Effect of oxygen concentration, culture format and donor variability on in vitro chondrogenesis of human adipose tissue-derived stem cells.

BACKGROUND: The chondrogenic differentiation potential of the easily accessible adipose tissue-derived stem cells (ASCs) is of particular interest within the field of tissue engineering for treating cartilage defects. However, no consensus has been reached as to which oxygen tension is more beneficial for the differentiation process. MATERIALS & METHODS: In this investigation, the impact of available oxygen was investigated to identify optimal conditions for human ASC chondrogenesis in vitro. Four physiologically relevant oxygen concentrations of 15, 10, 5 and 1% were compared with ambient air condition, and the ASCs originating from six unrelated donors were subjected to chondrogenic induction in high-density pellet cultures. RESULTS: The qualitative and quantitative assessment of accumulated extracellular matrix and the gene-expression analysis revealed marked interindividual differences, nevertheless the chondrogenic process was optimally supported in high-density pellet setup at ambient or 15% oxygen concentrations, irrespective of the origin of cells. The histochemical analysis based on alcian blue staining demonstrated that the differentiation took place in a gradient-like fashion, displaying highest levels in restricted regions, most often adjacent to the periphery. The two lowest hypoxic conditions, at 5 and 1% oxygen, seemed to have an inhibitory effect. CONCLUSION: The micropellet cultures at ambient or 15% oxygen concentration provided the most suitable environment for inducing chondrogenesis in ASCs. Furthermore, in light of the fact that the induction appeared in a zone-dependent manner, this format lends itself as a suitable model for further analysis of the relationship between chondrogenic differentiation and the gradient of nutrients.

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions.

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.