Pluripotency process unveiled

Scientists have identified a key component of cellular reprogramming that may aid in more efficiently creating induced pluripotent stem (iPS) cells, according to a study published online in Nature today (December 21). Mouse embryonic stem cellsImage: Wikimedia commons"This [research] is pretty astonishing," molecular biologist Xiangru Xu of linkurl:Yale University;http://www.yale.edu/ wrote in an email to The Scientist. "This study provides a specific epigenetic mechanism [for] the pluripotent cell production from differentiated human cells. I think this will ultimately help scientists to understand better and facilitating the yield of iPS cell production." By fusing mouse embryonic stem (ES) cells with human fibroblasts to create cells known as heterokaryons, stem cell biologist linkurl:Helen Blau;http://www.stanford.edu/group/blau/members-blau.html of the Baxter laboratory for Stem Cell Biology at Stanford University and her colleagues have developed a way to study the reprogramming process. Presumably because the researchers are "overwhelming" the fibroblast with ES cell factors, Blau said, heterokaryons initiate reprogramming of the human nucleus quickly and efficiently, with 70% of the cells expressing pluripotency markers by the second day after fusion -- a vast leap from less than 0.1% efficiency seen when pluripotency is induced with just four factors. Using this technique, the researchers examined the role of DNA demethylation, which has been found to be important in reprogramming fibroblasts into iPS cells. In the reprogrammed heterokaryons, Blau's group found, demethylation occurs at the promoter regions of two well-defined pluripotency genes, OCT4 and NANOG, and corresponded with an increase in the two genes' expression. The researchers observed demethylation in the absence of cell division and DNA replication, suggesting that it is an active process during reprogramming, "which is contrary to what people have been thinking," Blau said. To understand the mechanism of demethylation, Blau and her colleagues focused on a protein called activation-induced cytidine deaminase (AID), which had previously been detected in germ cells and has been suggested to play a role in global demethylation in zebrafish embryos. The team knocked down AID in the mouse ES cells and human fibroblasts with RNA interference 24 hours before fusing them. In the resulting heterokaryons, both OCT4 and NANOG gene expression was greatly reduced, as was demethylation. Overexpressing AID in these knockdown cells fully rescued NANOG and partially rescued OCT4 demethylation and expression. These results suggest that the demethylation of two key pluripotency genes is an essential part of cellular reprogramming, and that AID plays a critical role in this process. Finally, using chromatin immunoprecipitation (ChIP) to target the AID protein and detect its substrate, the researchers confirmed the direct role of AID in demethylation. Thus, in addition to identifying a key protein in cellular reprogramming, this research "is also important for advance our understanding of mammalian cell demethylation," Xu wrote. DNA demethylation is critical for mammalian development and has been shown to participate in cancer and aging, he added, but the cellular components involved aren't known. AID does not work alone, however, Blau said, and the exact mechanism of demethylation is still "pretty vague." It appears that AID somehow initiates a DNA repair pathway that replaces the methylated base with an unmethylated one, she said, but the details remain to be worked out. The next step, Blau said, is to see if AID works similarly in other methods of inducing pluripotency, such as nuclear transfer or factor-induced reprogramming, and if supplementing AID can increase the efficiency of these methods. "It'll be interesting to see how this works in iPS [cell formation] and whether it can enhance reprogramming to pluripotency." Finally, Blau hopes that the heterokaryon method will allow her team to continue to hack away at the mechanism of cellular reprogramming. So far, their luck has been good. "It's turning out to be a goldmine," Blau said. "The first major factor that we studied is the road block to reprogramming; it's key to DNA demethylation." **__Related stories:__***linkurl:One step to human pluripotency;http://www.the-scientist.com/blog/display/55949/ [28th August 2009]*linkurl:Pluripotency: the third option?;http://www.the-scientist.com/blog/display/55762/ [16th June 2009]*linkurl:Purely protein pluripotency;http://www.the-scientist.com/blog/display/55657/ [23rd April 2009]

Pluripotency process unveiled

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