The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291.

SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription.

The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis. Retroviral SOX9 transduction of passaged chondrocytes increased the endogenous rate of GAG synthesis and the total capacity for GAG synthesis assessed in monolayer culture with beta-xyloside. Both the endogenous rate and the total capacity of GAG biosynthesis were increased further in chondrogenic cell aggregate cultures. The GAG synthesized was predominantly CS and the hydrodynamic size of the newly synthesized chains was unchanged by SOX9 transduction. Aggrecan gene expression was increased in the SOX9-transduced chondrocytes and increased further in chondrogenic culture, but no comparable effects were found in SOX9 transduced dermal fibroblasts. However, the expression of CS glycosyltransferase and sulfotransferase genes in chondrocytes was unaffected by SOX9 transduction. Therefore SOX9 transduction in chondrocytes increased their CS synthetic capacity, but this was not accompanied by changes in the transcription of the CS biosynthetic enzymes and must occur by indirect regulation of enzyme activity through control of enzyme protein translation or enzyme organization.

Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage.

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 high-density 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 x 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.

The influence of donor and hypoxic conditions on the assembly of cartilage matrix by osteoarthritic human articular chondrocytes on Hyalograft matrices.

Resurfacing of cartilage defects using cell-seeded, biomaterial grafts is a promising approach for articular cartilage repair and in this study we investigated the ability of human chondrocytes from osteoarthritic joints to generate cartilage tissue under standard conditions in cultured over 21 days on Hyalograft matrices under normoxic (20% O(2)) and hypoxic (5% O(2)) conditions. The results showed that constructs were more chondrogenic when cultured under hypoxic conditions, which resulted in greater production of sulphated glycosaminoglycan and collagen type II within the constructs and the cells expressed higher levels of genes encoding cartilage matrix proteins and chondrocyte transcription factors. However, there were very wide differences in the chondrogenic potential amongst donors as the weight ratio of total sulphated glycosaminoglycan to DNA in constructs varied from above 200 to below 10. These results establish that the generation of cartilage from human OA chondrocytes on biodegradable supports is favoured in lowered oxygen, but that under standard conditions, even at low passage, there is a large variation in the chondrogenic potential amongst chondrocytes from different donors. Further analysis of this variation suggested that the gene expression ratio of COL2A1/COL1A1 mRNA in the chondrocytes in monolayer culture may predict their subsequent performance in forming cartilage matrix on the Hyalograft scaffold.