Project Summary Intra-amniotic infection, also referred to as chorioamnionitis, is a major etiological factor of preterm premature rupture of the membranes (pPROM), leading to preterm birth. Despite its prevalence and grave consequences, the pathology of intra-amniotic infection has yet to be completely understood due to a lack of tractable human- relevant models. Even though animal models of preterm birth have been successfully developed for testing medical interventions of intra-amniotic infection, they remain suboptimal for quantitative studies of dynamic bacterium-amnion interactions in the intrauterine cavity. The scarcity of preterm human amnion samples, especially from early/mid-gestation stages, also prevents these human tissues as experimental models for studying intra-amniotic infection and its functional link to pPROM. Altogether, there is a critical need for quantitative, tractable, human-relevant amnion membrane models for advancing fundamental understanding of intra-amniotic infection. The primary goal of this NIH R21 project is to specifically address this significant technological need, by developing a human-relevant amnion membrane model that can faithfully recapitulate the interaction between bacteria and amnion membrane tissues, and at the same time, allow high-resolution, quantitative experiments to study mechanisms underlying bacterial invasion of the amniotic cavity. In our preliminary study, we have unexpectedly discovered the amniogenic differentiation potency of human pluripotent stem cells (hPSCs) and successfully developed an hPSC-based, synthetic microfluidic embryogenesis platform in which key developmental landmarks during early human post-implantation development can be recapitulated successively in a highly controllable and scalable fashion. Importantly, we also observed sensitive inflammatory response of hPSC-derived amniotic cells to bacterial infection. Thus, in this research we propose to leverage the amnion differentiation potential of hPSCs, in conjunction with innovative microfluidics, to develop the first-of-its-kind human amnion membrane organ-on-chip system. We will further apply this tractable experimental system to quantitatively study the dynamics of bacterial invasion of the amniotic cavity and to elucidate the functional connection between inflammation-induced amniotic membrane remodeling and intra- amniotic bacterial trafficking. Successful accomplishment of this proposed research will lead to innovative technologies and methodologies for controllable, reproducible, and scalable manufacturing of human amnion membrane tissues, offering a tractable experimental system for studying related pregnancy complications, including intra-amniotic infection. The reproducibility and scalability of the human amnion membrane organ-on- chip system will make it a promising screening platform to explore complex interactions between the human amnion membrane, bacterial pathogens, drugs and toxic substances.