Telomere Manipulation in Adult Stem Cells

As seen above for T cells, modulation of telomerase expression can be a valuable tool for studying its function in various cell types. Along with other groups we could demonstrate that hTERT overexpression in hematopoietic progenitors such as CD34+ CB cells results in a significant elevation of telomerase activity that cannot prevent overall telomere shortening and is incapable of increasing the replicative capacity of these cells (Akimov et al. 2005, Elwood et al. 2004, Wang et al. 2005, Zimmermann et al. 2004). Interestingly, only cotransduction of hTERT with the human papillomavirus type 16 (HPV16) E6 and E7 oncogenes in CD34+ CB cells resulted in continuous proliferation, giving rise to permanent cell lines with a myelo-erythroid/mast cell progenitor phenotype. These cell lines exhibited stabilization of telomere length and minimal chromosomal aberrations in contrast to CB cell cultures expressing only HPV16 E6/E7, which went through a crisis period resulting in highly aneuploid cell lines (Akimov et al. 2005). An eventual stabilization of telomere length without changes in telomerase activity was observed during establishment of leukemic cell lines from normal cord blood cells, indicating that additional genetic or epigenetic alterations are required for telomere maintenance in immortalized human hematopoietic cells (Wang et al. 2005). Overall, evidence is accumulating that hTERT could function independently of its enzymatic activity as a prosurvival factor (Armstrong et al. 2005, Cao et al. 2002, Elwood et al. 2004, Folini et al. 2005, Gorbunova and Seluanov 2003, Massard et al. 2006, Yamada et al. 2003). In this context, recent data demonstrated that overexpression of hTERT in a cytokine-dependent, human hematopoietic progenitor cell line, as well as in normal CB CD34+ cells, resulted in protection from apoptosis in the absence of cytokine stimulation but did not favor unlimited replicative potential (Li et al. 2006).

Extensive CB CD34+ cell expansion for up to 18 weeks in long-term cultures with sustained telomerase activity and a minimal telomere loss could be demonstrated only on the basis of refined culture conditions, including special cytokine cocktails and stroma support (Gammaitoni et al. 2004). Similarly, the application of an optimized serum-free and cytokine-limited defined medium in CB AC133+ cell expansion provided a very early uncommitted HSC proliferation and was accompanied by high levels of telomerase activity to maintain telomere length (Yao et al. 2006). No matter which approach is used, in vitro expansion of HSCs for a prolonged period without telomere shortening has striking clinical implications for allogeneic transplantation, considering the limited HSC numbers in individual CB samples and other HSC sources (Jaroscak et al. 2003, Moore 2000).

Other stem cells which are prevalently found in the bone marrow are mesenchymal stem cells (hMSCs). These cells can differentiate along variable lineages, including bone, cartilage, adipose and muscle cells (Jiang et al. 2002). Unlike HSCs and other adult stem cells, hMSCs lack telomerase expression, which limits their replicative capacity in tissue culture (Simonsen et al. 2002, Zimmermann et al. 2003). Ectopic telomerase expression is able to expand the replicative capacity of hMSCs while the cells are retaining their functional characteristics (Shi et al. 2002, Simonsen et al. 2002). Nevertheless, premalignant changes were observed in the course of long-term cultures of hTERT overexpressing hMSCs (Serakinci et al. 2004), evoking some concerns about potential clinical applications (Keith 2004). The fact that stem cells and cancer cells share several common features, such as the same factors regulating self-renewal of normal HSCs and leukemic cells (Lessard and Sauvageau 2003), has led to the "cancer stem cell" hypothesis (reviewed in (Marx 2003) ). If there is a stem cell origin for certain cancers, these stem cells would already have telomerase and it would not need to be reactivated, although the enzyme activity may increase in later stages of carcinogenesis (reviewed in (Armanios and Greider 2005). In reverse, limited telomerase activity accompanied by a finite life span in adult stem cells may promote aging but may prevent cancer. On the other hand there is evidence that telomere shortening can induce chromosomal instability and cancer initiation (see Rudolph, this volume).

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