August 28, 2009 - Researchers at the Kyoto University Center for iPS Research and Application (CiRA) have found a new trick for improving the efficiency with which cells can be reprogrammed to pluripotency. In a report published in Cell Stem Cell, they showed that they were able to boost the rate of induced pluripotent stem (iPS) cell generation many fold by culturing cells in low-oxygen conditions.
It has been shown previously that environmental factors such as temperature and oxygen tension can affect the behavior and viability of stem cells in culture, which prompted the group to test whether reducing the oxygen to levels beneath that of ambient air, which is approximately 21% 02. Using a fluorescent protein to track the expression of a genetic marker of pluripotency, Yoshinori Yoshida, an assistant professor, and others in the laboratory of CiRA Director Shinya Yamanaka, found that when the conventional induction method of four transgenes (Oct3/4, Sox2, Klf4 and c-Myc) delivered by a retrovirus was used in mouse fibroblasts grown at just 5% oxygen, more than 7 times as many cells were reprogrammed by day 21 of culture than others cultured in room air. When only three of the iPS factors (omitting c-Myc) were used, the effect was even more dramatic, yielding a twenty-fold increase at the same 21-day mark.
Recent months have seen a multitude of alternative iPS techniques developed, so the group next looked at whether hypoxia would have similar effects using these as well. They found again that whether mouse iPS cells were made from neural stem cells, which naturally express SOX2, using only a pair of reprogramming factors, or from fibroblasts into which the conventional factors were introduced by plasmid or excisable transgenes, 5% 02 proved to be an iPS-friendlier environment. Further analysis of morphology and gene expression confirmed that the cells reprogrammed in low oxygen were essentially indistinguishable from those established under normoxic conditions.As a final test of the new technique, Yoshida generated human iPS cells as well. Although the increase in efficiency was not as pronounced as it was using mouse cells, he nevertheless showed that hypoxia improved the success rate by several times. These human iPS cells too showed all the hallmarks of pluripotency and formed embryoid bodies in culture and teratomas on transplantation into mice, indicating that they have the same potential as both iPS cells made using conventional methods and embryonic stem cells.