PROJECT SUMMARY/ABSTRACT During pregnancy, trophoblast cells from the placenta invade into the uterine spiral arteries and assist with the remodeling necessary for adequate blood supply to the fetus. Cytotrophoblast, an undifferentiated and proliferative cell subpopulation, can give rise to extravillous trophoblast (EVT) cells. EVT cells are terminally differentiated trophoblast cells that invade into and restructure the uterine compartment. Impaired EVT cell development leads to adverse pregnancy outcomes, including early pregnancy loss, preeclampsia, intrauterine growth restriction, and preterm birth. Human trophoblast stem (TS) can be captured, maintained in vitro under specific conditions, and differentiated to ST as well as EVT cells. Regulatory mechanisms underlying human TS cell self-renewal and early events in the differentiation of EVT cells have largely remained elusive. We have utilized single cell RNA sequencing (scRNA-seq) to identify cell populations and candidate regulators that arise during EVT cell differentiation. A transcription factor termed CCAAT/enhancer-binding protein beta (CEBPB) was identified as a candidate regulator of early events in EVT cell differentiation. CEBPB is expressed in an intermediate progenitor cell population arising during the early stages of EVT cell differentiation and in a transitional compartment within EVT cell columns of the first trimester human placenta. Depletion of CEBPB in human TS cells disrupts EVT cell differentiation. We hypothesize that CEBPB contributes to the regulation of early events in EVT cell differentiation. To investigate actions of CEBPB on trophoblast cell lineage development we will deplete CEBPB by using lentiviral-mediated shRNA in human TS cells and will evaluate EVT cell differentiation with sc-RNAseq (Aim 1). To characterize CEBPB gene regulatory networks defining EVT cell differentiation we will perform chromatin immunoprecipitation-sequencing (ChIP-seq) and single cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) at day 3 of EVT cell differentiation (Aim 2). We will evaluate the bioavailability of CEBPB by investigating upstream transcriptional regulation of CEBPB and post-translational modification (PTM) of CEBPB (Aim 3). The proposed research plan will provide me with a body of experimental work necessary for publications and preliminary data for R-series grants. I will utilize the expertise of the mentoring team as well as resources at the University of Kansas Medical Center for cultivation of professional development skills. These skills will be improved through trainee mentoring, data presentation, and scientific writing. During the R00 phase I will develop independence from my mentors by identifying targets of CEBPB and investigating their contributions to invasive trophoblast cell development.