Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements.
Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.
Wang
Chien-Yuan
CY
Institute of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan.
Chen
Ling-Lan
LL
Kuo
Pei-Yin
PY
Chang
Jia-Ling
JL
Wang
Yng-Jiin
YJ
Hung
Shih-Chieh
SC
D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002452:Cell Count; D020219:Chondrogenesis / Q000187:drug effects*; D003470:Culture Media / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D044967:Serum / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D015415:Biomarkers; D003470:Culture Media; D016207:Cytokines; D018836:Inflammation Mediators; D051028:bcl-2-Associated X Protein; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3