The hematopoietic system consists of two distinct but interacting compartments, the blood forming cells and the immediate microenvironmental cells that surround them. The extraordinary proliferative capacity of blood forming cells is based on a hierarchical network anchored by a population of pluripotent and self-renewing stem cells. It has been shown that these pluripotent stem cells reside in discrete niches within a hematopoietic organ. The intrinsic and extrinsic molecular mechanisms that control the behavior of stem cells must be tightly regulated in order to insure an orderly and life-long production of at least eight distinct lineages of mature blood cell populations. While much is known about the physical and molecular characteristics of stem cells there is a paucity of information about the cellular and molecular nature of stem cell niches. The proposed experiments are targeted towards a global and, comprehensive molecular analysis of the stem cell niche. We have developed and characterized a fetal liver-derived stromal cell line that supports highly enriched populations of murine stem cells for extended periods of in vitro, culture. When transplanted, these cultured stem cells retain a competitive repopulating activity that is both qualitatively and quantitatively equivalent to freshly purified cells. In collaboration with other investigators we have shown that this cell line, AFT024, also supports human stem cells. These cells retain both an immature stem cell phenotype and possess expanded biological activity measured both in vitro and in xenogeneic in vivo assay systems. We have hypothesized that AFT024 cells provide a complex molecular milieu that facilitates and balances stem cell fate decisions. We further propose that the molecules that constitute this milieu will be specifically expressed by these cells and not by non-supporting cell lines. We propose experiments that will provide a global genomic profile of all molecules expressed by AFT024 cells. At least a portion of these will contribute to the network of stem cell regulatory mechanisms. Our analyses will be extended to other murine stromal cell lines with the ability to support murine as well as human stem cells. We propose that a distinct pattern of gene expression will correlate with, and thus define the supporting phenotype. In addition, we propose to examine the changes in gene expression that result when the two compartments, stem cells and stromal cells, interact. Finally, we will investigate the functional roles of individual candidate regulatory molecules identified in our analyses We propose that molecules isolated from this screen will define novel molecular pathways by which the microenvironment interacts with stem cells Collectively, the proposed studies will provide insights into the molecular biology and function of the hematopoietic, microenvironment specifically and stem cell microenvironments in general.
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