Project Summary/Abstract: Hematopoiesis occurs at multiple sites during human prenatal development. In addition to the primary intraembryonic hematopoietic tissues, it is now recognized that the extra-embryonic tissues – the placenta and chorion – contain hematopoietic stem cells. The presence of hematopoietic stem cells in these tissues raises the possibility that they actively contribute to the pool of blood cells contained in the extra-embryonic tissues. In this proposal we focus on the human fetal membranes, specifically the chorion. Macrophages are present in the chorion and presumably act as sentinels and first responders to infection as well as invasion by maternal cells. The presence of these macrophages suggests that the chorion is not just an inert physical barrier. We hypothesize that the human chorion is an immunologically active barrier containing macrophages that develop in situ from hematopoietic stem cells that together with other cells in the chorion maintain the maternal-fetal interface to prevent infection and immune rejection of the fetus during pregnancy. In the first of three specific aims, we will investigate if chorionic hematopoietic stem cells serve as a local source of precursors for monocytic cells found in the chorion. Tissues across a range of gestational ages will be examined using flow cytometry, hematopoietic progenitor cultures, and a transplant model, using immunodeficient mice, to determine the types of hematopoietic precursors that exist in the chorion. Experiments will focus on demonstrating active myelopoiesis in the chorion. In the second aim, we will explore the role of the chorionic microenvironment in the development and function of macrophages. Mesenchymal stromal cells and cytotrophoblasts comprise the hematopoietic niche in the chorion, which will be studied in culture to test their effects on monocyte development and macrophage maturation. We hypothesize that these cell types support the development of M2 macrophages to foster an anti-inflammatory environment in the chorion. This will be more specifically tested in the third aim, in which we will investigate the immunomodulatory role of chorion cells in preventing rejection by maternal T-cells. Preliminary data show the presence of T cells in the chorion of both maternal and fetal origin. The origins and distribution of T-cells in the chorion will be further investigated at different gestations. Additionally, we will determine if the T-cells are active and if they are enriched in any specific T-cell subtypes. T-cells from the adjacent decidua will be analyzed for comparison as well as fetal T-cells from the spleen. We will explore the roles of chorionic macrophages, mesenchymal stromal cells, and cytotrophoblasts in the regulation of T-cell activation. Together these aims will show that chorionic macrophages develop locally from a population of stem cells in the chorion. Also, that these macrophages, in conjunction to other cell cellular populations ...