This Core will provide key support in the form of primary cells and cell lines that are integral to accomplishing the goals of every project in this U-54 application. The creation of this core acknowledges the fact that highly specialized technical skills are required to obtain the cell types with which we are working. Thus, we propose taking advantage of the unique combination of cell culture expertise in the Fisher and Reijo-Pera groups to support the work of all the participating investigators equally;i.e., 20% effort will be allocated to each project. The cells will come from two sources. The first is human placentas. Specifically, this tissue will be used for the isolation of highly purified preparations of primary cytotrophoblasts (CTBs) as well as villus explants. The second source of cells is mouse embryos, which will be used for the derivation of embryonic and trophoblast stem cells. In addition, quality control will be an important part of the Core's activities. For example, we will use cytokeratin expression, a unique feature of CTBs in human placental cell preparations, to document purity. We will also analyze the cells'ability to differentiate in vitro by monitoring the expression of stage-specific antigens that are upregulated with acquisition of an invasive phenotype (e.g., HLA-G). The quality of the mouse cells will be determined by assessing the differentiative capacity of control lines that are derived in parallel to study functional aberrations that appear as the result of genetic or experimental manipulations. Our specific plans for utilization of these cells are as follows. Control and mutant mouse embryonic stem cells will be provided to Project I with the goal of testing the hypothesis that gamete formation requires dazl function. Studies of the role of Notch family members in the initial stages of placentation will employ purified CTBs and villus explants (Project II). Likewise, experiments that are designed to identify the effect of endometrial stromal signals on CTB differentiation/invasion will also use human placental cells (Project III). Pilot Project I will utilize mouse trophoblast stem cells to understand the impact of in vitro fertilization on the differentiative capacity of these cells. Pilot Project II will also use mouse trophoblast stem cells, with the goal of determining the role of microRNAs in regulating cell fate decisions in the extraembryonic lineages. Because the availability of high-quality cells is a crucial requirement of every project, we envision the activities of this Core to be central to the success of this U-54 program.
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