Method for producing cartilage cells induced to be differentiated from stem cells
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 obtain embryoid bodies, wherein the induced pluripotent stem cells of step i) are obtained by reprogramming cord blood mononuclear cells;
ii) performing adherent culture of the embryoid bodies obtained in step i), to obtain outgrowth cells (OG cells);
iii) performing centrifugation of the OG cells obtained in step ii) so that the OG cells are isolated by sizes, and selecting light OG cells;
iv) inducing differentiation of the light OG cells selected in step iii) into chondrocytes; and
v) obtaining the chondrocytes produced by differentiation induction in step iv),
wherein the centrifugation and selection in step iii) is performed through the following steps a) to c):
a) centrifuging a medium containing the OG cells at 300 rpm to 800 rpm for 3 to 10 seconds, to classify precipitated cells as heavy OG cells;
b) centrifuging the supernatant after the centrifugation in step a) at 800 rpm to 1,200 rpm for 3 to 10 seconds, to classify precipitated cells as medium OG cells; and
c) centrifuging the supernatant after the centrifugation in step b) at 1,200 rpm to 2,000 rpm for 3 to 10 seconds, to classify precipitated cells as the light OG cells, and
wherein the light OG cells exhibit higher expression level of SOX9 and lower expression level of COL10 than the heavy OG cells and the medium OG cells.
2. The method according to claim 1 ,
wherein the adherent culture in step ii) is performed by culturing the OG cells on a gelatin-coated plate.
3. The method according to claim 1 ,
wherein the inducing differentiation in step iv) is performed in a medium containing human bone morphogenetic protein 2 and human transforming growth factor beta 3.
4. The method according to claim 3 ,
wherein the medium is additionally supplemented with an IGF2 inhibitor.