Method for producing cartilage cells induced to be differentiated from stem cells

Abstract

The present invention relates to a method for inducing differentiation, into chondrocytes, of cord blood mononuclear cell-derived induced pluripotent stem cells. In a case where a chondrogenic pellet produced by the method of the present invention is transplanted into a cartilage damage area in vivo, regeneration of cartilage can be effectively exhibited by differentiated chondrocytes. In such a case, an effective cartilage regeneration capacity can be exhibited as compared with a case where chondrocytes produced by differentiation induction with the addition of a recombinant growth factor are transplanted. Thus, the present invention can be usefully used for tissue engineering therapies.

1 . A method for producing chondrocytes obtained by differentiation induction from stem cells, comprising:
 i) culturing induced pluripotent stem cells (iPSCs) to generate embryoid bodies (EBs);   ii) culturing the EBs generated in step i) in a gelatin-coated medium, to obtain outgrowth cells (OG cells);   iii) transducing the OG cells obtained in step ii) with either or both of a minicircle vector that contains a base sequence encoding BMP2 and a minicircle vector that contains a base sequence encoding TGFβ3;   iv) inducing differentiation of the OG cells transduced in step iii) into chondrocytes; and   v) obtaining the chondrocytes produced by differentiation induction in step iv).   
     
     
         2 . A method for producing chondrocytes obtained by differentiation induction from stem cells, comprising:
 i) culturing iPSCs to generate EBs;   ii) culturing the EBs generated in step i) in a gelatin-coated medium, to obtain OG cells;   iii) transducing the OG cells obtained in step ii) with a minicircle vector that contains a base sequence encoding BMP2;   iv) transducing the OG cells obtained in step ii) with a minicircle vector that contains a base sequence encoding TGFβ3;   v) performing mixed culture of the OG cells transduced in step iii) and the OG cells transduced in step iv), so that the OG cells are induced to differentiate into chondrocytes; and   vi) obtaining the chondrocytes produced by differentiation induction in step v).   
     
     
         3 . The method according to  claim 1 ,
 wherein the minicircle vector that contains a base sequence encoding BMP2 is a non-viral vector, the non-viral vector,   (a) containing a gene expression cassette that contains a CMV promoter, a BMP2 gene consisting of the base sequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence;   (b) containing the att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); and   (c) not containing a replication origin and an antibiotic resistance gene.   
     
     
         4 . The method according to  claim 1 ,
 wherein the minicircle vector that contains a base sequence encoding TGFβ3 is a non-viral vector, the non-viral vector,   (a) containing a gene expression cassette that contains a CMV promoter, a TGFβ3 gene consisting of the base sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence;   (b) containing the att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); and   (c) not containing a replication origin and an antibiotic resistance gene.   
     
     
         5 . The method according to  claim 1 ,
 wherein the step of inducing differentiation of the OG cells into chondrocytes is performed by culturing the OG cells in a medium containing no recombinant growth factor for 3 to 30 days.   
     
     
         6 . A chondrocyte produced by the method of  claim 1 . 
     
     
         7 . A chondrocyte produced by the method of  claim 2 . 
     
     
         8 . The method according to  claim 2 ,
 wherein the minicircle vector that contains a base sequence encoding BMP2 is a non-viral vector, the non-viral vector,   (a) containing a gene expression cassette that contains a CMV promoter, a BMP2 gene consisting of the base sequence of SEQ ID NO: 1, and an SV40 polyadenylation sequence;   (b) containing the att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); and   (c) not containing a replication origin and an antibiotic resistance gene.   
     
     
         9 . The method according to  claim 2 ,
 wherein the minicircle vector that contains a base sequence encoding TGFβ3 is a non-viral vector, the non-viral vector,   (a) containing a gene expression cassette that contains a CMV promoter, a TGFβ3 gene consisting of the base sequence of SEQ ID NO: 2, and an SV40 polyadenylation sequence;   (b) containing the att attachment sequence of bacteriophage lambda, located outside the gene expression cassette of (a); and   (c) not containing a replication origin and an antibiotic resistance gene.   
     
     
         10 . The method according to  claim 2 , wherein the step of inducing differentiation of the OG cells into chondrocytes is performed by culturing the OG cells in a medium containing no recombinant growth factor for 3 to 30 days.