Ceramidase and cell differentiation

Abstract

The present invention relates to methods of producing chondrocytes, improving the phenotype of a chondrocyte population, promoting chondrogenesis, maintaining a cell population in a differentiated state or increasing the number of cells of a population in a differentiated state, producing a population of differentiated cells, and treating certain diseases or disorders. Each of these methods involves treating a cell population with, and/or culturing a cell population in, a ceramidase

1 . An in vivo method of producing chondrocytes, said method comprising:
 selecting a population of mammalian cells having the potential to differentiate into chondrocytes, and   treating the selected cell population with a ceramidase to transform one or more of the cells in the selected population into chondrocytes.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method according to  claim 1 , wherein the cells are human cells. 
     
     
         5 . The method according to  claim 1 , wherein the selected cell population comprises a population of bone marrow cells, mesenchymal stem cells, and/or fibroblasts. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The method according to  claim 5 , wherein the cells are human cells. 
     
     
         9 . The method according to  claim 8 , wherein the selected cell population comprises bone marrow cells. 
     
     
         10 . The method according to  claim 5 , wherein the selected cell population comprises mesenchymal stem cells. 
     
     
         11 . The method according to  claim 10 , wherein the selected cell population consists essentially of mesenchymal stem cells. 
     
     
         12 . The method according to  claim 10  further comprising:
 treating the selected cell population with TGFβ together with the ceramidase. 
 
     
     
         13 . The method according to  claim 5 , wherein the selected cell population comprises fibroblasts. 
     
     
         14 . The method according to  claim 13 , wherein the selected cell population consists essentially of fibroblasts. 
     
     
         15 . The method according to  claim 1 , wherein said treating comprises:
 culturing the selected cell population in a culture medium containing the ceramidase.   
     
     
         16 - 21 . (canceled) 
     
     
         22 . The method according to  claim 1  further comprising:
 treating the cells with TGFβ. 
 
     
     
         23 . A method of improving the phenotype of a chondrocyte population, said method comprising:
 selecting a population of mammalian chondrocytes characterized by low expression of collagen 2A1, Sox9, and aggrecan; elevated expression of collagen 10; low staining for proteoglycans with Alcian Blue or Safranin O; and/or low expression of other chondrocyte-specific growth factors, and   treating the selected cell population with a ceramidase; wherein, after said treating the selected population is characterized by elevated expression of collagen 2A1, Sox9, and aggrecan; low expression of collagen 10; enhanced staining for proteoglycans with Alcian Blue or Safranin O; and/or elevated expression of other chondrocyte-specific growth factors.   
     
     
         24 - 25 . (canceled) 
     
     
         26 . The method according to  claim 23 , wherein the chondrocytes are human chondrocytes. 
     
     
         27 . The method according to  claim 23 , wherein the chondrocytes are primary chondrocytes. 
     
     
         28 . The method according to  claim 23 , wherein the chondrocytes are articular chondrocytes, nasal chondrocytes, tracheal chondrocytes, meniscal chondrocytes, or aural chondrocytes. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method according to  claim 23 , wherein said treating comprises:
 culturing the selected cell population in a culture medium containing the ceramidase.   
     
     
         32 - 33 . (canceled) 
     
     
         34 . The method according to  claim 23 , wherein said treating is carried out on a solid support. 
     
     
         35 . (canceled) 
     
     
         36 . The method according to  claim 34 , wherein the solid support is a three-dimensional tissue scaffold. 
     
     
         37 . The method according to  claim 34 , wherein the solid support is a hydrogel. 
     
     
         38 - 99 . (canceled) 
     
     
         100 . A method of treating a joint disease or disorder, said method comprising:
 selecting a mammalian subject having a joint disease or disorder, and   administering a population of chondrocytes produced according to the method of  claim 1  to the subject to treat the joint disease or disorder.   
     
     
         101 . The method according to  claim 100 , wherein the joint disease or disorder is selected from the group consisting of osteoarthritis, Rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, and joint injury. 
     
     
         102 - 119 . (canceled) 
     
     
         120 . The method according to  claim 129 , wherein said administering the population of chondrocytes comprises:
 injecting the population of chondrocytes into the subject.   
     
     
         121 . The method according to  claim 129 , wherein said administering the population of chondrocytes comprises:
 performing said culturing in vivo to produce the differentiated cells within the subject.   
     
     
         122 . (canceled) 
     
     
         123 . The method according to  claim 121 , wherein said treating comprises:
 administering the ceramidase to the subject at a tissue site containing a population of stem cells capable of differentiating into chondrocytes.   
     
     
         124 . The method according to  claim 123 , wherein said administering comprises:
 implanting in the subject a tissue scaffold having the ceramidase disposed thereon and/or therein.   
     
     
         125 - 128 . (canceled) 
     
     
         129 . A method of treating a joint disease or disorder, said method comprising:
 selecting a mammalian subject having a joint disease or disorder, and   administering a population of chondrocytes produced according to the method of  claim 23  to the subject to treat the joint disease or disorder.   
     
     
         130 . The method according to  claim 129 , wherein the joint disease or disorder is selected from the group consisting of osteoarthritis, Rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, and joint injury. 
     
     
         131 . The method according to  claim 10 , wherein the mesenchymal stem cells are derived from bone marrow. 
     
     
         132 . The method of  claim 1 , wherein the ceramidase comprises an amino acid sequence selected from the group consisting of UniProt Q13510, UniProt Q9H715, UniProt Q96AS2, OMIM 228000, NCBI Gene 427, NCBI RefSeq NP 808592, NCBI RefSeq NP_004306, NCBI RefSeq NM_177924, NCBI RefSeq NM_004315, NCBI UniGene 427, NCBI Accession Q13510, NCBI Accession AAC73009, and a combination thereof. 
     
     
         133 . The method of  claim 1 , wherein the ceramidase comprises an amino acid sequence UniProt Q13510. 
     
     
         134 . The method of  claim 1 , wherein the ceramidase comprises an amino acid sequence NCBI RefSeq NP_808592.