Unified Cellular and Molecular Core

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All of the projects described in this Program Project Grant either require a constant and consistent supply of uterine artery endothelial cells (UAEC) (Projects I and II) or use human umbilical vein endothelial cells (HUVEC-C; immortalized cells from ATCC) as positive control for identifying human ES derived-endothelial cells and angiogenesis bioassays (Project III)or as effector cells for trophoblast differentiation (Project IV). The role of the core is to not only relieve the project personnel of the burden of preparing frozen stocks of these cells, but also to provide a high quality of cell preparation and to provide uniformity of data acquisition between the projects. Core C will also undertake to supervise technically difficult areas of analysis In collaboration with Project 1 the core will focus on optimization of siRNA delivery systems and in collaboration with Core B (Stem Cell Core), Core C will also advise immunohistochemical studies for evaluating integration of human ES derived-endothelial cells into local blood vessels in SCID mice as proposed in Project III. Thus our specific aims are to: Aim 1: Prepare, isolate, characterize, maintain and distribute UAEC frozen stocks for Projects I and II, and also for Projects III and IV when need arises. Aim 2: Maintain and distribute HUVEC-C as required mainly for Projects III and IV but also Projects I and II when the need arises. Aim 3: Supervise in vitro angiogenesis assays including mitogenesis, migration, capillary tube formation assays in Project III using human ES cell-derived endothelial cells, HUVEC or UAEC. Aim 4: Advise and supervise real time detecting NO using fluorescent plate reader and imaging of NO and Ca2+ in cells for Projects I, II, and , and also for Project IV when the need arises. Aim 5: Develop/optimize methods for more efficient delivery of siRNA to UAEC and further combine with FACS (sorting of cells) purification based on green fluorescent protein (GFP) co-delivery to transfected cells. Aim 6: Provide ongoing support for optimization of antibody based and quantitative methods for protein and RNA analysis, including RT-PCR and siRNA as provided during the prior funding period. Aim 7: Provide an available source of fresh reagents which have short half life or are significantly more economical when purchased in bulk. In summary, by acting as a Core, we will provide much needed technical expertise, essential cell supplies and quality assurance, so contributing to the ability to directly compare data from different projects and between cell types.