The hematopoietic stem cell population is responsible for the regulated production of all mature blood cells throughout fetal and adult life. Although much information is available concerning the molecular mechanisms that control the proliferation and differentiation of committed, more mature progenitor cells, little is known about the most primitive population. Moreover, the only way to evaluate the biological properties of hematopoietic stem cells is by in vivo engraftment into an ablated host. As a consequence, practically no molecular information about signaling pathways responsible for self-renewal decisions, the initiation of complex programs of commitment and differentiation, or the regulation of cell cycle quiescence vs. activity is available. As preliminary studies, the principal investigator and colleagues have developed assays that maintain purified stem cells in vitro (by use of the stromal cell line AFT024) and a procedure to analyze the differentiation potential of single cells maintained with stromal support. He also has identified several novel protein tyrosine phosphatase and zinc finger encoding genes.
Specific Aim 1 is to analyze gene expression patterns in undifferentiated stem cells and other primitive members of the hematopoietic hierarchy. The hypothesis to be tested is that the unique properties of stem cells predict the existence of gene products that are uniquely or preferentially expressed in these cell types. He proposes a comprehensive differential display strategy to rapidly and quantitatively identify gene expression patterns in purified stem cell populations. The design will allow the identification of (1) genes expressed in all stem/progenitor cells regardless of developmental source (i.e., genes that are expressed in mouse and human cells and in fetal and adult stem cells); (2) genes expressed in stem cells at different levels of ontogeny; and (3) genes whose expression varies with distinct stem cell states (such as quiescence or cycling), with stem cell proliferative history, or as a function of exposure to different stromal microenvironments. Next, he will exploit his ability to purify large numbers of stem cells from fetal liver to construct high-quality subtracted and normalized cDNA libraries. These will be used in a variety of functional screens to identify biological interesting genes.
Specific Aim 2 proposes to analyze the function of two novel protein tyrosine phosphatases and four novel zinc-finger containing sequences that the investigator has previously identified in purified stem/progenitor cells. As a first step, he will analyze the expression of these genes in various stem/progenitor populations. Next, he will use the in vitro stem cell assay developed in his laboratory to determine if inhibiting these genes products might have an effect on stem cell maintenance, self-renewal, commitment or differentiation. Lastly, he will use gene targeting and embryonic stem (ES) cell techniques to create null mutations in these genes. The phenotype resulting from these mutations will be analyzed in vivo and in vitro.
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