My central question: how does the human placenta develop and how are specialized trophoblast sub-types generated? Despite advances in placental biology, pregnancy disorders occur frequently with few treatment options available, resulting in considerable maternal and/or infant mortality and morbidity. The cause of these disorders is abnormal early placental development but ethically and practically this is hard to investigate. Developing in vitro models that are truly representative of normal first trimester trophoblast will allow investigation of lineage specification and differentiation. I will take an interdisciplinary approach to characterize the first trimester villous cytotrophoblast (VCT) population. Specifically, I aim to:1. Characterize the expression of trophoblast stem (TS) cell transcription factors and epigenetically regulated genes to identify the anatomical compartment(s) containing population(s) with stem cell characteristics2. Isolate VCT using a specific cell surface marker and perform an in-depth molecular characterization at a single cell level to identify putative TS cells3. Characterize cell signalling pathways in the VCT and in the putative TS cell population4. Establish cell culture conditions for TS cells5. Perform a detailed characterization of cells cultured in vitroThis proposal has arisen from preliminary results obtained working in Centre for Trophoblast Research, University of Cambridge. Working in a lab with extensive experience of trophoblast isolation (Prof. A.Moffett) and input from our collaborators, Dr. M.Hemberger and Prof. G.Burton who have experience in mouse TS cells and human placental function in vivo, respectively has been pivotal. I am thus in an ideal position to capitalize on this expertise together with other stem cell labs in Cambridge to develop essential tools and knowledge to understand early developmental processes during placentation. It also has important translational impact for women and their babies.