Chondrogenic differentiation of mesenchymal stem cells isolated from patients in late adulthood: the optimal conditions of growth factors.
There is a controversy about the capacity of the mesenchymal stem cells (MSCs) from aged individuals to proliferate and differentiate into cartilage. The purpose of this study was to investigate the optimal condition to culture human MSCs from the aged individuals (>50 years) for cartilage tissue engineering. We tested the hypothesis that effective proliferation and chondrogenesis can be achieved with human MSCs from aged individuals under appropriate conditions. To investigate the best condition for proliferation, MSCs were cultured in medium containing four concentrations subsets (0, 0.05, 0.5, 5 ng/mL) of recombinant human TGF-beta2 and FGF-2, either with or without fetal calf serum. The cell numbers were counted 0, 1, 3, and 7 days after growth factors were given. For the induction of chondrogenesis in 3-dimensional (3-D) culture, cells were cultured in pellets with chondrogenic medium containing combinations of various growth factors. After 4 weeks of culture, the pellets were fixed and evaluated with Safranin-O staining for proteoglycan and immunohistochemical staining for type II collagen. RT-PCR was also performed for the mRNAs of type I collagen, type II collagen, and cartilage oligomeric protein (COMP). In a monolayer culture, TGF-beta2 in concentrations of 0.5 and 5 ng/mL caused significant reduction in cell number irrespective of the presence of serum. FGF-2 of 5 ng/mL most effectively increased cell number even in the absence of serum. In a pellet culture, remarkable chondrocyte-like differentiation of cells was induced around the peripheral areas of a pellet with 5 ng/mL of TGF-beta2, accompanied by increased proteoglycan and type II collagen production. The addition of 100 ng/mL of IGF-I induced notable increase in proteoglycan contents. The results of RT-PCR mirrored those of histological studies. This study shows that an effective proliferation and chondrogenesis may be obtained with proper combinations of growth factors and mesenchymal stem cells from aged individuals.
Im
Gun-Il
GI
Department of Orthopedics, Dongguk University International Hospital, Goyang, Korea. gunil@duih.org
Jung
Nam-Hee
NH
Tae
Suk-Kee
SK
D000368:Aged; D000375:Aging / Q000473:pathology; D001483:Base Sequence; D002358:Cartilage, Articular / Q000166:cytology; D002454:Cell Differentiation / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects / Q000502:physiology; D024043:Collagen Type II / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000008:administration & dosage; D006133:Growth Substances / Q000008:administration & dosage; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta / Q000008:administration & dosage; D053781:Transforming Growth Factor beta2
D024043:Collagen Type II; D006133:Growth Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D016222:Fibroblast Growth Factor 2