The proliferative life-span of the stem cells that sustain hematopolesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD3810 phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilica cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These fiings strongly suggest that the proliferative potential of most, if not all, hematopoletic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopolesis as well as gene therapy.The requirement for primers and the undirectional 5' -3 3' nature of DNA synthesis by DNA polymerases results in incomplete replication of the terminal 3' strands of linear chromosomes (1-3). Eukaryotic chromosomes end in specialized nucleoprotein structures called telomeres (4) and in vertebrates, including humans, telomeres terminate in tan-

Abstract. The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, an enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reactionbased telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34 + CD71 lo CD45RA lo ) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34 + CD71 + ) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34 -cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use. STEM CELLS 2008;26:988 -996 Disclosure of potential conflicts of interest is found at the end of this article.

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