Licensing Factor Expression in Stem Cells

Roswell Park Cancer Institute Corp, Buffalo NY

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Abstract

Stem cells contribute to the repair and maintenance of the tissues in which they reside. These cells have long been recognized in tissues, such as blood and epithelia, which turn over rapidly. More recently, the presence of such cells has also been demonstrated in organs that were not thought to be capable of self-renewal including the brain. Stem cells from somatic source, like their embryonic counter parts, are recognized as a potential source of donor cells in many applications. Additionally, the role of these cells in tissue maintenance suggest that understanding their biology, in the whole organism, will have wide application to understanding age related dysfunction. However, a major obstacle has been that, for most tissues, there is no effective means of identifying or recovering these cells. Here it is proposed to take advantage of one property which all stem cell populations share, their potential for cell division, to mark these cells for identification, recovery, and genetic manipulation using the mouse as a model organism. The methods rely on two recent advances. First, an effective means pf marking living cells, using green fluorescent protein, is now available. Secondly, advances in the understanding of cell-cycle control have identified better gene markers for cells which retain the capacity for proliferation. These genes are part of the mechanism which licenses cells for DNA replication. The advantage of these genes as a means of marking stem cells is that, unlike previous markers for proliferation that relied on S-phase specific events, these factors are expressed in G1 or throughout the cell cycle. Further, preliminary studies demonstrate that they are not expressed in most differentiated cells. Consequently, any cell which is capable of proliferating, not just those undergoing DNA synthesis, can be identified by expression of these genes.. There are two specific short term objectives of the current proposal that aim to address the conceptual and technical feasibility of this approach. First, we will address the conceptual feasibility by taking advantage of the properties of the neural stem cell population present within the subventricular zone of the cerebrum to definitively determine whether most or all of the slowly cycling stem cell population of this tissue expresses licensing factor. Second we will utilize gene targeting to address the technical feasibility of the approach by determining whether EGFP fluorescence is readily detectable when expressed from the endogenous encoding Mcm2 or alternative licensing factors.