (Prime MDF) supplemented with the osteogenic differentiation factor DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in osteoblast medium supplemented with DC compound (8 μM TGF-β pathway inhibitor (Alk5 inhibitor II)). The proliferated cells were then cultured at a density of 1 x 10e5 cells / 0.95 cm dish. ２ The cells were seeded into 48-well plates at a concentration of 100 μM each, and cultured for 5 days in either the osteoblast medium alone or DC medium (step 2). The cell sheets were then suspended and transferred to low-adhesion 48-well plates. The third step involved 15 days of suspension culture in either the osteoblast medium alone (referred to as "1. Osteoblast Medium Only") or DC medium. The DC medium culture was compared between a DC compound containing only 8 μM TGF-β pathway inhibitor (Alk5 inhibitor II) and a DC compound containing 8 μM TGF-β pathway inhibitor and 100 nM statin compound (Pravastatin). The former was designated "2. DC Medium A," and the latter "3. DC Medium AP." The medium was replaced approximately every 3 days throughout all steps. After 15 days of suspension culture in the third step, the samples (cell clumps) were washed with PBS, fixed with 4% paraformaldehyde, and embedded in paraffin. Serial sections of 8 μm were prepared and TUNEL stained using the DeadEnd™ Fluorometric TUNEL System (Promega). The results are shown in Figure 7.

[0064] TUNEL-positive dead cells were significantly observed within the cell clusters prepared in osteoblast medium. In comparison, the number of TUNEL-stained dead cells was significantly reduced in cell clusters prepared in DC medium A or DC medium AP, which were osteoblast medium supplemented with DC compounds. These findings suggest that the addition of DC compounds (TGF-β pathway inhibitors or TGF-β pathway inhibitor I plus a statin compound) may suppress cell death associated with 3D culture and enable the creation of three-dimensional tissue structures containing functional cells (converted osteoblasts).

[0065] 2-3. Cell viability assay (measurement of viable cell count) (Fig. 8) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36 x 10e4 cells / cm. ２ The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 10 days in osteoblast medium (basal medium Prime MDF supplemented with bone differentiation inducer DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in the osteoblast medium supplemented with DC compound (8 μM TGF-β pathway inhibitor: Alk5 inhibitor II) (DC medium). The proliferated cells were then cultured at a density of 1 x 10e5 cells / 0.95 cm. ２The cells were seeded into 48-well plates at a concentration of 100 μM each, and cultured for 5 days in the two media mentioned above (step 2). The cell sheets were then suspended and transferred to low-adhesion 48-well plates. The third step involved suspension culture in either the osteoblast medium alone (referred to as "1. Osteoblast Medium Only") or DC medium for 15 days. The DC medium culture was compared between a DC compound containing only 8 μM TGF-β pathway inhibitor (Alk5 inhibitor II) and a DC compound containing 8 μM TGF-β pathway inhibitor and 100 nM statin compound (Pravastatin). The former was designated "2. DC Medium A," and the latter "3. DC Medium AP." The medium was replaced approximately every 3 days throughout all steps. After 2 and 15 days of suspension culture in the third step, samples (cell clumps) were washed with PBS and viable cell counts were measured by colorimetry using Cell Counting Kit-8 (Dojindo Laboratories). The results are shown in Figure 8. The vertical axis of the graph represents absorbance at 450 nm (OD450), which indicates the amount of orange formazan produced by the reduction of added WST-8 by viable cells. In other words, the amount of formazan quantified by OD450 corresponds to the number of viable cells. After 2 days of suspension culture, all three samples obtained had similar absorbance (approximately 1.5). However, the absorbance of cell clumps after 15 days of suspension culture in osteoblast medium alone was significantly reduced. On the other hand, the absorbance of cell clumps cultured in DC medium for 15 days, "2. DC Medium A" and "3. DC Medium AP," maintained the same or higher level compared to that on day 2 of culture. These results suggest that cell clumps prepared using only osteoblast medium undergo cell death during the suspension culture process, whereas cell clumps prepared using DC compound-supplemented medium may be able to avoid such cell death.

[0066] Example 33-1. Quantitation of osteoblast marker mRNA expression (FIG. 9) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36×10e4 cells / cm ２The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 12 days in osteoblast medium (basal medium Prime MDF supplemented with bone differentiation inducer DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in the osteoblast medium supplemented with DC compound (4 μM TGF-β pathway inhibitor: Alk5 inhibitor II) (DC medium). The proliferated cells were then cultured at a density of 2 x 10e5 cells / 1.9 cm dish. ２ The cells were seeded at a density of 1000 / well (24-well plate) and cultured for 5 or 9 days in the medium described above in the second step. The cell sheets were then detached and suspended, transferred to low-adhesion 24-well plates, and cultured in the medium described above for 5 or 10 days in the third step. The medium was changed approximately every 3 days throughout the entire process. The experimental groups for each culture step are shown in Figure 9.

[0067] After washing the obtained samples (cell clumps) with PBS, total RNA was recovered from the cell clumps using RNAiso, and cDNA was prepared using Rever Tra Ace qPCR RT Master Mix. SYBR Green Realtime PCR Master Mix, specific primers, and cDNA were mixed, and real-time RT-PCR was performed using StepOne systems to quantify the mRNA expression levels of osteoblast-specific markers OCN, ALP, and RUNX2. Among the cell clumps prepared by culturing only in osteoblast medium, experimental group 1 was evaluated as a control for mRNA expression. The vertical axis represents the relative mRNA value of each gene when the mRNA level of control cells is set to 1. In the group cultured in DC medium and cultured for the longest period (10 days) during the third step, the expression of osteoblast markers ALP and OCN mRNA was significantly elevated, with expression levels approximately three times higher than in the group cultured for 5 days during the third step. Meanwhile, the expression of RUNX2 mRNA, essential for differentiation of MSCs into preosteoblasts, was significantly reduced by culture in DC medium under both conditions. This suggests that cell clusters cultured in DC medium (cell clusters containing osteoblasts obtained by the direct conversion method) are composed of cells directly converted to mature osteoblasts without undergoing induction into preosteoblasts.

[0068] 3-2. Quantitative quantification of osteoblast marker mRNA expression (FIG. 10) Fibroblasts (derived from normal human gingiva) were cultured at a density of 0.36×10e4 cells / cm ２ The cells were seeded at a density of 2 x 10e5 cells / 10 cm dish and cultured for 12 days in osteoblast medium (basal medium Prime MDF supplemented with bone differentiation inducer DAG (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate)) or in the osteoblast medium supplemented with DC compound (4 μM TGF-β pathway inhibitor: Alk5 inhibitor II) (DC medium). The proliferated cells were then cultured at a density of 2 x 10e5 cells / 1.9 cm dish. ２The cells were seeded at a density of 1000 / well (24-well plate) and cultured in the above-mentioned medium for 9 days in the second step. The cell sheet was then detached, suspended, and transferred to a low-adhesion 24-well plate. The third step involved suspension culture in the above-mentioned medium for 2 or 10 days. Throughout the entire process, the medium was changed approximately every 3 days. The experimental groups for each culture step are shown in Figure 10.

[0069] After washing the resulting samples (cell clumps) with PBS, total RNA was extracted from the cell clumps using RNAiso, and cDNA was prepared using Rever Tra Ace qPCR RT Master Mix. SYBR Green Realtime PCR Master Mix, specific primers, and cDNA were mixed, and real-time RT-PCR was performed using StepOne systems to quantify the mRNA expression levels of the osteoblast-specific markers human OCN, human ALP, and human COL1 genes.

[0070] Among the cell clumps prepared by culturing only in osteoblast medium, experimental group 1 was evaluated as a control for mRNA expression. The vertical axis represents the relative mRNA value of each gene, with the mRNA level of the control cells set at 1. Cell clumps cultured in DC medium showed significantly increased mRNA expression of osteoblast marker genes ALP, OCN, and COL1 compared to those cultured in osteoblast medium. In particular, extending the culture period in the third step to 10 days, rather than 2 days, further promoted increased mRNA expression of the osteoblast marker genes. In other words, during the third step of culture, the content of osteoblast-specific ECM, OCN and COL, increased from the initial 2nd day to the 10th day of long-term culture. This suggests that the cell sheet was forming a spherical structure due to its own contractile force, and after the period required for the formation of a three-dimensional cell mass (1 to 3 days), an appropriate structure was formed with osteoblasts using the ECM produced by the osteoblasts as a scaffold, thereby improving the quality of the bone-like tissue containing osteoblasts.

[0071] Example 4: Evaluation of Drug Efficacy Using Pathological Model Animals (Figure 11) Animal experiments were conducted with the approval of the affiliated institution. Three-dimensional cell aggregates prepared using "DC Medium A" or "DC Medium AP" as described in Example 2 were transplanted into nude rat large calvarial defect model animals. Seven- to nine-week-old nude rats were used, and 1 ml of a triple-anesthesia mixture was administered per rat. After shaving, the hair on the skull was disinfected with an iodine cotton ball. An incision was made with a scalpel from between the eyes to between the ears, and the skull skin and periosteum of the skull were removed with a mucosal dissector. A midline defect was created under irrigation using a dental engine and a 5.8 mm outer diameter trephine burr. Nine cell aggregates were inserted into each defect to tightly fill it. The incision was then covered with Bioguide (Geistlich Japan: https: / / shop.geistlich.co.jp / collections / geistlich-bio-guide), an absorbable coating that had been pre-cut to the appropriate size, and the incision was closed with four sutures of 4-0 silk thread. For comparison, a "no transplant" group was prepared in which cells were not transplanted into the defect and the defect was covered with Bioguide. Eight weeks after transplantation, the animals were euthanized and μCT tomography was performed using an X-ray CT device. No bone regeneration was evident in groups #1 and #2 without transplantation. In the CT images of the non-transplant group #3, the bone at the base of the skull was visible, but opaque material extending from the defect stump was observed. This was thought to be bone fragments that were not completely removed during defect creation, and no significant bone regeneration was observed in the parietal skull defect. In other words, it was confirmed that bone regeneration did not occur in the non-transplant group. On the other hand, in the groups transplanted with cell aggregates prepared with "DC Medium A" or "DC Medium AP," opaque images were observed filling the defect. The opaque images showed natural continuity with the remaining bone and were considered to be images of bone regeneration accompanied by mineral deposition. Surprisingly, in "DC Medium A" #1 and "DC Medium AP" #1 and #3, bone regeneration was observed that completely filled the entire defect 8 weeks after transplantation, even in a nude rat large calvarial defect model.

[0072] Example 55-1. Alizarin staining (FIG. 12) Fibroblasts (derived from normal human gingiva) were cultured at a density of 2×10e4 cells / 1.9 cm. ２ The cells were seeded at a concentration of 1 / well (24-well plate) and cultured in either standard medium (DMEM supplemented with 10% FBS and 100 μM NEAA) or osteoblast medium (standard medium supplemented with bone differentiation inducer (100 nM dexamethasone, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate (DAG))). Furthermore, a group in which the osteoblast medium was supplemented with DC compound (4 μM TGF-β pathway inhibitor (ALK5 inhibitor II)) and a group in which the DC compound was supplemented with 1 mM VPA were also prepared. The medium was changed approximately every 3 days, and the cells were cultured for 28 days. After the completion of the culture, the culture medium was aspirated from the culture dish, washed twice with distilled water, and fixed in 10% formalin. After washing with sterile distilled water, Alizarin Red S staining solution was added and the cells were left to stand at room temperature for 20 minutes. After washing with sterile distilled water, the cells were observed with the naked eye and under a microscope. The red staining solution was then eluted with 10% cetylpyridinium chloride, and the absorbance (OD550 nm) was quantified. Cells cultured in normal medium were used as a control for evaluation. The results are shown in Figure 12.

[0073] Red staining in macroscopic and microscopic images indicates mineralized bone matrix. The vertical axis of the graph shows the relative OD550 values ​​when the OD550 of the control staining solution is set to 1. It can be seen that adding the DC compound to the osteoblast medium converted fibroblasts into osteoblasts that produce mineralized bone matrix. Furthermore, it can be seen that the addition of VPA improved the effect of the DC compound on conversion to osteoblasts that produce mineralized bone matrix.

[0074] 5-2. Alizarin staining and OCN mRNA expression (qPCR) (Figure 13) Fibroblasts (derived from normal human gingiva) were cultured at 2 x 10e4 cells / 1.9 cm. ２The cells were seeded at a concentration of 1000 / well (24-well plate) and cultured for 28 days in osteoblast medium (containing DAG) supplemented with DC compound (4 μM TGF-β pathway inhibitor (ALK5 inhibitor II)) or osteoblast medium supplemented with DC compound and 1 mM VPA. Alternatively, the cells were cultured for 7, 11, or 15 days in osteoblast medium supplemented with DC compound (4 μM TGF-β pathway inhibitor (ALK5 inhibitor II)), followed by 28 days in osteoblast medium supplemented with DC compound and 1 mM VPA. The medium was changed approximately every 3 days. The culture schedule is shown in Figure 13. After completion of the culture, the cells were fixed with 10% formalin. After washing with sterile distilled water, Alizarin Red S staining solution was added and the cells were left to stand at room temperature for 20 minutes. After washing with sterile distilled water, the cells were observed macroscopically and microscopically. The red stain was then eluted with 10% cetylpyridinium chloride, and the absorbance (OD550nm) was quantified. Cultures in normal medium were used as a control. The results are shown in Figure 13. At the end of the culture, the cells were washed with PBS, and total RNA was recovered from the cells using RNAiso. cDNA was then prepared using Rever Tra Ace qPCR RT Master Mix. SYBR Green Realtime PCR Master Mix, specific primers, and cDNA were mixed, and real-time RT-PCR was performed using StepOne systems to quantify the mRNA expression level of the osteoblast-specific marker OCN. The mRNA expression of cells cultured for 28 days in osteoblast medium supplemented with DC compound (4 μM ALK5 inhibitor II) was evaluated as a control. The vertical axis represents the relative value of OCN mRNA, with the mRNA level of the control cells set at 1. The results are shown in Figure 13.

[0075] Alizarin staining results indicate that the addition of DC compound to osteoblast medium converts fibroblasts into osteoblasts, which produce mineralized matrix, and that this effect is enhanced by the simultaneous addition of VPA. Furthermore, the amount of mineralized matrix production indicated that the addition of VPA after culturing in osteoblast medium containing DC compound further enhanced the osteoblast conversion effect. Furthermore, qPCR results indicated that the addition of VPA after 15 days of pre-culture in osteoblast medium containing DC compound significantly increased the expression of the osteoblast marker OCN mRNA. These results indicate that VPA enhances the osteoblast conversion efficiency induced by DC compound, and that this effect is further enhanced by adding VPA after pre-treatment with DC compound.

[0076] Three-dimensional bone-like tissue can be created by the method of the present invention, which involves the preparation of cell sheets through high-density culture of osteoblasts, followed by suspension culture of the three-dimensional cell masses. The method of the present invention allows for the production of sufficient amounts of osteoblasts in a state useful as a cell therapy (including transplant materials). The resulting osteoblasts can form three-dimensional bone-like tissue. Because the bone-like tissue does not contain cells in the process of differentiation, such as undifferentiated cells, and contains many mature osteoblasts, efficient bone formation and bone regeneration can be achieved using the bone-like tissue. According to the present invention, an effective treatment for the above-mentioned diseases can be provided by producing three-dimensional bone-like tissue containing osteoblasts and transplanting it into a bone defect site. This application is based on Patent Application No. 2024-103122 filed in Japan (filing date: June 26, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a three-dimensional cell mass containing osteoblasts, comprising a step of obtaining a three-dimensional cell mass containing osteoblasts from a cell sheet obtained by a step of culturing cells at a high density to form a cell sheet.

2. The method according to claim 1, comprising: a first step of culturing mammalian somatic cells under conditions that convert them into osteoblasts while increasing the number of somatic cells; a second step of forming a cell sheet by high-density culturing the cells that have undergone the first step under conditions that convert the somatic cells into osteoblasts; and a third step of obtaining a three-dimensional cell mass containing osteoblasts from the cell sheet obtained in the second step.

3. The method according to claim 2, further comprising, prior to the first step, a step (step 0) of culturing the somatic cells under conditions that do not induce direct conversion.

4. The first step of culture is at a seeding density of 1,000 to 32,000 cells / cm. ２ 3. The method of claim 2, wherein the method is carried out by 5. High density culture, or second-step high density culture, is performed at a seeding density of 20,000 to 400,000 cells / cm. ２ 3. The method according to claim 1 or 2, which is carried out as described above.

6. The method of claim 3, wherein the duration of step 0 is 1 to 7 days.

7. The method of claim 2, wherein the duration of the first step is 1 to 20 days.

8. The method of claim 2, wherein the duration of the second step is 1 to 20 days.

9. The method of claim 2, wherein the duration of the third step is 3 to 100 days.

10. The method according to claim 3, wherein the period of the series of steps 0 to 3 is 6 to 147 days.

11. The method according to claim 3, wherein the period of the series of steps 0 to 3 is 19 to 75 days.

12. The method of claim 2, wherein the direct conversion is by addition of a compound.

13. The method of claim 12, wherein the compound is at least one selected from the group consisting of (1) a TGF-β pathway inhibitor, (2) a statin compound, (3) a casein kinase 1 inhibitor, (4) a cAMP inducer, (5) a histone methyltransferase inhibitor, and (6) a histone deacetylase inhibitor (HDAC inhibitor).

14. The method of claim 12, wherein said compound is a TGF-β pathway inhibitor.

15. The method of claim 14, wherein said TGF-β pathway inhibitor is ALK5 inhibitor II.

16. The method of claim 2, wherein the direct conversion is by gene transfer.

17. The method according to claim 16, wherein the gene is at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc (M), L-Myc (L), GLIS family, Klf family, Lin-28, and Sox2.

18. The method of claim 16, wherein the genes are a combination of at least one bone-related gene selected from the group consisting of Runx2 (R), Osterix (O), and Dlx5 (D) and at least one reprogramming-related gene selected from the group consisting of the Oct family, c-Myc (M), L-Myc (L), GLIS family, Klf family, Lin-28, and Sox2.

19. The method of claim 2, wherein the mammalian somatic cells are fibroblasts.

20. The method according to claim 2, wherein the culture medium contains at least one of the following components in at least one of the first, second, and third steps (excluding the compound used in direct conversion): (1) a TGF-β pathway inhibitor, (2) a statin compound, (3) a casein kinase 1 inhibitor, (4) a cAMP inducer, (5) a histone methyltransferase inhibitor, and (6) a histone deacetylase inhibitor (HDAC inhibitor).

21. The method of claim 20, wherein in the second step, the medium contains a histone deacetylase inhibitor.

22. The method according to claim 1 or 2, wherein the three-dimensional cell mass expresses OCN (osteocalcin).

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