Telomeres play a central role in cell fate and aging by adjusting the cellular response to stress and growth stimulation on the basis of previous cell divisions and DNA damage. At least a few hundred nucleotides of telomere repeats must "cap" each chromosome end to avoid activation of DNA repair pathways. Repair of critically short or "uncapped" telomeres by telomerase or recombination is limited in most somatic cells and apoptosis or cellular senescence is triggered when too many "uncapped" telomeres accumulate. The chance of the latter increases as the average telomere length decreases. The average telomere length is set and maintained in cells of the germline which typically express high levels of telomerase. In somatic cells, telomere length is very heterogeneous but typically declines with age, posing a barrier to tumor growth but also contributing to loss of cells with age. Loss of (stem) cells via telomere attrition provides strong selection for abnormal and malignant cells, a process facilitated by the genome instability and aneuploidy triggered by dysfunctional telomeres. The crucial role of telomeres in cell turnover and aging is highlighted by patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Short telomeres in such patients are implicated in a variety of disorders including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, and cancer. Here the role of telomeres and telomerase in human aging and aging-associated diseases is reviewed.

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.

Although ribosomes are ubiquitously expressed and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein encoding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

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The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Little is known about the behavior of hematopoietic stem cells (HSCs) in primates because direct observations and competitive-repopulation assays are not feasible. Therefore, we used 2 different and independent experimental strategies, the tracking of transgene expression after retroviral-mediated gene transfer (N ‫؍‬ 11 baboons; N ‫؍‬ 7 rhesus macaques) and quantitation of the average telomere length of granulocytes (N ‫؍‬ 132 baboons; N ‫؍‬ 14 macaques), together with stochastic methods, to study HSC kinetics in vivo. The average replication rate for baboon HSCs is once per 36 weeks according to gene-marking analyses and once per 23 weeks according to telomere-shortening analyses. Comparable results were derived from the macaque data. These rates are substantially slower than the average replication rates previously reported for HSCs in mice (once per 2.5 weeks) and cats (once per 8.3 weeks). Because baboons and macaques live for 25 to 45 years, much longer than mice (ϳ2 years) and cats (12-18 years), we can compute that HSCs undergo a relatively constant number (ϳ80-200) of lifetime replications. Thus, our data suggest that the self-renewal capacity of mammalian stem cells in vivo is defined and evolutionarily conserved. IntroductionHematopoiesis is the ordered process by which hematopoietic stem cells (HSCs) proliferate and differentiate into mature blood cells. Through replication and differentiation, HSCs generate clones of progenitors, precursors, and mature cells, which support hematopoiesis throughout an animal's life. HSCs cannot be directly observed and are generally defined and identified by their function (their ability to reconstitute and maintain hematopoiesis).The most informative experimental approach for studying HSC kinetics is limiting-dilution competitive-repopulation experiments. Small numbers of cells with distinguishable phenotypes are transplanted into a recipient animal in which hematopoiesis has been ablated. After the HSCs engraft and restore blood cell production, the percentage of marrow progenitor cells or blood granulocytes of each phenotype is tracked over time, because this reflects the phenotype of contributing (differentiating) HSC clones. 1,2 Using such data, the frequency of HSCs in mice and cats was estimated, as were their average replication and differentiation rates. [3][4][5] Importantly, the estimated frequency of HSCs in mice derived with stochastic analyses was similar to frequencies estimated with independent methods. [6][7][8][9] In addition, the results from transplantation studies overlapped the results from studies of unperturbed hematopoiesis, including 5-bromodeoxyuridine (BrdU)-labeling experiments. 6,9,10 Given the significant differences in estimates of the frequency of HSCs (number of HSCs/number of nucleated marrow cells; 4-8/10 5 vs. 6/10 7 ) and the average HSC-replication rate (once/2.5 weeks vs once/8-10 weeks) between mice and cats, we hypothesized that underlying biologic principles might explain these discrepancies. For example, our data and addit...

Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allogeneic stem cell transplantation is a cause of morbidity and mortality from viral pneumonitis. Antiviral drugs given to reactivating patients have reduced the mortality from CMV but have toxic side effects and do not always prevent late CMV disease. Cellular immunotherapy to prevent CMV disease is less toxic and could provide prolonged protection. However, a practical approach to generating sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is required. This study describes a system for generating sufficient CMV-specific CTLs for adoptive immuno-