Musculoskeletal stem cell and medium for inducing differentiation of musculoskeletal stem cell

Abstract

The present disclosure relates to a novel musculoskeletal stem cell (MSSC) differentiated from an ESC (embryonic stem cell) or an iPSC (induced pluripotent stem cell). The musculoskeletal stem cell of the present disclosure can be easily induced from a human embryonic stem cell or a human-derived pluripotent stem cell and can be effectively differentiated not only into bone but also into cartilage, tendon and muscle. Accordingly, it can be usefully used for prevention or treatment of various musculoskeletal diseases.

What is claimed is: 
     
       1. A method of preparing musculoskeletal stem cells (MSSCs), the method comprising:
 culturing cells comprising embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in a culture medium composition, 
 wherein the culture medium composition comprises noggin, LIF (leukemia inhibitory factor), bFGF (basic fibroblast growth factor), Wnt signaling activator, ERK (extracellular signal-regulated kinase) signaling inhibitor and TGF-β/activin/nodal signaling inhibitor, 
 wherein the cells, after the culturing, are MSSCs and exhibit the following characteristics:
 positive for nestin (NES); 
 positive for Pax7; 
 positive for α-SMA; 
 negative for LIN28; and 
 negative for CD90. 
 
 
     
     
       2. The method of  claim 1 , wherein the cells cultured in the culture medium composition comprise ESCs and do not comprises iPSCs. 
     
     
       3. The method of  claim 1 , wherein the cells cultured in the culture medium composition comprise iPSCs and do not comprises ESCs. 
     
     
       4. The method of  claim 1 , wherein the cells cultured in the culture medium composition comprise both iPSCs and ESCs. 
     
     
       5. The method of  claim 1 , wherein the Wnt signaling activator comprises at least one selected from the group consisting of SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), kenpaullone (9-bromo-7,12-dihydro-indolo[3,2-d]-[1]benzazepin-6(5H)-one), CHIR99021 (9-bromo-7,12-dihydro-pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one), CP21R7 (3-(3-amino-phenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), H-89 (5-isoquinolinesulfonamide), purmorphamine (2-(1-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine) and IQ-1 (2-(4-acetyl-phenylazo)-2-[3,3-dimethyl-3,4-dihydro-2H-isoquinolin-(1E)-ylidene]-acetamide). 
     
     
       6. The method of  claim 1 , wherein the ERK signaling inhibitor comprises at least one selected from the group consisting of AS703026 (N-[(2S)-2,3-dihydroxypropyl]-3-[(2-fluoro-4-iodophenyl)amino]-isonicotinamide), AZD6244 (6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), ARRY-438162 (5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide), RDEA119 ((S)—N-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-6-methoxyphenyl)-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide), GDC0973 ([3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-3-hydroxy-3-[(2S)-piperidin-2-yl]-azetidin-1-yl-methanone), TAK-733 ((R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione), RO5126766 (3-[[3-fluoro-2-(methylsulfamoylamino)-4-pyridyl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one) and XL-518 ([3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-[(2S)-2-piperidinyl]-1-azetidinyl]methanone). 
     
     
       7. The method of  claim 1 , wherein the TGF-3/activin/nodal TGF-β/activin/nodal signaling inhibitor comprises at least one selected from the group consisting of E-616452 (2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine), A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) and SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide). 
     
     
       8. The method of  claim 1 , wherein the culturing is performed for at least 5 passages without changing the ingredients of the culture medium composition. 
     
     
       9. The method of  claim 1 , wherein the culturing is performed for 5 to 25 passages without changing the ingredients of the culture medium composition. 
     
     
       10. The method of  claim 8 , wherein the culturing is performed while maintaining the concentrations of the ingredients in the culture medium composition. 
     
     
       11. The method of  claim 1 , wherein the cells, after the culturing, further have a characteristic of positive for CD146. 
     
     
       12. The method of  claim 1 , wherein the cells, after the culturing, further have at least one of the following characteristics:
 negative for OCT4; 
 negative for NANOG; 
 negative for SOX2; and 
 negative for LIN28. 
 
     
     
       13. The method of  claim 12 , wherein the cells, after the culturing, further have a characteristic of negative for CD271. 
     
     
       14. The method of  claim 12 , wherein the cells, after the culturing, are configured to differentiate into mesoderm but not into ectoderm or endoderm. 
     
     
       15. The method of  claim 12 , wherein the cells, after the culturing, are configured to differentiate into muscle, bone, cartilage, tendon, or ligament. 
     
     
       16. The method of  claim 12 , wherein the cells, after the culturing, are not configured to differentiate into a nerve cell. 
     
     
       17. The method of  claim 12 , wherein the cells, after the culturing, are not configured to differentiate into an endothelial cell. 
     
     
       18. The method of  claim 12 , wherein the cells, after the culturing, comprise a cell deposited under the accession number KCLRF-BP-00460. 
     
     
       19. A method of in vitro differentiation of MSSCs, the method comprising,
 performing the method of  claim 1 , wherein the culture medium is referred to as a first culture medium; 
 collecting MSSCs from the cell composition; 
 in vitro culturing the collected MSSCs in a second culture medium; and 
 collecting cells differentiated from at least part of the MSSCs. 
 
     
     
       20. A method of preparing musculoskeletal cells, the method comprising:
 performing the method of  claim 1 , wherein the culture medium is referred to as a first culture medium; 
 collecting MSSCs from the cell composition; 
 in vitro culturing the collected MSSCs in a second culture medium; and 
 transplanting at least part of the MSSCs into a mass of tissues such that the at least part of the MSSCs differentiates into musculoskeletal cells in the mass of tissues. 
 
     
     
       21. A method of treating a musculoskeletal disease, the method comprising:
 performing the method of  claim 1 , wherein the culture medium is referred to as a first culture medium; 
 collecting MSSCs from the cell composition; and 
 administering, to a subject in need to such treatment, an effective amount of the collected MSSCs. 
 
     
     
       22. The method of  claim 2 , wherein the cells, after the culturing, further have the following characteristics:
 negative for OCT4; 
 negative for NANOG; 
 negative for SOX2; and 
 negative for LIN28. 
 
     
     
       23. The method of  claim 1 , wherein the cells, after the culturing, further have at least one of the following characteristics:
 negative for TDGF; 
 negative for NANOG; 
 negative for POU5F1; 
 negative for LEFTY1; and 
 negative for GDF3. 
 
     
     
       24. The method of  claim 1 , wherein the cells, after the culturing, further have at least one of the following characteristics:
 positive for CD44; 
 positive for CD51; 
 positive for CD73; 
 positive for CD105; 
 positive for CD146; 
 positive for CD166; 
 negative for CD90; and 
 negative for CD271. 
 
     
     
       25. The method of  claim 1 , wherein the cells, after the culturing, further have the following characteristics:
 positive for CD10; 
 negative for CD2; 
 negative for CD3; 
 negative for CD7; 
 negative for CD8; 
 negative for CD14; 
 negative for CD20; and 
 negative for CD56. 
 
     
     
       26. The method of  claim 1 , wherein the culturing is performed for at least 7 passages without changing the ingredients of the culture medium composition. 
     
     
       27. The method of  claim 1 , wherein the culturing is performed for at least 10 passages without changing the ingredients of the culture medium composition. 
     
     
       28. The method of  claim 1 , wherein the culturing is performed for 7 passages to 17 passages without changing the ingredients of the culture medium composition. 
     
     
       29. A musculoskeletal stem cell having the following characteristics:
 positive for nestin (NES);   positive for Pax7;   positive for α-SMA;   positive for CD146;   negative for LIN28; and   negative for CD90.   
     
     
       30. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for OCT4;   negative for NANOG; and   negative for SOX2.   
     
     
       31. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for TDGF;   negative for NANOG;   negative for POU5F1;   negative for LEFTY1; and   negative for GDF3.   
     
     
       32. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD44;   positive for CD51;   positive for CD73;   positive for CD105; and   positive for CD166.   
     
     
       33. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD10;   negative for CD2;   negative for CD3;   negative for CD7;   negative for CD8;   negative for CD14;   negative for CD20; and   negative for CD56.   
     
     
       34. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for DPPA4;   negative for mesodermal markers T and nodal;   positive for Pax6;   positive for LGR5;   negative for SOX9;   negative for MyoD;   positive for CD10;   positive for CD44;   positive for CD105; and   positive for CD166.   
     
     
       35. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell is configured to differentiate into muscle, bone, cartilage, tendon or ligament, wherein the musculoskeletal stem cell is not configured to differentiate into a nerve cell or an endothelial cell. 
     
     
       36. A composition comprising:
 the musculoskeletal stem cell of Claim 29; and   at least one selected from the group consisting of noggin, LIF (leukemia inhibitory factor), bFGF (basic fibroblast growth factor), Wnt signaling activator, ERK (extracellular signal-regulated kinase) signaling inhibitor and TGF-β/activin/nodal signaling inhibitor.   
     
     
       37. The composition of  Claim 36 , further comprising at least one of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). 
     
     
       38. The composition of  Claim 36 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for OCT4;   negative for NANOG; and   negative for SOX2.   
     
     
       39. The composition of  Claim 36 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for TDGF;   negative for NANOG;   negative for POU5F1;   negative for LEFTY1; and   negative for GDF3.   
     
     
       40. The composition of  Claim 36 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD44;   positive for CD51;   positive for CD73;   positive for CD105; and   positive for CD166.   
     
     
       41. The composition of  Claim 36 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD10;   negative for CD2;   negative for CD3;   negative for CD7;   negative for CD8;   negative for CD14;   negative for CD20; and   negative for CD56.   
     
     
       42. The composition of  Claim 36 , wherein the musculoskeletal stem cell is configured to differentiate into muscle, bone, cartilage, tendon or ligament, wherein the musculoskeletal stem cell is not configured to differentiate into a nerve cell or an endothelial cell. 
     
     
       43. The composition of  Claim 36 , comprising noggin, LIF (leukemia inhibitory factor), bFGF (basic fibroblast growth factor), Wnt signaling activator, ERK (extracellular signal-regulated kinase) signaling inhibitor and TGF-β/activin/nodal signaling inhibitor. 
     
     
       44. A composition comprising:
 the musculoskeletal stem cell of Claim 29; and   a pharmaceutically acceptable carrier.   
     
     
       45. The composition of  Claim 44 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for OCT4;   negative for NANOG; and   negative for SOX2.   
     
     
       46. The composition of  Claim 44 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 negative for TDGF;   negative for NANOG;   negative for POU5F1;   negative for LEFTY1; and   negative for GDF3.   
     
     
       47. The composition of  Claim 44 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD44;   positive for CD51;   positive for CD73;   positive for CD105; and   positive for CD166.   
     
     
       48. The composition of  Claim 44 , wherein the musculoskeletal stem cell further has at least one of the following characteristics:
 positive for CD10;   negative for CD2;   negative for CD3;   negative for CD7;   negative for CD8;   negative for CD14;   negative for CD20; and   negative for CD56.   
     
     
       49. The composition of  Claim 44 , wherein the pharmaceutical acceptable carriers is one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. 
     
     
       50. The composition of  Claim 44 , wherein the musculoskeletal stem cell is configured to differentiate into muscle, bone, cartilage, tendon or ligament, wherein the musculoskeletal stem cell is not configured to differentiate into a nerve cell or an endothelial cell. 
     
     
       51. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristic: positive for CD10. 
     
     
       52. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristic: positive for CD44. 
     
     
       53. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristic: positive for CD51. 
     
     
       54. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristic: positive for CD73. 
     
     
       55. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristics: positive for CD105. 
     
     
       56. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristics: positive for CD166. 
     
     
       57. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristics: positive for DPPA. 
     
     
       58. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristics: positive for Pax6. 
     
     
       59. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell further has the following characteristics: positive for LGR5. 
     
     
       60. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell is differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). 
     
     
       61. The musculoskeletal stem cell of  Claim 29 , wherein the musculoskeletal stem cell is configured to differentiate into musculoskeletal tissues in the absence of signaling or stimulation by mesenchymal stem cells for the differentiation into musculoskeletal tissues.