**5. Conclusion**

Adult stem cells have the unique capacity to self-renew and give rise to differentiated cells. To fulfil their lifelong function, these cells must be protected from cellular and genetic damage. Most adult stem cells are thought to enter a state of reversible cell cycle quiescence to preserve their role (Orford & Scadden, 2008). Indeed, activation of the cell cycle leads to premature stem cell exhaustion (Cheng *et al.*, 2000, Kippin *et al.*, 2005, Park *et al.*, 2003). However, most cells in the adult body have withdrawn from the cell cycle and can be induced to proliferate again, resulting in their eventual depletion (Bond *et al.*, 2004, Pajalunga *et al.*, 2007). Some somatic cells are able to resume proliferation and even selfrenew, for example differentiated T and B lymphocytes (Fearon *et al.*, 2001). It has thus been proposed that adult stem cells are distinguished by other mechanisms rather than cell cycle quiescence (Mikkers & Frisen, 2005). Yet, what kind of defining mechanisms or marker this property integrates, and if it is shared by various adult stem cells, is unclear at the moment.

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It has been long known that adult HSC can be isolated by their low retention of Pyronin Y, an RNA binding dye (Shapiro, 1981), suggesting that global mRNA transcriptional quiescence is a hallmark of quiescent stem cells. This observation however, has never been addressed further in terms of the precise molecular mechanism underlying global transcriptional quiescence. Analysis of the distinct phosphorylation patterns of the CTD of RNApII during the different stages of mRNA transcription can reveal initiation, paused transcription or productive elongation. Our analysis of various adult stem cell systems showed that down regulation of productive mRNA transcription elongation is a conserved, specific and early feature of adult stem cells (Freter *et al.*, 2010). In line with this, the CTD kinase CDK9 was absent and its inhibition improved cell survival during cellular stresses, suggesting a beneficial function of transcriptional quiescence for stem cell maintenance and survival. However, we could not induce activation of mRNA transcription by overexpression of CDK9 in vivo. It is thus not clear what the in vivo function of this down regulation is. New animal models with a constitutively active RNApII have to be developed to elucidate if transcriptional quiescence is necessary for adult stem cell maintenance or just a symptom of their quiescence.

Interestingly, we could observe some heterogeneity in terms of mRNA transcription elongation in the HSC pool, where only 27% of cells showed a clear absence of mRNA transcription elongation. Yet, identification and isolation of these transcriptionally quiescent cells depends on the availability of genetically encoded reporters of mRNA transcription. CDK9 activity reporters need to be specific for this kinase, and a large dynamic range has to be provided to clearly separate cell populations. A particular challenge will be to convert the negative observation of CTD Ser2 dephosphorylation into a signal-positive output, in order to avoid false-positive events by untransfected cells.

Taken together, we defined a novel molecular mechanism for adult stem cell quiescence, which may lead to the identification of pure stem cell-like cell populations from various sources, including heterogeneous adult stem cell populations or cancerous tissue. Even though some technical questions and functional tests are still to be answered, transcriptional quiescence is a novel and exciting mechanism to detect, isolate and characterize adult stem cells in an unprecedented purity from various sources.
