Hypoxic conditions increase hypoxia-inducible transcription factor 2 alpha and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients

Stem cells derived from the infrapatellar fat pad (IPFP) are a potential source of stem cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we investigated the effects of hypoxia on gene expression changes and chondrogenesis in stem cells from the IPFP removed from elderly patients with osteoarthritis at total knee replacement. Adherent colony-forming cells were isolated and cultured from the IPFP from total knee replacement. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured for 14 days as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemical staining were determined to assess chondrogenesis. IPFP-derived adherent colony-forming cells stained strongly for markers of adult mesenchymal stem cells, including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Cell aggregates of IPFP cells showed a chondrogenic response. In hypoxic conditions there was increased matrix accumulation of proteoglycan but less cell proliferation, which resulted in 3.5-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of hypoxia-inducible transcription factor (HIF)2 alpha and not HIF1 alpha, and the expression of key transcription factors SOX5, SOX6 and SOX9, and that of aggrecan, versican and collagens II, IX, X and XI, was also increased. These results show that cells with stem cell characteristics were isolated from the IPFP of elderly patients with osteoarthritis and that their response to chondrogenic culture was enhanced by lowered oxygen tension, which upregulated HIF2 alpha and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of cells derived from the IPFP.