Telomeres and disease PM Lansdorp Fertilized egg 6 Blastocyst 2 Inner cell mass 2 100 cell epiblast 4 150 primary gonadel cells 8 25 600 primary gonadel cells 3 Female germ line 75 000 oogonia 1 Oocytes Total cell divisions 25 Male germ line Stem cells (1–100) 9 Spermatocytes Total cell divisions 34–133 Figure 3 Estimated number of cell divisions in the female and male murine germline from one generation to the next (adapted from Drost and Lee, 1995). The number of cell divisions is given below the indicated developmental stages. Thus, to go from a fertilized egg to a blastocyst takes an estimated 6 divisions; 8 more divisions are needed to produce 150 primary gonadal cells; the latter go through 11 more divisions before cells commit to either the female germline (1 more cell division before mature oocytes is produced) or the male germline (in which the estimated number of divisions from stem cell to spermatocyte is 9). Production of spermatocytes in males continues throughout adult life. Estimates of the number of divisions at the level of spermatogonial stem cells are hampered by uncertainties that are similar to those discussed for haematopoietic and intestinal stem cells. For the (young) mice without telomerase in laboratory settings, it is estimated that at most 50 divisions separate one generation from the next. times, one would expect a severe gut phenotype in the ﬁrst generation of TERC KO mice unless intestinal stem cells could somehow avoid telomere loss or maintain telomeres in the absence of telomerase.4 More likely, intestinal cells, such as Lgr5 þ cells, that divide every 24 h (Barker et al, 2007) include a small subset of Lgr5 þ stem cells and a large proportion of Lgr5 þ progenitor cells similar to CD34 cells in human bone marrow.5 Only a very small fraction (o1%) of BM CD34 þ cells are probably true HSCs (Terstappen et al, 1991; Novelli et al, 1998), and only a minor fraction of sorted single Lgr5 þ cells is capable of initiating crypt formation in vitro (Sato et al, 2009). Telomerase deﬁciencies and other telomere disorders Uncertainties about the number of stem cells in various tissues and their turnover are important in the discussion of human telomerase deﬁciency disorders. It seems likely that most, if not all, phenotypic consequences of human telo- merase deﬁciency result from compromised telomere function that limits the proliferation of (stem) cells. If this is correct, the phenotypic manifestations of telomerase disorders could in fact instruct us about the turnover of stem cells in various tissues. This reasoning explains in part why telomeres and ‘telomerase disorders’ are of interest to a growing number of ‘stem cell’ researchers. The ﬁrst and most well-studied ‘telomere disorder’ is Dyskeratosis Congenita (DC). This is a rare disorder and fewer than a thousand patients have been described. 4Of note, a gut phenotype was observed in the ﬁrst generation of CAST/EiJ mice with short telomeres lacking telomerase (Hao et al, 2005). 5How this notion can be reconciled with the elegant lacZ lineage tracing experiments by Barker et al is not clear. Differences in Cre-induced recombination between cell types and/or variable detection of lacZ in slowly versus rapidly dividing cells could possibly contribute to the failure to detect differences in the frequency of lacZ positive crypts at variable time points after Lgr5-induced Cre expression. 2536 The EMBO Journal VOL 28 | NO 17 | 2009 Patients with DC typically present with three distinctive clinical characteristics: skin pigmentation abnormalities, nail dystrophy and abnormalities of the oral mucosa known as leukoplakia (Walne and Dokal, 2008; Savage and Alter, 2009). The most common fatal complications of DC are related to bone marrow failure, pulmonary ﬁbrosis and cancer (Walne and Dokal, 2008; Savage and Alter, 2009). The most frequent cancers are head and neck cancer (squamous cell carcinoma), cancer of the tongue and acute myeloid leukaemia (Alter et al, 2009). In addition, a variety of other clinical abnormalities have been described in DC pa- tients (reviewed in Kirwan and Dokal, 2008). Hepatic cirrho- sis has been described in DC and severe liver disease is not uncommon after HSC transplantation for marrow failure in patients with DC (Calado and Young, 2008). Several genes have now been implicated in DC; most notably, all three genes encoding components of the minimal (Cohen et al, 2007) telomerase enzyme complex (Walne and Dokal, 2008; Savage and Alter, 2009). Mutations were initially discovered in DKC1, located on the X chromosome (Heiss et al, 1998). A link with telomerase was ﬁrst suggested when the nucleo- lar protein encoded by DKC1, dyskerin, was found to stabilize telomerase RNA and maintain telomere length through telo- merase (Mitchell et al, 1999). Mutations in hTERC and hTERT themselves were later discovered in autosomal dominant DC (Vulliamy et al, 2001; Fogarty et al, 2003; Armanios et al, 2005). In total, B50% of patients with DC have a mutation in one of the three genes of the telomerase complex (Aubert and Lansdorp, 2008). It seems likely that mutations in other ‘telomere maintenance’ genes contribute to the remaining B50% of cases. This notion is supported by the recent discoveries of mutations in genes of DC patients such as TINF2, NHP2 and NOP10 (Walne et al, 2007; Savage et al, 2008; Vulliamy et al, 2008). Mutations in hTERT and hTERC have also been found in diseases other than DC, including other bone marrow failure syndromes such as aplastic anaemia (AA) (Yamaguchi et al, 2003, 2005; Marrone et al, 2004; Ly et al, 2005; Vulliamy et al, & 2009 European Molecular Biology Organization