PROJECT ABSTRACT Impacts of health-related issues are considered proportional to areas with dense air pollution. Air pollution composition is based highly on the area of pollution, however, are made up by general pollutants such as gases from exhaust, particulate matter, and compounds released from burning sources. Although environmental contaminants have been linked to health conditions in the past, direct work assessing air pollution exposure to prenatal bone development is inconclusive. Therefore, the purpose of this supplement is to 1) elucidate the impact of air pollutants on osteogenesis; 2) determine relevant miRNAs related to toxicant exposure and how it impacts bone development; 3) discern specific endocrine-disrupting chemicals found within air pollution; and 4) bring back findings to the communities that it may be impacting. This will be accomplished through direct exposure of pollution to osteogenically-differentiating human embryonic stem cells (hESCs), stained time-lapsed image quantification of mineralization phenotype, determining alterations of mRNA expression throughout differentiation, miRNA-sequencing analysis, and GC/MS data to quantify chemicals within samples. The work proposed within this supplement will be the first study of its kind to demonstrate perturbed osteogenesis in response to air pollution exposure. Additionally, this work will address the gap in knowledge of the dysregulation of miRNAs and resulting bone birth defects. In conjunction with an established human in vitro model and miRNA sequencing analysis, this supplement will continue to advance the evolving fields of molecular toxicology, reproductive toxicology, developmental toxicology, and bioinformatics, as was described in the parent grant. By completion of these studies, I expect to determine a causal relationship between prenatal air pollution exposure and skeletal birth defects. The implications of this work can reach beyond the science community, as these findings may aid in the development of prenatal check-up scans and help inform exposure policies in relation to congenital birth defects.