Characterization of the biology of normal and leukemic human hematopoietic stem cells

Studies in murine hematopoiesis have demonstrated that the hematopoietic system is a hierarchy maintained by rare self-renewing pluripotent hematopoietic stem cells (HSC). Long-term in vivo repopulation assays are the only definitive means by which to identify and characterize HSC. Research in human hematopoiesis has advanced significantly since the development of xenotransplantation assays using immune-deficient mouse recipients to detect primitive human hematopoietic cells with in vivo repopulating ability (SCID-repopulating cells, SRC). Here we establish the quantitative nature of the SRC assay, and use this assay to purify SRC based on surface marker expression, achieving a 1,500-fold enrichment within the Lin - CD34+CD38- fraction. Combined with gene transfer techniques to uniquely mark the progeny of individual SRC, these developments have facilitated detailed analysis of the clonal behaviour of human HSC, and have led to the demonstration of functional heterogeneity within the human stem cell compartment.; Recent studies have shown that human myeloid leukemia is also hierarchical and is sustained in vivo by rare leukemia stem cells (LSC). As with normal HSC, in vivo assays are required to study the unique biology of LSC. Here we develop an in vivo model for chronic myelogenous leukemia (CML) that allows characterization of CML stem cells and assessment of the contribution of secondary genetic changes to leukemic progression in this disease. Complementary in vitro systems modelled in primary human cells enable examination of the molecular events that occur during leukemogenesis. Here we report the transformation and immortalization of normal human hematopoietic cells following initiation of a pre-leukemic program by TLS-ERG expression. Our findings provide direct evidence for multiple cooperating events in leukemogenesis, and demonstrate the usefulness of this system for studying leukemic initiation and progression. We have also investigated the role of telomerase in normal and leukemic hematopoiesis. We demonstrate that telomerase overexpression fails to extend the replicative lifespan of human hematopoietic cells unless normal developmental pathways are disrupted. Elevated telomerase activity does not prevent telomere shortening even in transformed hematopoietic cells, implying tighter regulation of telomere length dynamics compared to other somatic cell types. Our findings further suggest that telomerase itself may contribute to leukemic progression.