During hematopoiesis, pluripotent stem cells and their progeny undergo proliferation and differentiation, resulting in hematopoietic cells of various lineages. In some systems, decisions that determine the fate of stem cells and their progeny have been shown to result from intercellular interactions with adjacent cells, modulated by several molecular families, including the Notch gene family. Our preliminary evidence supporting a role for the Notch receptor in regulating hematopoiesis includes the demonstration that a human homolog of Notch, TAN-1, is expressed in primitive hematopoietic precursors, and that expression of a constitutively active form of Notch inhibits granulocytic differentiation in murine 32D cells. We propose to further investigate the role of Notch homologs in hematopoiesis by determining the effects of activated forms of members of the Notch family on the growth and differentiation of murine multipotent hematopoietic cell lines, including granulocytic differentiation by 32D cells, granulocytic and monocytic differentiation by EDCP-mix cells, and myeloid and lymphoid differentiation by EML cells. We will also assess the expression of Notch homologs in human marrow precursor cells using RT-PCR, and will evaluate cell surface expression of Notch, and isolate hematopoietic precursors that express Notch homologs and assess their in vitro growth properties, using monoclonal antibodies. We will then determine the effect of Notch-mediated cellular interactions on hematopoietic differentiation by murine multipotent cells and by isolated human hematopoietic precursors, using peptides, fusion proteins, antibodies, and anti sense oligonucleotides to inhibit Notch interactions. We will also determine whether Notch can be activated using antibody and, as available, the Notch ligands, Delta and Serrate. These studies will provide insight into the role of this highly conserved receptor family in regulating mammalian hematopoiesis and should lay the groundwork for efforts aimed at exploiting altered Notch activity to manipulate human hematopoietic stem cell differentiation and proliferation in vitro. This ability may enable more effective ex vivo stem cell expansion and/or retroviral-mediated gene insertion in hematopoietic stem cells for therapeutic purposes.
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