Emerging evidence to support the use of endothelial progenitor cells (EPCs) for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression is compelling. However, there is no uniform definition of an EPC, which makes interpretation of these studies difficult. Although hallmarks of stem and progenitor cells are their ability to proliferate and to give rise to functional progeny, EPCs are primarily defined by the expression of cell-surface antigens. Here, using adult peripheral and umbilical cord blood, we describe an approach that identifies a novel hierarchy of EPCs based on their clonogenic and proliferative potential, analogous to the hematopoietic cell system. In fact, some EPCs form replatable colonies when deposited at the singlecell level. Using this approach, we also identify a previously unrecognized population of EPCs in cord blood that can achieve at least 100 population doublings, replate into at least secondary and tertiary colonies, and retain high levels of telomerase activity. Thus, these studies describe a clonogenic method to define a hierarchy of EPCs based on their proliferative potential, and they identify a unique population of high proliferative potentialendothelial colony-forming cells (HPPECFCs) in human umbilical cord blood.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Telomeres are specialized DNA͞protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 Kd subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA doublestranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells. V ertebrate telomeric DNA is composed of (T 2 AG 3 ) tandem repeats, with the number of repeats varying between different species (1, 2). The GT-rich strand of telomeric DNA is synthesized by telomerase, a specialized ribonucleoprotein reverse transcriptase. A minimal telomeric DNA length and, in some situations, an active telomerase are required for chromosome stability and cellular viability (3, 4), with failure to maintain telomere length or function leading to a form of replicative senescence in Tetrahymena, yeasts, and mammalian cells (5-7). Activation of telomerase is characteristic of most established mammalian cell lines and tumors (8). Correspondingly, experimental activation of telomerase also allows certain virustransformed human cell lines to bypass authentic cellular replicative senescence and crises and continue proliferation (4, 6, 9).Several lines of evidence have recently converged supporting the conclusion that the Ku heterodimer is critical for telomere maintenance in yeast. Saccharomyces cerevisiae null for Ku70 or Ku80 have defects in telomere silencing, abnormally short telomeres, and a senescence-like phenotype at 37°C (10-13). Strikingly, despite the relatively low sequence conservation between yeast and human Ku subunits at the amino acid level, the exogenous expression of human Ku subunits rescues Ku null yeast from cell death at 37°C (14). Direct evidence for the physical localization of the Ku heterodimer to yeast telomeres came from an in vivo crosslinking experiment linking Ku to telomeric DNA (15). Recently, Martin et al. (16) demonstrated in yeast that Ku80 colocalizes with the telomere binding protein Rap1 at telomeric foci. These types of experiments have not been possible to perform in mammalian cells because of the large abundance and uniform distribution of Ku throughout the mammalian nucleus.However, nothing has been reported concerning the Ku heterodimer's role at the mammalian telomere. Using an in vivo crosslinking method, we show that Ku is localized to mammalian telomeric repeats. We also determined that the DNA-dependent protein kinase catalytic component (DNA-PKcs) is not required in vivo for the association of Ku with telomeric repeats. Materials and MethodsCell Cultures....

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying a template sequence in the telomerase RNA. We studied the functional roles of specific residues in the Tetrahymena telomerase RNA template region. Unexpectedly, mutation of certain templating residues caused dramatic effects on specific aspects of the enzyme reaction, including loss of enzymatic fidelity and premature product dissociation. None of these fundamental changes in enzymatic action are explainable by altered base-pairing between the telomerase RNA and DNA substrate. These influences of specific template bases of the telomerase RNA on enzymatic properties of telomerase provide evidence for critical roles of these RNA residues in two active site functions--fidelity and DNA substrate/enzyme interaction.