Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure

<p>Abstract</p> <p>Background</p> <p>For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.</p> <p>Results</p> <p>In this study, we have compared the gene expression levels of a panel of twelve widely used reference genes during hypoxic culture, osteogenic and chondrogenic differentiation, and passaging of primary human ASCs. We found that several of the commonly used reference genes including 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were unsuitable for normalization in the conditions we tested, whereas tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB) were the most stable across all conditions.</p> <p>Conclusion</p> <p>When determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.</p

Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation

Dette er post-print versjonen av artikkelen. Den trykte versjonen kan leses her: http://www.sciencedirect.com/science/article/pii/S1873506111000286In the den, hibernating brown bears do not develop tissue atrophy or organ damage, despite almost no physical activity. Mesenchymal stem cells could play an important role in tissue repair and regeneration in brown bears. Our objective was to determine if adipose tissue-derived stem cells (ASCs) can be recovered from adipose tissue of wild Scandinavian brown bears and characterize osteogenic, chondrogenic, and adipogenic differentiation in the cells. Following immobilization of 8 wild brown bears 7-10 days after leaving the den in mid-April, adipose tissue biopsies (5-8 ml) were obtained subcutaneously from 7 bears. ASCs were recovered and characterized. Adipose stem cell cultures were established from 6 of 7 bears. Adipose tissue-derived stem cells from yearlings spontaneously formed bone-like nodules surrounded by cartilaginous deposits, suggesting differentiation into osteogenic and chondrogenic lineages. This ability appears to be lost gradually with age. This is the first study to demonstrate stem cell recovery and growth from brown bears, and it is the first report of ASCs spontaneously differentiating into osteocytes and chondrocytes. These findings could have implications for the use of hibernating brown bears as a model to study osteoporosis