THE INTERGENERATIONAL IMPACT OF ENVIRONMENTAL EXPOSURES: MOLECULAR INSIGHTS FROM MODELLING PLURIPOTENCY TRANSITIONS THROUGH THE HUMAN GERMLINE

How environmental exposures shape inheritance across generations is a fundamental question in biology, with far-reaching implications for human health. Despite evidence of such inheritance in animal models, its mechanisms and relevance in humans remain elusive due to key challenges: (i) the inaccessibility of developing germ cells from exposed individuals; (ii) the predominance in the field of methodological approaches based on the investigation of single molecular layers; (iii) the impossibility of studying multiple human generations in laboratory settings; (iv) the scarcity of multi-generational human data precisely linking environmental exposures and health outcomes. Considering the central role of endocrine signalling during development, in this PhD thesis, I tackle these challenges by focusing on the paradigmatic case of the intergenerational impact of endocrine disrupting chemicals (EDCs), widespread compounds that interfere with hormonal signalling causing several adverse health effects. To overcome these challenges, I first implemented in the lab publicly available protocols to differentiate human induced pluripotent stem cells into primordial germ cells-like cells (hPGCLCs) and pro-spermatogonia/oogonia. Following their comprehensively characterization at the transcriptomic, DNA methylation, and protein-marker levels and benchmarking against human fetal reference datasets, we confirmed their ability of recapitulating key aspects of human germ cell development. Since these cells also express key hormone receptors, we considered them suitable for studying the molecular and cellular responses to EDC exposure. We also investigated the role of lipid droplet (LD) accumulation and metabolism in these cells, as a relevant cellular component that interplays with transcriptomic and epigenomic layers. We noted that, interfering with LD accumulation in different time windows, impacts differentially on hPGCLC specification and differentiation. This provided a comprehensive view of germ cell state under physiological and perturbed conditions, becoming a starting point to move from approaches based on the investigation of a single-layer and a predominantly (epi)genetic-centric view toward a cell-centric view of inheritance. However, making any claim about inheritance, requires showing whether and how these changes in germ cells, the enduring link across generations, are transmitted to the next generation(s). To address this, we developed and characterized an innovative in vitro model capable of capturing transcriptomic and epigenetic dynamics across generations, thus making epigenetic inheritance experimentally tractable in humans. We then used this model to investigate the intergenerational impact of EDCs on human neurodevelopment. To this aim, we also leveraged epidemiological data from a multi-generational human cohort, the Swedish Environmental Longitudinal, Mother and child, Asthma and allergy study (SELMA), exposing our in vitro model to agonists of key hormonal pathways and to epidemiologically relevant EDCs, to evaluate their impact on the germline transcriptome and to identify EDC-induced molecular changes in the germline that are maintained upon conversion back to a pluripotent state, as potential mechanistic underpinnings of intergenerational effects of EDC exposure in humans. We found that these compounds seem to have relatively subtle effects on hPGCLCs, possibly reflecting an intrinsic resilience of these cells to perturbations, being the founder cells of the germ cell lineage. Ongoing work is addressing potential genotype-specific responses, by testing the effect of these compounds on multiple hiPSC lines, using a mosaic approach. We are also evaluating their impact at later developmental windows, leveraging in vitro differentiated prospermatogonia and oogonia. Overall, by integrating in vitro modelling, multi-layer molecular phenotyping and epidemiological data, this work aims to establish a framework for studying the intergenerational impact of chemical exposure on human health, to shed light on the extent to which environmentally induced and inherited changes influence human development and disease risk, ultimately providing scientific evidence to guide the reduction of adverse effects on present and future generations caused by widespread EDC environmental exposures.