Abstract
We previously developed a 14-day culture protocol under potentially GMP, chemically defined conditions, to generate chondroprogenitors from human embryonic stem cells (hESCs). In vivo work has confirmed the cartilage repair capacity of these cells in a nude rat osteochondral defect model. Aiming to enhance hESC-chondrogenesis, we screened a range of extracellular matrix (ECM) molecules for their ability to support differentiation of hESCs toward chondrocytes. We identified two novel ECM protein fragments that supported hESC-chondrogenesis: Fibronectin III (fibronectin 7-14 protein fragments, including the RGD domain, syndecan-binding domain, and heparin-binding domain) and fibrillin-1 (FBN1) fragment PF8 (encoded by exons 30-38, residues 1238-1605, which contains the RGD motif but not heparin-binding site). These two protein fragments support hESC-chondrogenesis compared with the substrates routinely used previously (a mixture of fibronectin and gelatin) in our directed chondrogenic protocol. We have identified recombinant fibronectin fragment (FN III) and FBNI fragment (PF8) as alternative coating substrates to promote expression of genes known to regulate chondrocytes and code for chondrocyte ECM components. These recombinant protein fragments are likely to have better batch to batch stability than full-length molecules, especially where extracted from tissue/serum.