Method for preparing cells

Abstract

To provide a cell preparation method, particularly a method, an inducer, and a kit for converting differentiated somatic cells into other somatic cells without gene transfer.SOLUTION: Provided is a method for preparing somatic cells characterized in that differentiated somatic cells of a mammal are cultured in a medium for inducing differentiation of somatic cells other than the differentiated somatic cells in the presence of a TGF-β pathway inhibitor, thereby converting the differentiated somatic cells into other somatic cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application is Japanese Patent Application No. 2015-207529, filed on October 21, 2015. We claim priority under the detailed statement (the entire disclosure thereof is incorporated herein by reference).

[0002] This invention primarily relates to a method for preparing cells. More specifically, it relates to a method for preparing cells by direct reprogramming. This invention also relates to an inducer for converting differentiated somatic cells into other somatic cells. [Background technology]

[0003] In recent years, regenerative medicine technologies that treat diseases by transplanting cells into patients to compensate for abnormalities in function or form have attracted attention.

[0004] For example, in the case of osteoblasts, transplanting osteoblasts to the lesion site is expected to promote bone formation and improve functional and morphological prognosis, for the purpose of repairing bone defects associated with bone tumors, trauma, osteomyelitis, etc., and bone defects after curettage of bone tumors, etc. In fact, autologous transplantation of bone marrow cells harvested from the patient's cancellous bone is being performed, and its effectiveness is known. In this case, it is thought that osteoblasts are differentiated from mesenchymal stem cells contained in the autologous bone marrow cells, contributing to bone formation and remodeling. On the other hand, the incidence of osteoporosis is increasing with the aging population, and fractures in the elderly can lead to prolonged bed rest. Osteoblast transplantation is thought to be able to promote the healing of fractures associated with osteoporosis and trauma, as well as intractable fractures and pseudofractures. Osteoblast transplantation may also be useful for rheumatoid arthritis, idiopathic femoral head necrosis, osteoarthritis, lumbar degenerative spondylosis, spinal stenosis, herniated disc, spondylolysis, spondylolisthesis, scoliosis, cervical spondylotic myelopathy, ossification of the posterior longitudinal ligament, spinal cord injury, osteoarthritis of the hip, osteoarthritis of the knee, slipped capital femoral epiphysis, osteomalacia, bone repair after surgery (such as sternal repair after cardiac surgery), repair of defects associated with artificial ankle joint surgery, osteomyelitis, and osteonecrosis.

[0005] On the one hand, periodontal disease is also called the fourth lifestyle disease, with an extremely high incidence rate and being the cause of various systemic diseases. As periodontal disease progresses, bone resorption of the alveolar bone occurs. Therefore, if osteoblasts can be efficiently supplied to the local bone resorption site, it is considered to lead to the regenerative treatment of the alveolar bone.

[0006] In addition, if the transplantation of osteoblasts is combined with bone transplantation, artificial bone transplantation, artificial joints or implants, the therapeutic effect may be enhanced.

[0007] In regenerative medicine technology, the supply means of cells used is one of the issues.

[0008] For example, as osteoblasts for transplantation purposes, bone marrow mesenchymal stem cells and bone marrow cells including bone marrow mesenchymal stem cells have been used so far. However, there are problems such as the collection of bone marrow being highly invasive to the patient and there being cases where a sufficient number of bone marrow cells cannot be supplied. On the other hand, if human embryonic stem cells (ES cells) are used, there is no need to collect bone marrow from the patient, and there is a possibility of supplying a sufficient number of osteoblasts. However, in addition to ethical issues, there is a risk of tumorigenesis of residual ES cells after transplantation. Also, if iPS cells are used, there is no need to collect bone marrow from the patient, and there is a possibility of supplying a sufficient number of osteoblasts, but there is a risk of tumorigenesis of residual iPS cells after transplantation .

[0009] Similar problems exist for other cells.

[0010] Non-Patent Document 1 conducts differentiation induction into osteoblasts by introducing the Lentivirus vector of Osterix into human ES cells + Osteogenic medium . Non-Patent Documents 2 and 3 obtain osteoblasts by differentiating mouse iPS cells through MSC and then inducing differentiation in Osteogenic medium.

[0011] Non-Patent Document 4 introduced the Adenovirus vector of Runx2 into mouse iPS cells and induced differentiation in Osteogenic medium to obtain osteoblasts. As shown in Non-Patent Documents 1 to 4, osteoblasts are prepared by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, so long-term culture is required and there is a risk of canceration.

[0012] Regarding the direct induction of differentiation into tissue cells without passing through iPS cells by introducing a group of genes of tissue-specific transcription factors into somatic cells (direct conversion (direct reprogramming)), for example, there are the following reports: Mouse fibroblasts → chondrocytes (introduction of SOX9 + Klf4 + c-Myc genes) Mouse fibroblasts → cardiomyocytes (introduction of GATA4 + Mef2c + Tbx5 genes) Mouse fibroblasts → hepatocytes (introduction of Hnf4α + (Foxa1 or Foxa2 or Foxa3) genes) Mouse fibroblasts → neural stem cells (such as introduction of Sox2 + FoxG1 genes), Mouse and human cells → hematopoietic stem cells, etc.

[0013] Patent Document 1 discloses a method for efficiently preparing (direct conversion) functional osteoblasts by introducing a predetermined group of genes into somatic cells. However, in the method of gene introduction, there is a possibility that cells may become tumorigenic due to the influence of the introduced genes and vectors. There are also problems such as the need for cost and time for safety verification. Therefore, a technique for inducing cells for transplantation without gene introduction is required.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Non-Patent Documents

[0015] [Non-Patent Document 1] Karner E et al. J Cell Physiol. 2009. [Non-Patent Document 2] Li F et al. J Cell Biochem. 2010. [Non-Patent Document 3] Biloussova G et al. Stem cells. 2011. [Non-Patent Document 4] Tashiro K et al. Stem cells. 2009. [Overview of the project] [Problems that the invention aims to solve]

[0016] The present invention primarily aims to provide a method for preparing cells, and in particular a technique for converting differentiated somatic cells into other somatic cells without gene transfer (direct conversion (direct reprogramming) technique). [Means for solving the problem]

[0017] The inventors have discovered that differentiated mammalian somatic cells can be converted into other somatic cells other than the original somatic cells by culturing them in culture media of various compositions in the presence of a TGF-β pathway inhibitor.

[0018] There are no reports of direct conversion (direct reprogramming) using TGF-β pathway inhibitors.

[0019] The present invention encompasses the following methods for preparing osteoblasts, osteoblast inducers, and kits.

[0020] Item 1, A method for preparing somatic cells, characterized by culturing differentiated mammalian somatic cells in a culture medium for inducing differentiation of other somatic cells, in the presence of a TGF-β pathway inhibitor, thereby converting the differentiated somatic cells into other somatic cells.

[0021] Item 2, The TGF-β pathway inhibitor is a TGF-β / SMAD pathway inhibitor as described in Item 1. The method.

[0022] The method according to item 3, wherein the TGF-β pathway inhibitor is D4476, SB431542, LY2157299, SD208, or ALK5 inhibitor II.

[0023] Item 4, the TGF-β pathway inhibitor is ALK5 inhibitor II, one of items 1-4 The method described in section [section number].

[0024] Item 5, The method according to any one of items 1 to 4, characterized by converting fibroblasts into mesenchymal cells.

[0025] Item 6, The method according to any one of items 1 to 4, characterized by converting fibroblasts or keratinocytes into osteoblasts.

[0026] Item 7, The method according to any one of items 1 to 4, characterized by converting fibroblasts or peripheral blood mononuclear cells into white adipocytes.

[0027] Item 8, The method according to any one of items 1 to 4, characterized by converting fibroblasts or keratinocytes into brown adipocytes.

[0028] Item 9, the method according to item 7 or 8, wherein the culture medium for inducing differentiation of somatic cells comprises a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist.

[0029] Item 10, The method according to any one of items 1 to 4, characterized by converting fibroblasts into chondrocytes.

[0030] Item 11, The method according to any one of items 1 to 4, characterized by converting fibroblasts into myoblasts.

[0031] Item 12, The method according to any one of items 1 to 4, characterized by converting fibroblasts into Schwann cells.

[0032] Item 13, The method according to any one of items 1 to 4, characterized by converting keratinocytes into urothelial cells.

[0033] Item 14, The method according to any one of items 1 to 4, characterized by converting fibroblasts into mesenchymal stem cells.

[0034] Item 15, Inducers for converting differentiated somatic cells into other somatic cells, including TGF-β pathway inhibitors.

[0035] Item 16, A kit for converting differentiated somatic cells into other somatic cells, comprising a TGF-β pathway inhibitor and a medium for inducing differentiation of the other somatic cells. [Effects of the Invention]

[0036] In this invention, other somatic cells can be provided from differentiated somatic cells through the action of a small molecule compound in a short period of time. Since the obtained somatic cells (e.g., mesenchymal stem cells or osteoblasts) can be easily induced from the recipient's own somatic cells, problems such as immunological rejection do not occur when the obtained somatic cells themselves or tissues produced from them are transplanted. Furthermore, this invention allows for the use of iPS cells and ES cells. Because it allows for the direct induction of somatic cells from other somatic cells, it avoids problems caused by pluripotent stem cells, such as cancer development. On the other hand, it is also possible to create and bank the cells in advance and use them for allotransplantation or xenotransplantation in patients. [Brief explanation of the drawing]

[0037] [Figure 1] The results of alizarin red S staining (staining diagram) are shown. [Figure 2] The results of alizarin red S staining (absorbance measurement) are shown. [Figure 3] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 4] The results of immunohistochemical staining for osteocallinin (staining diagram) are shown. [Figure 5] The results of alizarin red S staining (staining diagram and absorbance measurement) are shown. [Figure 6] The results of immunohistochemical staining of Runx2 (staining diagram) are shown. [Figure 7] The results of Bodipy staining (staining diagram) are shown. [Figure 8] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 9] The results of Alcian blue staining (staining diagram) are shown. [Figure 10] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 11] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 12] The results of the Oil Red O staining (staining diagram) are shown. [Figure 13] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 14] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 15A] The results of Bodipy staining (staining diagram) are shown. [Figure 15B] The results of Bodipy staining (fluorescence intensity measurement) are shown. [Figure 16] The TGF-β / SMAD pathway is schematically shown. [Figure 17] The TGF-β / SMAD pathway and the TGF-β / ERK pathway are schematically shown. [Figure 18] The TGF-β / SMAD pathway, TGF-β / JNK pathway, and TGF-β / p38 pathway are schematically shown. [Figure 19] The TGF-β / SMAD pathway and the TGF-β / RhoA pathway are schematically shown. [Figure 20] The results of alizarin red S staining (staining diagram) are shown. [Figure 21] The results of ALP staining (staining diagram) are shown. [Figure 22] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 23] The results of the Oil Red O staining (staining diagram) are shown. [Figure 24] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 25] The results of Alcian blue staining (staining diagram) are shown. [Figure 26] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 27] The results of alizarin red S staining (staining diagram) are shown. [Figure 28] The results of ALP staining (staining diagram) are shown. [Figure 29] The image shows a cross-sectional view (μCT analysis). The arrowheads indicate bone defects, and the arrows indicate regenerated bone tissue (callus). [Figure 30] This image shows a 3D reconstruction of a microCT scan. The image was taken 21 days after transplantation. Arrows indicate bone defects or regenerated bone tissue (callus). The upper panel shows a cross-section, and the lower panel shows a longitudinal section. [Figure 31]The results of histological analysis using hematoxylin-eosin staining (HE staining) and alizarin red S staining are shown. [Figure 32] This heatmap shows the expression levels of MSC marker genes as determined by DNA microarray analysis. [Figure 33] The results of alizarin red S staining (staining diagram) are shown. [Figure 34] The results of alizarin red S staining (absorbance measurement) are shown. [Figure 35] The results of alizarin red S staining and ALP staining (staining diagram) are shown. [Figure 36] The results of alizarin red S staining (absorbance measurement) are shown. [Figure 37] The results of ALP staining (staining diagram) are shown. [Figure 38] The results of alizarin red S staining (staining diagram) are shown. [Figure 39] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 40] The results of immunohistochemical staining for osteocalcin (OC) are shown (staining diagram and percentage of OC-positive cells among DAPI-positive cells). [Figure 41] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 42] The results of immunohistochemical staining for UCP1 (stained image) are shown. [Figure 43] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 44] The results of immunohistochemical staining for UCP1 (staining diagram) are shown. [Figure 45] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 46] The results of immunohistochemical staining for UCP1 are shown (stained image (A) and percentage of UCP1-positive cells among DAPI-positive cells (B)). [Figure 47] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 48] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 49] The results of Bodipy staining (staining diagram) are shown. [Figure 50] The results of Bodipy staining (percentage of Bodipy-positive cells) are shown. [Figure 51] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 52] The results of Bodipy staining (staining diagram) are shown. [Figure 53] The results of Bodipy staining (quantitative) are shown. [Figure 54] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 55] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 56] The results of gene expression level measurements using real-time RT-PCR are shown. [Figure 57] The results of gene expression level measurements using real-time RT-PCR are shown.

[0038] Figures 4, 6, and 7 also show the color-inverted images. [Modes for carrying out the invention]

[0039] This invention relates to a method for preparing other somatic cells using differentiated mammalian somatic cells as raw materials. In other words, this invention relates to a method for converting differentiated mammalian somatic cells into other somatic cells. "Convert" means transforming somatic cells into target somatic cells. One preferred embodiment of the method of this invention is a cell reprogramming process, also called "direct conversion" or "direct reprogramming," which is exemplified by the creation of iPS cells. This is a method of converting somatic cells into other somatic cells without any intermediate steps. In other words, it is a method of directly converting somatic cells into other somatic cells.

[0040] In a preferred embodiment of the method of the present invention, somatic cells are converted to other somatic cells without gene introduction. "Without gene introduction" means that the somatic cells are converted to other somatic cells without altering the original genomic sequence (primarily meaning the DNA base sequence) of the somatic cells. Alternatively, "without gene introduction" means that the somatic cells are converted to other somatic cells based on the function of the original endogenous genes of the somatic cells.

[0041] Differentiated somatic cells (raw material) The differentiated somatic cells of a mammal used as raw materials in the method of the present invention are not particularly limited as long as they are of mammalian origin. Somatic cells refer to cells other than germ cells.

[0042] Examples of somatic cells include fibroblasts, epithelial cells (such as skin epidermal cells, oral mucosal epithelial cells, airway mucosal epithelial cells, and intestinal mucosal epithelial cells), epidermal cells, gingival cells (gingival fibroblasts and gingival epithelial cells), dental pulp cells, white adipocytes, subcutaneous fat, visceral fat, muscle, and blood cells (for example, peripheral blood mononuclear cells). Preferably, these include fibroblasts, gingival cells, oral mucosal epithelial cells, dental pulp cells, adipocytes, epidermal keratinocytes, and blood cells.

[0043] In addition, mesenchymal stem cells (MSC), neural stem cells, hepatic stem cells, intestinal stem cells, skin stem cells, hair follicle stem cells, pigment cell stem cells This also includes somatic cells produced by differentiating, dedifferentiating, or reprogramming somatic stem cells such as other cells. Furthermore, it includes cells derived from various somatic cells by differentiating, dedifferentiating, or reprogramming them into other somatic cells. Finally, it includes somatic cells derived from germline cells by differentiating, dedifferentiating, or reprogramming them.

[0044] Furthermore, cultured cells can also be mentioned, as can somatic cells induced by differentiation, dedifferentiation, or reprogramming from cultured cells.

[0045] Examples of mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cattle, horses, and pigs. It is particularly preferable that the somatic cells are of human origin. The age of the individual from which the somatic cells originate is not limited; they may be adults, children, or fetuses. In this specification, cells derived from fetuses, as well as cells derived from the placenta, amniotic membrane, umbilical cord, etc., are also included in the definition of "somatic cells."

[0046] When transplanting prepared somatic cells into a living organism, it is preferable to use somatic cells derived from the recipient (autologous cells) to reduce the risk of infection and rejection. However, somatic cells made from the somatic cells of another person or another animal can be used for transplantation instead of autologous cells. Alternatively, other somatic cells can be created from somatic cells of another person or another animal that have been prepared in advance and used for transplantation. Or, somatic cells that have been prepared in advance from the somatic cells of another person or another animal can be used for transplantation. In other words, a somatic cell bank or a somatic cell progenitor cell bank can be created and made available for transplantation purposes. In such cases, blood type and MHC can be typed in advance to reduce the risk of rejection. It is possible to check the characteristics and tumorigenicity of somatic cells intended for transplantation in advance.

[0047] Other somatic cells to be prepared Other somatic cells prepared by the method of the present invention are somatic cells other than the somatic cells used as raw materials. Furthermore, somatic cells that differentiate from the somatic cells used as raw materials under physiological conditions (especially in vivo) are not included in "other somatic cells." For example, if mesenchymal stem cells are used as raw materials because they differentiate from mesenchymal stem cells into osteoblasts in vivo, then osteoblasts do not fall under the category of "other somatic cells."

[0048] Examples of somatic cells that can be prepared include mesenchymal stem cells (MSCs), osteoblasts, and adipose cells. Examples include cells (brown or white adipocytes), chondrocytes, myoblasts, urothelial cells, bone marrow stromal cells, tendinocytes, hepatocytes, bile duct epithelial cells, glial cells such as Schwann cells, nerve cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, and lymphatic endothelial cells.

[0049] Furthermore, epidermal cells, pigment cells, hair follicle cells, nail matrix cells, connective tissue cells, lung and airway cells (airway epithelial cells, alveolar epithelial cells, etc.), gastrointestinal epithelial cells, glandular cells, hematopoietic stem cells, lymphocytes, granulocytes, monocytes, macrophages, mast cells, megakaryocytes, platelets, erythroblasts, erythrocytes, and lymphoreticular cells. Examples include stem cells, antigen-presenting cells, mammary gland epithelial cells, kidney cells (such as glomerular cells, tubular epithelial cells, and urothelial cells), reproductive system cells, corneal cells, conjunctival cells, retinal cells, synovial cells, endocrine cells, pancreatic islet cells (such as insulin-producing cells (β cells)), exocrine cells, hepatic stem cells, pancreatic stem cells, intestinal stem cells, and salivary gland stem cells.

[0050] The combination of somatic cells used as raw materials and the somatic cells to be prepared is not particularly limited.

[0051] From the viewpoint of high efficiency in preparing somatic cells, it is preferable that both the somatic cells used as raw materials and the other somatic cells prepared are somatic cells belonging to the mesenchymal lineage, differentiated from mesenchymal stem cells.

[0052] Cells belonging to the mesenchymal lineage include mesenchymal stem cells (MSCs), fibroblasts, osteoblasts, and adipocytes. Examples include cells such as cysts, chondrocytes, myoblasts, bone marrow stromal cells, and tendon cells. Cells known to potentially differentiate from mesenchymal stem cells, such as hepatocytes, bile duct epithelial cells, glial cells, nerve cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, and lymphatic endothelial cells, are also included in the category of "cells belonging to the mesenchymal lineage."

[0053] Osteoblasts include preosteoblasts, immature osteoblasts, mature osteoblasts, and osteocytes. Adipocytes include white adipocytes and brown adipocytes. White adipocytes include adipose stem cells, preadipocytes, mature adipocytes, and hypertrophic adipocytes. Brown adipocytes include beige cells and Brite cells. Chondrocytes include immature chondrocytes and mature chondrocytes. This includes chondrocytes, hypertrophic chondrocytes, etc. Myoblasts include muscle satellite cells, immature myoblasts, mature myoblasts, muscle cells, myotubes, muscle fibers, etc. Similarly, all cells described herein include cells of different degrees of differentiation within the cell lineage represented by their name.

[0054] A specific example of an aspect of the present invention is the conversion of fibroblasts into mesenchymal stem cells (MSCs). Examples of methods include converting fibroblasts into osteoblasts, converting fibroblasts into adipocytes (for example, converting fibroblasts into white adipocytes, converting fibroblasts into brown adipocytes, etc.), converting fibroblasts into chondrocytes, converting fibroblasts into chondrocytes, converting fibroblasts into myoblasts, converting keratinocytes into urothelial cells, and converting blood cells (for example, peripheral blood mononuclear cells) into white adipocytes.

[0055] Furthermore, gingival cells, oral mucosal epithelial cells, dental pulp cells, adipocytes, blood cells, etc., are used as raw materials and converted into mesenchymal stem cells (MSCs), osteoblasts, adipocytes, chondrocytes, and myoblasts. Examples of methods are provided.

[0056] In this invention, the process also includes converting raw materials into intermediate cells (e.g., MSCs, MSC-like cells, other somatic stem cells, etc.) and then differentiating those intermediate cells into the final target cells (osteoblasts, adipocytes, chondrocytes, myoblasts, etc.). Since intermediate cells are also a type of somatic cell, this process is referred to as direct conversion in this specification.

[0057] Culture medium In the method of the present invention, differentiated somatic cells are cultured in a culture medium for inducing other somatic cells (differentiation induction medium). As the differentiation induction medium, known differentiation induction media can be used depending on the somatic cells to be prepared.

[0058] "Differentiation induction medium" refers to a culture medium used to induce somatic cells other than differentiated somatic cells. This refers to a culture medium containing [a specific component].

[0059] For example, as a culture medium for inducing osteoblasts, ascorbic acid (for example, at a concentration of 0.1%) ~1000 μg / ml, preferably 1~100 μg / ml); β-Glycerophosphate (for example, Concentration of approximately 0.1 to 1000 mM, preferably 1 to 100 mM); a group consisting of dexamethasone (approximately 1 nM to 10 mM, preferably 10 to 1000 mM) and glucocorticoids such as hydrocortisone. Examples include adding one or more components selected from the above to a standard liquid culture medium. Specific examples include adding 50 μg / ml ascorbic acid, 10 mM β-Glycerophosphate, and 100 nM dexamethasone (all final concentrations) to a standard medium such as DMEM supplemented with 10% FBS or 5% HS. However, the method is not limited to these examples.

[0060] For the culture medium used to induce white adipocytes, one or more components such as insulin (e.g., concentration of about 0.01 to 100 μg / mL, preferably about 0.1 to 10 μg / mL), 3-isobutyl-1-methylxanthine (IBMX) (e.g., concentration of about 0.01 to 100 mM, more preferably about 0.1 to 10 mM), and dexamethasone (e.g., concentration of about 0.01 to 100 μM, more preferably about 0.1 to 10 μM) are added to the standard culture medium. Examples are given. A specific example is 10% FBS-added DMEM + MDI medium (10% FBS-added DMEM with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexametazone, and 1 μg / mL insulin added). However, it is not limited to these.

[0061] From the viewpoint of achieving high efficiency in converting to white adipocytes, it is preferable to further add a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist (for example, at a concentration of about 0.01 to 100 μM, more preferably about 0.1 to 10 μM).

[0062] Examples of PPAR-γ agonists include thiazolidinedione compounds such as rosiglitazone, ciglitazone, GW1929, nTZDpa, pioglitazone hydrochloride, and troglitazone.

[0063] The culture medium for inducing brown adipose tissue includes: insulin (e.g., concentration of approximately 0.01-100 μg / mL, more preferably 0.1-10 μg / mL); 3-isobutyl-1-methylxanthine (IBMX) (e.g., concentration of approximately 0.01-100 mM, more preferably 0.1-10 mM); and dexametazone (e.g., concentration of approximately 0.01-100 μM). Examples include adding a concentration (more preferably about 0.1 to 10 μM) to a normal culture medium. Indomethacin (for example, concentration of about 0.001 to 10 mM, more preferably 0.01 to 1 mM) You may also add an amount (approximately mM).

[0064] From the perspective of high efficiency in converting to brown adipose tissue, further thyroid hormone (e.g., concentration of approximately 0.01-100 nM, more preferably 0.1-10 nM) and / or peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist (e.g., concentration of approximately 0.01-100 μM) are also recommended. It is preferable to add (more preferably about 0.1 to 10 μM), and it is preferable to add both. It is preferable.

[0065] Examples of thyroid hormones include triiodothyronine (T3) and thyroxine (T4).

[0066] Examples of PPAR-γ agonists include thiazolidinedione compounds such as rosiglitazone, ciglitazone, GW1929, nTZDpa, pioglitazone hydrochloride, and troglitazone.

[0067] Specific examples of culture media for inducing brown adipose tissue include: [1] FBS 10%, 0.5 mM IBMX, 12 5 nM Indomethacin, 1 microM Dexamethasone, 850 nM insulin, 1 nM Triiodothiol Examples include DMEM medium supplemented with triidothyronine (T3) and 1 μM rosiglitazone, and DMEM medium supplemented with 10% FBS, 850 nM insulin, 1 nM T3, and 1 μM rosiglitazone. [1] can be used on days 1-2, and [2] from day 3 onwards.

[0068] Furthermore, as a culture medium for inducing brown adipocytes from fibroblasts, 1 nM T3, 1 μM Rosiglitazone, 0.5 mM isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 DMEM supplemented with μg / mL insulin and 10% FBS can be used. For inducing brown adipocytes from keratinocytes, 1 nM T3, 1 μM Rosiglitazone, 0.5 mM can be used as the culture medium. Added isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL Insulin. You can use the HuMedia-KG2.

[0069] However, it is not limited to these.

[0070] Examples of PPAR-γ agonists include rosiglitazone, ciglitazone, GW1929, nTZDpa, pioglitazone hydrochloride, and troglitazone.

[0071] For inducing mesenchymal stem cells, the culture media described in the following references (1-1) to (1-4), for example, can be used, but are not limited to these. (1-1) MD Hoffman and DSW Benoit, J Tissue Eng Regen Med. 2013 Apr 1. doi: 10.1002 / term.1736 (1-2) CY Li, et al., Stem Cell Res Ther. 2015 Apr 13;6:55. doi: 10.1186 / s13287-015-0066-5. (1-3) B. Gharibi and FJ Hughes, Stem Cells Transl Med. 2012 Nov;1(11):771-82. doi: 10.5966 / sctm.2010-0031. Epub 2012 Oct 23 (1-4) SK Both et al. Tissue Eng. 2007 Jan;13(1):3-9. (1-5) F. Ng et al., Blood. 2008 Jul 15;112(2):295-307. doi: 10.1182 / blood-2007-07-103697. Epub 2008 Mar 10. (1-6) Hynes K. et al., J Dent Res. 2013 Sep;92(9):833-9.

[0072] Known culture media can be used to induce chondrocytes. The media described in the following references (2-1) to (2-4) can be used, but are not limited to these. (2-1) H. Outani, et al., PLOS One, 8(10): e77365. doi:10.1371 / journal.pone.0077365 (2-2)G.-I. Im, et al. Tissue Engineering. March 2006, 12(3): 527-536. doi:10.1089 / ten.2006.12.527. (2-3) H.J. Kim and G.-I. Im, Journal of Orthopedic Research,Volume 27, Issue 5, pages 612-619, May 2009 (2-4) AM Ibrahim et al., Microscopy Research and Technique, Volume 78, Issue 8, pages 667-675, August 2015.

[0073] Known culture media can be used to induce myoblasts. For example, Ham's / F10 medium supplemented with 5% FBS, 50 microG / ml Bovine Fetuin, 10 nG / ml hEGF, 1 nG / ml bFGF, 10 microG / ml Insulin, 0.4 microG Dexamethasone; DMEM medium supplemented with 10% FBS, 10 microM 5-azacytidine, 10 ng / mL VEGF, 10 ng / mL IGF-1, 10 ng / mL bFGF; and αMEM medium supplemented with 5% Horse serum, 10 ng / mL can be used. Contribution: Int J Mol Med. 2014 Jan;33(1):160-70. doi: 10.3892 / ijmm.2013.1555. Epub 2013 The culture media described in Nov 13. may be used, but are not limited to these.

[0074] Known culture media can be used to induce nerve cells. For example, [1] DMEM medium supplemented with 1 microM Retinoic acid (RA) and 5 microM FSK, [2] DMEM medium supplemented with 1 microM RA, 5 microM FSK, and 10 ng / mL bFGF, and [3] DMEM medium supplemented with 1 microM RA, 1 microM FSK, 10 ng / mL bFGF, and 100 ng / mL SHH are used, with [1] used on days 1-3, [2] on days 4-5, and on day 6. [3] can be used from the next step onward. Alternatively, the culture medium described in, for example, Yan Y. et al., Stem Cells Transl Med. 2013 Nov;2(11):862-70. can be used, but is not limited to these.

[0075] Known culture media can be used to induce glial and neuronal cells. For example, the media described in L. Hook et al., Neurochemistry International. Volume 59, Issue 3, September 2011, Pages 432-444 and Selvaraj V. et al., Front Biosci (Landmark Ed). 2012 Jan 1;17:65-89. can be used, but are not limited to these.

[0076] Any known culture medium can be used to induce glial cells. For example, the medium described in Duan L. et al., Stem Cells Transl Med. 2015 May;4(5):437-47. can be used, but is not limited to these.

[0077] Known culture media can be used to induce Schwann cells. For example, DMEM medium (Dulbecco's modified Eagle medium) supplemented with 10% FBS (fetal bovine serum) can be used. Add approximately 1-20 μM (especially 5) to a standard culture medium such as Dulbecco's Modified Eagle's Medium. Forskolin (approximately μM); bFGF (basic fibroblast growth factor) at approximately 2-50 ng / ml (especially around 10 ng / ml); approximately 2-50 ng / ml (especially around 10 ng / ml) A culture medium (Schwann cell induction medium) can be used that contains one or more components (preferably all of them), such as PDGF (Platelet-Derived Growth Factor) at approximately 50-1000 ng / ml (especially about 200 ng / ml) of human neuregulin-β1 (also known as heregulin, GGF (Glial growth factor)). (All of the above concentrations are final concentrations.) As an example, the culture medium described in the following references (3-1) to (3-2) (a culture medium capable of inducing Schwann cells from undifferentiated adipose stem cells) can be used. (3-1)Kingham PJ, DF Kalbermatten, D Mahay, et al: Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. ExpNeurol, 2007; 207:267-274. (3-2)Liu Y, Zhang Z, Qin Y, Wu H, Lv Q, Chen X, Deng W: A new method for Schwann-like cell differentiation of adipose derived stem cells. Neurosci Lett. 2013 Sep 13;551:79-83.

[0078] Known culture media can be used to induce cardiomyocytes. For example, the culture media described in the reference: YJ Nam et al., Proc Natl Acad Sci US A. 2013 Apr 2;110(14):5588-93. doi: 10.1073 / pnas.1301019110. Epub 2013 Mar 4. can be used. However, it is not limited to these.

[0079] For inducing vascular endothelial cells, known culture media can be used. For example, DMEM supplemented with 10% FBS and 100 ng / mL VEGF can be used. Alternatively, the method described in Chatterjee I. et al., Methods Mol Biol. 2015 Feb 17. PMID:25687301 can be used. It is possible. However, it is not limited to these.

[0080] Known culture media can be used to induce smooth muscle cells. For example, this can be done by the method described in Wang Y. et al., Biomaterials. 2014 Oct;35(32):8960-9. However, it is not limited to these.

[0081] Known culture media can be used to induce mast cells. For example, [1] DMEM medium supplemented with 15% FBS and 20 ng / mL VEGF, [2] IDMD medium supplemented with 10% FBS, 10 ng / mL IL-3, 100 ng / mL IL-6, 10 ng / mL Flt3L, 10 ng / mL TPO, and 10 ng / mL VEGF, [3] 10% Stem span, 100 ng / mL SCF, 100 ng / mL IL-6, 10 ng / mL Flt3L, 10 ng / mL TPO, and 10 ng / mL Using IDMD supplemented with VEGF, [1] was administered on days 1-8, [2] on days 9-18, and [3] from day 19 onward. These may be used, but are not limited to them.

[0082] For inducing β-cells (insulin-producing cells), known media can be used. For example, DMEM supplemented with 15% FBS, 100 ng / mL Activin A, 10 nM GLP-1, 10 mM Nicotinamide, 20 ng / mL EGF, 10 ng / mL bFGF, and ITS can be used. Alternatively, media described in Shahjalal HM et al., J Mol Cell Biol. 2014 Oct;6(5):394-408. and Noguchi H. et a., Curr Diabetes Rev. 2010 May;6(3):184-90. can be used. However, the media are not limited to these.

[0083] Any known culture medium can be used to induce hepatocytes. For example, the medium described in Y. Yu et al., Stem Cell Res. 2012 Nov;9(3):196-207 can be used. However, it is not limited to these.

[0084] Known culture media can be used to induce gastrointestinal cells. For example, the media described in Spence JR. et al. Nature. 2011 Feb 3;470(7332):105-9. and Wells JM and Spence JR. Development. 2014 Feb;141(4):752-60. can be used. However, they are not limited to these.

[0085] Known culture media can be used to induce lung and airway cells. For example, the media described in Ghaedi M. et al., J Clin Invest. 2013 Nov;123(11):4950-62. These may be used, but are not limited to them.

[0086] Known culture media can be used to induce urothelial cells. For example, the media described in Osborn SL et al., Stem Cells Transl Med. 2014; 3(5): 610-619. and Kang M. et al., Int. J. Mol. Sci. 2014, 15(5), 7139-7157 can be used. However, they are not limited to these.

[0087] Known culture media can be used to induce kidney cells. For example, see Lam AQ, et al. Semin Nephrol. 2014 Jul;34(4):445-61. and Lam AQ, and Bonventre Use the culture medium described in JV. Curr Opin Organ Transplant. 2015 Apr;20(2):187-92. It is possible to do these things. However, it is not limited to these.

[0088] For inducing hematopoietic stem cells, known culture media can be used. For example, the culture media described in Wang Y, et al., Proc Natl Acad Sci USA. 2005;102:19081-6. can be used. However, it is not limited to these.

[0089] For inducing blood cells, known culture media can be used. For example, the culture media described in Nakayama N. et al., Blood. 1998 Apr 1;91(7):2283-95. can be used. It is possible to do these things. However, it is not limited to these.

[0090] Known media can be used to induce lymphocytes. For example, media described in Carpenter L. et al., Blood. 2011 Apr 14;117(15):4008-11. and Vodyanik MA, et al., Blood. 2005;105:617-26. can be used. However, this is not limited to these. It is not something that can be determined.

[0091] Any known culture medium can be used to induce erythroblasts. For example, the medium described in Lu SJ, et al., Blood. 2008;112:4475-84. can be used. However, it is not limited to these.

[0092] Any known culture medium can be used to induce megakaryocytes. For example, the medium described in Takayama N. and Eto K. Methods Mol Biol. 2012;788:205-17. can be used. However, it is not limited to these.

[0093] Known culture media can be used to induce dendritic cells and macrophages. For example, the culture media described in Senju S. et al., Gene Ther. 2011 Sep;18(9):874-83. Land can be used. However, it is not limited to these.

[0094] Any known culture medium can be used to induce granulocytes. For example, the medium described in Morishima T. et al., J Cell Physiol. 2011 May;226(5):1283-91. can be used. However, it is not limited to these.

[0095] Any known culture medium can be used to induce retinal cells. For example, the medium described in Maeda T. et al., J Biol Chem. 2013 Nov 29;288(48):34484-93 can be used. However, it is not limited to these.

[0096] Any known culture medium can be used to induce corneal cells. For example, the medium described in Yu D. et al., Cell Biol Int. 2013 Jan;37(1):87-94. can be used. However, it is not limited to these.

[0097] Standard liquid culture media such as Dulbecco's Modified Eagle's Medium and EMEM (Eagle's minimal essential medium) can be used. Serum components (Fetal Bovine Serum (FBS), Human Serum (Serum)) and streptomycin can be added as needed. Antibacterial agents such as penicillin and components such as non-essential amino acid can be added.

[0098] TGF-β pathway inhibitors In the method of the present invention, somatic cells are cultured in a culture medium for inducing other somatic cells in the presence of a TGF-β pathway inhibitor.

[0099] "TGF-β pathway inhibitors" refer to TGF-β / SMAD pathway inhibitors, TGF-β / Erk pathway inhibitors, etc. This includes TGF-β / JNK pathway inhibitors, TGF-β / p38 pathway inhibitors, and TGF-β / RhoA pathway inhibitors. In other words, it includes molecules of the TGF-β receptor family, and their receptors. The TGF-β inhibitor of the present invention is an inhibitor that suppresses one or more of the molecules that constitute the TGF-β / SMAD pathway, TGF-β / Erk pathway, TGF-β / JNK pathway, TGF-β / p38 pathway, or TGF-β / RhoA pathway downstream of TGF-β superfamily cytokines or TGF-β receptor family molecules that act as GANDs.

[0100] "TGF-β pathway inhibitors" are not limited to small molecule compounds that are inhibitors in the narrow sense, but also include cytokine neutralizing antibodies, receptor antagonists, soluble receptors, antibodies, aptamers, peptides, mutant proteins and peptides and their analogs that bind to pathway proteins and inhibit their action, siRNA, shRNA, and microRNA that suppress the expression of pathway proteins.

[0101] TGF-β / SMAD pathway inhibitors TGF-β / SMAD pathway inhibitors inhibit the activity of proteins belonging to the TGF-β / SMAD pathway. This refers to compounds that can be inhibited. The TGF-β / SMAD pathway is a signaling pathway known to those skilled in the art, schematically shown in Figure 16 (quoted from Chen G et al, Int J Biol Sci, 2012).

[0102] The TGF-β / SMAD pathway is composed of proteins belonging to the TGF-β superfamily. Ligands that are affected (TGF-β1, TGF-β2, TGF-β3, activin-βA, activin-βB, activin Proteins belonging to the TGF-β type I receptor family and proteins belonging to the TGF-β type II receptor family that constitute heterodimeric receptors (e.g., activin-βC, activin-βE, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH(MIS), etc.) It is primarily composed of proteins belonging to the SMAD family, which are intracellular signaling molecules (effectors) (especially SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, or SMAD8).

[0103] In the TGF-β / SMAD pathway, when the ligand binds to the dimeric receptor, quinine The TGF-β type I receptor protein, a type of enzyme receptor, phosphorylates SMAD proteins, thereby transmitting signals downstream. Therefore, in this specification, the TGF-β superfamily cytokines, the TGF-β type I receptor family, and the TGF-β type II receptor family are referred to. Molecules that inhibit any of the SMAD family proteins (especially SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, or SMAD8) are called TGF-β / SMAD pathway inhibitors.

[0104] One form of TGF-β / SMAD pathway inhibitor is the ALK protein (ACVRL1 (ALK1), ACVR1 (ALK2), BMPR1A (ALK3), ACVR1B (ALK4), TGFBR1 (ALK5), BMPR1B) belonging to the TGF-β type I receptor family (also called the Activin receptor-like kinase (ALK) family). Examples include inhibitors of (ALK6) and ACVR1C (ALK7) (ALK inhibitors). Inhibitors of proteins belonging to the TGF-β type II receptor family (ACVR2A (ACTRII), ACVR2B (ACTRIIB), TGFBRII (AAT3), BMPR2 (PPH1)) are also given.

[0105] Specifically, these include the ALK5 inhibitors D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)1H-imidazol-2-yl]-benzamide), ALK5 Inhibitor II (2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; also known as RepSox), GW788388, SD-208 (2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine); and the ALK5 and TGFBRII (AAT3) inhibitors LY2109761 and LY2157299 (Galunisertiv, 4-[5,6-dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide), LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline); SM16, an inhibitor of ALK4 and ALK5. (4-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1-carboxamide), EW-7197, SB525334((6-[2-tert-butyl-5-(6-me SB431542 (4-[4-(1,3-Benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide), an ALK4, ALK5 and ALK7 inhibitor Examples include SB505124, A83-01; ALK2 and ALK3 inhibitors LDN-193189, PKC-412, Apigenin, DMH1, ML347; ALK1 and ALK2 inhibitor LDN-214117; ALK1, ALK2 and ALK3 inhibitor LDN-212854; and ALK1, ALK2, ALK3 and ALK6 inhibitor K02288.

[0106] As a TGF-β / SMAD pathway inhibitor, use a compound represented by formula (1) or (2) below, or a salt thereof, as described in Gellibert, F et al. J. Med. Chem. 2004, 47, 4494-4506. It is also possible.

[0107] [ka]

[0108] [In the formula, X is either CH or N. R1 is H, a methyl group, or a halogen (e.g., fluorine, chlorine, bromine, or iodine). R2 is either an H group or a methyl group.

[0109] From the perspective of high efficacy, ALK inhibitors possess at least inhibitory activity against ALK5. A substance that inhibits ALK4 and ALK5, or has specific inhibitory activity against ALK5 (among the ALK proteins, the substance that inhibits the protein) is preferred. It is preferable that the inhibitory activity is significantly high.

[0110] Specific examples of preferred ALK inhibitors include D4476, SB431542, SD208, LY2157299, and ALK5 Inhibitor II, with ALK5 Inhibitor II being particularly preferred. D4476, SB431542, SD208, and ALK5 Inhibitor II are highly efficient at converting fibroblasts into osteoblasts. LY2157299 and ALK5 Inhibitor II are highly efficient at converting fibroblasts into brown adipose tissue. .

[0111] Another embodiment of a TGF-β / SMAD pathway inhibitor is an inhibitor of the SMAD protein. In particular, inhibitors of SMAD2 and SMAD3, which are located downstream of ALK5, and especially SMAD4, are preferred. .

[0112] TGF-β / ERK pathway inhibitors TGF-β / ERK pathway inhibitors are compounds that can inhibit the activity of proteins belonging to the TGF-β / ERK pathway. The TGF-β / ERK pathway is schematically shown in Figure 17 (quoted from YE Zhang, “Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009). This is a signal path known to those skilled in the art.

[0113] The TGF-β / Erk pathway consists of ligands (TGF-β1, TGF-β2, TGF-β3, activin-βA, activin-βB, activin) that are proteins belonging to the TGF-β superfamily. n-βC, activin-βE, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B The TGF-β type I receptor family, which includes BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc., constitutes heterodimeric receptors. Proteins belonging to the family and proteins belonging to the TGF-β type II receptor family, as It is mainly composed of intracellular signaling molecules such as Raf, MEK1 / 2, and Erk1 / 2 proteins.

[0114] In the TGF-β / Erk pathway, when a ligand binds to a dimeric receptor, the signal is transmitted downstream to Raf, MEK1 / 2, and Erk1 / 2. Therefore, in this specification, the TGF-β super Molecules that suppress any of the following: cytokines, the TGF-β type I receptor family, the TGF-β type II receptor family, Raf, MEK1 / 2, or Erk1 / 2, are called TGF-β / Erk pathway inhibitors.

[0115] One example of a TGF-β / Erk pathway inhibitor is the aforementioned ALK protein inhibitor.

[0116] Other embodiments of TGF-β / Erk pathway inhibitors include Raf inhibitors, MEK1 inhibitors, and MEK2 inhibitors. Examples include inhibitors, inhibitors of Erk1, and inhibitors of Erk2. Among these, inhibitors of Erk1 and Erk2 are preferred.

[0117] TGF-β / JNK pathway inhibitors TGF-β / JNK pathway inhibitors are compounds that can inhibit the activity of proteins belonging to the TGF-β / JNK pathway. The TGF-β / JNK pathway is schematically shown in Figure 18 (quoted from YE Zhang, “Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009). This is a signal path known to those skilled in the art.

[0118] The TGF-β / JNK pathway consists of ligands (TGF-β1, TGF-β2, TGF-β3, activin-βA, activin-βB, activin) that are proteins belonging to the TGF-β superfamily. n-βC, activin-βE, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B Proteins belonging to the TGF-β type I receptor family and proteins belonging to the TGF-β type II receptor family that constitute heterodimeric receptors (such as BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.), as well as BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.), and It is mainly composed of intracellular signaling molecules such as TAB1 / 2, TAK1, TRAF6, MKK4, and JNK proteins.

[0119] In the TGF-β / JNK pathway, when a ligand binds to a dimeric receptor, the signal is transmitted downstream to TAB1 / 2, TAK1, TRAF6, MKK4, and JNK. Therefore, this specification refers to molecules that suppress any of the following: cytokines of the TGF-β superfamily, the TGF-β type I receptor family, the TGF-β type II receptor family, TAB1 / 2, TAK1, TRAF6, MKK4, or JNK. These are called TGF-β / JNK pathway inhibitors.

[0120] One example of a TGF-β / JNK pathway inhibitor is the aforementioned ALK protein inhibitor.

[0121] Examples of other embodiments of TGF-β / JNK pathway inhibitors include inhibitors of TAK1, MKK4, and JNK. Among these, JNK inhibitors are preferred.

[0122] TGF-β / p38 pathway inhibitors In the method of the present invention, somatic cells are cultured in a culture medium for inducing other somatic cells in the presence of a TGF-β / p38 pathway inhibitor.

[0123] TGF-β / p38 pathway inhibitors are compounds that can inhibit the activity of proteins belonging to the TGF-β / p38 pathway. The TGF-β / p38 pathway is schematically shown in Figure 18 (quoted from YE Zhang, “Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009). This is a signal path known to those skilled in the art.

[0124] The TGF-β / p38 pathway consists of ligands (TGF-β1, TGF-β2, TGF-β3, activin-βA, activin-βB, activin) that are proteins belonging to the TGF-β superfamily. n-βC, activin-βE, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B Proteins belonging to the TGF-β type I receptor family and proteins belonging to the TGF-β type II receptor family that constitute heterodimeric receptors (such as BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.), as well as BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.), and It is mainly composed of proteins belonging to the intracellular signaling molecules TAB1 / 2, TAK1, TRAF6, MKK3, MKK6, and the p38 family.

[0125] In the TGF-β / p-38 pathway, when a ligand binds to a dimeric receptor, it transmits signals downstream to TAK, MKK3 or MKK6, and the p-38 family (particularly p-38α and p-38β). Therefore, this specification refers to molecules that suppress any of the TGF-β superfamily cytokines, the TGF-β type I receptor family, the TGF-β type II receptor family, TAB1 / 2, TAK1, TRAF6, MKK3, MKK6, or p38 family proteins (particularly p38α and p38β). These are called TGF-β / SMAD pathway inhibitors.

[0126] One example of a TGF-β / p38 pathway inhibitor is the aforementioned ALK protein inhibitor.

[0127] Other embodiments of TGF-β / p38 pathway inhibitors include TAK1 inhibitors, MKK3 inhibitors, MKK6 inhibitors, and p38 inhibitors. Among these, p38 inhibitors are preferred.

[0128] TGF-β / RhoA pathway inhibitors In the method of the present invention, in a culture medium for inducing other somatic cells, the TGF-β / RhoA pathway Culturing is performed in the presence of an inhibitor.

[0129] TGF-β / RhoA pathway inhibitors are compounds that can inhibit the activity of proteins belonging to the TGF-β / RhoA pathway. The TGF-β / RhoA pathway is a signaling pathway known to those skilled in the art, schematically shown in Figure 19 (quoted from YE Zhang, “Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009).

[0130] The TGF-β / RhoA pathway is composed of proteins belonging to the TGF-β superfamily. Ligands that are affected (TGF-β1, TGF-β2, TGF-β3, activin-βA, activin-βB, activin The TGF-β type I receptor family (including Bin-βC, activin-βE, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.) constitutes heterodimeric receptors. Proteins belonging to the - family and proteins belonging to the TGF-β type II receptor family, In addition, intracellular signaling molecules such as Par6, PKCζ, Smurf1, and RhoA proteins are mainly involved. To accomplish.

[0131] In the TGF-β / RhoA pathway, when a ligand binds to a dimeric receptor, it transmits signals downstream to TAK, MKK3 or MKK6, and the p-38 family (particularly p-38α and p-38β). Therefore, in this specification, molecules that suppress any of the TGF-β superfamily cytokines, TGF-β type I receptor family, TGF-β type II receptor family, Par6, PKCζ, Smurf1, or RhoA are referred to as TGF-β / RhoA pathway inhibitors.

[0132] One example of a TGF-β / RhoA pathway inhibitor is an inhibitor of the aforementioned ALK protein. It will be shown.

[0133] Other embodiments of TGF-β / RhoA pathway inhibitors include RhoA inhibitors and PAK inhibitors. Among these, RhoA inhibitors are preferred.

[0134] Preferred examples of TGF-β pathway inhibitors include ALK5 or TGF-β family cytokines that act as its ligand, and TGF-β / SMAD pathways, TGF-β / Erk pathways, TGF-β / JNK pathways, TGF-β / p38 pathways, or TGF-β / RhoA pathways downstream of ALK5. Examples include inhibitors that suppress one or more of the molecules that make up the compound.

[0135] Of course, inhibitors that suppress two or more pathways are also acceptable. TGF-β receptor Inhibitors that suppress TGF-β family molecules or TGF-β superfamily cytokines that act as their ligands are preferred because they suppress multiple pathways within the TGF-β pathway.

[0136] The TGF-β pathway inhibitors are not limited to known TGF-β pathway inhibitors. All TGF-β pathway inhibitors developed in the future will be included in the TGF-β pathway inhibitors of the present invention.

[0137] TGF-β pathway inhibitors also include derivatives of the above compounds. For example, derivatives of D4476 described in WO00 / 61576 can be used.

[0138] "In the presence of a TGF-β pathway inhibitor" primarily refers to a configuration in which the TGF-β pathway inhibitor is contained in the culture medium, but is not limited to this as long as it does not impair the effects of the present invention. The concentration of the TGF-β pathway inhibitor in the culture medium can be appropriately set by those skilled in the art. Typically, it is 0.01 The concentration is approximately μM to 100 μM, particularly between 0.1 μM and 10 μM.

[0139] TGF-β pathway inhibitors can be used individually or in combination of two or more.

[0140] Furthermore, the present invention can be used in combination with other compounds, as long as it does not interfere with the effects of the present invention. For example, a TGF-β pathway inhibitor and a statin compound can be used in combination, from the viewpoint of high efficiency in preparing the target somatic cells.

[0141] The statin compounds broadly include HMG-CoA reductase inhibitors and are not particularly limited, but examples include simvastatin, atorvastatin, lovastatin, fluvastatin, pravastatin, cerivastatin, pitavastatin, rosuvastatin, dihydrocompactin, compactin, verbastatin, carvastatin, krillvastatin, dalvastatin, glenvastatin, fluindostatin, verostatin, mevastatin, rivastatin, sirivastatin, and CI-981. All statin compounds developed in the future will be included in the statin compounds of the present invention.

[0142] In a preferred embodiment of the present invention, the number of TGF-β pathway inhibitors and other compounds used in addition to the aforementioned culture medium components is 10 or less, more preferably 4 or less, even more preferably 3 or less, and particularly preferably 1 or 2.

[0143] culture In the method of the present invention, differentiated mammalian somatic cells are cultured in induction medium in the presence of a TGF-β pathway inhibitor.

[0144] Culturing can be carried out in a suitable container for storing cells and culture medium. The cultivation method involves culturing under conditions of approximately 37°C and a carbon dioxide concentration of approximately 5%. While examples of methods are provided, the method is not limited to these. Culturing under the above conditions can be carried out, for example, using a known CO2 incubator.

[0145] The incubation period is not particularly limited, as long as it does not impair the effects of the present invention. For example, from 24 hours to about 60 days, preferably 3 to 30 days, more preferably 10 to 20 days, particularly Preferably, this can be around 14 days.

[0146] TGF-β pathway inhibitors may be added only for a portion of the total culture period.

[0147] From the perspective of high efficacy, the culture can be performed in the presence of the compound in the induction medium for a predetermined period (for example, about 6 to 10 days, especially about 8 days) throughout the entire culture period, and then culture can be performed in the absence of the compound in the induction medium. In this case, the culture in the presence of the compound may be performed from the start of the culture throughout the entire culture period, or after the predetermined period of culture in the absence of the compound.

[0148] In particular, when preparing brown adipose tissue, brown adipose tissue can be efficiently prepared by culturing in the presence of the above compound in the induction medium for a predetermined period from the start of the culture, for example, about 6 to 10 days, especially about 8 days, and then culturing in the absence of the above compound in the induction medium.

[0149] Furthermore, differentiated mammalian somatic cells may be cultured in the presence of a TGF-β pathway inhibitor in a standard culture medium, and then cultured in the absence of a TGF-β pathway inhibitor in an induction medium. Alternatively, cells may be cultured in the presence of a TGF-β pathway inhibitor in a standard culture medium, then cultured in the absence of a TGF-β pathway inhibitor in a standard culture medium, and then cultured in the absence of a TGF-β pathway inhibitor in an induction medium. Alternatively, cells may be cultured in the presence of a TGF-β pathway inhibitor in a standard culture medium, then cultured in the presence of a TGF-β pathway inhibitor in an induction medium, and then cultured in the absence of a TGF-β pathway inhibitor in an induction medium. Thus, as long as the process includes both culturing in the presence of a TGF-β pathway inhibitor and culturing in an induction medium, these processes do not have to be simultaneous, and each may only be a part of the total culture period.

[0150] In the culture process, subculturing can be performed as needed. When subculturing, cells are harvested before or immediately after reaching confluence and seeded in fresh culture medium. In addition, the culture medium can be replaced as appropriate in the culture process of this invention.

[0151] Furthermore, even if conversion is not completely finished, cells in the process of being cultured can be collected and used for transplantation. In this case, the method of the present invention is expected to trigger conversion, causing irreversible epigenetic changes, which will lead to the cells converting to the target cells after transplantation.

[0152] Thus, the target somatic cells are prepared.

[0153] The preparation of the target somatic cells can be verified by known methods such as marker detection.

[0154] Examples are given below, but in each case, it is not always possible to confirm the result with just one marker; it is desirable to combine the detection of several markers for verification.

[0155] Comprehensive analysis of mRNA expression, such as transcriptome analysis of prepared somatic cells, and proteo By performing comprehensive analyses of protein expression, such as chromosome analysis, and comparing them with the results of analyses of target somatic cells derived from living organisms, it is also possible to evaluate whether the target somatic cells have been prepared.

[0156] For example, the fact that osteoblasts were obtained indicates the presence of the ALP (alkaline phosphatase) gene, male Theocalcin (OC) gene, osteopontin gene, Runx2 This can be confirmed by real-time PCR measurement of gene mRNA, staining with Alizarin Red S (production of calcified (mineralized) bone matrix), etc. Note that in this specification... Pre-osteoblasts, immature osteoblasts, mature osteoblasts, and osteocytes will all be referred to as "osteoblasts."

[0157] The acquisition of white adipocytes was possible because they were large and stained with Oil Red O or Bodipy stain. This can be identified by a distinctive morphology with acupunctate lipid droplets, as well as by the expression of the FABP4 gene, HS gene L, and AdipoQ gene.

[0158] The acquisition of brown adipose tissue was confirmed by staining with Oil Red O or Bodipy stain, which is a multilocular adipose tissue. It can be detected by its distinctive morphology, which includes fat droplets, and by the expression of genes such as UCP-1, CIDEA, KCNK3, PCG-1α, Cox8b, Otop, and ELOVL3. However, UCP-1 (Uncoupling protein 1) is a gene that is specifically expressed in brown adipose tissue. Furthermore, because it encodes a mitochondrial inner membrane protein that uncouples oxidative phosphorylation and is thought to play a fundamental role in the function of brown adipose tissue, it is one of the particularly preferred indicators of brown adipose tissue.

[0159] The acquisition of chondrocytes can be confirmed by, for example, the expression of the SOX9 gene, COL2A1 (Type II collagen) gene, aggrecan gene, COL11A1 (Type XI collagen) gene, and MMP13 gene, the production of cartilage matrix, and staining with Alcian blue, toluidine blue, and safranin O. It can be detected.

[0160] The presence of myoblasts can be detected by, for example, the expression of MyoD, Myogenin, Myf5, and MRF4 genes; the expression of skeletal muscle-type α-actin protein and skeletal muscle-type myosin protein; muscle-specific morphology (multinucleation, muscle fiber formation, myotube formation); and contractility.

[0161] The presence of cardiomyocytes can be detected, for example, by the expression of troponin T (cTnT) protein, tropomyosin protein, cardiomyocyte α-actin protein, and cardiomyocyte myosin protein; the expression of ATP2A2 gene, GJA1 gene, GJA5 gene, NPPA gene, and NPPB gene; and autonomous contractility.

[0162] The acquisition of smooth muscle cells indicates, for example, the alpha-smooth muscle actin (αSMA) protein, smooth It can be detected by the expression of muscle myosin protein.

[0163] Mesenchymal stem cells (MSCs) were obtained by staining with anti-Stro-1, anti-CD90, anti-CD106, anti-CD105, anti-CD146, anti-CD166, and anti-CD44 antibodies, but not by anti-CD19 and anti-CD45 antibodies. These can be detected by analyzing the expression patterns of cell surface antigens using multiple antibodies. Furthermore, the expression of MASP1 and FOSB genes, as well as Nestin protein, CXCL12 protein, and PDGF can also be used. The expression of receptor α / Sca-1 protein, CD51 / PDGF receptor α protein, etc., is examined to make a comprehensive determination. It can be determined. Furthermore, it can be detected by its ability to differentiate into various mesenchymal cells.

[0164] The presence of nerve cells can be detected, for example, by the expression and morphology of Tuj1, vGLUT1, and MAP2 proteins, as well as by neurotransmitter synthesis and action potentials.

[0165] The fact that glial cells were obtained means that, for example, in the case of oligodendroglia, the olig2 protein In the case of cysts and astrocytes, detection can be achieved through the expression of GFAP protein, and in the case of microglia, through the expression of OX42 protein.

[0166] The acquisition of Schwann cells was significant due to their morphology (bipolar or multipolar cell morphology with relatively small nuclei, and cell width-to-length ratio), and the presence of enzymes such as S100β, p75NTR, GFAP, Nestin, and NG2. This can be detected by the expression of a specific marker for Wuwan cells, or by the effect of neurite outgrowth and myelin formation ability on co-cultured nerve cells.

[0167] The acquisition of liver cells indicates, for example, the expression of albumin, cytochrome P450, and various enzymes. It can be detected through functions such as activity, drug metabolism, and LDL uptake.

[0168] The presence of bile duct epithelial cells can be detected, for example, by the presence of the following characteristics: AFP(-), Dlk(-), Alb(-), CK19(+), zEpCAM(+), Thy1(-).

[0169] The presence of vascular endothelial cells can be detected, for example, by the expression of CD31 antigen, Endoglin, VE-cadherin, VWF, TIE2, and ANGPT2.

[0170] The presence of lymphoid endothelial cells can be detected, for example, by the expression of D2-40 antigen, podoplanin, LYVE-1, and PROX-1.

[0171] The acquisition of tendon cells was, for example, due to the expression of Scleraxis (Scx) and tenomodulin (Tnmd). It can be detected that way.

[0172] Fibroblasts can be identified by the expression of genes such as COL1A1 and COL1A2.

[0173] Bone marrow stromal cells can be identified by the expression of cell surface markers, gene expression, etc.

[0174] The acquisition of lymphocytes, macrophages, granulocytes, hematopoietic stem cells, mast cells, etc., can also be confirmed by the expression of cell surface markers, gene expression, etc.

[0175] The presence of urothelial cells can be detected, for example, by the expression of Uroplakin1b, Uroplakin2, CK5, and CK17 genes, or by the formation of Asymmetric Unit Membranes.

[0176] Treatment or preventive agent; grafting material Somatic cells prepared by the method of the present invention can be used to prevent or treat various diseases or conditions by transplanting them into a living organism. In this specification, unless otherwise specified, the term “treatment” means an action taken while a patient is suffering from a particular disease or disorder, thereby reducing the severity of the disease or disorder, or reducing one or more of its symptoms, or delaying or slowing the progression of the disease or disorder. In this specification, “treatment” includes “prevention.”

[0177] Transplant materials refer to materials used to introduce somatic cells into a living organism. Transplant materials include materials used to prepare somatic cells in vitro and then transplant them into the same or another individual. Furthermore, transplant materials in which target somatic cells are induced to a partial differentiation stage in vitro and then ultimately induced into the target somatic cells in vivo after transplantation are also included in this invention.

[0178] For example, diseases treated with osteoblasts include bone tumors, bone defects associated with trauma or osteomyelitis, bone defects after curettage of bone tumors, fractures, osteoporosis, periodontal disease, and alveolar bone. Bone resorption, cleft lip and palate, rheumatoid arthritis, idiopathic osteonecrosis of the femoral head, osteoarthritis, lumbar degenerative spondylosis, spinal canal stenosis, lumbar disc herniation, spondylolysis, spondylolisthesis, scoliosis, cervical spondylotic myelopathy, ossification of the posterior longitudinal ligament, spinal cord injury, coxarthrosis, gonarthrosis, slipped capital femoral epiphysis, osteomalacia, reconstruction surgery for fracture sites destroyed by complex fractures such as mandibular reconstruction surgery, postoperative bone repair (such as sternal repair after cardiac surgery), repair of defects associated with artificial joint surgery, osteomyelitis, osteonecrosis, etc. In addition, if osteoblasts are transplanted, it may be possible to enhance the therapeutic effect in combination with bone transplantation, artificial bone transplantation, artificial joints or implants. Also, osteoblasts are 3 cultured using a two-dimensional scaffold or the like to produce various forms of bone tissue in vitro, and by transplanting the bone tissue, the above-mentioned diseases can also be treated. In addition, various diseases related to the deficiency, insufficiency or functional decline of osteoblasts are targeted.

[0179] Not limited to the treatment of diseases, it can also be used for cosmetic purposes. For example, by transplanting osteoblasts or bone tissue produced thereby to a site damaged by an accident or surgery, it is possible to produce a bone matrix to repair the damaged site and make it plump and less conspicuous. In this case, the treatment for humans is also referred to as treatment for convenience in this specification, and "patient" can be read as "healthy person" or "human", and "disease" can be read as "cosmetic".

[0180] Osteoblasts can be transplanted into patients as a cell preparation, or transplanted together with a substrate (scaffold) made of an artificial material such as hydroxyapatite or bioabsorbable ceramic, or cultured together with the scaffold and then transplanted. In these cases, the scaffold can be made into various three-dimensional shapes according to the transplantation purpose.

[0181] Furthermore, brown adipose tissue can be used to prevent or treat obesity, metabolic syndrome, or related diseases or conditions. Target diseases include type 1 diabetes, type 2 diabetes, diabetic complications (retinopathy, peripheral neuropathy, nephropathy, macrovascular complications, diabetic gangrene, osteoporosis, diabetic coma, etc.), impaired glucose tolerance, insulin resistance, acidosis, ketosis, ketoacidosis, obesity, central obesity and its complications, visceral obesity syndrome, hypertension, postprandial hyperlipidemia, cerebrovascular disease, arteriosclerosis, atherosclerosis, metabolic syndrome, dyslipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, and kidney disease. Diseases (diabetic nephropathy, nephrotic syndrome, etc.), arteriosclerosis, thrombotic diseases, myocardial infarction This includes thromboembolism, ischemic heart disease, angina pectoris, heart failure, cerebrovascular disorders (cerebral infarction, stroke, etc.), peripheral circulatory disorders, sensory disturbances, hyperuricemia, gout, infections (respiratory infections, urinary tract infections, gastrointestinal infections, skin infections, soft tissue infections, etc.), malignant tumors, cataracts, fatty liver, non-alcoholic steatohepatitis, and osteoporosis. It is believed that the burning of lipids by brown adipose tissue and the improvement of glucose and lipid metabolism abnormalities can provide preventive and therapeutic effects against these diseases.

[0182] Furthermore, brown adipose tissue can be used for cosmetic purposes to remove fat from areas such as the abdomen, jawline, and thighs. Brown adipose tissue can also be used as a transplant material for cosmetic procedures, such as introducing it into the breasts.

[0183] Administering brown adipose tissue reduces fat mass, particularly white adipose tissue such as visceral and subcutaneous fat, and also suppresses weight gain even when consuming a high-calorie diet. Therefore, it is useful for both the prevention and treatment of obesity, metabolic syndrome, or related diseases or conditions. The present invention can also be used not only for the prevention or treatment of diseases, but also for purposes such as health promotion and cosmetic purposes (e.g., removal of visceral and subcutaneous fat from the abdomen, jaw, arms, thighs, etc.). In this specification, procedures on humans are referred to as "treatment" for convenience, and "patient" can be read as "healthy person" or "human," and "disease" can be read as "health promotion" or "cosmetic purposes," etc.

[0184] Brown adipose tissue can also be used as a transplant material for cosmetic procedures, such as introducing it into the breasts.

[0185] White adipose tissue can be used to improve tissue morphology and prevent infection by transplanting it into tissue defects caused by trauma, burns, or surgery. For example, by transplanting white adipose tissue into the tissue defect after a mastectomy for breast cancer, the breast can be reconstructed. It can also be used as a transplant material for cosmetic procedures.

[0186] Myoblasts are used in various myopathic diseases, including Duchenne muscular dystrophy, muscular dystrophy, congenital / distal myopathy, myotonia syndromes such as myotonic dystrophy, mitochondrial diseases, periodic paralysis, and neurogenic muscle diseases such as Werdnig-Hoffmann disease, Charcot-Marie-Tooth disease, congenital myelin dysplasia, and amyotrophic lateral sclerosis (ALS). It is believed that this drug can provide preventive and therapeutic effects against collagen diseases such as dermatomyositis, polymyositis, polyarteritis nodosa, polymyalgia rheumatica, and mixed connective tissue disease, as well as inflammatory muscle diseases, endocrine muscle diseases, drug-induced muscle diseases caused by steroids and antihyperlipidemia drugs, sarcopenia, and fibrodysplasia ossificans progressive (FOP).

[0187] Regarding chondrocytes, it is believed that preventive and therapeutic effects can be obtained against cartilage damage and cartilage defects seen in cartilage diseases such as osteoarthritis, degenerative chondropathy, chondrodysplasia, rheumatoid arthritis, trauma, intervertebral disc injury, meniscal injury, osteochondritis dissecans, osteonecrosis, and neurogenic arthropathy.

[0188] Regarding mesenchymal stem cells, it is believed that osteoblasts, adipocytes, chondrocytes, myoblasts, etc., which can differentiate from mesenchymal stem cells, can provide preventive and therapeutic effects against many of the diseases mentioned above.

[0189] Regarding tendon cells, it is believed that preventive and therapeutic effects can be obtained against tendon ruptures caused by trauma or surgery.

[0190] Regarding nerve cells and glial cells, they are associated with neurodegenerative diseases (Parkinson's disease, Parkinsonian syndrome, Alzheimer's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, Shy-Drager syndrome, nigrostriatal degeneration, olivopontocerebellar atrophy, spinocerebellar ataxia, etc.), corticobasal degeneration, Lewy body disease, dystonia, Mage syndrome, late-onset cerebellar cortical atrophy, familial spastic paraplegia, motor neuron disease, McCard-Joseph disease, Pick's disease, stroke, and cerebrovascular disease. It is believed that this drug may provide preventive and therapeutic effects against various conditions, including: brain tumors, demyelinating diseases (multiple sclerosis, Guillain-Barré syndrome, acute disseminated encephalomyelitis, acute cerebellitis, transverse myelitis, etc.), brain tumors, cerebrospinal diseases associated with infection (meningitis, brain abscess, Creutzfeldt-Jakob disease, etc.), post-traumatic neurological disorders, neurological disorders caused by toxins or radiation, mental illnesses (schizophrenia, bipolar disorder, etc.), sleep disorders (narcolepsy, primary hypersomnia, recurrent hypersomnia, sudden onset hypersomnia, insomnia, etc.), and epilepsy.

[0191] Diseases treated with Schwann cells include: central nervous system defects or damage due to cerebral infarction, spinal cord injury, etc.; peripheral nervous system defects or damage due to trauma, neuritis, tumor resection, etc.; multiple sclerosis, neuromyelitis optica (Devic syndrome), concentric sclerosis (Balo disease), acute disseminated encephalomyelitis (ADEM), inflammatory generalized sclerosis (Schilder's disease), subacute sclerosing panencephalitis (SSPE), and progressive multifocal leukoencephalopathy (PML). These include central nervous system disorders; peripheral nervous system disorders such as Guillain-Barré syndrome, Fisher syndrome, and chronic inflammatory demyelinating polyradiculoneuritis; and diseases based on Schwann cell deficiency, deficiency, or dysfunction, such as Charcot-Marie-Tooth disease (CMT).

[0192] Regarding liver cells and bile duct epithelial cells, liver failure, fulminant hepatitis, cirrhosis, fatty liver disease, metabolic syndrome... It is considered that preventive and therapeutic effects against Cushings syndrome and the like can be obtained. It is also useful for drug toxicity tests, metabolism tests, and safety tests on the liver. It is also useful for screening and effect determination of drugs against diabetes, dyslipidemia, metabolic syndrome, hypertension, and the like.

[0193] Lung and airway cells are considered to have preventive and therapeutic effects against primary pulmonary hypertension, pulmonary fibrosis, emphysema, bronchiectasis, pulmonary sarcoidosis, interstitial pneumonia, cystic fibrosis, diffuse panbronchiolitis, obliterative bronchiolitis, pulmonary eosinophilic granulomatosis, chronic thromboembolic pulmonary hypertension, multiple pulmonary arteriovenous fistulas, and the like.

[0194] Kidney cells are considered to have preventive and therapeutic effects against renal failure, diabetic nephropathy, chronic glomerulonephritis, congenital renal hypoplasia, polycystic kidney, IgA nephropathy, nephrosclerosis, preeclampsia, and the like.

[0195] Urothelial cells are useful for forming a substitute bladder in cases of total bladder resection for bladder cancer, constructing a patch at the site where urothelium is deficient in vesicovaginal fistula, forming urothelium for partial supplementation when performing cystoplasty for cases with neurogenic bladder where the bladder has fibrosed and atrophied, and urothelial regeneration for severe interstitial cystitis (urothelial dysfunction).

[0196] Cardiomyocytes are considered to have preventive and therapeutic effects against myocardial infarction, ischemic heart disease, congestive heart failure, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, and the like.

[0197] Lymphatic endothelium is considered to have preventive and therapeutic effects against lymphedema and the like.

[0198] Smooth muscle cells are useful for regenerative treatment of the airway, digestive tract, and blood vessels.

[0199] Vascular endothelial cells are useful for regenerative treatment of blood vessels.

[0200] Hematopoietic stem cells and bone marrow stromal cells are useful for bone marrow failure, aplastic anemia, and immunodeficiency.

[0201] Lymphocytes, granulocytes, macrophages, and dendritic cells are useful for immunodeficiency and anti-tumor immunity. Megakaryocytes are useful for thrombocytopenia. Erythroblasts are useful for aplastic anemia.

[0202] This invention can also be used to treat diseases in pets such as dogs and cats, as well as livestock such as cattle, horses, pigs, sheep, and chickens, in addition to humans. In that case, "patient" or "human" should be read as "sick animal" or "animal."

[0203] This invention can also be used not only in regenerative medicine, but also in the development of drugs for various diseases (including biological agents and nucleic acid drugs), evaluation of drug side effects, and basic research such as elucidation of disease development and pathogenesis.

[0204] Inducer The present invention also provides inducers for converting differentiated somatic cells into other somatic cells, including TGF-β pathway inhibitors. Furthermore, it provides the use of TGF-β pathway inhibitors for converting differentiated somatic cells into other somatic cells.

[0205] In other words, the present invention provides novel applications for TGF-β pathway inhibitors.

[0206] The inducer may be a TGF-β pathway inhibitor alone, or it may be a composition containing a TGF-β pathway inhibitor. If it is a composition, it may further contain a suitable solvent (such as water or DMSO) for dissolving the TGF-β pathway inhibitor. [Examples]

[0207] Examples are shown below, but the present invention is not limited to these examples.

[0208] The structures of the compounds used in the examples are shown below.

[0209] [ka]

[0210] [ka]

[0211] [ka]

[0212] [ka]

[0213] Hereafter, in this specification and in the figures, "ALK5 Inhibitor II" will be referred to as "ALK5 Inhibitor". It may be written as "ALK5IH", "ALK5 IHII", or "ALK5 i II".

[0214] Example 1 (Figure 1) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 5 × 10⁻⁶ cells. 3 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either standard medium, calcification-inducing medium, or calcification-inducing medium with each compound added was added, as shown in the figure.

[0215] The calcification-inducing medium was prepared by adding 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to 10% FBS DMEM. The culture medium was replaced with fresh medium every 3-4 days and cultured until day 24.

[0216] On Day 24, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The wells were then washed three times with sterile distilled water. After that, Alizarin Red S staining solution was added and incubated at room temperature for 15 minutes. The stained wells were washed with sterile distilled water and photographed.

[0217] The concentrations of each compound are as follows: D4476: 2 μM ALK5 inhibitor II: 2 μM SB431542: 2 μM.

[0218] The results are shown in Figure 1. It can be seen that human fibroblasts were converted into osteoblasts capable of producing calcified bone matrix by culturing them in induction medium supplemented with D4476, ALK5 inhibitor II, or SB431542. In particular, ALK5 inhibitor II was the most efficient in converting fibroblasts into osteoblasts. It can be seen that they convert into blast cells.

[0219] TGF-β signaling is essential for osteoblast proliferation, survival, differentiation, and bone formation, and is known to promote osteoblast differentiation and bone formation (e.g., reference: Kasagi and Chen, Cell & Bioscience 2013, 3:4). The fact that conversion to osteoblasts can be achieved by inhibiting the TGF-β pathway using the method of the present invention was an unexpected result.

[0220] Example 2 (Figure 2) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). 5 x 10 3 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of calcification-inducing medium, or calcification-inducing medium with each compound added, was added as shown in the figure.

[0221] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. The cells were replaced with [the other cells] and cultured until day 24.

[0222] On Day 24, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was performed. After washing three times with sterile distilled water, Alizarin Red S staining solution was added and incubated at room temperature for 15 minutes. The stained solution was collected in a 96-well plate and its absorbance (OD: 550 nm) was measured using a spectrophotometer.

[0223] The concentrations of each compound are as follows: D4476: 2μM SB431542: 2μM ALK5 inhibitor II: 2 μM.

[0224] The results are shown in Figure 2. It can be seen that human fibroblasts were converted into osteoblasts capable of producing calcified bone matrix by culturing them in induction medium supplemented with D4476, ALK5 inhibitor II, or SB431542. In particular, ALK5 inhibitor II was the most efficient in converting fibroblasts into osteoblasts. It can be seen that they convert into blast cells.

[0225] Example 3 (Figure 3) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 5 × 10⁻⁶ cells. 3 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of calcification-inducing medium, or calcification-inducing medium with each compound added, was added as shown in the figure.

[0226] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. The cells were replaced with [the other cells] and cultured until day 24.

[0227] On Day 24, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was then mixed with Real-time PCR Master Mix, osteocalcin, and Alkaline Phosphatase. Alternatively, primers specific to the β-actin gene were mixed with Taqman pobe. qRT-PCR was performed using the AB7300 Real-time PCR system. mRNA levels of the osteocalcin and alcohol phosphates were quantified as a ratio to the β-actin gene mRNA, and the value for fibroblasts cultured in calcification-inducing medium alone was set to 1.

[0228] The concentrations of each compound are as follows: D4476: 2μM SB431542: 2μM ALK5 inhibitor II: 2 μM.

[0229] The results are shown in Figure 3. It can be seen that human fibroblasts converted into osteoblasts expressing osteocalcin and alkaline phosphatase genes when cultured in induction medium supplemented with D4476, ALK5 inhibitor II, or SB431542. In particular, ALK5 inhibitor II was shown to convert. It was found that type II is the most efficient at converting fibroblasts into osteoblasts.

[0230] Example 4 (Figure 4) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 5 × 10⁻⁶ cells. 3 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either standard medium or calcification-inducing medium supplemented with ALK5 inhibitor II was added.

[0231] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. The cells were replaced with [the other cells] and cultured until day 24.

[0232] On Day 24, the culture medium was aspirated and removed from each well and washed with PBS(-). After fixation with 4% paraformaldehyde, it was washed with PBS(-). After washing three times with PBS(-), Blocking One was added. Then, it was incubated at room temperature for 60 minutes.

[0233] The reaction was carried out overnight at 4°C with an anti-osteocalcin antibody, followed by three washes with wash buffer. The reaction was carried out at room temperature for 1 hour with an Alexa 488-conjugated anti-mouse Ig antibody, followed by five washes with wash buffer. Photographs were taken using a fluorescence microscope at 200x magnification.

[0234] The results are shown in Figure 4. By culturing in induction medium supplemented with ALK5 inhibitor II, This indicates that human fibroblasts were converted into osteoblasts that produce osteocalcin.

[0235] Example 5 (Figure 5) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) are cultured in standard culture medium (with 10% FBS added). The modified Dulbecco's minimum essential medium (DMEM) was used as a base for the turbidity control. This was then mixed into 5 × 10⁻¹⁴ saturates. 3Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and the cells were cultured in standard medium as shown in the figure. Add 500 μL / well of calcification-inducing medium, or calcification-inducing medium supplemented with ALK5 inhibitor II. I did it.

[0236] The calcification-inducing medium was prepared by adding 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to 10% FBS DMEM. The culture medium was replaced with fresh medium every 3-4 days and cultured until day 19.

[0237] On Day 19, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The sample was then washed three times with sterile distilled water. Afterward, Alizarin Red S staining solution was added, and the sample was incubated at room temperature for 15 minutes. The stained solution was collected in a 96-well plate, and the absorbance at 550 nm (OD) was measured using a spectrophotometer. 550 The following parameters were measured (Figure 5, bottom). The stained wells were then washed with sterile distilled water and photographed at 1x magnification and 40x magnification using an inverted microscope (Figure 5, top).

[0238] The results are shown in Figure 5. By culturing in induction medium supplemented with ALK5 inhibitor II, This indicates that human fibroblasts and adipose-derived mesenchymal stem cells were converted into osteoblasts capable of producing calcified bone matrix.

[0239] Example 6 (Figure 6) Human normal skin fibroblasts (HDFs) in standard culture medium (10% FB) The solution was turbidified in Dulbecco's modified minimum essential medium (DMEM) to which sulfur was added. This was then mixed with 5 × 10⁻⁶ of the solution.3 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either standard medium or calcification-inducing medium supplemented with ALK5 inhibitor II was added.

[0240] The calcification-inducing medium was prepared by adding 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to 10% FBS DMEM. The culture medium was replaced with fresh medium every 3-4 days and cultured until day 24.

[0241] On Day 19, the culture medium was aspirated and removed from each well and washed with PBS(-). After fixation with 4% paraformaldehyde, it was washed with PBS(-). After washing three times with PBS(-), Blocking One was added. Then, it was incubated at room temperature for 60 minutes.

[0242] The reaction was carried out overnight at 4°C with the addition of anti-Runx2 antibody, followed by three washes with wash buffer. The reaction was carried out at room temperature for 1 hour with the addition of Alexa 488-conjugated anti-mouse Ig antibody, followed by five washes with wash buffer. Photographs were taken using a fluorescence microscope at 200x magnification.

[0243] The figure shows cells cultured in normal medium and cells cultured in calcification-inducing medium supplemented with HDF and ALK5 inhibitor II as doBs.

[0244] The results are shown in Figure 6. By culturing in induction medium supplemented with ALK5 inhibitor II, This shows that human fibroblasts were converted into osteoblasts expressing Runx2.

[0245] Example 7 (Figure 7) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 10 × 10⁻¹⁴ cells. 3 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. On day 4, the culture supernatant was aspirated and removed, and culture was continued using induction medium (a) (see below) supplemented with 50 mM ALK5 Inhibitor II. On day 6, induction medium (a) was removed, and culture was continued using induction medium (b) (see below) supplemented with 50 mM ALK5 Inhibitor II. Subsequently, on days 9, 12, 14, and 16, the same process was carried out using induction medium (b) supplemented with 50 mM ALK5 Inhibitor II. The culture medium was repeatedly changed.

[0246] On Day 20, the culture medium was aspirated and removed, washed with PBS(-), and then fixed with 10% formalin. Sterilization was performed. The samples were washed three times with distilled water. After staining with Bodipy, the wells were washed with sterile distilled water and photographed at 100x and 200x magnification using an inverted microscope.

[0247] The results are shown in Figure 7. Both phase contrast and fluorescence imaging revealed white adipocytes containing large unilocular lipid droplets, indicating that human normal skin-derived fibroblasts were converted into white adipocytes.

[0248] The compositions of induction media (a) and (b) are as follows: [Induction medium (a)] DMEM (High Glucose): 500 mL FETAL BOVINE SERUM (FBS):50 mL MEM Non-Essential Amino Acids Solution:5 mL100 mM-Sodium Pyruvate Solution:5 mL Penicillin-Streptomycin Mixed Solution: 5 mL Insulin: 170 nM 3,3',5-Triiodo-L-thyronine: 1 nM Rosiglitazone: 1 uM IBMX (3-isobutyl-1-methylxanthine): 0.5 mM Indomethacin: 62.5 nM Dexamethasone: 1 uM [Induction Medium (b)] DMEM (High Glucose): 500 mL FOETAL BOVINE SERUM (FBS): 50 mL MEM Non-Essential Amino Acids Solution: 5 mL 100 mM-Sodium Pyruvate Solution: 5 mL Penicillin-Streptomycin Mixed Solution: 5 mL Insulin: 170 nM 3,3',5-Triiodo-L-thyronine: 1 nM.

[0249] Example 8 (Figure 8) Human dermal fibroblasts (HDFs) were suspended in normal medium (Dulbecco’s modified minimum essential medium supplemented with 10% FBS; DMEM). This was seeded into a 12-well plate at a concentration of 2×10 4 cells / well (day 0), and culture was started at 5% CO2 / 95% humidified air, 37°C. The next day, the culture supernatant was aspirated and removed, and Cyclic Pifithrin-α or 1000 μL / well of cartilage induction medium (StemPro Chondrogenesis Differentiation Kit: ThermoFisher Scientific) supplemented with ALK5 inhibitor II was added.

[0250] The culture medium was replaced with fresh medium every two days, and the cells were cultured until day 18.

[0251] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to Sox9 and Aggrican or β-actin genes, and Taqman pobe. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of Sox9 and Aggrican genes were compared to the β-actin gene mRNA. The ratio was quantified, and the value for fibroblasts cultured in normal culture medium was set to 1.

[0252] The concentrations of each compound are as follows: Cyclic Pifithrin-α: 5μM ALK5 inhibitor II: 2 μM.

[0253] The results are shown in Figure 8. By culturing in cartilage induction medium in the presence of ALK5 inhibitor II, Human normal skin-derived fibroblasts are converted into chondrocytes expressing Sox9 and Aggrican genes. It was confirmed that the conversion is possible. Cyclic Pifithrin-α is known to promote reprogramming from fibroblasts to iPS cells, but it was found that it does not promote conversion from fibroblasts to chondrocytes.

[0254] It is known that activin-mediated TGF-β receptor signaling positively regulates MSC proliferation and promotes the differentiation of MSCs into osteoblasts and chondrocytes (e.g., WG Li and XXXu, Chin J Traumatol. 2005;8(6):349-51., F. Ng et al., Blood. 2008;112(2):295-307.). The fact that conversion to chondrocytes can be achieved by inhibiting the TGF-β pathway using the method of the present invention was an unexpected result.

[0255] Example 9 (Figure 9) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 2 × 10⁻⁶ cells. 4 Seeds were seeded in a 12-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and cartilage induction medium or 2 μM was added. 1000 μL / well of cartilage induction medium supplemented with ALK5 inhibitor II was added.

[0256] The culture medium was replaced with fresh medium every two days, and the cells were cultured until day 18. These cells were washed twice with PBS(-) and then once with 3% acetic acid solution, and then processed by Nacalai Tesque. Alcian blue staining solution with a pH of 2.5 was added and the samples were stained at room temperature for 1 hour. After washing three times with PBS(-), the samples were observed under a microscope.

[0257] The results are shown in Figure 9. By culturing in cartilage induction medium in the presence of ALK5 inhibitor II, It was confirmed that fibroblasts derived from normal human skin can be converted into cartilage that produces cartilage matrix that stains blue.

[0258] Example 10 (Figure 10) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 2 × 10⁻⁶ cells. 4 Seeds were seeded in a 12-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 2 μM ALK5 inhibitor II was added. Skeletal muscle cell proliferation medium (5% FBS, 50 microG / ml Bovine Fetuin, 10) supplemented with 2 μM D4476 1000 μL / well of Ham's / F10 medium supplemented with nG / ml hEGF, 1 nG / ml bFGF, 10 microG / ml insulin, and 0.4 microG dexamethasone was added. The culture medium was replaced with fresh medium every two days and cultured until day 28.

[0259] On Day 28, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was then subjected to Real-time PCR Master Mix, MyoD1, and Myogenin or β-actin gene synthesis. Child-specific primers and Taqman pobe were mixed. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of MyoD1 and Myogenin genes were quantified as a ratio to β-actin gene mRNA, and the values ​​for fibroblasts cultured in normal medium were set to 1.

[0260] The results are shown in Figure 10. The cells were cultured in skeletal muscle cell proliferation medium in the presence of ALK5 inhibitor II. This allows for the conversion of human normal skin-derived fibroblasts into myoblasts expressing Myogenin and MyoD1 genes. And so, it was confirmed that conversion is possible.

[0261] Example 11 (Figure 11) Human normal dermal fibroblast (HDF) cells were divided into 5 × 10⁻¹⁴ cells. 3 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0). The following day, the culture medium in each well was discarded and replaced with fresh medium at a concentration of 500 μl / well. The osteoblast induction medium consists of Dulbecco's modified Eagle's medium (DMEM), 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone, and 10% fetal bovine serum (FBS) added.

[0262] As shown in the figure, the statin compounds Simvastatin (SS) or Pravastatin (PrS (Final concentration 100 nM) was further added to the culture medium.

[0263] After 28 days of culture, the culture medium was removed from each well and washed with PBS(-). ISOGEN II was extracted from the cells. Total RNA was recovered, and cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix, along with a Taqman probe and primers specific to the human alkaline phosphatase (ALP) gene, were added, and Real-time RT-PCR was performed using the AB7300 Real-time PCR system.

[0264] The results are shown in Figure 14 as relative values, with the value for normal human fibroblasts set to 1. It can be seen that adding SS and PrS along with D4476 induced ALP expression more strongly than D4476 alone.

[0265] Example 12 (Figure 12) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ cells. 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. 500 μL / well of culture medium, adipocyte induction medium, or adipocyte induction medium with added compounds was added.

[0266] The adipocyte induction medium is 10% FBS-supplemented DMEM + MDI medium (10% FBS-supplemented DMEM with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin added).

[0267] The concentrations of the additives are as follows: T3: 1 nM Rosiglitazone: 1 μM D4476: 2 μM Pifithrin alpha [p53 inhibitor]: 5 μM SB431542: 2 μM ALK5 Inhibitor II: 2 μM.

[0268] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 14.

[0269] On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The sample was then washed three times with sterile distilled water, followed by the addition of Oil Red O staining solution and incubation at room temperature for 15 minutes. Afterwards, it was washed with sterile distilled water and photographed at 100x magnification using a phase-contrast microscope.

[0270] The results are shown in Figure 12. By adding one of the following to the culture in addition to T3 and Rosiglitazone, D4476, SB431542, or ALK5 Inhibitor II, fibroblasts converted to brown adipocytes. It's clear that it was done.

[0271] Example 13 (Figure 13) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ cells. 4 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), under 5% CO2 / 95% humid air. The culture was started at 37°C. The following day, the culture supernatant was aspirated and removed, and the culture was prepared in a standard medium as shown in the figure. 500 μL / well of adipocyte induction medium, or adipocyte induction medium with each compound added, was added.

[0272] The adipocyte induction medium is 10% FBS-supplemented DMEM + MDI medium (10% FBS-supplemented DMEM with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin added).

[0273] The concentrations of the additives are as follows: T3: 1 nM Rosiglitazone: 1 μM D4476: 2 μM Pifithrin alpha [p53 inhibitor]: 5 μM SB431542: 2 μM ALK5 Inhibitor II: 2 μM.

[0274] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 14. On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was extracted using Rever Tra Ace qPCR RT Master Mix. Synthesized. This cDNA was mixed with Real-time PCR Master Mix and the CIDEA gene or β-actin gene. Child-specific primers and Taqman probes were mixed. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA level of the CIDEA gene was defined as the ratio to the β-actin gene mRNA. The values ​​were quantified and calculated with the value of fibroblasts cultured in normal medium set to 1.

[0275] The results are shown in Figure 13. It can be seen that fibroblasts were converted into brown adipocytes expressing CIDEA gene mRNA by adding either D4476, SB431542, or ALK5 Inhibitor II to the culture in addition to T3 and Rosiglitazone.

[0276] Example 14 (Figure 14) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). to 1 × 10 4Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humid air. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either standard medium, adipocyte induction medium, or adipocyte induction medium with various small molecule compounds added was added, as shown in the figure.

[0277] The adipocyte induction medium is 10% FBS-supplemented DMEM + MDI medium (10% FBS-supplemented DMEM with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin added).

[0278] The concentrations of the additives are as follows: T3: 1 nM Rosiglitazone: 1 μM D4476: 2 μM. Pifithrin alpha [p53 inhibitor]: 5 μM PD0325901: 1 μM SB431542: 2 μM ALK5 Inhibitor II: 2 μM.

[0279] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 14.

[0280] On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then the cells were transferred to ISOGEN II. Total RNA was extracted. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to AdipoQ or β-actin genes, and Taqman pobe. An AB7300 Real-time PCR system was used. qRT-PCR was performed. The mRNA level of the AdipoQ gene was quantified as a ratio to the β-actin gene mRNA, and the value for fibroblasts cultured in normal medium was set to 1.

[0281] The results are shown in Figure 14. It can be seen that fibroblasts were converted into brown adipocytes expressing AdipoQ gene mRNA by adding one of the following to the culture: T3 and Rosiglitazone, along with D4476, PD0325901, SB431542, or ALK5 Inhibitor II.

[0282] Example 15 (Figure 15) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). to 1 × 10 4 Seeds were seeded in a 24-well plate at a concentration of cells / well (day 0), and then incubated in 5% CO2 / 95% humidified conditions. Culture was started in air at 37°C. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of standard medium, adipocyte induction medium, or adipocyte induction medium with various small molecule compounds added was added, as shown in the figure.

[0283] The adipocyte induction medium is 10% FBS-supplemented DMEM + MDI medium (10% FBS-supplemented DMEM with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin added).

[0284] The concentrations of the additives are as follows: T3: 1 nM Rosiglitazone: 1 μM D4476: 2 μM SB431541: 2 μM ALK5 inhibitor II: 2 μM.

[0285] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 14.

[0286] On Day 14, the culture medium was aspirated and removed from each well, and washed with PBS(-). The cells were then fixed with 4% paraformaldehyde, washed with PBS(-), reacted in BODIPY 493 / 503 (Invitrogen) / PBS solution at room temperature for 5 minutes, and washed three times with PBS. Photographs were taken at 200x magnification using a fluorescence microscope, and fluorescence intensity was measured.

[0287] The results are shown in Figure 15A (fluorescence microscope image) and Figure 15B (fluorescence intensity). In addition to T3 and Rosiglitazone, one of the following was added to the culture: D4476, SB431541, or ALK5 inhibitor II. This indicates that fibroblasts were converted into brown adipocytes containing lipid droplets that stain with BODIPY.

[0288] Example 16 (Figure 20) Human normal skin fibroblasts (HDFs) or human mesenchymal stem cells (MSCs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ units. 4 cells / well Seeds were seeded in a 24-well plate at the specified concentration (day 0), and culture was opened at 37°C with 5% CO2 / 95% humidified air. It began. The next day, the culture supernatant was aspirated and removed, and the culture medium was prepared as shown in the diagram. 500 μL / well was added either calcification-inducing medium (OB medium) or calcification-inducing medium (OB medium + ALK5 i II) supplemented with ALK5 inhibitor II (ALK5 i II) at a concentration of 4 μM.

[0289] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. It was replaced with and cultured until day 18.

[0290] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was confirmed. The wells were washed three times with sterile distilled water. The wells after Alizarin Red S staining were washed with sterile distilled water. The image was then taken using an inverted microscope at 40x magnification.

[0291] The results are shown in Figure 20. By culturing in calcification-inducing medium supplemented with ALK5 inhibitor II, This indicates that human skin-derived fibroblasts were converted into osteoblasts that produce large amounts of calcified bone matrix.

[0292] Example 17 (Figure 21) Human normal skin fibroblasts (HDFs) in standard culture medium (10% FB) The solution was turbidified in Dulbecco's modified minimum essential medium (DMEM) to which sulfur was added. This solution was then mixed with 1 × 10⁻⁶ of the solution. 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. 500 μL / well of culture medium, calcification-inducing medium, or calcification-inducing medium supplemented with ALK5 inhibitor II and / or TGF-β was added.

[0293] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. It was replaced with and cultured until day 13.

[0294] On Day 13, the culture medium was aspirated and removed from each well, washed with PBS(-), and then fixed with fixative. The wells were washed three times with sterile distilled water. After ALP staining, the wells were washed with sterile distilled water and photographed at 40x magnification using an inverted microscope.

[0295] The concentrations of the compounds and cytokines added to the culture medium are as follows: ALK5 inhibitor II: 4 μM TGF-β: 50 ng / ml.

[0296] The results are shown in Figure 21. By culturing in calcification-inducing medium supplemented with ALK5 inhibitor II, Furthermore, human skin-derived fibroblasts were converted into osteoblasts with high ALP activity. I understand.

[0297] Example 18 (Figure 22) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ units. 4Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either standard medium, calcification-inducing medium, or calcification-inducing medium supplemented with 4 μM ALK5 inhibitor II was added, as shown in the figure.

[0298] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. It was replaced with and cultured until day 18.

[0299] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to osteocalcin, alcohol phosphate, or β-actin genes, and Taqman pobe. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of the osteocalcin (Oc) and alcohol phosphate (ALP) genes were quantified as a ratio to the β-actin gene mRNA level, and the value for fibroblasts cultured in calcification-inducing medium was set to 1.

[0300] The results are shown in Figure 22. By culturing in calcification-inducing medium supplemented with ALK5 inhibitor II, Furthermore, human skin-derived fibroblasts express osteocalcin and ALP genes. It can be seen that it converted to the . In the figure, * and ** indicate that p < 0.05 and p < 0.01, respectively, relative to the calcification-inducing medium (OB medium). OB medium. Values ​​are means ± SD (n=4).).

[0301] Example 19 (Figure 23) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 2 × 10⁻⁶ samples. 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and 500 μL / well of either normal medium, MDI medium (adipocyte induction medium for fibroblasts), or MDI medium with 4 μM ALK5 inhibitor II added (MDI medium + ALK5 i II) was added, as shown in the figure.

[0302] The MDI medium was prepared by adding 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin to 10% FBS DMEM. The culture medium was replaced with fresh medium every 3-4 days and cultured until day 14.

[0303] On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was confirmed. The samples were washed three times with sterile distilled water. After staining with Oil Red O, the wells were washed with sterile distilled water and photographed at 40x and 100x magnification using an inverted microscope.

[0304] The results are shown in Figure 23. It can be seen that human skin-derived fibroblasts converted into white adipocytes that accumulate large amounts of lipid droplets when cultured in MDI medium supplemented with ALK5 inhibitor II.

[0305] Example 20 (Figure 24) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) were turbidified in normal medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This mixture was then divided into 1 × 10⁻⁶ units. 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. Then, 500 μL / well of either standard medium, MDI medium, or MDI medium supplemented with ALK5 inhibitor II at a concentration of 4 μM was added.

[0306] MDI medium (3-isobutyl-1-methylxanthine / dexamethasone / insulin-containing medium) is prepared by adding 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin to 10% FBS DMEM. The culture medium is refreshed every 3-4 days. The culture was replaced with a suitable alternative and incubated until day 14.

[0307] On Day 14, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was then subjected to Real-time PCR Master Mix and AdipoQ, FABP4, or β-actin gene synthesis. Primers specific to the offspring and Taqman pobe were mixed. An AB7300 Real-time PCR system was used. qRT-PCR was performed. mRNA levels of the AdipoQ and FABP4 genes were compared to the β-actin gene mRNA level. The value was quantified as a ratio to the total, and the value of fibroblasts cultured in MDI medium was set to 1.

[0308] The results are shown in Figure 24. By culturing in MDI medium supplemented with ALK5 inhibitor II, human skin-derived fibroblasts converted into white adipocytes expressing the AdipoQ gene and FABP4 gene. It's clear that it was done.

[0309] Example 21 (Figure 25) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ units. 4 Seeds were seeded in the center of a fibronectin-coated 24-well plate at a concentration of cells / well (day 0), 5% Culture was started in CO2 / 95% humidified air at 37°C. The next day, the culture supernatant was aspirated and removed, as shown in the figure. As described, 500 μL / well was added to either standard culture medium, chondrocyte medium, or chondrocyte medium with ALK5 inhibitor II added at a concentration of 4 μM (Chondrocyte medium + ALK5 i II).

[0310] The cartilage induction medium is 1% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 1% insulin-transferrin-selenium, 10 ng / ml BMP-2, 10 ng / ml TGF-β, 10 ng / ml GDF5, and 10 ng / nl b-FGF. The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 21.

[0311] On Day 21, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The samples were then washed three times with sterile distilled water. After Alcian blue staining, the wells were washed with sterile distilled water and photographed at 100x magnification using an inverted microscope.

[0312] The results are shown in Figure 25. By culturing in cartilage induction medium supplemented with ALK5 inhibitor II This indicates that human skin-derived fibroblasts were converted into chondrocytes that produce large amounts of cartilage matrix.

[0313] Example 22 (Figure 26) Human normal skin fibroblasts (HDFs) or human adipose-derived mesenchymal stem cells (ADSCs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ units. 4 Seeds were seeded in the center of a fibronectin-coated 24-well plate at a concentration of cells / well (day 0), 5% Culture was started in CO2 / 95% humidified air at 37°C. The next day, the culture supernatant was aspirated and removed, as shown in the figure. As described, 500 μL / well of either standard medium, cartilage induction medium, or cartilage induction medium supplemented with ALK5 inhibitor II at a concentration of 4 μM was added.

[0314] The cartilage induction medium is 1% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 1% insulin-transferrin-selenium, 10 ng / ml BMP-2, 10 ng / ml TGF-β, 10 ng / ml GDF5, and 10 ng / nl b-FGF. The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 21.

[0315] On Day 21, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to Aggrecan, Type II collagen, or β-actin genes, and Taqman pobe. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of Aggrecan (ACAN) and Type II collagen genes were quantified as ratios to the β-actin gene mRNA level, and the cells were cultured in cartilage induction medium. The value of the fibroblasts was set to 1 for calculation.

[0316] The results are shown in Figure 26. By culturing in cartilage induction medium supplemented with ALK5 inhibitor II Human skin-derived fibroblasts express the Aggrecan gene and type II collagen gene in chondrocytes. It appears that it was converted to [something].

[0317] Example 23 (Figure 27) Human normal gingival fibroblasts (GFs) were incubated in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). 1 × 10 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, as shown in the figure. 500 μL / well of normal medium, calcification-inducing medium, or calcification-inducing medium supplemented with 4 μM ALK5 inhibitor II was added.

[0318] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. It was replaced with and cultured until day 18.

[0319] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was confirmed. The wells were washed three times with sterile distilled water. The wells after Alizarin Red S staining were washed with sterile distilled water. The image was then taken using an inverted microscope at 40x magnification.

[0320] The results are shown in Figure 27. By culturing in calcification-inducing medium supplemented with ALK5 inhibitor II, This indicates that human gingival fibroblasts were converted into osteoblasts that produce large amounts of calcified bone matrix.

[0321] Example 24 (Figure 28) Human normal gingival fibroblasts (GFs) were incubated in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). 1 × 10 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, as shown in the figure. 500 μL / well of normal medium, calcification-inducing medium, or calcification-inducing medium supplemented with 4 μM ALK5 inhibitor II was added.

[0322] The calcification-inducing medium is 10% FBS DMEM supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium is replaced with fresh medium every 3-4 days. It was replaced with and cultured until day 18.

[0323] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS(-), and then fixed with fixative. The wells were washed three times with sterile distilled water. After ALP staining, the wells were washed with sterile distilled water and photographed at 40x magnification using an inverted microscope.

[0324] The results are shown in Figure 28. By culturing in calcification-inducing medium supplemented with ALK5 inhibitor II, Furthermore, human gingival fibroblasts were converted into osteoblasts with high ALP activity. I understand.

[0325] Example 25 (Figure 29) The animal experiment was conducted with the approval of the affiliated institution. An 8-week-old male NOD / SCID mouse (Charles River) was anesthetized. A partial bone defect of approximately 7 mm in diameter was created in the left femoral shaft using a dental drill under water irrigation. Successfully achieved. HDFs were cultured for 13 days in the presence of ALK5 inhibitor II using the same method as in Example 16 described below. The nourished cells (CdOBs; Chemical-mediated directly converted osteoblasts) are processed by Matri Suspend with gel (BD Bioscience, San Jose, CA) and apply 5 to the bone surface of the bone defect and its surrounding area. ×10 5 Cells were transplanted at a concentration of cells / mouse. Similarly, after creating a bone defect, fibroblasts were transplanted. Mice were also prepared with the mice suspended and transplanted in Matrigel. After 21 days, the mice were euthanized, their thighs were removed, and they were fixed with neutral formalin before being scanned using an X-ray CT device (Scan Xmate-L090, Com Scan Techno). Microcomputed tomography (μCT) was performed using a device (Yokohama, Japan).

[0326] Figure 29 shows a cross-sectional image obtained using μCT. It can be seen that CdOB promoted bone formation in the bone defect area within the mouse body.

[0327] Example 26 (Figure 30) Figure 30 shows a 3D reconstruction of the μCT image from Example 25. Cells cultured with ALK5 inhibitor II were shown to have osteogenic ability in vivo. CdOB in mice This indicates that bone formation was performed in the area of ​​the bone defect.

[0328] Example 27 (Figure 31) The animal experiments were conducted with the approval of the affiliated institution. The transplantation experiment was performed in the same manner as in Example 25. Mice with transplanted fibroblasts were also prepared. After 21 days, the mice were euthanized, and the thigh was removed in the same manner as in Example 25. The tissue was fixed with neutral formalin, and then the bone tissue was examined using SCEM (Leica Microsystem). The samples were embedded in a compound and rapidly frozen. After slicing into 6 μm sections, serial sections were stained with hematoxylin and eosin (H&E) and Alizarin Red S.

[0329] The results (40x microscopic image) are shown in Figure 31. It can be seen that CdOB promoted bone formation in the bone defect area within the mouse body.

[0330] Example 28 (Figure 32) Human normal dermal fibroblast (HDF) cells were cultured in 60 mm culture dishes and cultured in standard medium (-). HDF cells were also cultured for 3 or 7 days in standard medium supplemented with 4 μM ALK5 inhibitor II. Total RNA was recovered from these cells using ISOGEN II. mRNA expression patterns of each cell were analyzed genome-wide using Affymetrix DNA chips. Expression levels of MSC markers were expressed. The heat map is shown in Figure 32. By culturing in a medium supplemented with ALK5 inhibitor II... This indicates that fibroblasts converted into mesenchymal stem cells (MSCs).

[0331] Example 29 (Figure 33) Human normal skin fibroblasts (HDFs) were turbidified in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 1 × 10⁻⁶ cells. 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. 500 μL / well of culture medium, calcification-inducing medium, or calcification-inducing medium with each small molecule compound added was placed in the well.

[0332] The calcification-inducing medium consists of 10% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. This is a DMEM to which 100 nM Dexamethasone has been added.

[0333] The compounds added to the culture medium and their concentrations are as follows: I-BET151: 2μM Pifthrin-α: 5 μM PD0325901: 2μM 2-Me-5HT:2μM CX4945: 2μM CHIR99021: 2μM Forskolin: 2μM DZnep: 50nM D4476: 2μM SB431542: 2μM ALK5 i II: 2μM.

[0334] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 28.

[0335] On Day 28, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was performed. After washing three times with sterile distilled water, Alizarin Red S staining solution was added and the samples were left at room temperature for 15 minutes. The samples were incubated. The stained solution was collected in a 96-well plate and used in the experiment of Example 30. The wells were then washed with sterile distilled water and photographed.

[0336] The results are shown in Figure 33.

[0337] Example 30 (Figure 34) In the experiment of Example 29, the solution after staining with Alizarin Red S was collected in a 96-well plate, and the absorbance (OD550nm) was measured using a spectrophotometer. The results are shown in Figure 34.

[0338] The results shown in Figures 33 and 34 indicate that the TGF-β pathway inhibitors D4476 and SB431542 Both ALK5 i II, when added to calcification-inducing medium, promote the transformation of fibroblasts into osteoblasts. It was found that it induces conversion to cells. However, it was found that fibroblasts can be converted into iPS cells. Compounds other than TGF-β pathway inhibitors that have been reported to promote induction to (I-BET) Pifthrin-α, PD0325901, 2-Me-5HT, CHIR, Forskolin and DZnep) are used in TGF-β pathways. It was found that even when cultured under the same conditions as with the WA inhibitor, conversion from fibroblasts to osteoblasts was not induced. Among TGF-β pathway inhibitors, ALK5 i II in particular... This most strongly induced the conversion of fibroblasts to osteoblasts.

[0339] Example 31 (Figure 35) Human normal skin-derived fibroblasts (HDFs) were suspended in a standard culture medium (DMEM supplemented with 10% FBS). This is 1 x 10 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. 500 μL / well of either standard medium, calcification-inducing medium (OB medium), or calcification-inducing medium supplemented with each small molecule compound or cytokine was added.

[0340] The calcification-inducing medium consists of 10% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. This is a DMEM to which 100 nM Dexamethasone has been added.

[0341] The compounds added to the culture medium and their concentrations are as follows: D4476: 1 or 4 μM LY2157299: 1 or 4 μM LY364947: 1 or 4 μM SB431542: 1 or 4 μM SB525334: 1 or 4 μM SD208: 1 or 4 μM ALK5 i II: 1 or 4 μM TGF-β: 10 ng / ml.

[0342] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 21.

[0343] On Day 21, the culture medium was aspirated and removed from some wells, washed with PBS(-), and then fixed with fixative. The wells were washed three times with sterile distilled water. The wells were washed with sterile distilled water after ALP staining and then photographed.

[0344] On Day 21, the culture medium was aspirated and removed from the remaining wells, washed with PBS(-), and fixed with 10% formalin. After washing three times with sterile distilled water, Alizarin Red S staining solution was added, and the specimens were left at room temperature for 15 minutes. The samples were incubated for a period of time. The stained solution was collected in a 96-well plate and used in the experiment described in Example 32. It was used for this purpose. Afterwards, the wells were washed with sterile distilled water and photographed.

[0345] The results are shown in Figure 35.

[0346] Example 32 (Figure 36) In the experiment of Example 31, the solution after staining with Alizarin Red S was collected in a 96-well plate, and the absorbance (OD550nm) was measured using a spectrophotometer. The results are shown in Figure 36.

[0347] The results shown in Figures 35 and 36 indicate that human fibroblasts can be converted to osteoblasts by culturing them in calcification-inducing medium supplemented with various TGF-β pathway inhibitors. In particular, SD208 and ALK5 i II were found to be highly effective in converting human fibroblasts to osteoblasts. Among these, ALK5 i II was the most potent in converting human fibroblasts to osteoblasts. It was found that it could be converted.

[0348] When cultured in calcification-inducing medium supplemented with TGF-β, human fibroblasts did not convert to osteoblasts.

[0349] Example 33 (Figure 37) Human normal skin-derived fibroblasts from various individuals (HDF45 (purchased from KURABO), HDF69 (purchased from PromoCell), HDF22 (purchased from PromoCell)) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 1 × 10⁶ cells. 4 Cells were seeded into a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C with 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day. As shown in the figure, 500 μL / well of either standard medium, calcification-inducing medium, or calcification-inducing medium supplemented with 4 μM ALK5 i II was added.

[0350] The calcification-inducing medium consists of 10% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. This is a DMEM to which 100 nM Dexamethasone has been added.

[0351] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until Day 10 or 15.

[0352] On Day 10, the culture medium was aspirated and removed from each well, washed with PBS(-), and then fixed with fixative. The specimens were washed three times with sterile distilled water. After ALP staining, the wells were washed with sterile distilled water and photographed at 40x magnification using an inverted microscope.

[0353] The results are shown in Figure 37.

[0354] Example 34 (Figure 38) Using human normal skin-derived fibroblasts (HDF45, HDF69, HDF22) from various individuals. Then, the same culture procedure as in Example 33 was performed.

[0355] On Day 15, the culture medium was aspirated and removed from each well, washed with PBS(-), and then solidified with 10% formalin. The staining was performed. The wells were washed three times with sterile distilled water. After staining with Alizarin Red S, the wells were washed with sterile distilled water. The image was then taken using an inverted microscope at 40x magnification. The results are shown in Figure 38.

[0356] The results in Figures 37 and 38 show that human normal skin fibroblasts from different individuals, when cultured in calcification-inducing medium supplemented with ALK5 i II, converted to osteoblasts. It was found that...

[0357] Example 35 (Figure 39) Human normal skin-derived fibroblasts (HDFs) were turbidified in a standard culture medium (DMEM supplemented with 10% FBS). . This is 1 x 10 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, as shown in the figure. Add calcification-inducing medium (OB medium), or each small molecule compound and / or cytokine. 500 μL / well of the added calcification-inducing medium was added.

[0358] The calcification-inducing medium consists of 10% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. This is a DMEM to which 100 nM Dexamethasone has been added.

[0359] The concentrations of the added small molecule compounds or cytokines are as follows: ALK5 i II: 4 μM 1α,25-dihydroxy Vitamin D3 (VD3): 5 nM Human insulin-like growth factor-1 (IGF-1): 100 ng / ml.

[0360] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 18.

[0361] On Day 18, the culture medium was aspirated and removed from each well, washed with PBS, and total RNA was extracted from the cells using ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, and primers specific to Alkaline Phosphatase, Osteocalcin, Bonesialoprotein, or β-actin genes, along with Taqman pobe. QRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of Alkaline Phosphatase, Osteocalcin (OC), and Bonesialoprotein genes were quantified as ratios to the β-actin gene mRNA level, and the values ​​were obtained from fibroblasts cultured in calcification-inducing medium supplemented with ALK5 i II. It was calculated as 1.

[0362] The results are shown in Figure 39. ALP mRNA expression was significantly increased in cells cultured in calcification-inducing medium supplemented with ALK5 i II. OC mRNA expression was elevated in cells cultured in calcification-inducing medium supplemented with ALK5 i II compared to cells cultured in calcification-inducing medium supplemented with ALK5 i II, but was even more significantly elevated in cells cultured in calcification-inducing medium supplemented with ALK5 i II and VD3 compared to cells cultured in calcification-inducing medium supplemented with ALK5 i II. BSP mRNA expression was elevated in cells cultured in calcification-inducing medium. Compared to normal cells, the levels were elevated in cells cultured in calcification-inducing medium supplemented with ALK5 i II. Compared to cells cultured in calcification-inducing medium supplemented with ALK5 i II, cells cultured in calcification-inducing medium supplemented with ALK5 i II and IGF-1 The levels were significantly higher in cells cultured in the added calcification-inducing medium. Values ​​are mean ± SD (n=4).

[0363] Example 36 (Figure 40) Human normal skin-derived fibroblasts (HDFs) were turbidified in a standard culture medium (DMEM supplemented with 10% FBS). . This is 1 x 10 4 Cells were seeded in a 24-well plate at a concentration of cells / well (day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and the cells were mixed with either standard medium or ALK5 i II, VD3, and IGF-1 at concentrations of 4 μM, 5 nM, and 100 ng / ml, respectively, to induce calcification. 500 μL / well of lead-in medium was added.

[0364] The calcification-inducing medium consists of 10% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate. This is a DMEM to which 100 nM Dexamethasone has been added.

[0365] The culture medium was replaced with fresh medium every 3-4 days, and the cells were cultured until day 18.

[0366] On Day 18, the culture medium was aspirated and removed from each well and washed with PBS(-). After fixation with 4% paraformaldehyde, it was washed with PBS(-). After washing three times with PBS(-), Blocking One was added. Then, it was incubated at room temperature for 60 minutes.

[0367] After adding anti-osteocalcin antibody and reacting overnight at 4°C, the mixture was washed three times with wash buffer. After adding Alexa 488-conjugated anti-mouse Ig antibody and reacting at room temperature for 1 hour, the mixture was washed. Cells were washed three times with h buffer. Nuclear staining was then performed using DAPI, and images were taken at 200x magnification using a fluorescence microscope. Additionally, the number of OC-positive cells / DAPI count was calculated using BZ-H3C and BZ-H3CM (KEYENCE). I calculated it.

[0368] The results are shown in Figure 40. In HDFs, many cell nuclei were observed to be stained with DAPI, but anti-OC anti Very few cells in the body were markedly stained. On the other hand, many cells were strongly stained with anti-OC antibody against CdOB. When calculating the ratio of cells stained with anti-OC antibody to the number of cells whose nuclei were stained with DAPI as the denominator, approximately 87% of human fibroblasts were stained with osteocalcin. This indicates that the cells were converted into osteoblasts that express the protein. In the figure, ** indicates the cells that do not express HDF. Then show that p < 0.01 (**p < 0.01 vs. the HDF.).

[0369] Example 37 (Figure 41) Human normal skin fibroblasts (HDFs) were suspended in standard culture medium (Dulbecco's modified minimum essential medium; DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and as shown in the figure, group 1 Standard culture medium was added at a rate of 1 mL / well. Group 2 was given adipocyte induction medium without rosiglitazone. (Adipocyte induction medium (R-)) was added at a concentration of 1 mL / well to group 3. Adipocyte induction medium was added at a concentration of 1 mL / well to group 4. Adipocyte induction medium (R-) was added at a concentration of 4 μM to group 4. ) was added at a concentration of 1 mL / well. Groups 5-9 were treated with ALK5 inhibitor II at a concentration of 4 μM. Cell induction medium was added at a rate of 1 mL / well.

[0370] The adipocyte induction medium is prepared by adding (1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL Insulin, and 10% FBS). It is DMEM.

[0371] Adipocyte induction medium (R-) contains (1 nM T3, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX (DMEM supplemented with 0.5 μM dexamethasone, 1 μg / mL insulin, and 10% FBS) .

[0372] The culture medium was replaced with fresh medium every two days. In groups 5-9, as shown in the figure, the cells were cultured in adipocyte induction medium supplemented with ALK5 inhibitor II only for the periods of 0-2, 0-4, 0-6, 0-8, and 0-10, respectively, and thereafter cultured in adipocyte induction medium without ALK5 inhibitor II. On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and the cells were removed. Total RNA was extracted using the Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using the Rever Tra Ace qPCR RT Master Mix. This cDNA was then mixed with the Real-time PCR Master Mix, primers specific to the UCP-1 gene or β-actin gene, and a Taqman probe. The mixture was then combined. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA level of the UCP1 gene was quantified as a ratio to the β-actin gene mRNA level, and the mixture was cultured in standard medium. The value of nourished fibroblasts was set to 1 for calculation.

[0373] The results are shown in Figure 41. The cells were cultured in adipocyte induction medium supplemented with ALK5 inhibitor II. This resulted in fibroblasts being converted into brown adipose tissue that strongly expresses the UCP1 gene. I understand. In particular, adding ALK5 inhibitor II for 0-8 days, and then removing ALK5 inhibitor II... It was found that culturing fibroblasts in adipocyte-inducing medium for 4 days most effectively induced the conversion of fibroblasts into brown adipocytes.

[0374] Example 38 (Figure 42) Human normal skin-derived fibroblasts (HDFs) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4 Seeds were seeded in a 12-well plate at a concentration of cells / well (Day 0), under 5% CO2 / 95%. Culture was started in humidified air at 37°C. The following day, the culture supernatant was aspirated and removed, and group 1 was normally Group 2 received 1 mL / well of culture medium (Control). Group 2 received 1 mL / well of adipocyte induction medium. Groups 3-8 were fed adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 μM, at a rate of 1 mL / well. Added.

[0375] The adipocyte induction medium is supplemented with (1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL Insulin, and 10% FBS). The culture medium was replaced with fresh PBS (DMEM). In groups 3-7, as shown in the figure, the cells were cultured in adipocyte induction medium supplemented with ALK5 inhibitor II only for the periods of 0-2, 0-4, 0-6, 0-8, and 0-10, respectively, and thereafter cultured in adipocyte induction medium without ALK5 inhibitor II. On Day 14, the culture medium was aspirated and removed from each well, and PBS ( Washed with PBS(-). After fixing with 4% paraformaldehyde, washed with PBS(-), and incubated with Perm Buffer (PBS with 0.2% Triton-X) for 15 minutes. Washed three times with PBS(-). After purification, Blocking One was added and incubated at room temperature for 60 minutes. Anti-UCP-1 antibody (RD MAB6158) was added and reacted at room temperature for 2 hours, followed by three washes with wash buffer. CF488-conjugated anti-mouse Ig antibody (Biotum 20014) was added and reacted at room temperature for 2 hours, followed by three washes with PBS(-). Nuclear staining was performed with Lifetechnology's SlowFade Gold antifade reagent with DAPI, and images were taken at 100x magnification using a fluorescence microscope.

[0376] Fluorescence microscope images are shown in Figure 42. Cells in groups 1 and 2 were hardly stained with anti-UCP1 antibody. In groups 3-8, many cells were stained with anti-UCP1 antibody. In particular, group 6 had the highest density of cells stained with anti-UCP1 antibody. Lipid cells treated with ALK5 inhibitor II By culturing in fibroblast-inducing medium, fibroblasts can develop brown adipose tissue that highly expresses the UCP1 protein. It can be seen that the cells were converted. In particular, by adding ALK5 inhibitor II for 0-8 days and then culturing for 4 days in adipocyte induction medium without ALK5 inhibitor II, more It was found that this method can strongly induce the conversion of fibroblasts into brown adipose tissue.

[0377] Example 39 (Figure 43) Human normal skin-derived fibroblasts (HDFs) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4 Seeds were seeded in a 12-well plate at a concentration of cells / well (Day 0), under 5% CO2 / 95%. The culture was started in humidified air at 37°C. The next day, the culture supernatant was aspirated and removed, and the culture medium was changed to normal medium and lipids. Adipose cell induction medium (BA medium), or adipose cell induction medium supplemented with the compounds ALK5 inhibitor II, LY2157299, SB431542, and D4476, was added at a concentration of 1 mL / well. The compound concentrations were 4 μM and 8. The concentration is μM, 12 μM, or 16 μM. The adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL Insulin, and 10% FBS). The culture medium is replaced with fresh medium every two days. The culture medium was replaced with the appropriate substance and cultured from Day 1 to Day 8. Subsequently, from Day 9 to Day 14, the culture was performed in a medium that did not contain ALK5 inhibitor II, LY2157299, SB431542, or D4476. Day 14 Next, the culture medium was aspirated and removed from each well, washed with PBS(-), and total RNA was extracted from the cells using the Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was then mixed with Real-time PCR Master Mix and UCP-1 gene. Primers specific to the gene or β-actin gene were mixed with the Taqman probe. AB7300 qRT-PCR was performed using a Real-time PCR system. The mRNA level of the UCP1 gene was measured using β-A The values ​​were quantified as a ratio to the cutin gene mRNA level, and were obtained from fibroblasts cultured in normal culture medium. It was calculated with value set to 1.

[0378] The results are shown in Figure 43. Choose one of the following: ALK5 inhibitor II, LY2157299, SB431541, or D4476. By culturing in the added adipocyte-inducing medium, fibroblasts strongly express the UCP1 gene. It can be seen that it was converted into brown adipose tissue. In particular, ALK5 inhibitor II was the most effective. LY2157299 strongly induced the conversion of fibroblasts into brown adipose tissue. It can be seen that it comes next.

[0379] Example 40 (Figure 44) Human normal skin-derived fibroblasts (HDFs) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and group 1 was selected as shown in the figure. Group 1 was given normal culture medium, Group 2 was given adipocyte induction medium, and Groups 3-6 were given the compounds ALK5 inhibitor II (4 μM), LY2157299 (8 μM), SB431542 (4 μM), and D4476 (4 μM), respectively. Cell induction medium was added at a concentration of 1 mL / well. The adipocyte induction medium contained (1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL). The culture medium was DMEM supplemented with insulin and 10% FBS. The culture was replaced with fresh medium every two days and cultured from Day 1 to Day 8. Subsequently, from Day 9 to Day 14, ALK5 inhibitor was used. Adipocytes were cultured in adipocyte induction medium that did not contain II, LY2157299, SB431542, or D4476. On Day 14, the culture medium was aspirated from each well and washed with PBS(-). 4% paraformaldehyde After fixation with hi-, the samples were washed with PBS(-) and incubated with Perm Buffer (PBS with 0.2% Triton-X) for 15 minutes. After washing three times with PBS(-), Blocking One was added and incubated at room temperature for 60 minutes. Anti-UCP-1 antibody (RD MAB6158) was added and reacted at room temperature for 2 hours. After incubation, the samples were washed three times with Wash buffer. CF488-conjugated anti-mouse Ig antibody (Biotum 20014) was added and the samples were reacted at room temperature for 2 hours, then washed three times with PBS (-). Nuclear staining was performed with Lifetechnology's SlowFade Gold antifade reagent with DAPI, followed by fluorescence microscopy. I took a photograph at 100x magnification.

[0380] The results are shown in Figure 44 (fluorescence microscope image). In all groups, a large number of cell nuclei stained with DAPI were observed. In groups 1 and 2, very few cells were stained with anti-UCP1 antibody. On the other hand, in groups 3-6, a large number of cells stained with anti-UCP1 antibody were observed. In particular, group 3 showed the highest density of cells stained with anti-UCP1 antibody, followed by group 4. Therefore, lipid cells treated with one of the following were found to be highly stained with ALK5 inhibitor II, LY2157299, SB431541, or D4476. By culturing fibroblasts in cell-inducing medium, brown adipose cells expressing the UCP1 protein are produced. It can be seen that it converted to fibroblasts. In particular, AKL5 inhibitor II had the strongest effect on fibroblasts. It induced the conversion of cells into brown adipose tissue, and LY2157299 followed suit. I understand.

[0381] Example 41 (Figure 45) Human normal skin-derived fibroblasts (HDFs) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4 Seeds were seeded in a 12-well plate at a concentration of cells / well (day 0), under 5% CO2 / 95%. The culture was started in humidified air at 37°C. The next day, the culture supernatant was aspirated and removed, and the culture medium was changed to normal medium and lipids. Fat cell induction medium, or the compound ALK5 inhibitor II (4 μM) or LY2157299 (8 μM) Adipose cell induction medium containing (1 nM T3, 1) was added at a rate of 1 mL / well. The culture medium is DMEM supplemented with μM rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL insulin, and 10% FBS. The culture medium is changed every two days. The culture was replaced with fresh material and incubated from Day 1 to Day 8. Then, from Day 9 to Day 14... Next, ALK5 inhibitor II was cultured in a medium that did not contain LY2157299. On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then the cells were extracted using the Qiagen RNA easy Mini Kit. Total RNA was extracted using this method. From this RNA, Rever Tra Ace qPCR RT Master Mix was used. Then, cDNA was synthesized. This cDNA was mixed with Real-time PCR Master Mix, the UCP-1 gene, and CIDEA. Mix primers specific to the gene, KCNK3 gene, or β-actin gene with Taqman probes. The results were obtained by performing qRT-PCR using the AB7300 Real-time PCR system. The mRNA level of the UCP1 gene was quantified as a ratio to the β-actin gene mRNA, and the value for fibroblasts cultured in normal medium was set to 1.

[0382] The results are shown in Figure 45. It can be seen that culturing fibroblasts in adipocyte induction medium supplemented with either ALK5 inhibitor II or LY2157299 converted them into brown adipocytes expressing the UCP1, CIDEA, and KCNK3 genes. In particular, ALK5 inhibitor II was effective. This indicates that it more strongly induced the conversion of fibroblasts into brown adipocytes.

[0383] Example 42 (Figure 46) Human normal skin-derived fibroblasts (HDFs) were suspended in standard culture medium (DMEM supplemented with 10% FBS). This was then divided into 3 × 10⁻⁶ cells. 4Cells were seeded into a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and the cells were cultured in standard medium and lipids. Add 1 mL / well of cell induction medium, or adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 μM. The adipocyte induction medium is DMEM (supplemented with 1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, 1 μg / mL Insulin, and 10% FBS). Replace the culture medium with fresh medium every two days. The cells were cultured from Day 1 to Day 8. Then, from Day 9 to Day 14, they were treated with ALK5 inhibitor II. The cultures were incubated in a culture medium without [specific ingredient]. On Day 14, the culture medium was aspirated and removed from each well, and washed with PBS(-). After fixation with 4% paraformaldehyde, the samples were washed with PBS(-) and incubated with Perm Buffer (PBS with 0.2% Triton-X) for 15 minutes. After washing three times with PBS(-), Blocking One was added and incubated at room temperature for 60 minutes. Anti-UCP-1 antibody (RD MAB6158) was used. In addition, after reacting at room temperature for 2 hours, the mixture was washed three times with wash buffer. CF488-conjugated anti-mouse Ig antibody (Biotum 20014) was added and reacted at room temperature for 2 hours, then washed three times with PBS(-). The samples were washed multiple times. Nuclear staining was performed using Lifetechnology's SlowFade Gold antifade reagent with DAPI. Subsequently, images were taken using a fluorescence microscope at 100x magnification. The percentage of UCP1-positive cells among DAPI-positive cells was calculated using Keyence BZ-H3A software.

[0384] The results are shown in Figure 46. In HDFs cultured in normal medium and in adipocyte-inducing medium without ALK5 inhibitor II, numerous cell nuclei stained with DAPI were observed, but very few cells stained with anti-UCP1 antibody. On the other hand, in HDFs cultured in adipocyte-inducing medium without ALK5 inhibitor II, Many cells cultured in adipocyte-inducing medium were strongly stained by anti-UCP1 antibody. Calculating the ratio of cells stained with anti-UCP1 antibody to cells stained with DAPI (denominator) revealed that over 90% of fibroblasts converted to brown adipocytes, which highly express UCP1 protein, when cultured in adipocyte-inducing medium supplemented with ALK5 inhibitor II.

[0385] Example 43 (Figure 47) Normal human epidermal keratinocytes (NHEK(AD)) were infused in serum-free liquid medium for normal human epidermal keratinocyte proliferation (HuMedia-KG2; Kurabo). This mixture was then divided into 3 × 10⁻⁶ units. 4 Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, and HuMedia-KG2 medium (Ctrl medium) was used. K-adipocyte induction medium (adipocyte induction medium for keratinocytes), or ALK5 inhibitor Add 1 mL / well of K-adipocyte induction medium supplemented with II (4 μM) or LY2157299 (8 μM). Added.

[0386] The K-adipocyte induction medium is HuMedia-KG2 (containing 1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL Insulin).

[0387] The culture medium was replaced with fresh medium every two days, and the cells were cultured from Day 1 to Day 8. Subsequently, from Day 9 to Day 14, the cells were cultured in a medium that did not contain ALK5 inhibitor II or LY2157299. On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then the cells were extracted from Qiagen. Total RNA was extracted using the RNA easy Mini Kit. From this RNA, Rever Tra Ace qP cDNA was synthesized using CR RT Master Mix. This cDNA was then mixed with Real-time PCR Master Mix, primers specific to the UCP-1 gene, CIDEA gene, or β-actin gene, and a Taqman probe. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of the gene and the CIDEA gene were quantified as a ratio to the β-actin gene mRNA level, and the values ​​for fibroblasts cultured in normal medium were set to 1.

[0388] The results are shown in Figure 47. By culturing normal human epidermal keratinocytes in K-adipocyte induction medium supplemented with either ALK5 inhibitor II or LY2157299, the UCP1 gene and CIDEA gene were expressed. It appears that the cells were converted into brown adipose tissue.

[0389] Example 44 (Figure 48) Normal human epidermal keratinocytes (NHEK(AD)) were infused in serum-free liquid medium for normal human epidermal keratinocyte proliferation (HuMedia-KG2; Kurabo). This mixture was then divided into 3 × 10⁻⁶ units. 4Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The culture supernatant was aspirated and removed the following day, and HuMedia-KG2 medium (Ctrl medium) was used. 1 mL / well of either K-osteoblast induction medium (osteoblast induction medium for keratinocytes) or K-osteoblast induction medium supplemented with compound ALK5 inhibitor II (4 μM) was added. The K-osteoblast induction medium was HuMedia-KG2 supplemented with 50 μg / mL ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone. The culture medium was replaced with fresh medium every two days and cultured until Day 14. Thereafter, from Day 14 to Day 28, all cells were cultured in a medium that did not contain ALK5 inhibitor II. On Day 28, the culture medium was aspirated and removed from each well, and PBS ( After washing with (-), total RNA was extracted from the cells using the Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix was mixed with primers and a Taqman probe specific to the Runx2 gene or β-actin gene. qRT-PCR was performed using the AB7300 Real-time PCR system. The Runx2 gene mRNA level was quantified as a ratio to the β-actin gene mRNA level, and the value for fibroblasts cultured in normal medium was set to 1.

[0390] The results are shown in Figure 48. The cells were cultured in K-osteoblast-inducing medium supplemented with ALK5 inhibitor II. As a result, normal human epidermal keratinocytes were converted into osteoblasts expressing the Runx2 gene. This can be understood. Example 45 (Figure 49) Human normal skin fibroblasts (HDFs) were placed in Control medium. I made it vague. This is 10 4 cells / mm 2 Seeds were seeded in a 48-well plate at the specified concentration (Day 0), and cultivation was started at 37°C in 5% CO2 / 95% humidified air. On Day 4, the culture supernatant was aspirated and removed, and the wells were divided into 5 groups. The culture medium was divided and added as shown in the diagram. That is, Group 1: Control medium Group 2: WA medium Group 3: WA medium supplemented with 1 μM Rosiglitazone Group 4: WA medium supplemented with 16 μM ALK5 inhibitor II Group 5: WA medium supplemented with 16 μM ALK5 inhibitor II and 1 μM rosiglitazone.

[0391] The composition of the culture medium is as follows: The control medium is Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS, 100 mM non-essential amino acids (NEAA), 100 U / ml penicillin, and 100 μg / mL streptomycin. WA medium contains 10% FBS, 100 mM non-essential amino acids (NEAA), 100 U / ml penicillin, This is Dulbecco's modified minimum essential medium (DMEM) supplemented with 100 μg / mL streptomycin, 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 62.5 nM indomethacin, 1 μM dexamethasone, and 170 nM insulin.

[0392] The culture was continued at 5% CO2 / 95% humidified air and 37°C. On the 6th, 8th, and 10th days, the same composition was used. Each culture medium was replaced with fresh medium.

[0393] On the 12th day, BODIPY staining was performed as follows. (1) After removing the culture medium from the wells, wash each well once with PBS. (2) Add 50 μL of 4% formaldehyde solution to each well and fix at room temperature overnight. (3) Wash twice with PBS. (4) Add 50 μL of BODIPY staining solution to each well and leave at room temperature for 30 minutes. (5) After removing the staining solution from the wells, let stand at room temperature for 30 minutes.

[0394] Phase-contrast and green fluorescence images taken with a fluorescence microscope are shown in Figure 49. Most of the cells were not stained with Bodipy, and the group cultured in WA medium supplemented with Rosiglitazone was also affected. Very weakly stained cells are present at a low frequency in WA culture with ALK5 inhibitor II added. In the group cultured in soil, a greater number of cells were observed to be more strongly stained. ALK5 inhibitor II In the group cultured in WA medium supplemented with both ALK5 inhibitor II and rosiglitazone, the most cells were strongly stained with BODIPY. Therefore, it can be seen that human fibroblasts converted to white adipocytes when cultured in WA medium supplemented with ALK5 inhibitor II. In particular, it can be seen that ALK5 inhibitor II more strongly induced the conversion of fibroblasts to white adipocytes in the presence of rosiglitazone.

[0395] Example 46 (Figure 50) Human normal skin fibroblasts (HDFs) were placed in Control medium. I made it vague. This is 10 4 cells / mm 2 Seeds were seeded in a 48-well plate at the specified concentration (Day 0), and cultivation was started at 5% CO2 / 95% humidified air and 37°C. On Day 4, the culture supernatant was aspirated and removed, the wells were divided into 7 groups, and the culture medium shown in the figure was added. That is, Group 1: Control medium Group 2: WA medium Group 3: WA medium supplemented with 1 μM Rosiglitazone Group 4: WA medium supplemented with 8 μM ALK5 inhibitor II Group 5: WA medium supplemented with 16 μM ALK5 inhibitor II Group 6: WA medium supplemented with 8 μM ALK5 inhibitor II and 1 μM rosiglitazone. Group 7: WA medium supplemented with 16 μM ALK5 inhibitor II and 1 μM rosiglitazone.

[0396] The composition of the culture medium is the same as in Example 45.

[0397] The culture was continued at 5% CO2 / 95% humidified air and 37°C. On the 6th, 8th, and 10th days, the same composition was used. Each culture medium was replaced with fresh medium.

[0398] On the 12th day, the cells were stained with BODIPY in the same manner as in Example 45, and lipid droplets were fluorescently stained. Images were taken at 100x magnification using a fluorescence microscope BZ-9000 (Keyence). The number of BODIPY-positive cells was calculated using BZ-II Analyzer software (Keyence, Osaka, Japan), and comparisons were made between each group.

[0399] The results (mean ± standard deviation) are shown in Figure 50. Cells cultured in Control medium were hardly stained with BODIPY, but were stained with 1 μM Rosiglitazone or 16 μM ALK5 inhibitor II. In groups cultured in WA medium supplemented with either of the inhibitors, stained cells were present at a low frequency. In groups cultured in WA medium co-supplemented with 8 μM or 16 μM ALK5 inhibitor II and 1 μM rosiglitazone, a higher number of stained cells were observed. Therefore, it can be seen that culturing in WA medium supplemented with ALK5 inhibitor II converted human fibroblasts into white adipocytes. In particular, in the presence of rosiglitazone, ALK5 inhibitor II converted fibroblasts more strongly. This indicates that the conversion of blast cells into white adipocytes was induced.

[0400] Example 47 (Figure 51) Human normal skin fibroblasts (HDFs) were placed in Control medium. I made it vague. This is 10 4 cells / mm 2 Seeds were seeded in a 6-well plate at the specified concentration (Day 0), and cultivation was started at 37°C in 5% CO2 / 95% humidified air. On Day 4, the culture supernatant was aspirated and removed, the wells were divided into 5 groups, and the culture medium shown in the figure was added, as in Example 45. The composition of the culture medium was the same as in Example 45.

[0401] The culture was continued at 5% CO2 / 95% humidified air and 37°C. On the 6th, 8th, and 10th days, the same composition was used. Each culture medium was replaced with fresh medium.

[0402] On the 12th day, RNA was recovered from the cells. TaqMan probes and primers specific to the AdipoQ, FABP4 (mature adipocyte marker), and PPAR-γ (adipocyte progenitor marker) genes were used, respectively. Next, real-time PCR was performed to quantify the mRNA.

[0403] The results (mean) (relative value) are shown in Figure 51. AdipoQ and FABP4 were mildly induced in cultures with ALK5 i II alone, and strongly induced in cultures with co-addition of ALK5 i II and Rosiglitazone. PPAR- γ expression was mildly induced even when cultured in WA medium, moderately induced with the addition of either ALK5 i II or Rosiglitazone, and strongly induced with the co-addition of ALK5 i II and Rosiglitazone.

[0404] Therefore, it can be seen that human fibroblasts converted to white adipocytes by culturing them in WA medium supplemented with ALK5 inhibitor II, especially in the presence of rosiglitazone. AKL5 inhibitor II more strongly induces the conversion of fibroblasts into white adipocytes. It is clear that he guided them.

[0405] Example 48 (Figure 52) Human normal skin fibroblasts (HDFs) were placed in Control medium. I made it vague. This is 10 4 cells / mm 2 Seeds were seeded in a 48-well plate at the specified concentration (Day 0), and cultivation was started at 5% CO2 / 95% humidified air and 37°C. On Day 4, the culture supernatant was aspirated and removed, the wells were divided into 10 groups, and the culture medium shown in the figure was added. That is, Group 1: WA medium Group 2: WA medium supplemented with 16 μM ALK5 inhibitor II (ALK5 i II). Group 3: WA medium supplemented with 16 μM of LY2157299 Group 4: WA medium supplemented with 16 μM SB431542 Group 5: WA medium supplemented with 16 μM of D4476 Group 6: WA medium supplemented with 1 μM rosiglitazone. Group 7: WA medium supplemented with 16 μM ALK5 inhibitor II (ALK5 i II) and 1 μM rosiglitazone. Group 8: WA medium supplemented with 16 μM LY2157299 and 1 μM Rosiglitazone. Group 9: WA medium supplemented with 16 μM SB431542 and 1 μM Rosiglitazone. Group 10: WA medium supplemented with 16 μM D4476 and 1 μM Rosiglitazone.

[0406] The composition of the culture medium is the same as in Example 45.

[0407] The culture was continued at 5% CO2 / 95% humidified air and 37°C. On the 6th, 8th, and 10th days, the same composition was used. Each culture medium was replaced with fresh medium.

[0408] On the 12th day, the lipid droplets were stained with BODIPY in the same manner as in Example 45, and then fluorescently stained. Fluorescence microscopy Images were taken at 100x magnification using a BZ-9000 (Keyence) scanner. The number of adipocytes was calculated using BZ-II Analyzer software (Keyence, Osaka, Japan), and comparisons were made between groups. The results are shown in Figure 52.

[0409] Cells cultured in WA medium supplemented with ALK5 inhibitor II, LY2157299, SB431542, or D4476 individually had lipid droplets stained with Bodipy in only a small number of cells. However, when either ALK5 inhibitor II, LY2157299, SB431542, or D4476 was added along with Rosiglitazone, Numerous stained cells were observed in the group cultured in WA medium. Therefore, it can be seen that human fibroblasts convert to white adipocytes when cultured in WA medium supplemented with a TGF-β pathway inhibitor. Furthermore, when both a TGF-β pathway inhibitor and rosiglitazone were added... When cultured in WA medium, human fibroblasts converted to white adipocytes more efficiently than with TGF-β pathway inhibitors alone. In particular, among TGF-β pathway inhibitors, AKL5 inhibitor II was the most potent in converting fibroblasts to white adipocytes. It's clear they were manipulated.

[0410] Example 49 (Figure 53) Human normal skin fibroblasts (HDFs) were mixed in Control medium. I did this. 4 cells / mm 2 Seeds were seeded in a 6-well plate at the specified concentration (Day 0), and cultivation was started at 5% CO2 / 95% humidified air and 37°C. On Day 4, the culture supernatant was aspirated and removed, and the wells were divided into 10 groups. Then, similar to Example 48, the culture medium shown in the figure was added. The composition of the culture medium was the same as in Example 45.

[0411] The culture was continued at 5% CO2 / 95% humidified air and 37°C. On the 6th, 8th, and 10th days, the same composition was used. Each culture medium was replaced with fresh medium.

[0412] On the 12th day, the cells were stained with BODIPY in the same manner as in Example 48, and lipid droplets were fluorescently stained. Images were taken at 100x magnification using a fluorescence microscope BZ-9000 (Keyence). The number of BODIPY-positive cells, the area stained with BODIPY, and the intensity of staining by BODIPY were calculated using BZ-II Analyzer software (Keyence, Osaka, Japan), and comparisons were made between each group.

[0413] The results (mean ± standard deviation) are shown in Figure 53. Cells cultured in Control medium were hardly stained with BODIPY, but were stained with 1 μM Rosiglitazone or 16 μM ALK5 inhibitor II. Cells cultured in WA medium supplemented with any of these substances accumulated small amounts of fat. 16 μM ALK5 inhibitor II, LY2157299, SB431542, and D4476 all induced fat accumulation more strongly when co-added with rosiglitazone than with rosiglitazone alone. ALK5 inhibitor II and 1 μM In the group cultured in WA medium co-supplemented with Rosiglitazone, the largest amount of fat was accumulated. Cells were observed. Therefore, Rosiglitazone containing a TGF-β pathway inhibitor was included. By culturing in WA medium, it was found that human fibroblasts were efficiently converted into white adipocytes. In particular, among TGF-β pathway inhibitors, AKL5 inhibitor II was the most effective. This indicates that it strongly induced the conversion of fibroblasts into white adipocytes.

[0414] Example 50 (Figure 54) Human normal skin fibroblasts (HDFs) were placed in Control medium. I made it vague. This is 10 4 cells / mm 2 Seeds were seeded in a 6-well plate at the specified concentration (Day 0), and cultivation was started at 5% CO2 / 95% humidified air and 37°C. On Day 4, the culture supernatant was aspirated and removed, the wells were divided into 5 groups, and the culture medium shown in the figure was added. That is, Group 1: WA medium supplemented with 1 μM Rosiglitazone Group 2: WA medium supplemented with 1 μM rosiglitazone and 16 μM ALK5 inhibitor II (ALK5 i II). Group 3: WA medium supplemented with 1 μM Rosiglitazone and 16 μM LY2157299 (LY). Group 4: WA medium supplemented with 1 μM Rosiglitazone and 16 μM SB431542(SB) Group 5: WA medium supplemented with 1 μM rosiglitazone and 16 μM D4476 (D4).

[0415] The culture medium composition is the same as in Example 45. The culture was performed at 37°C with 5% CO2 / 95% humidified air. The culture medium was then replaced with fresh medium of the same composition on the 6th, 8th, and 10th days.

[0416] On the 12th day, RNA was recovered from the cells and, as in Example 47, AdipoQ, FABP4 (mature lipid cells) were used. mRNA levels of the cellular marker and the PPAR-γ (adipocyte progenitor cell marker) gene were quantified.

[0417] The results (mean) are shown in Figure 54. Cells in groups 2-5 moderately to highly expressed mRNA of one or more of the following genes: AdipoQ, FABP4, or PPAR-γ. Cells treated with ALK5 inhibitor II expressed all three of these genes most strongly.

[0418] Therefore, it can be seen that ALK5 inhibitor II, LY2157299, SB431542, and D4476 all converted human fibroblasts into white adipocytes. In particular, among TGF-β pathway inhibitors, ALK5 inhibitor II was the most potent in converting fibroblasts into white adipocytes. It's clear that John was being manipulated.

[0419] Example 51 (Figure 55) Human fibroblasts aHDF45 were placed in a 24-well plate in a 1.0 × 10⁶ arrangement. 4 Seed cells in 500 μL / well portions. The following day, the wells were divided into two groups. One group had its culture medium replaced with the Control medium described below, and the other group had its culture medium replaced with the SC medium described below, to which 4 μM Alk5 i II had been added. The cultures were incubated at 37°C, 5% CO2 / 95% air for 14 days. They were nourished. The culture medium was changed twice a week. Control medium: Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS, 100 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 100 U / ml penicillin, and 100 μg / mL streptomycin. SC (Schwann cell) medium: 10% FBS, 10 ng / mL rhbFGF (basic fibroblast growth factor), 5.7 μg / mL Forskolin, 200 ng / mL rhHeregulin beta-1, 5 ng / mL rhPDGF-AA, 100 mM non-essential amino acids (NEAA), 1 mM Sodium Pyruvate, 100 U / ml penicillin, and Dulbecco's modified minimum essential medium (DMEM) supplemented with 100 μg / mL streptomycin.

[0420] RNA was extracted from each cell on days 8 and 14 using the RNeasy Mini QIAcube Kit (QIAGEN), and cDNA was synthesized by reverse transcription using ReverTra Ace qPCR RT Master Mix (TOYOBO). Real-time RT-PCR analysis of EGR2 and S100β gene mRNA expression was performed using the obtained cDNA with StepOnePLUS (Applied Biosystems).

[0421] The results are shown in Figure 55. It can be seen that human fibroblasts converted to Schwann cells co-expressing S100β and EGR2 when cultured in SC medium supplemented with 4 μM Alk5 i II.

[0422] Example 52 (Figure 56) Normal human epidermal keratinocytes (NHEK(AD)) were inoculated into serum-free liquid medium for normal human epidermal keratinocyte proliferation (HuMedia-KG2; Kurabo). This was then divided into 3 × 10⁻⁶ units. 4 Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed, and 1 mL / well of HuMedia-KG2 medium, human epidermal basal cell proliferation medium (CnT-PR; CELLnTEC), or human epidermal basal cell proliferation-like medium supplemented with 1 μM or 10 μM ALK5 inhibitor II was added. The culture medium was replaced with fresh medium every two days and cultured until day 21. On day 21, the culture medium was aspirated and removed from each well, washed with PBS(-), and total RNA was extracted from the cells using the Qiagen RNA easy Mini Kit. cDNA was synthesized from this RNA using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to the Uroplakin 1b gene, Uroplakin 3b gene, or β-actin gene, and a Taqman probe. qRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA levels of the Uroplakin 1b and Uroplakin 3b genes were quantified as a ratio to the β-actin gene mRNA level, and the value of human epidermal keratinocytes cultured in HuMedia-KG2 medium was set to 1. The results are shown in Figure 56. It can be seen that human epidermal keratinocytes converted to urothelial cells expressing the Uroplakin 1b and Uroplakin 3b genes by culturing them in CnT-PR medium supplemented with ALK5 inhibitor II.

[0423] Example 53 Human normal peripheral blood mononuclear cells (PBMCs) were turbidified in control medium (RPMI1640 medium supplemented with 10% FBS). This was then divided into 2.5 × 10⁶ units. 5 Cells were seeded in a 12-well plate at a concentration of cells / well (Day 0), and culture was started at 37°C in 5% CO2 / 95% humidified air. The following day, the culture supernatant was aspirated and removed. Group 1 was given 1 mL / well of control medium. Group 2 was given 1 mL / well of P-adipocyte induction medium (adipocyte induction medium for peripheral blood mononuclear cells). Group 3 was given 1 mL / well of P-adipocyte induction medium supplemented with 4 μM ALK5 inhibitor II. The P-adipocyte induction medium was RPMI1640 medium supplemented with 10% FBS, 30 U / ml recombinant hIL-2, 1 nM T3, 1 μM rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 μM dexamethasone, and 1 μg / mL insulin. Every two days, 50% of the culture medium was replaced with fresh medium.

[0424] On Day 14, the culture medium was aspirated and removed from each well, washed with PBS(-), and then the cells were extracted from Qiagen. Total RNA was extracted using the RNA easy Mini Kit. From this RNA, cDNA was synthesized using the Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with the Real-time PCR Master Mix, primers specific to the FABP4 gene or β-actin gene, and a Taqman probe. QRT-PCR was performed using the AB7300 Real-time PCR system. The mRNA level of the FABP4 gene was quantified as a ratio to the mRNA level of the β-actin gene, and the value for human normal peripheral blood mononuclear cells cultured in standard medium was set to 1.

[0425] The results are shown in Figure 57. The cells were cultured in P-adipocyte induction medium supplemented with ALK5 inhibitor II. As a result, normal human peripheral blood mononuclear cells are converted into white adipocytes that express the FABP4 gene. It became clear that this was the case.

Claims

1. A method for preparing somatic cells, characterized by culturing differentiated mammalian somatic cells in a culture medium for inducing differentiation of other somatic cells, in the presence of a TGF-β pathway inhibitor, thereby directly converting the differentiated somatic cells into other somatic cells without the need for pluripotent stem cells, the method being selected from the following embodiments (1) to (5): (1) The differentiated somatic cells are fibroblasts, the other somatic cells are mesenchymal stem cells, and the culture medium is a medium for inducing mesenchymal stem cells. (2) The differentiated somatic cells are fibroblasts, the other somatic cells are Schwann cells, and the culture medium is a medium for inducing Schwann cells (forskolin, bFGF, PDGF, and heregulin added to normal culture medium). (3) The differentiated somatic cells are fibroblasts or peripheral blood mononuclear cells, the other somatic cells are white adipocytes, and the culture medium is a culture medium for inducing white adipocytes (a standard culture medium to which one or more components selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone are added). (4) The differentiated somatic cells are fibroblasts, the other somatic cells are chondrocytes, and the culture medium is a culture medium for inducing chondrocytes (ascorbic acid, insulin-transferrin-selenium, BMP-2, TGF-β, GDF5, and β-FGF added to a normal culture medium). (5) The differentiated somatic cells are fibroblasts, the other somatic cells are myoblasts, and the culture medium is a culture medium for inducing myoblasts (Bovine Fetuin, hEGF, bFGF, insulin, and dexamethasone added to a normal culture medium).

2. The method according to claim 1, wherein the TGF-β pathway inhibitor is D4476, SB431542, LY2157299, SD208, or ALK5 inhibitor II.

3. The method according to claim 1 or 2, wherein the TGF-β pathway inhibitor is ALK5 inhibitor II.

4. The method according to any one of claims 1 to 3, wherein the culture medium further comprises a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist.

5. An inducer comprising a TGF-β pathway inhibitor for directly converting differentiated somatic cells into other somatic cells in a culture medium without the need for pluripotent stem cells, wherein the combination of the differentiated somatic cells, the other somatic cells, and the culture medium is selected from the following embodiments (1) to (5): (1) The differentiated somatic cells are fibroblasts, the other somatic cells are mesenchymal stem cells, and the culture medium is a medium for inducing mesenchymal stem cells. (2) The differentiated somatic cells are fibroblasts, the other somatic cells are Schwann cells, and the culture medium is a medium for inducing Schwann cells (forskolin, bFGF, PDGF, and heregulin added to normal culture medium). (3) The differentiated somatic cells are fibroblasts or peripheral blood mononuclear cells, the other somatic cells are white adipocytes, and the culture medium is a culture medium for inducing white adipocytes (a standard culture medium to which one or more components selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone are added). (4) The differentiated somatic cells are fibroblasts, the other somatic cells are chondrocytes, and the culture medium is a culture medium for inducing chondrocytes (ascorbic acid, insulin-transferrin-selenium, BMP-2, TGF-β, GDF5, and β-FGF added to a normal culture medium). (5) The differentiated somatic cells are fibroblasts, the other somatic cells are myoblasts, and the culture medium is a culture medium for inducing myoblasts (Bovine Fetuin, hEGF, bFGF, insulin, and dexamethasone added to a normal culture medium).

6. A kit for directly converting differentiated somatic cells into other somatic cells without going through pluripotent stem cells, comprising a TGF-β pathway inhibitor and a culture medium for inducing differentiation of the other somatic cells, selected from the following embodiments (1) to (5): (1) The differentiated somatic cells are fibroblasts, the other somatic cells are mesenchymal stem cells, and the culture medium is a medium for inducing mesenchymal stem cells. (2) The differentiated somatic cells are fibroblasts, the other somatic cells are Schwann cells, and the culture medium is a medium for inducing Schwann cells (forskolin, bFGF, PDGF, and heregulin added to normal culture medium). (3) The differentiated somatic cells are fibroblasts or peripheral blood mononuclear cells, the other somatic cells are white adipocytes, and the culture medium is a culture medium for inducing white adipocytes (a standard culture medium to which one or more components selected from the group consisting of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone are added). (4) The differentiated somatic cells are fibroblasts, the other somatic cells are chondrocytes, and the culture medium is a culture medium for inducing chondrocytes (ascorbic acid, insulin-transferrin-selenium, BMP-2, TGF-β, GDF5, and β-FGF added to a normal culture medium). (5) The differentiated somatic cells are fibroblasts, the other somatic cells are myoblasts, and the culture medium is a culture medium for inducing myoblasts (Bovine Fetuin, hEGF, bFGF, insulin, and dexamethasone added to a normal culture medium).

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