Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment.

We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.