WO2017069222A1 - 細胞の調製方法 - Google Patents
細胞の調製方法 Download PDFInfo
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- WO2017069222A1 WO2017069222A1 PCT/JP2016/081187 JP2016081187W WO2017069222A1 WO 2017069222 A1 WO2017069222 A1 WO 2017069222A1 JP 2016081187 W JP2016081187 W JP 2016081187W WO 2017069222 A1 WO2017069222 A1 WO 2017069222A1
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Definitions
- the present invention mainly relates to a method for preparing cells. Specifically, it relates to a method for preparing cells by direct reprogramming. The present invention also relates to an inducer for converting differentiated somatic cells to other somatic cells.
- osteogenesis can be promoted by transplanting osteoblasts in the affected area to repair bone defects associated with bone tumors, trauma, osteomyelitis, etc., and bone defects after curettage such as bone tumors.
- functional and morphological prognosis is improved.
- a treatment for autologous transplantation of bone marrow cells collected from a patient's cancellous bone is performed, and its effectiveness is known.
- osteoblasts are induced to differentiate from mesenchymal stem cells contained in autologous bone marrow cells and contribute to bone formation and remodeling.
- the prevalence of osteoporosis is increasing with the aging of the population.
- osteoblast transplantation can promote the healing of fractures associated with osteoporosis, trauma, etc., intractable fractures and pseudo-fractures.
- Osteoblast transplantation also includes rheumatoid arthritis, sudden femoral head necrosis, osteoarthritis, lumbar osteoarthritis, spinal stenosis, disc herniation, spondylolysis, spondylolisthesis, scoliosis, cervical spine Myelopathy, posterior longitudinal ligament ossification, spinal cord injury, hip osteoarthritis, knee osteoarthritis, femoral slippage, osteomalacia, bone repair after surgery (sternal repair after cardiac surgery, etc.) ), May be useful for repairing defects associated with artificial ankle surgery, osteomyelitis, osteonecrosis, and the like.
- periodontal disease is also called the fourth lifestyle-related disease, has a very high morbidity rate, and causes various systemic diseases. As periodontal disease progresses, bone resorption of alveolar bone occurs, and if osteoblasts can be efficiently supplied to the local bone resorbing part, it is considered that this will lead to regenerative treatment of alveolar bone.
- osteoblast transplantation is used in combination with bone transplantation, artificial bone transplantation, artificial joints or implants, the therapeutic effect may be enhanced.
- the means of supplying cells to be used is one of the problems.
- bone marrow mesenchymal stem cells and bone marrow cells containing bone marrow mesenchymal stem cells have been used as osteoblasts for transplantation purposes.
- the collection of bone marrow has problems such as the invasion to a patient is large and a sufficient number of bone marrow cells may not be supplied.
- ES cells human embryonic stem cells
- ES cells human embryonic stem cells
- iPS cells it is not necessary to collect bone marrow from the patient, and a sufficient number of osteoblasts may be supplied.
- there is a risk that the remaining iPS cells may become tumor after transplantation.
- Non-Patent Document 1 introduces the differentiation of osteoblasts into human ES cells by introducing Osterix Lentivirus vector + Osteogenic medium.
- Non-Patent Documents 2 and 3 obtain osteoblasts by inducing differentiation from mouse iPS cells through Osteogenic medium via MSC.
- Non-Patent Document 4 introduces Runx2 Adenovirus vector into mouse iPS cells and induces differentiation in Osteogenic medium to obtain osteoblasts. As shown in Non-Patent Documents 1 to 4, osteoblasts are produced by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells. There was a danger.
- Patent Document 1 discloses a method for efficiently preparing (direct conversion) functional osteoblasts by introducing a predetermined gene group into somatic cells.
- cells may become tumorous due to the effect of the introduced gene or vector.
- problems such as cost and time required for safety verification. Therefore, there is a demand for a technique for inducing cells for transplantation without introducing a gene.
- the present invention mainly provides a cell preparation method, particularly a technique for converting differentiated somatic cells into other somatic cells without performing gene transfer (direct conversion (direct reprogramming)). For the purpose.
- the present inventor cultivated mammalian somatic cells in a medium of various compositions in the presence of a TGF- ⁇ pathway inhibitor, so that the somatic cells of the raw material other than the somatic cells of the raw material It was found that it can be converted into other somatic cells.
- the present invention includes the following osteoblast preparation method, osteoblast inducer and kit.
- Item 1 Differentiated somatic cells of a mammal are cultured in a medium for inducing differentiation of other somatic cells other than the differentiated somatic cells in the presence of a TGF- ⁇ pathway inhibitor.
- Item 2 The method according to Item 1, wherein the TGF- ⁇ pathway inhibitor is a TGF- ⁇ / SMAD pathway inhibitor.
- Item 3 The method according to Item 1 or 2, wherein the TGF- ⁇ pathway inhibitor is D4476, SB431542, LY2157299, SD208, or ALK5 inhibitor II.
- Item 4 The method according to any one of Items 1 to 4, wherein the TGF- ⁇ pathway inhibitor is ALK5 inhibitor II.
- Item 5 The method according to any one of Items 1 to 4, wherein the fibroblast is converted into a mesenchymal cell.
- Item 6 The method according to any one of Items 1 to 4, wherein fibroblasts or keratinocytes are converted into osteoblasts.
- Item 7 The method according to any one of Items 1 to 4, wherein the fibroblasts or peripheral blood mononuclear cells are converted into white adipocytes.
- Item 8 The method according to any one of Items 1 to 4, wherein the fibroblasts or keratinocytes are converted into brown adipocytes.
- Item 9 The method according to Item 7 or 8, wherein the medium for inducing differentiation of somatic cells contains a Peroxisome® Proliferator-Activated Receptor- ⁇ (PPAR- ⁇ ) agonist.
- PPAR- ⁇ Peroxisome® Proliferator-Activated Receptor- ⁇
- Item 10 The method according to any one of Items 1 to 4, wherein the fibroblast is converted into a chondrocyte.
- Item 11 The method according to any one of Items 1 to 4, wherein the fibroblast is converted into a myoblast.
- Item 12 The method according to any one of Items 1 to 4, wherein the fibroblasts are converted into Schwann cells.
- Item 13 The method according to any one of Items 1 to 4, wherein keratinocytes are converted into urothelial cells.
- Item 14 The method according to any one of Items 1 to 4, wherein the fibroblast is converted into a mesenchymal stem cell.
- Item 15 An inducer for converting differentiated somatic cells into other somatic cells, including a TGF- ⁇ pathway inhibitor.
- Item 16 A kit for converting differentiated somatic cells into other somatic cells, comprising a TGF- ⁇ pathway inhibitor and a medium for inducing differentiation of the other somatic cells.
- somatic cells can be provided in a short period of time from somatic cells differentiated by the action of a low molecular compound. Since the obtained somatic cells (for example, mesenchymal stem cells and osteoblasts) can be easily derived from the somatic cells of the subject to be transplanted, immunity can be obtained even when the obtained somatic cells themselves or tissues produced therefrom are transplanted. There will be no problems such as academic rejection. In addition, since somatic cells can be directly induced from somatic cells without going through iPS cells or ES cells, problems caused by pluripotent stem cells such as canceration can be avoided. On the other hand, it is possible to create a bank in advance and use it for allo-transplantation and xenotransplantation to patients.
- somatic cells for example, mesenchymal stem cells and osteoblasts
- the result (stained drawing) of alizarin red S staining is shown.
- the result (absorbance measurement) of alizarin red S staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained figure) of the immunostaining of Osteocalsin is shown.
- the result (stained drawing and absorbance measurement) of alizarin red S staining is shown.
- the result (stained figure) of the immunostaining of Runx2 is shown.
- the results of Bodipy staining (stained diagram) are shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained drawing) of Alcian blue staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result of oil ⁇ Red O staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the results of Bodipy staining (stained diagram) are shown.
- the results of Bodipy staining (fluorescence intensity measurement) are shown.
- a TGF- ⁇ / SMAD pathway is schematically shown.
- a TGF- ⁇ / SMAD pathway and a TGF- ⁇ / ERK pathway are schematically shown.
- TGF- ⁇ / SMAD pathway TGF- ⁇ / JNK pathway and TGF- ⁇ / p38 pathway are schematically shown.
- the TGF- ⁇ / SMAD pathway and the TGF- ⁇ / RhoA pathway are schematically shown.
- the result (stained drawing) of alizarin red S staining is shown.
- the result (stained drawing) of ALP staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result of oil ⁇ Red O staining (stained diagram) is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained drawing) of Alcian blue staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained drawing) of alizarin red S staining is shown.
- the result (stained drawing) of ALP staining is shown.
- a tomographic image of ⁇ CT ( ⁇ CT analysis) is shown. Arrowheads indicate bone defects (Bone defect), and arrows indicate regenerated bone tissue (Regenerated bone issue (Callus)).
- the 3D reconstruction image of ⁇ CT is shown. It is an image of 21 days after transplantation (21 days after transplantation). Arrows indicate bone defects (Bone defect) or regenerated bone tissue (Regenerated bone tissue (Callus)).
- the upper part is a cross-sectional view (Cross section), and the lower part is a longitudinal section.
- the result of histological analysis by hematoxylin and eosin staining (HE staining) and alizarin red S staining (Alizarin Red Staining) is shown.
- the heat map showing the expression level of the MSC marker gene (MSC marker genes) by DNA microarray analysis is shown.
- the result (stained drawing) of alizarin red S staining is shown.
- the result (absorbance measurement) of alizarin red S staining is shown.
- the results (stained diagram) of alizarin red S staining and ALP staining are shown.
- the result (absorbance measurement) of alizarin red S staining is shown.
- the result (stained drawing) of ALP staining is shown.
- the result (stained drawing) of alizarin red S staining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the results of immunostaining of osteocalsin (OC) are shown (stained diagram and percentage of OC positive cells in DAPI positive cells).
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained image) of UCP1 immunostaining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the result (stained diagram) of UCP1 immunostaining is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the results of immunostaining of UCP1 (stained image (A) and percentage of UCP1-positive cells in DAPI-positive cells (B)) are shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the results of Bodipy staining are shown.
- the results of Bodipy staining (Bodipy positive cell ratio) are shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the results of Bodipy staining (stained diagram) are shown.
- the results (quantitative) of Bodipy staining are shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- the measurement result of the gene expression level by real-time RT-PCR method is shown.
- FIGS. 4, 6 and 7 also show the color reversal images.
- the present invention relates to a method for preparing other somatic cells using mammalian differentiated somatic cells as a raw material.
- the present invention relates to a method for converting mammalian differentiated somatic cells into other somatic cells.
- Convert means the transformation of somatic cells into the desired somatic cells.
- somatic cells can be transferred to other cells without going through a cell reprogramming step represented by the production of iPS cells, also called “direct conversion” or “direct reprogramming”. It is a method of converting into somatic cells. In other words, the somatic cell can be directly converted to another somatic cell.
- somatic cells are converted to other somatic cells without introducing a gene.
- “Without gene introduction” means that the original genome sequence of a somatic cell (mainly a DNA base sequence) is converted into another somatic cell without change.
- “without introduction of a gene” means being converted into another somatic cell based on the function of the original endogenous gene of the somatic cell.
- the differentiated somatic cell of the mammal used as a raw material in the method of the present invention is not particularly limited as long as it is derived from a mammal.
- a somatic cell means a cell other than germ cells.
- somatic cells examples include fibroblasts, epithelial cells (skin epidermal cells, oral mucosal epithelial cells, airway mucosal epithelial cells, intestinal mucosal epithelial cells, etc.), epidermal cells, gingival cells (gingival fibroblasts, gingival epithelium) Cell), dental pulp cell, white fat cell, subcutaneous fat, visceral fat, muscle, blood cell (for example, peripheral blood mononuclear cell), etc., preferably fibroblast, gingival cell, oral mucosal epithelial cell, dental pulp cell , Fat cells, keratinocytes (keratinocytes), blood cells and the like.
- somatic stem cells such as mesenchymal stem cells (Mesenchymal stem cells: MSC), neural stem cells (Neural stem cells), hepatic stem cells (hepatic stem cells), intestinal stem cells, skin stem cells, hair follicle stem cells, pigment cell stem cells
- MSC mesenchymal stem cells
- neural stem cells neural stem cells
- Neuronal stem cells hepatic stem cells
- intestinal stem cells skin stem cells
- pigment cell stem cells examples also include somatic cells produced by induction, dedifferentiation, or reprogramming.
- derived to another somatic cell is also mentioned.
- somatic cells derived from germline cells by differentiation induction, dedifferentiation, or reprogramming are also included.
- cultured cells are exemplified, and somatic cells derived from cultured cells by differentiation induction, dedifferentiation, or reprogramming are also included.
- Mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cows, horses, pigs, and the like.
- the somatic cell is particularly preferably derived from a human.
- the age of an individual from which a somatic cell is derived is not limited, and may be an adult, a child, or a fetus.
- cells derived from fetuses and cells derived from placenta, amniotic membrane, umbilical cord and the like are also included in “somatic cells”.
- somatic cells autologous cells
- somatic cells autologous cells
- somatic cells autologous cells
- somatic cells not somatic cells, but somatic cells made from somatic cells of other people or other animals can be used for transplantation.
- other somatic cells prepared from others or other animal somatic cells prepared in advance can be used for transplantation.
- somatic cells previously prepared from somatic cells of other people or other animals can be used for transplantation. That is, a bank of somatic cells or somatic progenitor cells can be created and used for transplantation purposes.
- the blood group and MHC can be typed in advance.
- the character of the somatic cell for transplantation and tumorigenicity can be confirmed in advance.
- somatic cells prepared by the method of the present invention are somatic cells other than somatic cells used as raw materials.
- somatic cells that differentiate from somatic cells used as raw materials under physiological conditions are not included in “other somatic cells”. For example, in order to differentiate from mesenchymal stem cells into osteoblasts in vivo, osteoblasts do not fall under “other somatic cells” when using mesenchymal stem cells as a raw material.
- somatic cells to be prepared include mesenchymal stem cells (MSC), osteoblasts, adipocytes (brown adipocytes or white adipocytes), chondrocytes, myoblasts, urothelial cells, and bone marrow.
- MSC mesenchymal stem cells
- osteoblasts adipocytes (brown adipocytes or white adipocytes)
- chondrocytes myoblasts
- myoblasts urothelial cells
- bone marrow examples include glial cells such as stromal cells, tendon cells, hepatocytes, bile duct epithelial cells, Schwann cells, nerve cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, lymphatic endothelial cells and the like.
- epidermal cells pigment cells, hair follicle cells, nail mother 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, lymphoid cells, antigen-presenting cells, mammary epithelial cells, kidney cells (glomerular cells, tubular epithelial cells, urine) Urothelial cells such as urothelial cells), genital cells, corneal cells, conjunctival cells, retinal cells, synovial cells, endocrine cells, islet cells (insulin producing cells ( ⁇ cells), etc.), exocrine cells, liver Examples include stem cells, pancreatic stem cells, intestinal stem cells, salivary gland stem cells.
- somatic cells used as raw materials and somatic cells to be prepared is not particularly limited.
- somatic cells used as raw materials and other prepared somatic cells are somatic cells belonging to the mesenchymal system that all differentiate from mesenchymal stem cells. It is preferable.
- Examples of cells belonging to the mesenchymal system include mesenchymal stem cells (MSC), fibroblasts, osteoblasts, adipocytes, chondrocytes, myoblasts, bone marrow stromal cells, tendon cells and the like.
- MSC mesenchymal stem cells
- fibroblasts fibroblasts
- osteoblasts adipocytes
- chondrocytes myoblasts
- bone marrow stromal cells tendon cells and the like.
- cells that are known to be able to differentiate from mesenchymal stem cells such as hepatocytes, bile duct epithelial cells, glial cells, nerve cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, lymphatic endothelial cells are also Included in “cells belonging to the leaf system”.
- Osteoblasts include pre-osteoblasts, immature osteoblasts, mature osteoblasts, bone cells and the like.
- the fat cells include white fat cells and brown fat cells, and the white fat cells include adipose stem cells, preadipocytes, mature adipocytes, enlarged fat cells, and the like.
- Brown adipocytes include beige cells, Brite cells, and the like.
- Chondrocytes include immature chondrocytes, mature chondrocytes, hypertrophic chondrocytes and the like.
- the myoblasts include myo satellite cells, immature myoblasts, mature myoblasts, myocytes, myotube cells, myofibers and the like.
- the cells described in this specification include cells having different levels of differentiation in the cell line represented by the name.
- embodiments of the present invention include a method of converting fibroblasts into mesenchymal stem cells (MSC), a method of converting fibroblasts into osteoblasts, a method of converting fibroblasts into adipocytes (for example, , A method of converting fibroblasts into white adipocytes, a method of converting fibroblasts into brown adipocytes, etc.), a method of converting fibroblasts into chondrocytes, a method of converting fibroblasts into chondrocytes, fibers
- Examples include a method of converting blasts into myoblasts, a method of converting keratinocytes into urothelial cells, a method of converting blood cells (for example, peripheral blood mononuclear cells) into white adipocytes, and the like.
- the target cell osteoblast, Differentiation into adipocytes, chondrocytes, myoblasts, etc.
- a cell in the middle of the process is one of somatic cells, it is referred to as a direct conversion in this specification even through this cell.
- differentiated somatic cells are cultured in a medium (differentiation induction medium) for inducing other somatic cells other than differentiated somatic cells.
- a medium differentiate induction medium
- the differentiation-inducing medium a known differentiation-inducing medium can be used depending on the somatic cell to be prepared.
- “Medium for inducing other somatic cells other than differentiated somatic cells” and “differentiation-inducing medium” are mainly to differentiate pluripotent stem cells (ES cells, iPS cells, etc.) into the somatic cells.
- ES cells pluripotent stem cells
- iPS cells iPS cells
- ascorbic acid for example, a concentration of about 0.1 to 1000 ⁇ g / ml, preferably about 1 to 100 ⁇ g / ml
- ⁇ -Glycerophosphate for example, a concentration of about 0.1 to 1000 mM, Preferably, about 1 to 100 mM
- Dexamethasone about 1 to 10 to about 10 mm, preferably about 10 to about 1000 to mM
- a component one or more selected from the group consisting of glucocorticoids such as hydrocortisone
- insulin As a medium for inducing white adipocytes, insulin (Insulin) (for example, a concentration of about 0.01 to 100 ⁇ g / mL, preferably about 0.1 to 10 ⁇ g / mL); 3-isobutyl-1-methylxanthine (3 -Isobutyl-1-methylxanthine (IBMX) (for example, a concentration of about 0.01 to 100 ⁇ mM, more preferably about 0.1 to 10 mM); Dexamethasone (for example, a concentration of about 0.01 to 100 ⁇ m, more preferably about 0.1 to 10 ⁇ m) The component (1 type or 2 types or more) is added to a normal medium.
- IBMX 3-isobutyl-1-methylxanthine
- IBMX 3-isobutyl-1-methylxanthine
- Dexamethasone for example, a concentration of about 0.01 to 100 ⁇ m, more preferably about 0.1 to 10 ⁇ m
- 10% FBS-added DMEM + MDI medium 10% FBS-added DMEM supplemented with 0.5 ⁇ M 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1 ⁇ g / mL Insulin
- IBMX 3-isobutyl-1-methylxanthine
- IBMX 0.5 ⁇ M dexamethasone
- Peroxisome ⁇ Proliferator-Activated Receptor- ⁇ (PPAR- ⁇ ) agonist for example, a concentration of about 0.01 to 100 ⁇ M, more preferably about 0.1 to 10 ⁇ M
- PPAR- ⁇ Peroxisome ⁇ Proliferator-Activated Receptor- ⁇
- PPAR- ⁇ agonists examples include thiazolidinedione compounds such as Rosiglitazone, Ciglitazone, GW1929, nTZDpa, Pioglitazone Hydrochloride, Troglitazone and the like.
- insulin As a medium for inducing brown adipocytes, insulin (Insulin) (for example, a concentration of about 0.01 to 100 ⁇ g / mL, more preferably about 0.1 to 10 ⁇ g / mL); 3-isobutyl-1-methylxanthine (3- Isobutyl-1-methylxanthine (IBMX) (for example, a concentration of about 0.01 to 100 ⁇ mM, more preferably about 0.1 to 10 mM); Dexametazone (for example, a concentration of about 0.01 to 100 ⁇ m, more preferably about 0.1 to 10 ⁇ m) is usually used The thing added to the culture medium is illustrated. Indometacin (for example, a concentration of about 0.001 to 10 ⁇ mM, more preferably about 0.01 to 1 ⁇ mM) may be added.
- IBMX 3-isobutyl-1-methylxanthine
- IBMX 3-isobutyl-1-methylxanthine
- thyroid hormone for example, a concentration of about 0.01-100 nM, more preferably about 0.1-10 nM
- PPAR- ⁇ PeroxisomeoxiProliferator-Activated Receptor- ⁇
- the concentration is about 0.01 to 100 ⁇ M, more preferably about 0.1 to 10 ⁇ M
- thyroid hormones examples include triiodothyronine (T3) and thyroxine (T4).
- PPAR- ⁇ agonists examples include thiazolidinedione compounds such as Rosiglitazone, Ciglitazone, GW1929, nTZDpa, Pioglitazone Hydrochloride, Troglitazone and the like.
- the medium for inducing brown adipocytes include [1] FBS 10%, 0.5 mM IBMX, 125 nM Indomethacin, 1 microM Dexamethasone, 850 nM insulin, 1 nM triiodothyronine (Triiodothyronine, T3), Examples include DMEM medium supplemented with 1 ⁇ Rosiglitazone, and [2] 10% FBS, 850 nM insulin, 1 nM T3, and 1 ⁇ M Rosiglitazone. [1] can be used on the first and second days, and [2] can be used on and after the third day. .
- 1 nM T3, 1 ⁇ M Rosiglitazone As media for inducing brown adipocytes from fibroblasts, 1 nM T3, 1 ⁇ M Rosiglitazone, (0.5 mM isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS Added DMEM can be used.
- PPAR- ⁇ agonists examples include Rosiglitazone, Ciglitazone, GW1929, nTZDpa, Pioglitazone Hydrochloride, Troglitazone and the like.
- Examples of the medium for inducing mesenchymal stem cells include, but are not limited to, the media described in the following documents (1-1) to (1-4).
- a known medium can be used as a medium for inducing chondrocytes.
- the media described in the following documents (2-1) to (2-4) can be used, but are not limited thereto.
- a known medium can be used as a medium for inducing myoblasts.
- 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; 10% FBS, 10onemicroM 5- DMEM medium supplemented with azacytidine, 10 ng / mL VEGF, 10 ng / mL IGF-1, 10 ng / mL bFGF; 5% Horse serum, ⁇ MEM medium supplemented with 10 ng / mL, or the like can be used.
- a known medium can be used as a medium for inducing nerve cells.
- a known medium can be used.
- a known medium can be used as a medium for inducing glial cells and nerve cells.
- the medium described in 89. can be used. However, it is not limited to these.
- a medium for inducing glial cells As a medium for inducing glial cells, a known medium can be used. For example, a medium described in the literature: Duan L. et al., Stem Cells Transl Med. 2015 May; 4 (5): 437-47. Can be used. However, it is not limited to these.
- a known medium can be used as a medium for inducing Schwann cells.
- a normal medium such as DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum), forskolin of about 1 to 20 ⁇ M (particularly about 5 ⁇ M); BFGF (basic fibroblast growth factor) of about 50 ng / ml (especially about 10 ng / ml); PDGF (about 10 ng / ml, especially about 2 to 50 ng / ml) Platelet-Derived Growth Factor):
- a medium preferably all) (a Schwann cell induction medium) can be used (all of these concentrations are final concentrations).
- a medium described in the following documents (3-1) to (3-2) (a 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 differentiated into a Schwann cell phenotype and promote neurite outgrowth in vitro. ExpNeurol, 2007; 207: 267-274.
- the medium for inducing cardiomyocytes a known medium can be used.
- Doi: 10.1073 / pnas.1301019110. Epub 2013 Mar 4. can do.
- a known medium can be used.
- DMEM supplemented with 10% FBS and 100 ng / mL VEGF can be used.
- it can carry out by the method as described in Chatterjee I. et al., Methods Mol Biol. 2015 Feb 17 PMID: 25687301. However, it is not limited to these.
- a known medium can be used as a medium for inducing smooth muscle cells.
- it can be carried out by the method described in Wang Y. et al., Biomaterials. 2014 Oct; 35 (32): 8960-9.. However, it is not limited to these.
- a known medium can be used as a medium for inducing mast cells.
- DMEM medium supplemented with 15% FBS, 20 ng / mL VEGF, [2] 10% FBS, 10 ng / mL IL-3, 100 ng / mL IL-6, 10 ng / mL Flt3L, 10 IDMD medium with ng / mL / TPO, 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, Using IDMD supplemented with 10 ng / mL VEGF, [1] can be used on days 1 to 8, [2] on days 9 to 18, and [3] on or after day 19. However, it is not limited to these.
- a known medium can be used as a medium for inducing ⁇ cells (insulin producing cells).
- ⁇ cells insulin producing cells
- 15% FBS 100 ng / mL Activin A, 10 nM GLP-1, 10 mM Nicotinamide, 20 ng / mL EGF, 10 ng / mL bFGF, ITS added DMEM can be used.
- the described media can be used. However, it is not limited to these.
- a known medium can be used as a medium for inducing hepatocytes.
- a 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.
- known media can be used as a medium for inducing cells of the digestive tract.
- the medium 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 do. However, it is not limited to these.
- Known media can be used as a medium for inducing lung and airway cells.
- the medium described in Ghaedi M. et al., J Clin Invest. 2013 Nov; 123 (11): 4950-62. Can be used. However, it is not limited to these.
- a known medium can be used.
- Kang M. et al., Int. J. Mol. Sci. 2014, 15 (5), 7139-7157 Can be used. However, it is not limited to these.
- Lam AQ et al. Semin Nephrol. 2014 Jul; 34 (4): 445-61.
- Lam AQ et al. Semin Nephrol. 2014 Jul; 34 (4): 445-61.
- Lam AQ and Bonventre JV. Curr Opin Organ Transplant. 2015 Apr; 20 (2): 187-92.
- Medium can be used. However, it is not limited to these.
- Known media can be used as a medium for inducing hematopoietic stem cells.
- the medium 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.
- a known medium can be used as a medium for inducing blood cells.
- the medium described in Nakayama N. et al., Blood. 1998 Apr 1; 91 (7): 2283-95. Can be used. However, it is not limited to these.
- a known medium can be used as the medium for inducing lymphocytes.
- a known medium can be used.
- Vodyanik MA, et al., Blood. 2005; 105: 617-26. Can do.
- a known medium can be used as a medium for inducing erythroblasts.
- the medium described in LuLSJ, et al., Blood. 2008; 112: 4475-84. Can be used. However, it is not limited to these.
- a known medium can be used as a medium for inducing megakaryocytes.
- 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.
- known media can be used as the medium for inducing dendritic cells and macrophages.
- the medium described in Senju S. et al., Gene Ther. 2011 Sep; 18 (9): 874-83. Can be used. However, it is not limited to these.
- a known medium can be used as a medium for inducing granulocytes.
- 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.
- the medium for inducing retinal cells a known medium can be used.
- 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.
- the medium for inducing corneal cells a known medium can be used.
- the medium described in YuYD.et al., Cell Biol Int. 2013 Jan; 37 (1): 87-94. Can be used. However, it is not limited to these.
- a normal liquid medium such as Dulbecco's Modified Eagle's Medium or EMEM (Eagle's minimal essential medium) can be used.
- serum components Fetal (Bovine Serum (FBS), Human Serum (Serum)
- antibacterial agents such as streptomycin and penicillin
- components such as Non-Essential Amino Acid
- the cells are cultured in the medium for inducing other somatic cells in the presence of a TGF- ⁇ pathway inhibitor.
- TGF- ⁇ pathway inhibitor '' means TGF- ⁇ / SMAD pathway inhibitor, TGF- ⁇ / Erk pathway inhibitor, TGF- ⁇ / JNK pathway inhibitor, TGF- ⁇ / p38 pathway inhibitor and TGF- ⁇ Includes / RhoA pathway inhibitors. That is, the TGF- ⁇ receptor family molecule, the TGF- ⁇ superfamily cytokine serving as its ligand, the TGF- ⁇ / SMAD pathway, TGF- ⁇ / Erk pathway, TGF- ⁇ / An inhibitor that suppresses any one or more of the molecules constituting the JNK pathway, TGF- ⁇ / p38 pathway, and TGF- ⁇ / RhoA pathway is the TGF- ⁇ inhibitor of the present invention.
- TGF- ⁇ pathway inhibitors are not limited to low-molecular compounds, which are inhibitors in the narrow sense, but have the ability to bind to and inhibit the effects of cytokine neutralizing antibodies, receptor antagonists, soluble receptors, and pathway proteins.
- TGF- ⁇ / SMAD pathway inhibitor refers to a compound that can inhibit the activity of a protein belonging to the TGF- ⁇ / SMAD pathway.
- the TGF- ⁇ / SMAD pathway is a signal pathway known to those skilled in the art schematically shown in FIG. 16 (cited from Chen G et al, Int J Biol Sci, 2012).
- the TGF- ⁇ / SMAD pathway is a ligand composed of proteins belonging to the TGF- ⁇ superfamily (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E, 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.
- TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E nodal
- TGF- ⁇ / SMAD pathway when a ligand binds to a dimeric receptor, the kinase-type receptor TGF- ⁇ type I receptor protein phosphorylates SMAD protein and transmits a signal downstream . Therefore, in this specification, any of TGF- ⁇ superfamily cytokine, TGF- ⁇ type I receptor family, TGF- ⁇ type II receptor family, SMAD family protein (especially SMAD2, SMAD3, SMAD4, SMAD1, SMAD5, or SMAD8) Molecules that suppress this are called TGF- ⁇ / SMAD pathway inhibitors.
- ALK proteins ACVRL1 (ALK1), ACVR1 (ALK2), BMPR1A belonging to the TGF- ⁇ type I receptor family (also called Activin receptor like kinase (ALK) family) (ALK3), ACVR1B (ALK4), TGFBR1 (ALK5), BMPR1B (ALK6), ACVR1C (ALK7)) inhibitors (ALK inhibitors) are exemplified.
- inhibitors of proteins belonging to the TGF- ⁇ type II receptor family ACVR2A (ACTRII), ACVR2B (ACTRIIB), TGFBRII (AAT3), BMPR2 (PPH1) are exemplified.
- ALK5 inhibitor 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); LY2109761, LY2157299 (Galunisertiv, 4- [5,6-dihydro-), inhibitors of ALK5 and TGFBRII (AAT3) 2- (6-methyl-2-pyridinyl) -4H-pyrrolo [1,2-b] pyrazol-3-yl] -6-
- TGF- ⁇ / SMAD pathway inhibitor a compound represented by the following formula (1) or (2) or a salt thereof described in Gellibert, F et al. J. Med. Chem. 2004, 47, 4494-4506 It can also be used.
- R 1 is H, a methyl group or halogen (for example, fluorine, chlorine, bromine or iodine).
- R 2 is H or a methyl group.
- ALK inhibitor those having at least an inhibitory activity against ALK5 (ALK5 inhibitor) are preferable from the viewpoint of high effect. From the viewpoint that the effect is particularly high, ALK4 and ALK5, or those having specific inhibitory activity against ALK5 (among ALK proteins, those having a markedly high inhibitory activity on the protein) are 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 in converting fibroblasts into osteoblasts.
- LY2157299 and ALK5 Inhibitor II are highly efficient in converting fibroblasts into brown adipocytes.
- TGF- ⁇ / SMAD pathway inhibitor an inhibitor of SMAD protein is exemplified.
- TGF- ⁇ / ERK pathway inhibitor refers to a compound capable of inhibiting the activity of a protein belonging to the TGF- ⁇ / ERK pathway.
- the TGF- ⁇ / ERK pathway is a signal pathway known to those skilled in the art schematically shown in FIG. 17 (cited from Y. E. Zhang, “Non-smad pathways in TGF- ⁇ signaling” Cell Research 19: 128, 2009).
- TGF- ⁇ / Erk pathway is a ligand composed of proteins belonging to the TGF- ⁇ superfamily (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.) Proteins belonging to the TGF- ⁇ type I receptor family and proteins belonging to the TGF- ⁇ type II receptor family, and intracellular signaling molecules Raf, MEK1 / 2, Erk1 / 2 proteins Is mainly composed.
- TGF- ⁇ / Erk pathway when a ligand binds to a dimeric receptor, a signal is transmitted downstream to Raf, MEK1 / 2, and Erk1 / 2. Therefore, in the present specification, a molecule that suppresses any of TGF- ⁇ superfamily cytokine, TGF- ⁇ type I receptor family, TGF- ⁇ type IIRreceptor family, Raf, MEK1 / 2, Erk1 / 2, Called ⁇ / Erk pathway inhibitor.
- the aforementioned ALK protein inhibitor is exemplified.
- Raf inhibitor As another embodiment of the TGF- ⁇ / Erk pathway inhibitor, Raf inhibitor, MEK1 inhibitor, MEK2 inhibitor, Erk1 inhibitor and Erk2 inhibitor are exemplified. Of these, an inhibitor of Erk1 and an inhibitor of Erk2 are preferred.
- TGF- ⁇ / JNK pathway inhibitor refers to a compound that can inhibit the activity of a protein belonging to the TGF- ⁇ / JNK pathway.
- the TGF- ⁇ / JNK pathway is a signal pathway known to those skilled in the art schematically shown in FIG. 18 (cited from Y. E. Zhang, “Non-smad pathways in TGF- ⁇ signaling” Cell Research 19: 128, 2009).
- TGF- ⁇ / JNK pathway is a ligand composed of proteins belonging to the TGF- ⁇ superfamily (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.) Proteins belonging to the TGF- ⁇ type I receptor family and proteins belonging to the TGF- ⁇ type II receptor family, and intracellular signaling molecules TAB1 / 2, TAK1, TRAF6, MKK4, JNK protein is mainly composed.
- TGF- ⁇ / JNK pathway when a ligand binds to a dimeric receptor, signals are transmitted downstream to TAB1 / 2, TAK1, TRAF6, MKK4, and JNK. Therefore, in the present specification, a molecule that suppresses any of TGF- ⁇ superfamily cytokine, TGF- ⁇ type I receptor family, TGF- ⁇ type II receptor family, TAB1 / 2, TAK1, TRAF6, MKK4, JNK, Called TGF- ⁇ / JNK pathway inhibitor.
- the aforementioned ALK protein inhibitor is exemplified.
- TGF- ⁇ / JNK pathway inhibitor an inhibitor of TAK1, an inhibitor of MKK4, and an inhibitor of JNK inhibitor are exemplified. Of these, inhibitors of JNK are preferred.
- the cells are cultured in the medium for inducing other somatic cells in the presence of a TGF- ⁇ / p38 pathway inhibitor.
- TGF- ⁇ / p38 pathway inhibitor means a compound that can inhibit the activity of a protein belonging to the TGF- ⁇ / p38 pathway.
- the TGF- ⁇ / p38 pathway is schematically shown in Figure 18 (Y. ⁇ ⁇ E. Zhang, “Non-smad pathways in TGF- ⁇ signaling” Cell Research 19: 128, 2009). It is.
- TGF- ⁇ / p38 pathway is a ligand composed of proteins belonging to the TGF- ⁇ superfamily (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.) Proteins belonging to the TGF- ⁇ type I receptor family and proteins belonging to the TGF- ⁇ type II receptor family, and intracellular signaling molecules TAB1 / 2, TAK1, TRAF6, MKK3, Proteins belonging to MKK6, p38 family are mainly composed.
- TGF- ⁇ / p-38 pathway when a ligand binds to a dimeric receptor, it signals downstream to the TAK, MKK3 or MKK6, p-38 family (especially p-38 ⁇ , p-38 ⁇ ). Communicated. Therefore, in the present specification, TGF- ⁇ superfamily cytokine, TGF- ⁇ type I receptor family, TGF- ⁇ type II receptor family, TAB1 / 2, TAK1, TRAF6, MKK3, MKK6, p38 family proteins (particularly p38 ⁇ , p38 ⁇ Molecules that suppress either of these are called TGF- ⁇ / SMAD pathway inhibitors.
- the aforementioned ALK protein inhibitor is exemplified.
- an inhibitor of TAK1, an inhibitor of MKK3, an inhibitor of MKK6, and an inhibitor of p38 can be mentioned.
- an inhibitor of p38 is preferable.
- the cells are cultured in a medium for inducing other somatic cells in the presence of a TGF- ⁇ / RhoA pathway inhibitor.
- TGF- ⁇ / RhoA pathway inhibitor means a compound capable of inhibiting the activity of a protein belonging to TGF- ⁇ / RhoA pathway.
- the TGF- ⁇ / RhoA pathway is schematically shown in FIG. 19 (Y. E. Zhang, “Non-smad pathways in TGF- ⁇ signaling” Cell Research 19: 128, 2009). It is.
- TGF- ⁇ / RhoA pathway is a ligand composed of proteins belonging to the TGF- ⁇ superfamily (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, Activin- ⁇ A, Activin- ⁇ B, Activin- ⁇ C, Activin- ⁇ E, nodal, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, AMH, etc.)
- Mainly consists of proteins belonging to the TGF- ⁇ type I receptor family and proteins belonging to the TGF- ⁇ type II ⁇ ⁇ receptor family, and intracellular signaling molecules Par6, PKC ⁇ , Smurf1, and RhoA. Configure.
- TGF- ⁇ / RhoA pathway when a ligand binds to a dimeric receptor, it is signaled downstream to the TAK, MKK3 or MKK6, p-38 family (especially p-38 ⁇ , p-38 ⁇ ).
- TAK MKK3 or MKK6, p-38 family (especially p-38 ⁇ , p-38 ⁇ ).
- MKK3 or MKK6, p-38 family especially p-38 ⁇ , p-38 ⁇ .
- a molecule that suppresses any of the TGF- ⁇ superfamily cytokine, TGF- ⁇ type I receptor family, TGF- ⁇ type II receptor family, Par6, PKC ⁇ , Smurf1, RhoA, Called RhoA pathway inhibitor when a ligand binds to a dimeric receptor, it is signaled downstream to the TAK, MKK3 or MKK6, p-38 family (especially p-38 ⁇ , p-38 ⁇ ).
- the aforementioned ALK protein inhibitor is exemplified.
- RhoA inhibitors As another embodiment of the TGF- ⁇ / RhoA pathway inhibitor, there may be mentioned a RhoA inhibitor and a PAK inhibitor. Of these, RhoA inhibitors are preferred.
- TGF- ⁇ pathway inhibitors include ALK5 or its ligand TGF- ⁇ family of cytokines, and TGF- ⁇ / SMAD pathway, TGF- ⁇ / Erk pathway, TGF- ⁇ / JNK downstream of ALK 5.
- TGF- ⁇ / SMAD pathway TGF- ⁇ / Erk pathway
- TGF- ⁇ / JNK downstream of ALK 5 examples thereof include inhibitors that suppress any one or more of molecules constituting a pathway, a TGF- ⁇ / p38 pathway, or a TGF- ⁇ / RhoA pathway.
- TGF- ⁇ receptor family molecule or a TGF- ⁇ superfamily cytokine serving as a ligand thereof is preferable because it suppresses a plurality of pathways among TGF- ⁇ pathways.
- the TGF- ⁇ pathway inhibitor is not limited to known TGF- ⁇ pathway inhibitors. All TGF- ⁇ pathway inhibitors developed in the future are included in the TGF- ⁇ pathway inhibitors of the present invention.
- TGF- ⁇ pathway inhibitors also include derivatives of the above compounds.
- the derivative of D4476 described in WO00 / 61576 can be used.
- TGF- ⁇ pathway inhibitor In the presence of a TGF- ⁇ pathway inhibitor means an embodiment in which a TGF- ⁇ pathway inhibitor is mainly contained in the medium, but is not limited to this as long as the effects of the present invention are not impaired.
- a person skilled in the art can appropriately set the concentration of the TGF- ⁇ pathway inhibitor in the medium. Usually, it is about 0.01 to 100 ⁇ M, particularly about 0.1 to 10 ⁇ M.
- TGF- ⁇ pathway inhibitors can be used singly or in combination of two or more.
- TGF- ⁇ pathway inhibitor and a statin compound can be used in combination from the viewpoint that the target somatic cells are prepared with high efficiency.
- Statin compounds widely include HMG-CoA reductase inhibitors, and are not particularly limited, but examples include simvastatin, atorvastatin, lovastatin, fluvastatin, pravastatin, cerivastatin, pitavastatin, rosuvastatin, dihydrocompactin, compactin, bervastatin, carbastatin, Examples include crilvastatin, dalvastatin, glenvastatin, fluindostatin, verostatin, mevastatin, rivastatin, cilivastatin, CI-981 and the like. All the statin compounds developed in the future are included in the statin compounds of the present invention.
- the number of TGF- ⁇ pathway inhibitors and other compounds used in addition to the above-mentioned medium components is 10 or less, more preferably 4 or less, still more preferably 3 or less, particularly preferably. 1 type or 2 types.
- mammalian differentiated somatic cells are cultured in an induction medium in the presence of a TGF- ⁇ pathway inhibitor.
- Culturing can be performed in a suitable container for storing cells and media.
- suitable culture techniques include a technique of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5%, but is not limited thereto. Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
- the culture period is not particularly limited as long as the effects of the present invention are not impaired.
- it can be about 24 to 60 days, preferably 3 to 30 days, more preferably about 10 to 20 days, and particularly preferably about 14 days.
- the TGF- ⁇ pathway inhibitor may be added only during a part of the whole culture period. It can be.
- the whole culture period is cultured in the presence of the compound in the induction medium for a predetermined period (for example, about 6 to 10 days, particularly about 8 days), and then the non-existence of the compound in the induction medium.
- the culture can be performed in the presence.
- the culture in the presence of the compound may be from the start of the culture during the whole culture period or after the culture in the absence of the compound for a predetermined period.
- brown adipocytes when preparing brown adipocytes, 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, particularly about 8 days, in the entire culture period, and then induction medium By culturing in the absence of the above compound, brown adipocytes can be efficiently prepared.
- differentiated somatic cells of a mammal may be cultured in the absence of a TGF- ⁇ pathway inhibitor in an induction medium after culturing in the presence of a TGF- ⁇ pathway inhibitor in a normal medium.
- a TGF- ⁇ pathway inhibitor in a normal medium After culturing in the presence of a TGF- ⁇ pathway inhibitor in normal medium, culturing in the absence of a TGF- ⁇ pathway inhibitor in normal medium, and then in the absence of TGF- ⁇ pathway inhibitor in the induction medium You may culture below.
- culturing in the presence of a TGF- ⁇ pathway inhibitor in a normal medium culturing in the presence of a TGF- ⁇ pathway inhibitor in an induction medium, and then in the absence of a TGF- ⁇ pathway inhibitor in the induction medium.
- subculture In culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before or immediately after reaching the confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can be changed as appropriate.
- the conversion is triggered by the method of the present invention, and an irreversible epigenomic change occurs, so that it can be expected that the cell is converted to the target cell after transplantation.
- the desired somatic cell is prepared.
- the preparation of the desired somatic cell can be verified by a known method such as marker detection.
- osteoblasts were obtained by measuring real-time PCR of ALP (alkaline phosphatase) gene, osteocalcin (OC) gene, osteopontin gene, Runx2 gene mRNA, staining with alizarin red S (lime) Production of mineralized (mineralized) bone matrix).
- ALP alkaline phosphatase
- OC osteocalcin
- osteopontin osteopontin gene
- Runx2 gene mRNA
- Obtaining white adipocytes can be confirmed by the unique morphology with large single-celled lipid droplets stained with Oil ⁇ RedOO and Bodipy staining, and the expression of the FABP4 gene, HS gene L, and AdipoQ gene it can.
- brown adipocytes were obtained is due to the unique morphology with multilocular lipid droplets stained with OilORed O staining and Bodipy staining, UCP-1 gene, CIDEA gene, KCNK3 gene, PCG-1 ⁇ gene, Cox8b It can be detected by expression of a gene, Otop gene, ELOVL3 gene and the like.
- UCP-1 Uncoupling protein 1
- UCP-1 is a gene that is specifically expressed in brown adipocytes, and encodes a mitochondrial inner membrane protein that uncouples oxidative phosphorylation and plays a fundamental role in brown adipocyte function. Therefore, it is one of the particularly preferable indices for brown adipocytes.
- Chondrocytes were obtained from, for example, SOX9 gene, COL2A1 (Type II collagen) gene, aggrecan gene, COL11A1 (Type II XI collgaen) gene, MMP13 gene expression, cartilage matrix production, Alcian blue staining, toluidine blue staining It can be detected by safranin O staining and the like.
- Myoblasts were obtained by, for example, expression of MyoD gene, Myogenin gene, Myf5 gene, MRF4 gene; skeletal muscle ⁇ -actin protein, skeletal muscle myosin protein expression, muscle-specific morphology (multinuclear, muscle fiber) Formation, myotube formation), contractility, and the like.
- Cardiomyocytes were obtained, for example, expression of troponin T (cTnT) protein, tropomyosin protein, cardiac ⁇ -actin protein, cardiac myosin protein; ATP2A2 gene, GJA1 gene, GJA5 gene, NPPA gene, NPPB gene expression, It can be detected by autonomous contractility.
- cTnT troponin T
- Obtaining smooth muscle cells can be detected by, for example, expression of ⁇ -smooth muscle actin ( ⁇ SMA) protein and smooth muscle myosin protein.
- ⁇ SMA smooth muscle actin
- MSC Mesenchymal stem cells
- Tuj1 protein vGLUT1 protein
- MAP2 protein synthesis of neurotransmitter
- action potential synthesis of neurotransmitter
- glial cells can be detected by, for example, expression of oligo2 protein in the case of oligodendroglia, GFAP protein in the case of astrocytes, and OX42 protein in the case of microglia.
- Schwann cells were obtained because of their morphology (bipolar or multipolar cell morphology with relatively small nuclei, ratio of cell width to cell length), Schwann such as S100 ⁇ , p75NTR, GFAP, Nestin, NG2, etc. It can be detected by detecting the expression of a cell-specific marker, the effect of neurite outgrowth on the co-cultured neurons, and the ability to form myelin.
- hepatocytes have been obtained can be detected by functions such as expression of albumin, cytochrome P450, various enzyme activities, drug metabolism, LDL uptake and the like.
- bile duct epithelial cells have been obtained can be detected by, for example, the characters AFP (-), Dlk (-), Alb (-), CK19 (+), zEpCAM (+), Thy1 (-).
- vascular endothelial cells can be detected by, for example, expression of CD31 antigen, expression of Endoglin, VE-cadherin, VWF, TIE2, and ANGPT2.
- lymphatic endothelial cells can be detected by, for example, expression of D2-40 antigen, expression of Podoplanin®, LYVE-1, and PROX-1.
- the fact that tendon cells are obtained can be detected by, for example, expression of Scleraxis® (Scx) and tenomodulin® (Tnmd).
- Fibroblasts can be confirmed by the gene expression of COL1A1 and COL1A2.
- Bone marrow stromal cells can be confirmed from expression of cell surface markers, gene expression, and the like.
- lymphocytes macrophages, granulocytes, hematopoietic stem cells, mast cells and the like were obtained.
- urothelial cells have been obtained can be detected by, for example, gene expression of Uroplakin1b, Uroplakin2, CK5, and CK17, and formation of an asymmetric unit Membrane.
- treatment intends a treatment performed while a patient is suffering from a particular disease or disorder, whereby the severity of the disease or disorder, or one or It means that a plurality of its symptoms are alleviated or the progression of the disease or disorder is delayed or slowed down.
- treatment includes “prevention”.
- the transplant material refers to a material that introduces somatic cells into the living body.
- the transplant material includes a material that is prepared in vitro and then transplanted to the same or another individual.
- the transplant material which induces the target somatic cell in the middle of differentiation in vitro and finally leads to the target somatic cell in vivo after transplantation is also included in the present invention.
- the diseases to be treated with osteoblasts include bone tumors, bone defects associated with trauma and osteomyelitis, etc., bone defects after curettage such as bone tumors, fractures, osteoporosis, periodontal disease , Alveolar bone resorption, cleft lip and palate, rheumatoid arthritis, idiopathic femoral head necrosis, osteoarthritis, lumbar spondylotic spondylosis, spinal canal stenosis, intervertebral disc herniation, spondylolysis, spondylolisthesis, scoliosis, Of fracture sites destroyed by complex fractures such as cervical spondylotic myelopathy, posterior longitudinal ligament ossification, spinal cord injury, hip osteoarthritis, knee osteoarthritis, femoral head slippery, osteomalacia, mandibular reconstruction Examples include reconstruction, bone repair after surgery (such as sternum repair after heart surgery), repair of defects associated with artificial ankle surgery, osteo
- osteoblasts are transplanted, there is a possibility that the therapeutic effect can be enhanced in combination with bone grafts, artificial bone grafts, artificial joints and implants. It is also possible to treat the above-mentioned diseases by culturing osteoblasts using a three-dimensional scaffold or the like to produce various forms of bone tissue outside the body and transplanting the bone tissue. In addition, various diseases related to osteoblast deficiency, deficiency or reduced function are targeted.
- treatment for humans is also referred to as treatment in this specification for convenience, and “patient” can be read as “healthy person” or “human”, and “disease” can be read as “beauty”.
- Osteoblasts can be transplanted to patients as cell preparations, transplanted with a base material (scaffold) made of artificial material such as hydroxyapatite or bioabsorbable ceramic, or transplanted after culturing with scaffolds be able to.
- the scaffold can have various three-dimensional shapes depending on the purpose of implantation.
- Brown adipocytes can also be used for the prevention or treatment of obesity, metabolic syndrome, or diseases or conditions related to these.
- Target diseases include type I diabetes, type II diabetes, diabetic complications (retinopathy, peripheral neuropathy, nephropathy, macrovascular disorder, 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 disorder, arteriosclerosis, atherosclerosis, metabolic syndrome Dyslipidemia, hypertriglyceridemia, hypercholesterolemia, hypoHDLemia, kidney disease (diabetic nephropathy, nephrotic syndrome, etc.), arteriosclerosis, thrombotic disease, myocardial infarction, ischemic heart disease, angina Disease, heart failure, cerebrovascular disorder (cerebral infarction, stroke, etc.), peripheral blood flow disorder,
- Brown adipocytes can also be used for cosmetic purposes to remove fat such as around the abdomen, jaw, and thighs. Brown adipocytes can also be used as a transplant material for cosmetic treatments introduced into the breast or the like.
- brown adipocytes reduces fat mass, especially white fat cells such as visceral fat and subcutaneous fat, and also suppresses weight gain when a high-calorie diet is consumed, so obesity, metabolic syndrome, or these It is useful in both prevention and treatment of related diseases or conditions.
- the present invention is not limited to prevention or treatment of diseases, but can also be used for purposes such as health promotion and beauty (for example, removal of visceral fat and subcutaneous fat such as abdomen, jaw, arm, and thigh).
- treatment for humans is also referred to as treatment in this specification for convenience, and “patient” can be read as “healthy person” or “human”, and “disease” can be read as “health promotion” or “beauty”.
- Brown adipocytes can also be used as a transplant material for cosmetic treatments introduced into the breast or the like.
- transplantation of white adipocytes into a tissue defect caused by trauma, burns, surgery, etc. can improve the tissue morphology and prevent infection.
- breasts can be reconstructed by transplanting white adipocytes into the defect after surgery to remove breast cancer. It can also be used as a transplant material for cosmetic treatment.
- myoblasts Duchenne muscular dystrophy, muscular dystrophy, congenital / distal myopathy, myotonia syndrome such as myotonic dystrophy, myogenic diseases such as mitochondrial disease, periodic limb paralysis, Weldnig-Hoffmann disease, Charco Neurogenic myopathy such as Marie-Tooth disease, congenital myelination dysplasia, amyotrophic lateral sclerosis (ALS), dermatomyositis, polymyositis, polyarteritis nodosa, polymyalgia rheumatica, Collagen diseases such as mixed connective tissue disease, inflammatory myopathy, endocrine myopathy, drug-induced myopathy due to steroids and hyperlipidemia, sarcopenia, progressive ossifying fibrodysplasia (FOP), etc. It is thought that prevention and treatment effects can be obtained.
- ALS amyotrophic lateral sclerosis
- FOP progressive ossifying fibrodysplasia
- osteoarthritis For chondrocytes, osteoarthritis, osteochondrosis, dysplasia arthritis, rheumatoid arthritis, trauma, intervertebral disc injury, meniscus injury, osteolytic osteochondritis, osteonecrosis, neurogenic arthropathy, etc. It is thought that prevention and treatment effects can be obtained for cartilage damage, cartilage defect, etc. found in cartilage diseases.
- mesenchymal stem cells osteoblasts, adipocytes, chondrocytes, myoblasts and the like that can be differentiated from mesenchymal stem cells are considered to have preventive and therapeutic effects on the above-mentioned many diseases.
- tendon cells it is considered that a prophylactic and therapeutic effect against tendon rupture associated with trauma or surgery can be obtained.
- neurodegenerative diseases (Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, Shy-Drager syndrome, nigrostriatal degeneration , Olive Bridge cerebellar atrophy, spinocerebellar ataxia, corticobasal degeneration, Lewy body disease, dystonia, mage syndrome, late cerebellar cortical atrophy, familial spastic paraplegia, motor neuron disease, Card Joseph disease, Pick disease, stroke, cerebrovascular disorder, demyelinating disease (multiple sclerosis, Guillain-Barre syndrome, acute disseminated encephalomyelitis, acute encephalitis, transverse myelitis, etc.), brain tumor, associated with infection Cerebrospinal disease (meningitis, brain abscess, Creutzfeld-Jakob disease, etc.), neurological dysfunction after trauma, neuropathy caused by toxic substances
- Schwann cells include central nervous loss or damage due to cerebral infarction, spinal cord injury, peripheral nerve deficiency or damage due to trauma, neuritis, tumor resection, etc .; multiple sclerosis Optic myelitis (Devic syndrome), concentric sclerosis (Balo disease), acute disseminated encephalomyelitis (acute disseminated encephalomyelitis, ADEM), inflammatory diffuse sclerosis (Schilder disease), infectious subacute sclerosis panencephalitis ( diseases of the central nervous system such as subacute sclerosing panencephalitis (SSPE), progressive multifocal leukoencephalopathy (PML); peripheral such as Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory demyelinating polyradiculoneuropathy Nervous system diseases; Schwann cell deficiency such as Charcot-Marie-Tooth disease (CMT), diseases based on deficiency or reduced function.
- CMT Charcot-Marie-Tooth disease
- hepatocytes and bile duct epithelial cells can have preventive and therapeutic effects on liver failure, fulminant hepatitis, cirrhosis, steatohepatitis, metabolic syndrome and the like. It is also useful for drug toxicity tests, metabolic tests, and safety tests for the liver. In addition, it is useful for screening drugs and determining effects on diabetes, dyslipidemia, metabolic syndrome, hypertension and the like.
- Lung and airway cells are primary pulmonary hypertension, pulmonary fibrosis, emphysema, bronchiectasis, pulmonary sarcoidosis, interstitial pneumonia, cystic fibrosis, diffuse panbronchiolitis, obstructive bronchiolitis, lung Prophylactic and therapeutic effects are expected for eosinophilic granulomatosis, chronic thromboinfarction pulmonary hypertension, multiple pulmonary arteriovenous fistulas, etc.
- Kidney cells are considered to have preventive and therapeutic effects on renal failure, diabetic nephropathy, chronic glomerulonephritis, hypoplastic kidney, cystic kidney, IgA nephropathy, nephrosclerosis, pregnancy toxemia and the like.
- Urinary epithelial cells can be used to form a surrogate bladder in patients who have had a total cystectomy for bladder cancer, to construct a patch to a site where the urothelium is missing in the vaginal fistula, and to a patient who has fibrosis and atrophy of the neurogenic bladder.
- it is useful for the formation of urothelium for partial compensation, urinary epithelium regeneration for severe interstitial cystitis (urinary tract epithelial dysfunction), and the like.
- cardiomyocytes can prevent and treat myocardial infarction, ischemic heart disease, congestive heart failure, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, and the like.
- lymphatic endothelium can prevent and treat lymphedema.
- Smooth muscle cells are useful for regenerative treatment of the respiratory tract, digestive tract and blood vessels.
- Vascular endothelial cells are useful in the regenerative treatment of blood vessels.
- Hematopoietic stem cells and bone marrow stromal cells are useful for bone marrow failure, aplastic anemia and immunodeficiency.
- 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.
- the present invention can also be used for treating diseases of domestic animals such as pets such as dogs and cats and cattle, horses, pigs, sheep and chickens as well as humans.
- pets such as dogs and cats and cattle, horses, pigs, sheep and chickens
- patient or “human” is read as “patient” or “animal”.
- the present invention can be used not only for regenerative medicine but also for basic research such as development of drugs (including biological preparations and nucleic acid drugs) for various diseases, evaluation of side effects of drugs, elucidation of occurrence and pathological conditions.
- the present invention also provides an inducing agent for converting differentiated somatic cells to other somatic cells, including a TGF- ⁇ pathway inhibitor. Also provided is the use of a TGF- ⁇ pathway inhibitor to convert differentiated somatic cells to other somatic cells.
- the present invention provides a novel use of a TGF- ⁇ pathway inhibitor.
- the inducer may be a TGF- ⁇ pathway inhibitor alone or a composition containing a TGF- ⁇ pathway inhibitor.
- a composition it may further contain a suitable solvent (water, DMSO, etc.) for dissolving the TGF- ⁇ pathway inhibitor.
- ALK5 ⁇ Inhibitor II may be referred to as“ ALK5 Inhibitor ”,“ ALK5IH ”,“ ALK5 IHII ”, or“ ALK5 i II ”.
- Example 1 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, a calcification induction medium, or a calcification induction medium supplemented with each compound was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 m ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 24.
- the concentration of each compound is as follows: D4476: 2 ⁇ M ALK5 inhibitor II: 2 ⁇ M SB431542: 2 ⁇ M.
- the TGF- ⁇ signal is essential for osteoblast proliferation, survival, differentiation and osteogenesis, and is known to promote the osteoblast differentiation and osteogenesis (eg, literature: Kasagi and Chen, Cell & Bioscience 2013, 3: 4). It was an unexpected result that the method of the present invention can achieve the conversion to osteoblasts by suppressing the TGF- ⁇ pathway.
- Example 2 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a calcification induction medium or a calcification induction medium added with each compound was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 24.
- the culture solution was aspirated and removed from each well, washed with PBS (-), and fixed with 10% formalin. After washing 3 times with sterilized distilled water, Alizarin Red S staining solution was added and incubated at room temperature for 15 minutes. The stained solution after staining was collected in a 96-well plate and the absorbance (OD: 550 nm) was measured with an absorptiometer.
- the concentration of each compound is as follows: D4476: 2 ⁇ M SB431542: 2 ⁇ M ALK5 inhibitor II: 2 ⁇ M.
- Example 3 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a calcification induction medium or a calcification induction medium added with each compound was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 24.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR-Master Mix, Osteocalcin and Alkaline Phosphatase or ⁇ -actin gene-specific primers and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system.
- the mRNA levels of Osteocalcin and Alkaline Phosphatase genes were quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured only in a calcification induction medium was calculated as 1.
- the concentration of each compound is as follows: D4476: 2 ⁇ M SB431542: 2 ⁇ M ALK5 inhibitor II: 2 ⁇ M.
- Example 4 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium or a calcification induction medium supplemented with ALK5 inhibitor II was added.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 24.
- Anti-Osteocalcin antibody was added and reacted at 4 ° C overnight, and then washed 3 times with Wash buffer.
- Alexa 488-conjugated anti-mouse Ig antibody was added and reacted at room temperature for 1 hour, then washed 5 times with WashWbuffer. Photographs were taken at a magnification of 200 using a fluorescence microscope.
- Example 5 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSC) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) It was suspended. This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of normal medium, calcification induction medium, or calcification induction medium supplemented with ALK5 inhibitor II was added as shown in the figure.
- DMEM modified minimum essential medium
- Calcification induction medium is 10% FBS DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 m ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 19.
- the culture medium was removed from each well by suction, washed with PBS ( ⁇ ), and fixed with 10% formalin. Washed 3 times with sterile distilled water. Thereafter, alizarin red S staining solution was added and incubated at room temperature for 15 minutes. The dyed solution after staining was collected in a 96-well plate, and the absorbance at 550 nm (OD 550 ) was measured with an absorptiometer (bottom of FIG. 5). The wells after staining were washed with sterilized distilled water and photographed at a magnification of 1 ⁇ and a magnification of 40 ⁇ with an inverted microscope (upper part of FIG. 5).
- Example 6 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium or a calcification induction medium supplemented with ALK5 inhibitor II was added.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 m ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 24.
- Anti-Runx2 antibody was added and reacted overnight at 4 ° C., and then washed 3 times with Wash buffer.
- Alexa 488-conjugated anti-mouse Ig antibody was added and reacted at room temperature for 1 hour, then washed 5 times with WashWbuffer. Photographs were taken at a magnification of 200 using a fluorescence microscope.
- the cells cultured in the normal medium and induced in the mineralization induction medium with HDF and ALK5 inhibitor II added are shown as dOBs in the figure.
- Example 7 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 10 ⁇ 10 3 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the 4th day, the culture supernatant was removed by aspiration, and an induction medium (a) (described below) supplemented with 50 mM ALK5 Inhibitor II was added for culturing.
- DMEM Dulbecco's modified minimum essential medium
- induction medium (a) was removed, and the induction medium (b) (described below) supplemented with 50 mM ALK5 Inhibitor II was added to continue the culture. Thereafter, on days 9, 12, 14, and 16, medium exchange was repeated in the same manner using an induction medium (b) supplemented with 50 mM ALK5 Inhibitor II.
- the culture solution was removed by suction, washed with PBS (-), and fixed with 10% formalin. Washed 3 times with sterile distilled water. After staining with Bodipy, the wells were washed with sterilized distilled water, and photographed with an inverted microscope at 100 ⁇ and 200 ⁇ magnification.
- composition of induction media (a) and (b) is as follows: [Induction medium (a)] DMEM (High Glucose): 500 mL FETAL 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 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
- Example 8 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 2 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 1000 ⁇ L / well of a cartilage induction medium (StemPro Chondrogenesis Differentiation Kit: ThermoFisher Scientific) supplemented with Cyclic Pifithrin- ⁇ or ALK5 inhibitor II was added.
- DMEM Dulbecco's modified minimum essential medium
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR-Master Mix, Sox9 and Aggrican or ⁇ -actin genes specific primers and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA levels of Sox9 and Aggrican genes were quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- the concentration of each compound is as follows: Cyclic Pifithrin- ⁇ : 5 ⁇ M ALK5 inhibitor II: 2 ⁇ M.
- TGF- ⁇ receptor signal Activin-mediated TGF- ⁇ receptor signal is known to positively regulate MSC proliferation, and TGF- ⁇ receptor signal promotes differentiation from MSC to osteoblasts and chondrocytes (for example, , Literature: WG Li and XXXu, Chin J Traumatol. 2005; 8 (6): 349-51., Literature: F. Ng et al., Blood. 2008; 112 (2): 295-307.). It was an unexpected result that the method of the present invention can achieve the conversion to chondrocytes by suppressing the TGF- ⁇ pathway.
- Example 9 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 2 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and a cartilage induction medium or a cartilage induction medium supplemented with 2 ⁇ M ALK5 inhibitor II was added at 1000 ⁇ L / well.
- DMEM Dulbecco's modified minimum essential medium
- the culture medium was replaced with a fresh one and cultured until day 18.
- the cells were washed twice with PBS ( ⁇ ) and once with a 3% acetic acid solution, and then added with PH2.5 Alcian Blue staining solution of Nacalai Tesque, and stained for 1 hour at room temperature. After washing 3 times with PBS (-), it was observed with a microscope.
- Example 10 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 2 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air.
- DMEM Dulbecco's modified minimum essential medium
- the culture supernatant was removed by aspiration, and skeletal muscle cell growth medium supplemented with 2 ⁇ M ALK5 inhibitor II and 2 ⁇ M D4476 (5% FBS, 50 microG / ml Bovine Fetuin, 10 nG / ml hEGF, 1 nG / ml bFGF, 10 1000 ⁇ L / well of Ham's / F10 medium supplemented with microG / ml Insulin and 0.4 microG Dexamethasone was added. Once every two days, the culture medium was replaced with a fresh one and cultured until day 28.
- 2 ⁇ M ALK5 inhibitor II and 2 ⁇ M D4476 5% FBS, 50 microG / ml Bovine Fetuin, 10 nG / ml hEGF, 1 nG / ml bFGF, 10 1000 ⁇ L / well of Ham's / F10 medium supplemented with microG / ml Insulin and
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR-Master Mix, MyoD1 and Myogenin or ⁇ -actin gene-specific primers and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA levels of MyoD1 and Myogenin genes were quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- Example 11 Human normal skin fibroblast cell line HDFs were seeded on a 24-well plate at a concentration of 5 ⁇ 10 3 cells / well (day 0). On the next day, the culture medium in each well was discarded and replaced with a new medium of 500 ⁇ l / well.
- the osteoblast induction medium is Dulbecco's modified Eagle's medium (DMEM), 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone plus 10% fetal bovine serum (FBS).
- DMEM Dulbecco's modified Eagle's medium
- FBS fetal bovine serum
- statin compound Simvastatin (SS) or Pravastatin (PrS) final concentration 100 nM was further added to the medium.
- Real-time PCR Master-Mix and Taqman probe and Primers specific for the human alkaline phosphatase (ALP) gene were added, and Real-time RT-PCR was performed using AB7300 Real-time PCR system.
- Example 12 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, an adipocyte induction medium, or an adipocyte induction medium supplemented with a compound or the like was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- the adipocyte induction medium is 10% FBS-added DMEM + MDI medium (0.5% mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1% ⁇ g / mL Insulin added 10% FBS-added DMEM).
- the additive concentrations are as follows: T3: 1 nM Rosiglitazone: 1 ⁇ M D4476: 2 ⁇ M Pifithrin alpha [p53 inhibitor]: 5 ⁇ M SB431542: 2 ⁇ M ALK5 Inhibitor II: 2 ⁇ M.
- Example 13 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humid air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, an adipocyte induction medium, or an adipocyte induction medium supplemented with each compound and the like was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- the adipocyte induction medium is 10% FBS-added DMEM + MDI medium (0.5% mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1% ⁇ g / mL Insulin added 10% FBS-added DMEM).
- the additive concentrations are as follows: T3: 1 nM Rosiglitazone: 1 ⁇ M D4476: 2 ⁇ M Pifithrin alpha [p53 inhibitor]: 5 ⁇ M SB431542: 2 ⁇ M ALK5 Inhibitor II: 2 ⁇ M.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix, primers specific to CIDEA gene or ⁇ -actin gene, and Taqman probe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of CIDEA gene was quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- Example 14 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humid air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, an adipocyte induction medium, or an adipocyte induction medium supplemented with each small molecule compound or the like was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- the adipocyte induction medium is 10% FBS-added DMEM + MDI medium (0.5% mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1% ⁇ g / mL Insulin added 10% FBS-added DMEM).
- the additive concentrations are as follows: T3: 1 nM Rosiglitazone: 1 ⁇ M D4476: 2 ⁇ M.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix and primers specific to AdipoQ or ⁇ -actin gene and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of AdipoQ gene was quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- fibroblasts were converted to brown adipocytes expressing the mRNA of the AdipoQ gene by adding and culturing either D4476, PD0325901, SB431542 or ALK5KInhibitor II.
- Example 15 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, an adipocyte induction medium, or an adipocyte induction medium supplemented with each small molecule compound or the like was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- the adipocyte induction medium is 10% FBS-added DMEM + MDI medium (0.5% mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1% ⁇ g / mL Insulin added 10% FBS-added DMEM).
- the additive concentrations are as follows: T3: 1 nM Rosiglitazone: 1 ⁇ M D4476: 2 ⁇ M SB431541: 2 ⁇ M ALK5 inhibitor II: 2 ⁇ M.
- the culture broth was aspirated and removed from each well and washed with PBS (-). Thereafter, the cells were fixed with 4% paraformaldehyde, washed with PBS ( ⁇ ), reacted with BODIPY 493/503 (Invitrogen) / PBS solution at room temperature for 5 minutes, and washed three times with PBS. Photographs were taken at a magnification of 200 times using a fluorescence microscope, and the fluorescence intensity was measured.
- FIG. 15A fluorescence microscope image
- FIG. 15B fluorescence intensity
- Example 16 Human dermal fibroblasts (HDFs) or human mesenchymal stem cells (MSC) suspended in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) . This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. The next day, the culture supernatant was removed by aspiration and normal medium (Normal medium), calcification induction medium (OB medium), or ALK5 inhibitor II (ALK5 i II) was added at a concentration of 4 ⁇ M as shown in the figure. Calcification induction medium (OB medium + ALK5 i II) was added at 500 ⁇ L / well.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 18.
- the culture medium was aspirated and removed from each well, washed with PBS (-), and fixed with 10% formalin. Washed 3 times with sterile distilled water. The wells after staining with Alizarin Red S were washed with sterilized distilled water and photographed with an inverted microscope at a magnification of 40 times.
- Example 17 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of normal medium, mineralization induction medium, or mineralization induction medium supplemented with ALK5 inhibitor II and / or TGF- ⁇ was added as shown in the figure. .
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 13.
- concentrations of the compound and cytokine added to the medium are as follows.
- ALK5 inhibitor II 4 ⁇ M TGF- ⁇ : 50 ng / ml.
- Example 18 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSCs) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) It was suspended. This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air.
- DMEM modified minimum essential medium
- the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, a mineralization induction medium, or a mineralization induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added as shown in the figure.
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 18.
- RNA was extracted from the cells with 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 Master Mix, Osteocalcin, Alkaline Phosphatase or ⁇ -actin gene-specific primers and Taqman pobe. QRT-PCR was performed using AB7300 Real-time PCR system.
- the mRNA level of Osteocalcin (Oc) and Alkaline Phosphatase (ALP) genes was quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in calcification induction medium was calculated as 1.
- Example 19 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSCs) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) It was suspended. This was seeded on a 24-well plate at a concentration of 2 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration and normal medium (Normal medium), MDI medium (adipocyte induction medium for fibroblasts), or ALK5 inhibitor II was added at a concentration of 4 ⁇ M as shown in the figure. MDI medium (MDI medium + ALK5 i II) was added at 500 ⁇ L / well.
- DMEM Dulbecco's modified minimum essential medium
- the MDI medium is 10% FBS-DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 mM ⁇ dexamethasone, and 1 mg / mL insulin. Once every 3 to 4 days, the culture medium was replaced with a fresh one and cultured until day 14.
- IBMX 3-isobutyl-1-methylxanthine
- ⁇ dexamethasone 0.5 mM ⁇ dexamethasone
- Results are shown in FIG. By culturing in MDI medium supplemented with ALK5 ⁇ inhibitor II, it can be seen that human skin-derived fibroblasts have been converted to white fat cells that accumulate a large amount of lipid droplets.
- Example 20 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSCs) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) (DMEM) Normal medium). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of normal medium, MDI medium, or MDI medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- ALK5 inhibitor II a concentration of 4 ⁇ M was added as shown in the figure.
- MDI medium (3-isobutyl-1-methylxanthine / dexamethasone / insulin-containing medium) is supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, and 1 ⁇ g / mL Insulin in 10% FBS DMEM It is a thing. Once every 3 to 4 days, the culture medium was replaced with a fresh one and cultured until day 14.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix, and primers specific to AdipoQ, FABP4 or ⁇ -actin gene and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA levels of AdipoQ and FABP4 genes were quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in MDI medium was calculated as 1.
- results are shown in FIG. It can be seen that human skin-derived fibroblasts were converted to white adipocytes expressing AdipoQ gene and FABP4 gene by culturing in MDI medium supplemented with ALK5 inhibitor II.
- Example 21 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSCs) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) It was suspended. This was seeded at the center of a fibronectin coated 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air.
- HDFs Human dermal fibroblasts
- ADSCs human adipose derived stem cells
- DMEM modified minimum essential medium
- Cartilage induction medium is 1% FBS DMEM with 50 ⁇ g / ml ascorbic acid, 1% insulin-transferrin-selenium, 10ng / ml BMP-2, 10ng / ml TGF- ⁇ , 10ng / ml GDF5, 10ng / nl b-FGF It is what was added. Once every 3-4 days, the culture solution was replaced with a fresh one and cultured until day 21.
- the culture solution was aspirated and removed from each well, washed with PBS (-), and fixed with 10% formalin. Washed 3 times with sterile distilled water. The wells after staining with Alcian blue were washed with sterilized distilled water and photographed with an inverted microscope at a magnification of 100 times.
- Results are shown in FIG. By culturing in a cartilage induction medium supplemented with ALK5 Ainhibitor II, it can be seen that human skin-derived fibroblasts have been converted to chondrocytes that produce a large amount of cartilage matrix.
- Example 22 Human dermal fibroblasts (HDFs) or human adipose derived stem cells (ADSCs) in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS) It was suspended. This was seeded at the center of a fibronectin coated 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air.
- HDFs Human dermal fibroblasts
- ADSCs human adipose derived stem cells
- DMEM modified minimum essential medium
- the culture supernatant was removed by aspiration, and 500 ⁇ L / well of normal medium, cartilage induction medium, or cartilage induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added as shown in the figure.
- Cartilage induction medium is 1% FBS DMEM with 50 ⁇ g / ml ascorbic acid, 1% insulin-transferrin-selenium, 10ng / ml BMP-2, 10ng / ml TGF- ⁇ , 10ng / ml GDF5, 10ng / nl b-FGF It is what was added. Once every 3-4 days, the culture solution was replaced with a fresh one and cultured until day 21.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR-Master Mix, and Aggrecan, Type II collgaen or ⁇ -actin gene-specific primers and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of Aggrecan (ACAN) and Type II collagen gene was quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in the cartilage induction medium was calculated as 1.
- ACAN Aggrecan
- Type II collagen gene was quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in the cartilage induction medium was calculated as 1.
- Results are shown in FIG. It can be seen that by culturing in a cartilage induction medium supplemented with ALK5 ⁇ ⁇ inhibitor II, human skin-derived fibroblasts were converted to chondrocytes expressing the Aggrecan gene and type 2 collagen gene.
- Example 23 Human normal gingival fibroblasts (GFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, a mineralization induction medium, or a mineralization induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 18.
- the culture medium was aspirated and removed from each well, washed with PBS (-), and fixed with 10% formalin. Washed 3 times with sterile distilled water. The wells after staining with Alizarin Red S were washed with sterilized distilled water and photographed with an inverted microscope at a magnification of 40 times.
- Example 24 Human normal gingival fibroblasts (GFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, a mineralization induction medium, or a mineralization induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added as shown in the figure.
- DMEM Denbecco's modified minimum essential medium
- Calcification induction medium is 10% FBS-DMEM supplemented with 50 ⁇ g / ml ascorbic acid, 10 mM ⁇ -glycerophosphate, 100 nM Dexamethasone. Once every 3-4 days, the culture medium was replaced with a fresh one and cultured until day 18.
- the culture solution was aspirated and removed from each well, washed with PBS ( ⁇ ), and fixed with a fixative. Washed 3 times with sterile distilled water. The wells after ALP staining were washed with sterilized distilled water and photographed with an inverted microscope at a magnification of 40 times.
- Example 25 Animal experiments were performed with the approval of the institution. Eight week old male NOD / SCID mice (Charles River) were anesthetized. A partial bone defect having a diameter of about 7 mm was created in the left femoral shaft using a dental drill under water injection. Cells (CdOBs; Chemical-mediated directly converted osteoblasts) cultured for 13 days in the presence of ALK5 inhibitor II were suspended together with Matrigel (BD Bioscience, San Jose, Calif.) In the same manner as in Example 16 described below. The bone defect and the surrounding bone surface were transplanted at a concentration of 5 ⁇ 10 5 cells / mouse. Similarly, mice were prepared in which bone defects were made and fibroblasts were suspended in Matrigel and transplanted.
- CdOBs Chemical-mediated directly converted osteoblasts
- mice 21 days later, the mice were euthanized, the thighs were excised, fixed with neutral formalin, and then micro-computed tomography ( ⁇ CT using X-ray CT device (Scan Xmate-L090, Com Scan Techno, Yokohama, Japan) )
- Fig. 29 shows a tomographic image of ⁇ CT. It can be seen that CdOB formed bone at the bone defect site in the mouse body.
- Example 26 (FIG. 30) An image obtained by three-dimensionally constructing the ⁇ CT image of Example 25 is shown in FIG. Cells cultured with the addition of ALK5 inhibitor II were shown to have bone forming ability in vivo. It can be seen that CdOB formed bone at the bone defect site in the mouse body.
- Example 27 Animal experiments were performed with the approval of the institution. A transplantation experiment was conducted in the same manner as in Example 25 above. In addition, mice transplanted with fibroblasts were also prepared. After 21 days, the mice were euthanized, the thighs were excised in the same manner as in Example 25, fixed with neutral formalin, and the bone tissue was embedded with SCEM (Leica Microsystem) compound and snap frozen. After slicing into 6 ⁇ m sections, serial sections were stained with hematoxylin and eosin (H & E) staining and Alizarin Red S.
- H & E hematoxylin and eosin
- Example 28 Human normal dermal fibroblast cell line HDFs were cultured in a 60 mm culture dish and cultured in a normal medium ( ⁇ ). HDFs were cultured for 3 or 7 days in a normal medium supplemented with 4 ⁇ M ALK5 inhibitor II. Total RNA was recovered from these cells using ISOGEN II. The mRNA expression pattern of each cell was analyzed genome-wide using an Affymetrix DNA chip. A heat map showing the expression level of the MSC marker is shown in FIG. It can be seen that fibroblasts were converted to mesenchymal stem cells (MSCs) by culturing in a medium supplemented with ALK5 inhibitor II.
- MSCs mesenchymal stem cells
- Example 29 Human dermal fibroblasts (HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of a normal medium, a calcification induction medium, or a calcification induction medium added with each small molecule compound was added as shown in the figure.
- DMEM Dulbecco's modified minimum essential medium
- Calcification induction medium is DMEM supplemented with 10% FBS, 50 ⁇ g / ml ascorbic acid, 10 ⁇ m ⁇ -glycerophosphate, 100 nM Dexamethasone.
- I-BET151 2 ⁇ M Pifthrin- ⁇ : 5 ⁇ M PD0325901: 2 ⁇ M 2-Me-5HT: 2 ⁇ M CX4945: 2 ⁇ M CHIR99021: 2 ⁇ M Forskolin: 2 ⁇ M DZnep: 50nM D4476: 2 ⁇ M SB431542: 2 ⁇ M ALK5 i II: 2 ⁇ M.
- the culture medium was aspirated and removed from each well, washed with PBS (-), and fixed with 10% formalin. After washing 3 times with sterile distilled water, Alizarin Red S staining solution was added and incubated at room temperature for 15 minutes. The stained solution after staining was collected in a 96-well plate and used in the experiment of Example 30. The well was then washed with sterile distilled water and photographed.
- Example 30 (FIG. 34) In the experiment of Example 29, the solution after staining with the Alizarin Red S staining solution was collected on a 96 well plate, and the absorbance (OD550 nm) was measured with an absorptiometer. The results are shown in FIG.
- the TGF- ⁇ pathway inhibitors D4476, SB431542, and ALK5 i II are all added to the calcification-inducing medium to convert fibroblasts into osteoblasts. It turns out that it induces a conversion.
- compounds other than TGF- ⁇ pathway inhibitors reported to promote induction from fibroblasts to iPS cells I-BET, Pifthrin- ⁇ , PD0325901, 2-Me-5HT, CHIR ⁇ , Forskolin, DZnep
- ALK5 iII among others, induced the strongest fibroblast-to-osteoblast conversion.
- Example 31 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, aspirate the culture supernatant and add 500 ⁇ L / well of normal medium, calcification induction medium (OB medium), or calcification induction medium supplemented with each small molecule compound or cytokine as shown in the figure. It was.
- OB medium calcification induction medium
- Calcification induction medium is DMEM supplemented with 10% FBS, 50 ⁇ g / ml ascorbic acid, 10 ⁇ m ⁇ -glycerophosphate, 100 nM Dexamethasone.
- the compounds added to the medium and their concentrations are as follows. D4476: 1 or 4 ⁇ M LY2157299: 1 or 4 ⁇ M LY364947: 1 or 4 ⁇ M SB431542: 1 or 4 ⁇ M SB525334: 1 or 4 ⁇ M SD208: 1 or 4 ⁇ M ALK5 i II: 1 or 4 ⁇ M TGF- ⁇ : 10 ng / ml.
- the culture solution was aspirated and removed from some wells, washed with PBS (-), and fixed with a fixative. Washed 3 times with sterile distilled water. The wells after ALP staining were washed with sterile distilled water and photographed.
- the culture medium was removed from the remaining wells by suction, washed with PBS (-), and fixed with 10% formalin. After washing 3 times with sterile distilled water, Alizarin Red S staining solution was added and incubated at room temperature for 15 minutes. The stained solution after staining was collected in a 96-well plate and used in the experiment of Example 32. The well was then washed with sterile distilled water and photographed.
- Example 32 (FIG. 36) In the experiment of Example 31, the solution after staining with the Alizarin Red S staining solution was collected on a 96-well plate, and the absorbance (OD550 nm) was measured with an absorptiometer. The results are shown in FIG.
- human fibroblasts can be converted into osteoblasts by culturing in a calcification induction medium supplemented with various TGF- ⁇ pathway inhibitors.
- SD208 and ALK5 i II can strongly convert human fibroblasts into osteoblasts.
- ALK5 i II was found to convert human fibroblasts to osteoblasts most potently.
- Example 33 Human normal skin-derived fibroblasts (HDF45 (purchased from KURABO), HDF69 (purchased from PromoCell), HDF22 (purchased from PromoCell)) from various individuals suspended in normal medium (DMEM supplemented with 10% FBS) did. This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of normal medium, calcification induction medium, or calcification induction medium supplemented with 4 ⁇ M of ALK5 i II was added as shown in the figure.
- normal medium DMEM supplemented with 10% FBS
- Calcification induction medium is DMEM supplemented with 10% FBS, 50 ⁇ g / ml ascorbic acid, 10 ⁇ m ⁇ -glycerophosphate, 100 nM Dexamethasone.
- the culture solution was aspirated and removed from each well, washed with PBS ( ⁇ ), and fixed with a fixative. Washed 3 times with sterile distilled water. After ALP staining, the wells were washed with sterile distilled water and photographed with an inverted microscope at a magnification of 40 times.
- Example 34 ( Figure 38) The same culture as in Example 33 was performed using human normal skin-derived fibroblasts (HDF45, HDF69, HDF22) derived from various individuals.
- any human normal skin-derived fibroblasts derived from different individuals can be converted into osteoblasts by culturing in a mineralization induction medium supplemented with ALK5 i II. It was.
- Example 35 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / well of calcification induction medium (OB medium) or calcification induction medium supplemented with each small molecule compound and / or cytokine was added as shown in the figure. .
- OB medium calcification induction medium
- calcification induction medium supplemented with each small molecule compound and / or cytokine was added as shown in the figure. .
- Calcification induction medium is DMEM supplemented with 10% FBS, 50 ⁇ g / ml ascorbic acid, 10 ⁇ m ⁇ -glycerophosphate, 100 nM Dexamethasone.
- the concentration of the added small molecule compound or cytokine is as follows.
- ALK5 i II 4 ⁇ M 1 ⁇ , 25-dihydroxy Vitamin D3 (VD3): 5 nM Human insulin-like growth factor-1 (IGF-1): 100 ng / ml.
- RNA was extracted from the cells with ISOGEN II. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Master Mix, Alkaline Phosphatase, Osteocalcin, Bonesialoprotein or ⁇ -actin gene specific primers and Taqman pobe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system.
- the mRNA levels of Alkaline Phosphatase, Osteocalcin (OC), and Bonesialoprotein genes were quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in a calcification induction medium supplemented with ALK5 i II was calculated as 1.
- ALP mRNA expression was significantly increased in cells cultured in calcification induction medium supplemented with ALK5 i II.
- OC mRNA expression was increased in cells cultured in calcification induction medium supplemented with ALK5 i II compared to cells cultured in calcification induction medium, but mineralization induction medium supplemented with ALK5 i II As compared with the cells cultured in the above, the cells cultured in the mineralization induction medium supplemented with ALK5 i II and VD3 further increased significantly.
- Example 36 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 24-well plate at a concentration of 1 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. The next day, the culture supernatant was removed by aspiration, and 500 ⁇ L / min of normal medium or calcification induction medium supplemented with ALK5 i II, VD3, and IGF-1 at concentrations of 4 ⁇ M, 5 nM, and 100 ng / ml, respectively. Well added.
- DMEM normal skin-derived fibroblasts
- Calcification induction medium is DMEM supplemented with 10% FBS, 50 ⁇ g / ml ascorbic acid, 10 ⁇ m ⁇ -glycerophosphate, 100 nM Dexamethasone.
- Anti-Osteocalcin antibody was added and reacted at 4 ° C overnight, and then washed 3 times with Wash buffer.
- Alexa 488-conjugated anti-mouse Ig antibody was added and reacted at room temperature for 1 hour, and then washed 3 times with Wash buffer. Thereafter, nuclear staining was performed using DAPI, and photographs were taken at a magnification of 200 times using a fluorescence microscope. The number of OC-positive cells / DAPI was calculated using BZ-H3C and BZ-H3CM (KEYENCE).
- Example 37 Human dermal fibroblasts (HDFs) were suspended in normal medium (Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was aspirated and removed, and 1 mL / well of normal medium was added to Group 1 as shown in the figure. Group 2 was added with 1 mL / well of an adipocyte induction medium (adipocyte induction medium (R-)) not containing Rosiglitazone.
- adipocyte induction medium adipocyte induction medium (R-)
- Group 3 was supplemented with 1 mL / well of adipocyte induction medium.
- Group 4 was supplemented with 1 mL / well of adipocyte induction medium (R-) supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M.
- Groups 5-9 were supplemented with 1 mL / well of adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M.
- the adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM M3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- the adipocyte induction medium (R-) is (DMEM supplemented with 1 nM T3, 0.5 mM mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- the culture medium was replaced with fresh one once every 2 days.
- groups 5 to 9 as shown in the figure, adipocyte induction with addition of ALK5 inhibitor II during the period of 0-2 days, 0-4 days, 0-6 days, 0-8 days, 0-10 days, respectively
- the cells were cultured in a medium, and then cultured in an adipocyte induction medium to which ALK5 inhibitor II was not added.
- the culture broth was aspirated and removed from each well, washed with PBS ( ⁇ ), and total RNA was extracted from the cells using Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever, Tra, Ace, qPCR, RT, Master, and Mix.
- Example 38 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. The next day, the culture supernatant was removed by aspiration, and 1 mL / well of normal medium was added to Group 1 (Control). Group 2 was supplemented with 1 mL / well of adipocyte induction medium. Groups 3 to 8 were added 1 mL / well of adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M.
- DMEM normal skin-derived fibroblasts
- the fat cell induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM M3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin, 10% and 10% FBS).
- the culture medium was replaced with fresh one once every 2 days.
- groups 3-7 as described in the figure, adipocytes supplemented with ALK5 inhibitor II during the period of 0-2 days, 0-4 days, 0-6 days, 0-8 days, 0-10 days respectively.
- the cells were cultured in an induction medium, and then cultured in an adipocyte induction medium to which ALK5 Inhibitor II was not added.
- the fluorescence microscope image is shown in FIG. Group 1 and group 2 cells were hardly stained with anti-UCP1 antibody. In groups 3 to 8, many cells stained with anti-UCP1 antibody were observed. Particularly in group 6, the density of cells stained with anti-UCP1 antibody was the highest. It can be seen that fibroblasts were converted to brown adipocytes highly expressing UCP1 protein by culturing in an adipocyte induction medium supplemented with ALK5 inhibitor II. In particular, it can be seen that the conversion of fibroblasts into brown adipocytes is most strongly induced by adding ALK5 inhibitor II for 0-8 days and then culturing in an adipocyte induction medium not containing ALK5 inhibitor II for 4 days.
- Example 39 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 1 mL / well of normal medium, adipocyte induction medium (BA medium), or adipocyte induction medium supplemented with compounds ALK5 inhibitor II, LY2157299, SB431542, and D4476 was added.
- DMEM normal skin-derived fibroblasts
- Compound addition concentrations are 4 ⁇ M, 8 ⁇ M, 12 ⁇ M, or 16 ⁇ M.
- the adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- IBMX 3-isobutyl-1-methylxanthine
- dexamethasone 1 ⁇ g / mL Insulin and 10% FBS.
- RNA was extracted from the cells using Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix. Real-time PCR Master Mix, primers specific to UCP-1 gene or ⁇ -actin gene, and Taqman probe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of the UCP1 gene was quantified as a ratio to the ⁇ -actin gene mRNA level, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- Example 40 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was aspirated and removed, as shown in the figure, normal medium for group 1, adipocyte induction medium for group 2, compounds ALK5 inhibitor II (4 ⁇ M), LY2157299 (8 ⁇ M), SB431542 (4 ⁇ M), and D4476 (4 ⁇ M) were added to each adipocyte induction medium at 1 mL / well.
- DMEM normal skin-derived fibroblasts
- the adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- DMEM fetal calf serum
- IBMX 3-isobutyl-1-methylxanthine
- dexamethasone 1 ⁇ g / mL Insulin and 10% FBS.
- FIG. 44 fluorescence microscope image. In all groups, many cell nuclei stained with DAPI were observed. In Group 1 and Group 2, few cells were stained with anti-UCP1 antibody. On the other hand, in groups 3 to 6, many cells stained with anti-UCP1 antibody were observed. In particular, group 3 had the highest density of cells stained with anti-UCP1 antibody, followed by group 4. Therefore, it can be seen that by culturing in an adipocyte induction medium supplemented with any of ALK5 inhibitor II, LY2157299, SB431541, and D4476, fibroblasts were converted to brown adipocytes expressing UCP1 protein. In particular, it can be seen that AKL5 inhibitor II strongly induced the conversion of fibroblasts into brown adipocytes, followed by LY2157299.
- Example 41 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (day 0), and culture was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 1 mL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with compound ALK5 inhibitor II (4 ⁇ M) or LY2157299 (8 ⁇ M) was added.
- DMEM normal skin-derived fibroblasts
- the adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- DMEM fetal calf serum
- IBMX 3-isobutyl-1-methylxanthine
- dexamethasone 1 ⁇ g / mL Insulin and 10% FBS.
- RNA was synthesized using Rever Tra Ace qPCR RT Master Mix. This cDNA was mixed with Real-time PCR Master Mix, primers specific to UCP-1 gene, CIDEA gene, KCNK3 gene or ⁇ -actin gene and Taqman probe. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of UCP1 gene was quantified as a ratio to ⁇ -actin gene mRNA, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- Results are shown in FIG.
- adipocyte induction medium supplemented with either ALK5 inhibitor II or LY2157299.
- the fibroblasts were converted to brown adipocytes expressing the UCP1 gene, CIDEA gene, and KCNK3 gene.
- AKL5 inhibitor II more strongly induced the conversion of fibroblasts into brown adipocytes.
- Example 42 Human normal skin-derived fibroblasts (HDFs) were suspended in a normal medium (DMEM supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 1 mL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M was added.
- DMEM normal skin-derived fibroblasts
- the adipocyte induction medium is (DMEM supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin and 10% FBS).
- DMEM fetal calf serum
- IBMX 3-isobutyl-1-methylxanthine
- dexamethasone 1 ⁇ g / mL Insulin and 10% FBS.
- Example 43 (FIG. 47) Normal human epidermal keratinocytes (Human Epidermal Keratinocyte; NHEK (AD)) were suspended in a serum-free liquid medium (HuMedia-KG2; Kurabo) for normal human epidermal keratinocyte proliferation. This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air.
- a serum-free liquid medium HuMedia-KG2; Kurabo
- K-adipocyte induction medium adipocyte induction medium for keratinocytes
- ALK5 inhibitor II 4 ⁇ M
- LY2157299 8 ⁇ M
- the K-adipocyte induction medium is (HuMedia-KG2 supplemented with 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone, 1 ⁇ g / mL Insulin).
- the culture medium was replaced with a fresh one and cultured from Day 1 to Day 8. Thereafter, from Day 9 to Day 14, the ALK5 inhibitor II was also cultured in a medium not containing LY2157299.
- the culture broth was aspirated and removed from each well, washed with PBS ( ⁇ ), and total RNA was extracted from the cells using Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever, Tra, Ace, qPCR, RT, Master, and Mix. Real-time PCR Master Mix, and primers specific to UCP-1 gene, CIDEA gene or ⁇ -actin gene and Taqman probe were mixed with this cDNA.
- QRT-PCR was performed using AB7300 Real-time PCR system.
- the mRNA level of UCP1 gene and CIDEA gene was quantified as a ratio to ⁇ actin gene mRNA level, and the value of fibroblasts cultured in a normal medium was calculated as 1.
- Example 44 (FIG. 48) Normal human epidermal keratinocytes (Human Epidermal Keratinocyte; NHEK (AD)) were suspended in a serum-free liquid medium (HuMedia-KG2; Kurabo) for normal human epidermal keratinocyte proliferation. This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air.
- a serum-free liquid medium HuMedia-KG2; Kurabo
- the culture supernatant is removed by aspiration, and HuMedia-KG2 medium (Ctrl medium), K-osteoblast induction medium (osteoblast induction medium for keratinocytes), or K- supplemented with compound ALK5 inhibitor II (4 ⁇ M) Osteoblast induction medium was added at 1 mL / well.
- the K-osteoblast induction medium is HuMedia-KG2 supplemented with 50 ⁇ g / mL ascorbic acid, 10 mM ⁇ -glycerol phosphate, and 100 nM dexamethasone. Once every 2 days, the culture medium was replaced with a fresh one and cultured until Day 14.
- Example 45 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded in a 48-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 5 groups, and the medium described in the figure was added.
- HDFs Human normal dermal fibroblasts
- Group 1 Control medium group 2: WA medium group 3: WA medium supplemented with 1 ⁇ M Rosiglitazone
- Group 4 WA medium supplemented with 16 ⁇ M ALK5 inhibitor II
- Group 5 WA medium supplemented with 16 ⁇ M ALK5 inhibitor II and 1 ⁇ M Rosiglitazone.
- the composition of the 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 ⁇ g / mL streptomycin.
- WA medium is 10% FBS, 100 mM non-essential amino acids (NEAA), 100 U / ml penicillin, 100 ⁇ g / mL streptomycin, 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 62.5 nM Indomethacin, 1 ⁇ M Dexamethasone, and Dulbecco's modified minimum essential medium supplemented with 170 nM insulin; DMEM.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- BODIPY staining was performed as follows. (1) After removing the culture medium in the well, each well is washed once with PBS. (2) Add 50 ⁇ L of 4% formaldehyde solution to each well and fix at room temperature overnight. (3) Wash twice with PBS. (4) Add 50 ⁇ L of BODIPY staining solution to each well and let stand at room temperature for 30 minutes. (5) After removing the staining solution from the well, leave it at room temperature for 30 minutes.
- FIG. 49 shows a phase contrast image and a green fluorescence image taken with a fluorescence microscope.
- Most cells cultured in the Control medium are not stained with Bodipy, and the cells cultured in the WA medium supplemented with Rosiglitazone have a very low frequency of cells that are stained very weakly.
- WA medium supplemented with ALK5 inhibitor II more cells stained more intensely are observed.
- WA medium supplemented with both ALK5inhibitorII and Rosiglitazone the most cells were strongly stained with BODIPY. Therefore, it can be understood that human fibroblasts were converted to white adipocytes by culturing in WA medium supplemented with ALK5Kinhibitor II.
- AKL5 inhibitor II more strongly induced the conversion of fibroblasts into white adipocytes.
- Example 46 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded in a 48-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 7 groups, and the medium described in the figure was added.
- HPFs Human normal dermal fibroblasts
- Group 1 Control medium group 2: WA medium group 3: WA medium group added with 1 ⁇ M Rosiglitazone 4: WA medium group added with 8 ⁇ M ALK5 inhibitor II: WA medium group added with 16 ⁇ M ALK5 inhibitor II 6: WA medium supplemented with 8 ⁇ M ALK5 inhibitor II and 1 ⁇ M Rosiglitazone.
- Group 7 WA medium supplemented with 16 ⁇ M ALK5 inhibitor II and 1 ⁇ M Rosiglitazone.
- composition of the medium is the same as in Example 45.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- Example 45 On the 12th day, it was stained with BODIPY in the same manner as in Example 45, and the lipid droplets were fluorescently stained. Images were taken at a magnification of 100 using a fluorescence microscope BZ-9000 (Keyence). The number of BODIPY positive cells was calculated with BZ-II Analyzer software (Keyence, Osaka, Japan) and compared between groups.
- Results are shown in FIG.
- Most cells cultured in Control medium were not stained with BODIPY, but there was a low frequency of stained cells in the group cultured in WA medium supplemented with either 1 ⁇ M Rosiglitazone or 16 ⁇ M ALK5 Inhibitor II. did.
- the group cultured in WA medium supplemented with 8 ⁇ M or 16 ⁇ M ALK5 inhibitor II and 1 ⁇ M Rosiglitazone more cells were stained. Therefore, it can be understood that human fibroblasts were converted to white adipocytes by culturing in WA medium supplemented with ALK5Kinhibitor II.
- AKL5 inhibitor II more strongly induced the conversion of fibroblasts into white adipocytes.
- Example 47 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded on a 6-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 5 groups, and the medium described in the figure was added as in Example 45. The composition of the medium is the same as in Example 45.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- Real-time PCR was used to quantify mRNA using TaqMan probes and Primers specific for AdipoQ, FABP4 (mature adipocyte marker), and PPAR- ⁇ (adipocyte precursor marker) genes, respectively.
- results are shown in FIG.
- AdipoQ and FABP4 were slightly induced by ALK5 i II supplemented culture and strongly induced by co-addition of ALK5 iII and Rosiglitazone.
- Mild expression of PPAR- ⁇ was also induced when cultured in WA medium, moderate expression was induced by addition of either ALK5 i II or Rosiglitazone, and expression was strongly induced by co-addition of ALK5 i II and Rosiglitazone.
- human fibroblasts were converted to white adipocytes by culturing in WA medium supplemented with ALK5 inhibitor II.
- AKL5 inhibitor II more strongly induced the conversion of fibroblasts into white adipocytes.
- Example 48 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded in a 48-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 10 groups, and the medium described in the figure was added.
- HPFs Human normal dermal fibroblasts
- Group 1 WA medium group 2: WA medium group supplemented with 16 ⁇ M ALK5 inhibitor II (ALK5 i II) 3: WA medium group supplemented with 16 ⁇ M LY2157299 4: WA medium group 5 supplemented with 16 ⁇ M SB431542 : WA medium group supplemented with 16 ⁇ M D4476 6: WA medium supplemented with 1 ⁇ M Rosiglitazone.
- Group 7 WA medium supplemented with 16 ⁇ M ALK5 inhibitor II (ALK5 i II) and 1 ⁇ M Rosiglitazone.
- Group 8 WA medium supplemented with 16 ⁇ M LY2157299 and 1 ⁇ M Rosiglitazone.
- Group 9 WA medium supplemented with 16 ⁇ M SB431542 and 1 ⁇ M Rosiglitazone.
- Group 10 WA medium supplemented with 16 ⁇ M D4476 and 1 ⁇ M Rosiglitazone.
- composition of the medium is the same as in Example 45.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- Example 45 On the 12th day, it was stained with BODIPY in the same manner as in Example 45, and the lipid droplets were fluorescently stained. Images were taken at a magnification of 100 using a fluorescence microscope BZ-9000 (Keyence). The number of adipocytes was calculated with BZ-II Analyzer software (Keyence, Osaka, Japan) and compared between groups. The results are shown in FIG.
- Example 49 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded on a 6-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 10 groups, and the medium described in the figure was added as in Example 48. The composition of the medium is the same as in Example 45.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- Example 48 On the 12th day, it was stained with BODIPY in the same manner as in Example 48, and the lipid droplets were fluorescently stained. Images were taken at a magnification of 100 using a fluorescence microscope BZ-9000 (Keyence). The number of BODIPY positive cells, the area stained with BODIPY, and the intensity of staining with BODIPY were calculated using BZ-II Analyzer software (Keyence, Osaka, Japan), and comparison was made between the groups.
- Results are shown in FIG.
- Cells cultured in Control medium were hardly stained with BODIPY, but cells cultured in WA medium supplemented with either 1 ⁇ M Rosiglitazone or 16 ⁇ M ALK5 Inhibitor II accumulated a small amount of fat.
- 16 ⁇ M ALK5 inhibitor II, LY2157299, SB431542 and D4476 were all co-added with Rosiglitazone to induce fat accumulation stronger than Rosiglitazone alone.
- the cells that accumulated the largest amount of fat were observed.
- Example 50 Human normal dermal fibroblasts (HDFs) were suspended in Control medium. This was seeded on a 6-well plate at a concentration of 10 4 cells / mm 2 (day 0), and culture was started at 37 ° C. with 5% CO 2 /95% humidified air. On day 4, the culture supernatant was removed by aspiration, the wells were divided into 5 groups, and the medium described in the figure was added.
- HPFs Human normal dermal fibroblasts
- Group 1 WA medium supplemented with 1 ⁇ M Rosiglitazone 2: WA medium group supplemented with 1 ⁇ M Rosiglitazone and 16 ⁇ M ALK5 inhibitor II (ALK5 i II): 1 ⁇ M Rosiglitazone and 16 ⁇ M LY2157299 (LY) added WA medium group 4: 1 WA medium group supplemented with 1 ⁇ M Rosiglitazone and 16 ⁇ M SB431542 (SB) 5: 1 WA medium supplemented with 1 ⁇ M Rosiglitazone and 16 ⁇ M D4476 (D4).
- ALK5 inhibitor II ALK5 inhibitor II
- SB SB431542
- the composition of the medium is the same as in Example 45.
- the culture was continued at 37 ° C with 5% CO 2 /95% humidified air.
- the medium was replaced with a fresh medium having the same composition.
- Results are shown in FIG. Cells in groups 2 to 5 were moderate to highly expressed mRNA of one or more genes of AdipoQ, FABP4, or PPAR- ⁇ . Cells supplemented with ALK5 inhibitor II expressed the most strongly of all three genes.
- ALK5 inhibitor II all convert human fibroblasts into white adipocytes.
- AKL5 inhibitor II most strongly induced the conversion of fibroblasts into white adipocytes.
- Example 51 Human fibroblast aHDF45 was seeded in a 24-well plate at 1.0 ⁇ 10 4 cells / 500 ⁇ L / well. The next day, the wells were divided into two groups, one group was replaced with the following Control medium, and the other group was replaced with the following SC medium supplemented with 4 ⁇ M Alk5 i II. The cells were cultured for 14 days at 37 ° C. and 5% CO 2 /95% air. The 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 ⁇ g / mL streptomycin.
- SC (Schwann cell) medium 10% FBS, 10 ng / mL rhbFGF (basic fibroblast growth factor), 5.7 ⁇ g / mL Forskolin, 200 ng / mL rhHeregulin beta-1, 5 ng / mL rhPDGF-AA, 100 mM non- Dulbecco's modified minimum essential medium; DMEM supplemented with essential amino acids (NEAA), 1 mM Sodium Pyruvate, 100 U / ml penicillin, and 100 ⁇ g / mL streptomycin.
- NEAA essential amino acids
- Example 52 Normal human epidermal keratinocytes (Human Epidermal Keratinocyte; NHEK (AD)) were suspended in a serum-free liquid medium (HuMedia-KG2; manufactured by Kurabo) for normal human epidermal keratinocyte proliferation. This was seeded on a 12-well plate at a concentration of 3 ⁇ 10 4 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air.
- the culture supernatant is removed by aspiration, and the human epidermal basal supplemented with HuMedia-KG2 medium, human epidermal basal cell growth medium (CnT-PR; manufactured by CELLnTEC), or ALK5 inhibitor II at a concentration of 1 ⁇ M or 10 ⁇ M
- Each cell growth-like medium was added at 1 mL / well.
- the culture medium was replaced with a fresh one and cultured until day 21.
- the culture medium was aspirated and removed from each well, washed with PBS ( ⁇ ), and then total RNA was extracted from the cells using Qiagen RNA easy Mini Kit. From this RNA, cDNA was synthesized using Rever Tra Ace qPCR RT Master Mix.
- Example 53 Human peripheral blood mononuclear cells (PBMCs) were suspended in a control medium (RPMI1640 medium supplemented with 10% FBS). This was seeded on a 12-well plate at a concentration of 2.5 ⁇ 10 5 cells / well (Day 0), and cultivation was started at 37 ° C. with 5% CO 2/95% humidified air. On the next day, the culture supernatant was removed by aspiration, and 1 mL / well of Control medium was added to Group 1. Group 2 was supplemented with 1 mL / well of P-adipocyte induction medium (adipocyte induction medium for peripheral blood mononuclear cells).
- RPMI1640 medium RPMI1640 medium supplemented with 10% FBS
- Group 3 was supplemented with 1 mL / well of P-adipocyte induction medium supplemented with ALK5 inhibitor II at a concentration of 4 ⁇ M.
- P-adipocyte induction medium is 10% FBS, 30 U / ml recombinant hIL-2, 1 nM T3, 1 ⁇ M Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.5 ⁇ M dexamethasone and 1 ⁇ g / mL RPMI1640 medium supplemented with insulin. Once every two days, 50% of the culture was replaced with fresh one.
- RNA was extracted from the cells using Qiagen's RNAeasy MiniKit. From this RNA, cDNA was synthesized using Rever, Tra, Ace, qPCR, RT, Master, and Mix. Real-time PCR Master Mix, and primers specific to FABP4 gene or ⁇ -actin gene and Taqman probe were mixed with this cDNA. QRT-PCR was performed using AB7300 Real-time PCR system. The mRNA level of FABP4 gene was quantified as a ratio to the level of ⁇ -actin gene mRNA, and the value of human normal peripheral blood mononuclear cells cultured in a normal medium was calculated as 1.
- Results are shown in FIG. It was revealed that human normal peripheral blood mononuclear cells were converted to white adipocytes expressing the FABP4 gene by culturing in a P-adipocyte induction medium supplemented with ALK5 inhibitor II.
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Abstract
Description
本出願は、2015年10月21日に出願された、日本国特許出願第2015-207529号明細書(その開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
マウス線維芽細胞→軟骨細胞(SOX9 + Klf4 + c-Myc遺伝子を導入)
マウス線維芽細胞→心筋細胞(GATA4 + Mef2c + Tbx5遺伝子を導入)
マウス線維芽細胞→肝細胞(Hnf4α+(Foxa1またはFoxa2またはFoxa3)遺伝子を導入)
マウス線維芽細胞→神経幹細胞(Sox2 + FoxG1遺伝子を導入など)、
マウス、ヒト細胞→造血幹細胞など。
本発明の方法において原材料として用いる哺乳動物の分化した体細胞としては、哺乳動物由来であれば、特に限定されない。体細胞とは、細胞のうち生殖細胞以外の細胞を意味する。
本発明の方法により調製される他の体細胞は、原材料として用いる体細胞以外の体細胞である。また、原材料として用いる体細胞から、生理的な条件下(特に、生体内)で分化する体細胞は、「他の体細胞」には含まれない。例えば、生体内で間葉系幹細胞から骨芽細胞へと分化するため、間葉系幹細胞を原材料として用いる場合は、骨芽細胞は「他の体細胞」には該当しない。
本発明の方法において、分化した体細胞を、分化した体細胞以外の他の体細胞を誘導するための培地(分化誘導培地)中で培養する。分化誘導培地としては、目的の調製される体細胞に応じて、公知の分化誘導培地を使用することができる。
(1-1) M.D. Hoffman and D.S.W. Benoit, J Tissue Eng Regen Med. 2013 Apr 1. doi: 10.1002/term.1736
(1-2) C-Y Li, et al., Stem Cell Res Ther. 2015 Apr 13;6:55. doi: 10.1186/s13287-015-0066-5.
(1-3) B. Gharibi and F.J. Hughes, Stem Cells Transl Med. 2012 Nov;1(11):771-82. doi: 10.5966/sctm.2010-0031. Epub 2012 Oct 23
(1-4) S.K. 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。
(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 Orthopaedic Research,Volume 27, Issue 5, pages 612-619, May 2009
(2-4) A.M. Ibrahim et al., Microscopy Research and Technique, Volume 78, Issue 8, pages 667-675, August 2015。
(3-1)Kingham PJ, DF Kalbermatten, D Mahay,et al: Adipose-derived stem cells differentiate into a Schwann cell phenotype andpromote 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.。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-βパスウェイ阻害剤の存在下に培養する。
TGF-β/SMADパスウェイ阻害剤は、TGF-β/SMADパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/SMADパスウェイは図16(Chen G et al, Int J Biol Sci, 2012より引用)に模式的に示す、当業者に公知のシグナル経路である。
R1はH、メチル基又はハロゲン(例えば、フッ素、塩素、臭素又はヨウ素)である。
R2はH又はメチル基である。]
TGF-β/ERKパスウェイ阻害剤は、TGF-β/ERKパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/ERKパスウェイは図17(Y. E. Zhang,“Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009より引用)に模式的に示す、当業者に公知のシグナル経路である。
TGF-β/JNKパスウェイ阻害剤は、TGF-β/JNKパスウェイに属するタンパク質の活性を阻害できる化合物を意味する。TGF-β/JNKパスウェイは図18(Y. E. Zhang,“Non-smad pathways in TGF-β signaling” Cell Research 19: 128, 2009より引用)に模式的に示す、当業者に公知のシグナル経路である。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-β/p38パスウェイ阻害剤の存在下に培養する。
本発明の方法において、他の体細胞を誘導するための培地中で、TGF-β/RhoAパスウェイ阻害剤の存在下に培養する。
本発明の方法において、哺乳動物の分化した体細胞を誘導培地中、TGF-βパスウェイ阻害剤の存在下に培養する。
本発明の方法により調製される体細胞は、生体に移植することで、種々の疾患又は状態の予防又は治療に使用することができる。本明細書において、特に明示のない限り、「治療」という用語は、患者が特定の疾患又は障害を患っている間に行う処置を意図し、これによって疾患若しくは障害の重症度、又は1つ若しくは複数のその症状が軽減されるか、又は疾患若しくは障害の進行が遅延又は減速することを意味する。本明細書において、「治療」には「予防」を含むものとする。
本発明は、TGF-βパスウェイ阻害剤を含む、分化した体細胞を他の体細胞にコンヴァートさせるための誘導剤をも提供する。また、分化した体細胞を他の体細胞にコンヴァートさせるための、TGF-βパスウェイ阻害剤の使用をも提供する。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2 μM
ALK5 inhibitor II: 2 μM
SB431542: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2μM
SB431542: 2μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地、または各化合物を加えた石灰化誘導培地を、500 μL/well加えた。
D4476: 2μM
SB431542: 2μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSC)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを5×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 inhibitor IIを加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを10×103 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。4日日に培養上清を吸引除去し、50 mM ALK5 Inhibitor IIを添加した誘導培地(a)(下記)を加えて培養を行った。6日目に誘導培地(a)を除去し、50 mM ALK5 Inhibitor IIを添加した誘導培地(b)(下記)を加えて培養を続けた。その後、9,12,14,16日目に、50 mM ALK5 Inhibitor IIを添加した誘導培地(b)を用いて同様に培地交換を繰り返した。
[誘導培地(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 mLInsulin:170 nM
3,3',5‐Triiodo‐L‐thyronine:1 nM
Rosiglitazone:1 uM
IBMX(3-isobutyl-1-methylxanthine):0.5 mMIndomethacin:62.5 nM
Dexamethasone:1 uM
[誘導培地(b)]
DMEM (High Glucose):500 mL
FOETAL BOVINE SERUM (FBS):50 mL
MEM Non‐Essential Amino Acids Solution:5 mL100 mM‐Sodium Pyruvate Solution:5 mL
Penicillin‐Streptomycin Mixed Solution:5 mLInsulin:170 nM
3,3',5‐Triiodo‐L‐thyronine:1 nM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、Cyclic Pifithrin-αまたはALK5 inhibitor IIを添加した軟骨誘導培地(StemPro Chondrogenesis Differentiation Kit : ThermoFisher Scientific社製)を1000 μL/well加えた。
Cyclic Pifithrin-α:5μM
ALK5 inhibitor II: 2μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、軟骨誘導培地、または2μM ALK5 inhibitor IIを添加した軟骨誘導培地を、1000 μL/well加えた。
この細胞をPBS(-)で2回洗浄したのち3%酢酸溶液で1回洗浄したのちナカライテスク社のPH2.5 アルシアンブルー染色液を加えて1時間、室温で染色した。PBS(-)で3回洗浄したのち顕微鏡で観察した。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104 cells/wellの濃度で12-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、2μM ALK5 inhibitor II と2μM D4476を添加した骨格筋細胞増殖培地(5% FBS, 50 microG/ml Bovine Fetuin, 10 nG/ml hEGF, 1 nG/ml bFGF, 10 microG/ml Insulin, 0.4 microG Dexamethasoneを添加したHam's/F10培地)を1000 μL/well加えた。2日に1度、培養液をフレッシュなものに置換し、day 28まで培養した。
ヒト正常皮膚線維芽細胞株HDFsを、5×103 細胞/ウェルの濃度で24 well plateに播種した(day 0)。翌日、各wellの培養液を棄て、500 μl/wellの新しい培地に交換した。骨芽細胞誘導培地はDulbecco’s modified Eagle’s medium (DMEM)、50 μg/mlアスコルビン酸、10 mM β-glycerophosphate、100 nM Dexamethasoneに10% ウシ胎仔血清(FBS)を添加したものである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
Pifithrin alpha[p53阻害剤]: 5 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2 /95% humid air 37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
Pifithrin alpha[p53阻害剤]: 5 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
Day 14に、各wellから培養液を吸引除去し、PBS(-)で洗浄後、細胞からISOGEN IIにてtotal RNAを抽出した。このRNAから、Rever Tra Ace qPCR RT Master Mixを用いてcDNAを合成した。このcDNAにReal-time PCR Master Mixと、CIDEA遺伝子またはβアクチン遺伝子に特異的なprimersとTaqman probeを混和した。AB7300 Real-time PCR systemを用いてqRT-PCRを行った。CIDEA遺伝子のmRNAレベルをβアクチン遺伝子mRNAに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2 /95% humid air 37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各小分子化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM。
Pifithrin alpha[p53阻害剤]: 5 μM
PD0325901: 1 μM
SB431542: 2 μM
ALK5 Inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを1×104 cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、脂肪細胞誘導培地、または各小分子化合物等を加えた脂肪細胞誘導培地を、500 μL/well加えた。
T3: 1 nM
Rosiglitazone: 1 μM
D4476: 2 μM
SB431541: 2 μM
ALK5 inhibitor II: 2 μM。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト間葉系幹細胞(human mesenchymal stem cells; MSC)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地(Normal medium)、石灰化誘導培地(OB medium)、またはALK5 inhibitor II(ALK5 i II)を4 μMの濃度で加えた石灰化誘導培地(OB medium+ ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIおよび/またはTGF-βを加えた石灰化誘導培地を、500 μL/well加えた。
ALK5 inhibitor II: 4 μM
TGF-β: 50 ng/ml。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを2×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地(Normal medium)、MDI medium(線維芽細胞用の脂肪細胞誘導培地)、またはALK5 inhibitor IIを4 μMの濃度で加えたMDI培地(MDI medium + ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)(Normal medium)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、MDI培地、またはALK5 inhibitor IIを4 μMの濃度で加えたMDI培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度でfibronectin coated 24-well plateの中心部に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、軟骨誘導培地(Chondrocyte medium)、またはALK5 inhibitor IIを4 μMの濃度で加えた軟骨誘導培地(Chondrocyte medium + ALK5 i II)を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)またはヒト脂肪由来間葉系幹細胞(human adipose derived stem cells; ADSCs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度でfibronectin coated 24-well plateの中心部に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、軟骨誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた軟骨誘導培地を、500 μL/well加えた。
ヒト正常歯肉由来線維芽細胞(human gingival fibroblasts; GFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
ヒト正常歯肉由来線維芽細胞(human gingival fibroblasts; GFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 inhibitor IIを4 μMの濃度で加えた石灰化誘導培地を、500 μL/well加えた。
動物実験は所属機関の認可を得て行った。8週齢オスのNOD/SCIDマウス(Charles River)を麻酔した。注水下に歯科ドリルを用いて左大腿骨骨幹に直径約7mmの部分骨欠損を作成した。後述の実施例16と同様の方法で、HDFsをALK5 inhibitor II存在下で13日間培養した細胞(CdOBs; Chemical-mediated directly converted osteoblasts)を、マトリゲル (BD Bioscience, San Jose, CA)とともに懸濁し、骨欠損部とその周辺の骨表面に5×105cells/マウスの濃度で移植した。また、同様に骨欠損を作ったのち、線維芽細胞をマトリゲルに懸濁し移植したマウスも準備した。21日後にマウスを安楽死させ、大腿を切除し、中性ホルマリンで固定後、X-ray CT device (Scan Xmate-L090, Com Scan Techno, Yokohama, Japan)を用いてマイクロ・コンピューター断層撮影(μCT)を行った。
実施例25のμCT像を3次元構築したイメージを図30に示す。ALK5 inhibitor IIを添加して培養した細胞は、生体内で骨形成能を有することが示された。CdOBがマウスの体内で骨欠損部において骨形成を行ったことがわかる。
動物実験は所属機関の認可を得て行った。上記実施例25と同様に移植実験を行った。また、線維芽細胞を移植したマウスも準備した。21日後にマウスを安楽死させ、実施例25と同様に大腿を切除し、中性ホルマリンで固定後、骨組織をSCEM (Leica Microsystem) compoundで包埋し、急速凍結した。6 μmの切片にスライス後、連続切片をヘマトキシリン・エオジン (H&E)染色およびAlizarin Red Sで染色した。
ヒト正常皮膚線維芽細胞株HDFsを、60 mm培養ディシュに培養し、通常培地で培養した(-)。また、HDFsを4 μMのALK5 inhibitor IIを加えた通常培地で3または7日間培養した。これらの細胞からISOGEN IIにてtotal RNAを回収した。各細胞のmRNA発現パターンをAffymetrix 社のDNAチップを用いてゲノムワイドに解析した。MSCマーカーの発現量を表したヒートマップを図32に示す。ALK5 inhibitor IIを添加した培地で培養することにより線維芽細胞が間葉系幹細胞(MSCs)にコンヴァートしたことが分かる。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBSを添加したDulbecco’s modified minimum essential medium; DMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、または各小分子化合物を加えた石灰化誘導培地を、500 μL/well加えた。
I-BET151: 2μM
Pifthrin-α: 5μM
PD0325901:2μM
2-Me-5HT:2μM
CX4945:2μM
CHIR99021:2μM
Forskolin:2μM
DZnep:50nM
D4476:2μM
SB431542:2μM
ALK5 i II:2μM。
実施例29の実験において、Alizarin Red S染色液で染色した後の液を、96 well plateに回収し、吸光度計にて吸光度(OD550nm)を測定した。
結果を図34に示す。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地(OB培地)、または各小分子化合物あるいはサイトカインを添加した石灰化誘導培地を、500 μL/well加えた。
D4476: 1または4 μM
LY2157299: 1または4 μM
LY364947: 1または4 μM
SB431542: 1または4 μM
SB525334: 1または4 μM
SD208: 1または4 μM
ALK5 i II: 1または4 μM
TGF-β: 10 ng/ml。
実施例31の実験において、Alizarin Red S染色液で染色した後の液を、96 well plateに回収し、吸光度計にて吸光度(OD550nm)を測定した。
結果を図36に示す。
さまざまな個人に由来するヒト正常皮膚由来線維芽細胞(HDF45(KURABOより購入)、HDF69(PromoCellより購入)、HDF22(PromoCellより購入))を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、通常培地、石灰化誘導培地、またはALK5 i IIを4μM添加した石灰化誘導培地を、500 μL/well加えた。
さまざまな個人に由来するヒト正常皮膚由来線維芽細胞(HDF45、HDF69、HDF22)を用いて、実施例33と同様の培養を行った。
結果を図38に示す。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図中に記載のとおり、石灰化誘導培地(OB培地)、または各小分子化合物および/またはサイトカインを添加した石灰化誘導培地を、500 μL/well加えた。
ALK5 i II: 4 μM
1α,25-dihydroxy Vitamin D3 (VD3): 5 nM
Human insulin-like growth factor-1 (IGF-1): 100 ng/ml。
Values are means ± S.D. (n=4).)。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBSを添加したDMEM)に縣濁した。これを1×104cells/wellの濃度で24-well plateに播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、またはALK5 i II、VD3、IGF-1を、それぞれ4 μM 、5 nM、100 ng/mlの濃度で添加した石灰化誘導培地を、500 μL/well加えた。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)を通常培地(10% FBS を添加したDulbecco's modified minimum essential medium; DMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図に示すように、群1には通常培地を1 mL/well 加えた。群2にはRosiglitazoneを含まない脂肪細胞誘導培地(脂肪細胞誘導培地(R-))を1 mL/well 加えた。群3には脂肪細胞誘導培地を1 mL/well 加えた。群4にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地(R-)を1 mL/well 加えた。群5~9にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地を1 mL/well加えた。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、群1には通常培地を1 mL/well 加えた(Control)。群2には脂肪細胞誘導培地を1 mL/well 加えた。群3~8にはALK5 inhibitor II を4 μMの濃度で添加した脂肪細胞誘導培地を1 mL/well加えた。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地(BA medium)、または化合物 ALK5 inhibitor II、LY2157299、SB431542、D4476を添加した脂肪細胞誘導培地を、1mL/well 加えた。化合物の添加濃度は4 μM、8 μM、12 μM、または16 μMである。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9 - Day 14までは、ALK5 inhibitor II、LY2157299、SB431542、D4476をいずれも含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(-)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、UCP-1遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。UCP1 遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、図のように群1には通常培地を、群2には脂肪細胞誘導培地を、群3~6には化合物ALK5 inhibitor II (4 μM)、LY2157299 (8 μM)、SB431542 (4 μM)、D4476 (4 μM)をそれぞれ加えた脂肪細胞誘導培地を、1mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9からDay 14は、ALK5 inhibitor II、LY2157299、SB431542、D4476 をいずれも含まない脂肪細胞誘導培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(-)で洗浄した。4%パラホルムアルデヒドで固定後、PBS(-)にて洗浄し、Perm Buffer(0.2% Triton-X 添加PBS)を加えて15 分間インキュベートした。PBS(-)にて3 回洗浄した後、Blocking One を加えて、室温で60 分間インキュベートした。抗UCP-1 抗体(RD MAB6158)を加えて室温で2 時間反応させた後、Wash buffer にて3 回wash した。CF488-conjugated anti-mouse Ig 抗体(Biotum 20014)を加えて室温で2 時間反応させた後、PBS (-) にて3 回wash した。Lifetechnology社製SlowFade Gold antifade reagent with DAPIで核染色したのち蛍光顕微鏡を用いて倍率100 倍で写真撮影を行った。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地、あるいは化合物 ALK5 inhibitor II(4μM)またはLY2157299(8μM)を加えた脂肪細胞誘導培地を、1 mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および 10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9 - Day 14までは、ALK5 inhibitor IIもLY2157299を含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(-)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、UCP-1遺伝子、CIDEA遺伝子、KCNK3遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。UCP1 遺伝子のmRNA レベルをβ アクチン遺伝子mRNAに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
ヒト正常皮膚由来線維芽細胞(HDFs)を通常培地(10% FBS を添加したDMEM)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、通常培地、脂肪細胞誘導培地、またはALK5 inhibitor IIを、4 μMの濃度で加えた脂肪細胞誘導培地を、1 mL/well 加えた。脂肪細胞誘導培地は、(1 nM T3, 1 μM Rosiglitazone, 0.5 mM 3-isobutyl-1-methylxanthine(IBMX)、0.5 μM dexamethasone、1 μg/mL Insulin および10% FBSを添加したDMEM)である。2 日に1 度、培養液をフレッシュなものに置換し、Day 1 - Day 8まで培養した。その後、Day 9 - Day 14までは、ALK5 inhibitor IIを含まない培地で培養した。Day 14 に、各well から培養液を吸引除去し、PBS(-)で洗浄した。4%パラホルムアルデヒドで固定後、PBS(-)にて洗浄し、Perm Buffer(0.2% Triton-X 添加PBS)を加えて15 分間インキュベートした。PBS(-)にて3 回洗浄した後、Blocking One を加えて、室温で60 分間インキュベートした。抗UCP-1 抗体(RD MAB6158)を加えて室温で2 時間反応させた後、Wash buffer にて3 回wash した。CF488-conjugated anti-mouse Ig 抗体(Biotum 20014)を加えて室温で2 時間反応させた後、PBS(-) にて3 回wash した。Lifetechnology社製SlowFade Gold antifade reagent with DAPIで核染色したのち蛍光顕微鏡を用いて倍率100 倍で写真撮影を行った。撮影した画像をKeyence BZ-H3A softwareを用いてDAPI陽性細胞中のUCP1陽性細胞のパーセンテージを算出した。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地(Ctrl medium)、K-脂肪細胞誘導培地(ケラチノサイト用脂肪細胞誘導培地)、または、ALK5 inhibitor II(4 μM)あるいはLY2157299(8 μM)を添加したK-脂肪細胞誘導培地を、1mL/well 加えた。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地(Ctrl medium)、K-骨芽細胞誘導培地(ケラチノサイト用骨芽細胞誘導培地)、または化合物 ALK5 inhibitor II(4 μM)を添加したK-骨芽細胞誘導培地を、1mL/well 加えた。K-骨芽細胞誘導培地は、50 μg/mL ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone を添加したHuMedia-KG2である。2 日に1 度、培養液をフレッシュなものに置換し、 Day 14まで培養した。その後、Day 14 - Day 28までは、いずれの細胞もALK5 inhibitor IIを含まない培地で培養した。Day 28 に、各well から培養液を吸引除去し、PBS(-)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、Runx2遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。Runx2遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、通常培地で培養した線維芽細胞の値を1として算出した。
実施例45 (図49)
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、図中に記載の培地を加えた。すなわち、
群1:Control培地
群2:WA培地
群3:1 μM Rosiglitazoneを添加したWA培地
群4:16 μMのALK5 inhibitor IIを添加したWA培地
群5:16 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
Control培地は、10% FBS、100 mM non-essential amino acids (NEAA), 100 U/ml penicillin, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEMである。
WA培地は、10% FBS、100 mM non-essential amino acids (NEAA), 100 U/ml penicillin, 100 μg/mL streptomycin、0.5 mM IBMX(3-isobutyl-1-methylxanthine)、62.5 nM Indomethacin、1 μM Dexamethasone、および170 nM Insulinを添加したDulbecco’s modified minimum essential medium; DMEMである。
(1)ウェル内の培養液を除去後、PBSで各ウェルを1回洗浄。
(2)各ウェルに4%ホルムアルデヒド液を50μL加え、室温で一晩固定。
(3)PBSで2 回洗浄。
(4)各ウェルにBODIPY染色液を50μLずつ加え、室温で30 分間静。
(5)ウェル内から染色液を除去後、室温で30 分間静置。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを7群に分け、図中に記載の培地を加えた。すなわち、
群1:Control培地
群2:WA培地
群3:1 μM Rosiglitazoneを添加したWA培地
群4:8 μMのALK5 inhibitor IIを添加したWA培地
群5:16 μMのALK5 inhibitor IIを添加したWA培地
群6:8 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
群7:16 μMのALK5 inhibitor II と1 μM Rosiglitazoneを添加したWA培地。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、実施例45と同様に図中に記載の培地を加えた。培地の組成は実施例45と同じである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で48-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを10群に分け、図中に記載の培地を加えた。すなわち、
群1:WA培地
群2:16 μMのALK5 inhibitor II(ALK5 i II)を添加したWA培地
群3:16 μMのLY2157299を添加したWA培地
群4:16 μMのSB431542を添加したWA培地
群5:16 μMのD4476を添加したWA培地
群6:1 μM Rosiglitazoneを添加したWA培地。
群7:16 μMのALK5 inhibitor II (ALK5 i II)と1 μM Rosiglitazoneを添加したWA培地。
群8:16 μMのLY2157299と1 μM Rosiglitazoneを添加したWA培地。
群9:16 μMのSB431542と1 μM Rosiglitazoneを添加したWA培地。
群10:16 μMのD4476と1 μM Rosiglitazoneを添加したWA培地。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを10群に分け、実施例48と同様に、図中に記載の培地を加えた。培地の組成は実施例45と同じである。
ヒト正常皮膚由来線維芽細胞(human dermal fibroblasts; HDFs)をControl培地に縣濁した。これを104 cells/mm2の濃度で6-well plateに播種し(第 0日)、5% CO2/95% humidified air、37℃で培養を開始した。第4日に培養上清を吸引除去し、ウェルを5群に分け、図中に記載の培地を加えた。すなわち、
群1:1 μM Rosiglitazone を添加したWA培地
群2:1 μM Rosiglitazone と16 μMのALK5 inhibitor II(ALK5 i II)を添加したWA培地
群3:1 μM Rosiglitazone と16 μMのLY2157299(LY)を添加したWA培地
群4:1 μM Rosiglitazone と16 μMのSB431542(SB)を添加したWA培地
群5:1 μM Rosiglitazone と16 μMのD4476(D4)を添加したWA培地。
ヒト線維芽細胞aHDF45を24ウェルプレートに1.0×104 cells/500 μL/wellずつ播種した。翌日、ウェルを2群に分け、1群は培地を下記のControl培地に交換し、別の1群は4 μMのAlk5 i IIを添加した下記のSC培地に交換した。37℃、5% CO2/95% 大気下で14日間培養した。培地交換は週2回行った。
Control培地:10% FBS、100 mM non-essential amino acids (NEAA), 1 mM Sodium Pyruvate、100 U/ml penicillin, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEM。
SC(Schwann cell)培地: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, および 100 μg/mL streptomycinを添加したDulbecco’s modified minimum essential medium; DMEM。
正常ヒト表皮角化細胞(Human Epidermal Keratinocyte ; NHEK(AD))を正常ヒト表皮角化細胞増殖用無血清液体培地(HuMedia-KG2 ; Kurabo社製)に縣濁した。これを 3×104 cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、HuMedia-KG2培地、ヒト表皮基底細胞増殖用培地(CnT-PR;CELLnTEC社製)、またはALK5 inhibitor IIを1 μMまたは10 μMの濃度で添加したヒト表皮基底細胞増殖様培地を、それぞれ1 mL/well 加えた。2 日に1 度、培養液をフレッシュなものに置換し、day 21まで培養した。Day 21 に各well から培養液を吸引除去し、PBS(-)で洗浄後、細胞からQiagen社製 RNA easy Mini Kitを用いてtotal RNA を抽出した。このRNA から、Rever Tra Ace qPCR RT Master Mix を用いてcDNA を合成した。このcDNA にReal-time PCR Master Mix と、Uroplakin 1b遺伝子、Uroplakin 3b遺伝子またはβ アクチン遺伝子に特異的なprimers とTaqman probe を、混和した。AB7300 Real-time PCR system を用いてqRT-PCR を行った。Uroplakin 1b 遺伝子とUroplakin 3b遺伝子のmRNA レベルをβ アクチン遺伝子mRNAレベルに対する比として定量し、HuMedia-KG2培地で培養したヒト表皮角化細胞の値を1として算出した。その結果を図56に示す。ALK5 inhibitor IIを添加したCnT-PR培地で培養することにより、ヒト表皮角化細胞が、Uroplakin 1b遺伝子とUroplakin 3b遺伝子を発現する尿路上皮細胞にコンヴァートしたことがわかる。
ヒト正常末梢血単核細胞(human peripheral blood mononuclear cells; PBMCs)をcontrol medium(10% FBS を添加したRPMI1640培地)に縣濁した。これを 2.5×105cells/well の濃度で12-well plate に播種し(Day 0)、5% CO2/95% humidified air、37℃で培養を開始した。翌日、培養上清を吸引除去し、群1にはControl mediumを1 mL/well 加えた。群2にはP-脂肪細胞誘導培地(末梢血単核細胞用脂肪細胞誘導培地)を1 mL/well加えた。群3にはALK5 inhibitor II を4 μMの濃度で添加したP-脂肪細胞誘導培地を1 mL/well加えた。P-脂肪細胞誘導培地は、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および1 μg/mL Insulinを添加したRPMI1640培地である。2 日に1 度、培養液の50%をフレッシュなものに置換した。
Claims (15)
- 哺乳動物の分化した体細胞を、前記分化した体細胞以外の他の体細胞を分化誘導するための培地中で、TGF-βパスウェイ阻害剤の存在下に培養して、前記分化した体細胞を他の体細胞にコンヴァートさせることを特徴とする、体細胞を調製する方法。
- TGF-βパスウェイ阻害剤が、D4476、SB431542、LY2157299、SD208、又はALK5 inhibitor IIである、請求項1に記載の方法。
- TGF-βパスウェイ阻害剤が、ALK5 inhibitor IIである、請求項1又は2に記載の方法。
- 線維芽細胞を間葉系の細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 線維芽細胞又はケラチノサイトを骨芽細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 線維芽細胞又は末梢血単核細胞を白色脂肪細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 線維芽細胞又はケラチノサイトを褐色脂肪細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 体細胞を分化誘導するための培地が、Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ)アゴニストを含む、請求項6又は7に記載の方法。
- 線維芽細胞を軟骨細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 線維芽細胞を筋芽細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項記載の方法。
- 線維芽細胞をシュワン細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- ケラチノサイトを尿路上皮細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 線維芽細胞を間葉系幹細胞にコンヴァートさせることを特徴とする、請求項1~3のいずれか1項に記載の方法。
- TGF-βパスウェイ阻害剤を含む、分化した体細胞を他の体細胞にコンヴァートさせるための誘導剤。
- 分化した体細胞を他の体細胞にコンヴァートさせるためのキットであって、TGF-βパスウェイ阻害剤及び前記他の体細胞を分化誘導するための培地を含むキット。
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US20190024054A1 (en) | 2019-01-24 |
US11851681B2 (en) | 2023-12-26 |
JP7228217B2 (ja) | 2023-02-24 |
JP2022185031A (ja) | 2022-12-13 |
JP7475723B2 (ja) | 2024-04-30 |
JPWO2017069222A1 (ja) | 2018-08-16 |
US20240124842A1 (en) | 2024-04-18 |
CN109072183A (zh) | 2018-12-21 |
KR20180073631A (ko) | 2018-07-02 |
EP3378933A4 (en) | 2019-04-03 |
CN109072183B (zh) | 2023-01-20 |
EP3378933A1 (en) | 2018-09-26 |
CN116024164A (zh) | 2023-04-28 |
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