A Modified Aggregate Culture for Chondrogenesis of Human Adipose-Derived Stem Cells Genetically Modified with Growth and Differentiation Factor 5

Adipose-derived stem cells (ADSCs) are an attractive cell source for tissue engineering, and recently a modified aggregate culture of human ADSCs (hADSCs) was established based on preparation of three-dimensional (3D) cell aggregates in growth factor–enriched low serum medium using the hanging droplet method. Growth and differentiation factor 5 (GDF5) plays a critical role in chondrogenesis and cartilage development. In the present study, we examine (1) whether the modified aggregate culture is feasible for chondrogenic induction of hADSCs, (2) whether overexpressed GDF5 can promote chondrogenesis, and (3) the gene expression profile during chon-drogenesis in this aggregate culture. hADSCs were infected with an adenovirus carrying the GDF5 gene (Ad-GDF5). Cells were cultured with chondrogenic media either in a modified aggregate culture or in an attached micromass culture that served as a control. The chondrogenic phenotype was assessed bymorphology (n = 8), bio-chemistry (n = 3), and histology (n = 2). Expression of 12 genes was determined by quantitative real-time polymer-ase chain reaction (n = 3). We found that ADSCs cultured in the modified aggregates exhibited denser pellets and higher content of sulfated glycosaminoglycan (sGAG) compared with those cultured in the micromass. Infection of cells with Ad-GDF5 increased the aggregate size and sGAG content. It also up-regulated expression of GDF5, aggrecan, and leptin and down-regulated expression of COL I, while expression of COL II and COL 10 remained unchanged. We concluded that the modified aggregate culture is feasible for chondrogenic induction of human ADSCs. Infection with Ad-GDF5 appears to promote the chondrogenesis. These findings suggest that genetic modification of ADSCs with GDF5 in the modified aggregate culture could be useful for treating diseases with cartilage defects. Key words: aggregate culture; cartilage regeneration; chondrogenesis; GDF5; genetic modification; human adipose-derived stem cel