Exposure to environmental chemical contaminants may lead to adverse effects on human pregnancy and fetal development. The toxics under suspicion may act directly on the fetus but also via their effects on the placenta which is the interface structure between mother and fetus whose exchange functions, as well as endocrine function play a physiological crucial role in the success of pregnancy and fetal development. However, the nature of the deleterious effects of environmental contaminants on the placenta and genetic targets as well as molecular and cellular mechanisms involved are poorly known. The placental key cell is the trophoblast. These cells differentiate spontaneously in vitro into syncytiotrophoblast cells which are the site of all exchange and hormonal functions of the placenta. Primary cultures of trophoblast cells at different terms of pregnancy represent a unique source of human placental material that can be exposed in vitro to environmental pollutants and thus enabling to characterize the placental response to these contaminants throughout pregnancy in human species. Using cultures of placental cells in the first and last trimesters of pregnancy is an approach to analyze the response of placental tissues from exposure to environmental contaminants throughout pregnancy. Therefore the aim of our project is to establish the molecular basis of placental toxicology using two pollutants, benzo(a)pyrene and MEHP, a metabolite of DEHP phthalate. The project will be carried out on 1) in vitro models with cultures of first trimester (early and end late-term) and cultures of term placenta which will take into account the complexity of the human placenta for its development and its physiology throughout pregnancy and on 2) a dynamic model reproducing the physiological conditions of placenta at term. This project concerning placental toxicology will use biological techniques specific to the human placental physiology and analytical techniques of mass spectrometry. This entirely innovative project is based on the internationally recognized expertise of placental physiology for partner 1 and on approaches to experimental toxicology and analysis by mass spectrometry of proteins and metabolites for partner 2. The feasibility of this project is provided by 1 / specific equipment to Institut Universitaire «Médicament, Toxicologie, Chimie et Environnement» (IMTCE) 2 / technological expertise of involved teams in the Faculty of Pharmacy 3 / Foundation PremUp (www.premup.org) that allows access to placental biological samples whatever the term of pregnancy. This project will establish close links between both teams in the IMTCE of Paris-Descartes, whose objective is to animate interfaces between chemistry, biology and pharmaco-toxicology that are at the heart of this project. These data can be integrated in the long term in a multidisciplinary frame, involving cellular and molecular toxicology, medical practice (monitoring of pregnancy and child development) and epidemiology. Based on scientific considerations and on a multidisciplinary approach, the objective is to provide recommendations on the environment of the pregnant woman.

For patients suffering from inherited retinal diseases (IRDs), gene and cell therapies offer hope of preserving or restoring vision. Retinal imaging is crucial, to first phenotype patients to determine which cells are degenerated and devise an appropriate therapeutic path; then in the lab for cell therapy development, and finally back in the clinic to monitor therapeutic success in patients who have been treated with gene or cell therapy. To date, the imaging tools used in the clinic do not provide sufficient resolution for visualizing individual cells non-invasively, constituting a major roadblock for the development of gene and cell therapies. My group develops novel optical imaging tools for noninvasive cellular imaging such as full field optical coherence tomography (FFOCT), an interferometric technique, and adaptive optics ophthalmoscopy (AOO), which corrects ocular aberrations, to achieve diffraction limited resolution of in vivo retina. Recently we also devised a dynamic FFOCT method to detect metabolic contrast using intracellular organelle motion to indicate cell activity. These new noninvasive, all optical tools have the potential to provide, for the first time, simultaneous subjective and objective retinal function measurements. Here, I propose to adapt these existing optical setups based on dynamic FFOCT and AOO to allow retinal stimulation with visible light to enable functional testing of i) patients with IRDs in order to evaluate surviving cells and orient their therapy path; ii) in vitro retinal organoids derived from induced pluripotent stem cells; ii) patients being treated in our clinical centre with novel gene and cell therapies to check that vision is being successfully preserved or restored. The new imaging methods developed here will achieve quantitative functional assessment of cellular activity in vivo and in vitro, lifting the major imaging obstacles for successful application of gene and cell therapies in the clinic.

In the human placenta, organ responsible for fetal growth and development, the multinucleated cellular layer, named syncytiotrophoblast (ST) plays a major role. Indeed ST bathing in maternal blood is the site of nutrient exchange and hormone synthesis. ST expands and regenerates all along pregnancy via the recruitment by fusion of underlying mononucleated cytotrophoblast cells. The apical membrane of ST is formed of microvilli which can brake away in the intervillous space. In addition, large ST pieces, called sprouts, are also released in the maternal blood all along pregnancy. Preeclampsia one of the main causes of severe premature births and Intra-Uterine Growth Retardation (IUGR) are two major pregnancy pathologies from placental origin with major socio-economical and human costs. They are associated with a large release of membrane trophoblast necrotic material as well as an abnormal regeneration of the ST. These ST fragments are inflammatory for the maternal endothelial cells. Therefore extensive membrane ruptures occur in physiological and pathological conditions, and must be compensated by efficient processes of membrane repair. Our current understanding of the mechanisms of membrane repair of the human placenta is very poor. Recently, Partner-1 (A. Brisson) has elucidated the function of Annexin-A5 in membrane repair. Annexin-A5 discovered originally in the placenta is the prototype member of the annexins, a family of soluble proteins that share the property of binding to negatively-charged lipid membranes, principally those containing phosphatidylserine (PS) in a Ca2+-dependant manner. According to J. Rand’s hypothesis, proposed fifteen years ago, PS molecules are exposed at the ST membrane surface and Annexin-A5 forms a layer covering the ST surface, which prevents blood coagulation in the intervillous space. Despite its potential interest, this hypothesis is weakly supported by low-resolution immune-histological data. The recent finding that Annexin-A5 occupies a central place in the machinery of membrane repair, which is of vital importance in cell’s life, constitutes a major breakthrough in Annexin-A5 research. This study concludes that Annexin-A5 promotes membrane repair via the formation of 2D arrays at the level of damaged membranes, which prevents the expansion of membrane wound and facilitates the final step of membrane resealing. In view of 1) the role of Annexin-A5 in membrane repair, 2) the extensive membrane damages occurring at the placenta ST membrane and 3) the high Annexin-A5 content of the placenta, we postulate that Annexin-A5 is involved in membrane repair of the ST membrane. The overall aim of the PlacentA5 project is thus to elucidate whether Annexin-A5 is involved in membrane repair of placental ST, and what is its exact role in normal and pathological placentas. The strength of this project is the task force joining two leader teams with unique complementary expertise. Partner 1 (A. Brisson) is a leader in imaging and structure-function studies of Annexin-A5 assemblies. Partner 2 (D. Evain-Brion) is an international reference team in the field of human placenta physiology and human trophoblast differentiation, with a unique expertise in setting in vitro models of human trophoblast differentiation. This project is developed within the RTRS PremUp allowing normal and patholigical placenta collections.