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Differentiation needs REST; pluripotency does not

January 8, 2010 - Pluripotency, the ability to differentiate into cells of any adult tissue lineage, is one of the defining characteristics of both embryonic stem (ES) cells and induced pluripotent (iPS) stem cells. Research into the genetic regulation of the pluripotent state has revealed a genetic network centering on the pluripotency factors - Oct4, Sox2 and Nanog - that sustains this differentiative capacity. Many other genes expressed in pluripotent stem cells, however, are of unknown function, and it is critical to work out their roles and relationships if we are to gain a fuller understanding of how this state is maintained.

A new study by Yasuhiro Yamada, a visiting professor at Kyoto University's Center for iPS Cell Research and Application (CiRA) who also serves as an associate professor at Gifu University, in collaboration with Hitomi Aoki, also of Gifu University, and colleagues, settles a longstanding question about the function of a factor known as REST, which is expressed in ES cells. The team found that while REST is not required for the maintenance of pluripotency itself, it plays an important role in ES cell differentiation. This work was published in the Cell Stem Cell.

The expression of the REST gene, which also functions in neuronal regulation, in ES cells has been recognized for several years, but there have been conflicting reports of its putative function in this context. To address this question, Yamada, Aoki, et al. generated mice and ES cell lines carrying REST alleles that could be deleted conditionally, allowing them to knock the gene out at specific developmental time points or under certain conditions.

After testing the ES cells to confirm that they lacked REST's protein product, they examined the cells' morphology, gene activity, and behavior. As previously reported, the expression of Oct4, Sox2 and Nanog was unaffected in the REST knockouts, and such cells were able to form tumors known as teratomas on injection into nude mice, and even to contribute to chimeric mouse development, two of the gold standard tests of pluripotency. Importantly, even ES cells that were allowed to grow in culture for several days with REST intact showed no change in the expression of the pluripotency factors alkaline phosphatase, Oct4, or Nanog, following the conditional deletion of the gene. By all indications, pluripotency does not require REST activity.

What Yamada, Aoki and colleagues did notice, however, was that the REST-/- cells showed changes in genes related to early differentiation events, specifically the transcription factors Gata4 and Gata6, which drive differentiation into primitive endoderm. Embryoid bodies - three-dimensional cellular clusters formed from ES cells - that formed from REST-negative ES cells showed lower numbers of Gata4+ cells on their peripheries, which could be rescued by the introduction of exogenous REST.

In looking for the root of this effect, the team noted that the expression of several pluripotency genes actually increased in REST-knockout ES cells. Pluripotent cells show a mutually exclusive relationship between factors driving self-renewal and those that drive the cells to differentiate, so the team reasoned that the suppression of differentiation might in fact be a consequence of a delay in the repression of pluripotency factors. On closer examination, they found that Nanog was indeed upregulated in REST conditional knockout cells, suggesting a link to the reduced expression of Gata4.

As a final test, they used an inducible REST EScell line to observe the effects of its forced expression on ES cell colonies. Switching on this gene caused changes in cell morphology and gene expression consistent with a shift from self-renewal to differentiation. In particular, the number of Gata4-expressing cells at the edges of embryoid bodies, which was reduced in REST-negative colonies, was increased in the inducible REST-ES cell line.

These latest findings add a new piece to the puzzle of the regulatory network underlying pluripotency, the understanding of which will be crucial to the translation of fundamental stem cell science into future applications.

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