Hematopoietic stem cells (HSC) possess the ability to self-renew, differentiate, and produce massive numbers of blood cells of all lineages. If the molecular mechanisms governing the decision to expand vs. commit to differentiation could be fully understood, the knowledge could be applied to expand stem cells ex-vivo and to do successful gene therapy using retroviral vectors. Currently, all culture procedures lead to a progressive loss of pluripotent stem cells, so engineered HSC populations cannot yet be returned to an ablated donor with the confidence that they will sustain long-term multilineage blood cell production. The proposed studies will define culture conditions that can be used to expand primitive progenitors without differentiation. The long-term xenograft system with clonal marking is crucial in these studies because it allows identification of individual stem cells that have cycled during the culture period (identified by their ability to accept a retroviral vector), and have retained the ability to produce both lymphoid and myeloid progeny. They have used this system to show that human stem cells lose the capacity to sustain long-term engraftment if cultured for 72 hours without binding to fibronectin (FN). They hypothesize that engagement of the integrins 24^D1 to FN maintains the primitive phenotype of HSC by sustained activation and nuclear localization of MAPK proteins, resulting in phosphorylation of GATA-2, induction of c-ets-1 and maintenance of expression of c-myb. In suspension, MAPK will remain in the cytoplasm, c-ets-1 will not be induced, and c-myb levels will drop, causing a loss of expression of CD34, flt3 and c-kit. They will verify their hypotheses at the molecular level while determining the best conditions to induce HSC expansion. They predict that culture on FN with stimulatory cytokines, neutralization of TGF^D1, and reduction in levels of the cyclin-dependent kinase inhibitors (CDKI) p27kip1 and p15INK4 will cause division of HSC without induction of pathways of differentiation. They will measure the molecular and cellular effects of these conditions on progenitors (CD34+, colony-forming cells, and B cell precursors) and long-lived HSC (CD34+38-, extended long-term culture initiation cells (e-LTCIC), (e-LTCIC), and immune deficient mouse repopulating cells). Expansion of stem cells will be measured as an increase in the number of transduced e-LTCIC bearing the same clonal integration site, assessed by single colony inverse PCR, and increased number of T cells and myeloid progeny bearing the same proviral integration site following long-term engraftment in bnx mice, indicating that cones of both lineages arose from the same stem cell. The results of the proposed studies will define in vitro culture conditions which induce stem cell division without forcing lineage commitment during retroviral-mediated transduction or ex-vivo stem cell expansion, and will give insight into the molecular changes that precede lineage commitment.
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