We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in highdensity cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8 -1 mm in thickness from 0.5 ؋ 10 6 cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.