Modeling Human Trophoblast Differentiation and Function Using Pluripotent Stem Cells - PROJECT SUMMARY/ABSTRACT Trophoblast is the major epithelial cell type in the placenta and provides an essential maternal-fetal interface during pregnancy. Understanding the specification and differentiation of the trophoblast lineage is vital for improved diagnosis and treatment of placental complications, including implantation failure, preeclampsia, and preterm birth. Trophoblast has historically been difficult to study in humans due to limited access to first-trimester placental tissue and the inability of animal models to fully recapitulate human placental development. To enable dissection of early mechanisms of human trophoblast development, we will leverage “naïve” human pluripotent stem cells (hPSCs), which exhibit molecular signatures of pluripotent cells in pre-implantation embryos. We recently showed that naïve hPSCs readily differentiate into human trophoblast stem cells (hTSCs), which can in turn differentiate into specialized extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs) or self- organize into 3D organoids that encompass a diversity of trophoblast cell types. Here, we will combine these 2D and 3D models of trophoblast development with epigenomic and single cell approaches to dissect the mechanisms of human trophoblast differentiation and function. Importantly, we have already validated key preliminary findings in human first-trimester placental tissues and will extend this in vivo validation to all the major findings of this project. Aim 1 will establish an epigenome roadmap during the transition from naïve hPSCs into hTSCs and their subsequent differentiation into specialized trophoblast fates. This work will identify functional cis-regulatory elements (CREs) and test the hypothesis that trophoblast specification from naïve hPSCs is primary mediated by transitions between unmarked and actively marked CREs. Aim 2 will define a core transcriptional circuitry governing human trophoblast specification, starting from the results of a genome-wide CRISPR/Cas9 knockout screen in hTSCs. We will test the hypothesis that transcription factors (TFs) enriched at enhancers and promoters of hTSC-specific essential genes constitute an upstream circuitry of trophoblast inducers and integrate these trophoblast inducers and their target CREs into a dynamic gene regulatory network during trophoblast specification. Aim 3 will investigate candidate regulators of trophoblast differentiation into EVT and STB lineages, which perform specialized functions during pregnancy. We hypothesize that the G protein- coupled receptor CCR7 and the TF TEAD1 are required for EVT differentiation. By generating trophoblast organoids from CCR7 and TEAD1 knockout hPSC lines, we will delineate a genetic hierarchy of factors regulating EVT differentiation and invasion. We will also investigate novel regulators of STB differentiation based on their ability to promote cell cycle exit in hTSCs and their enrichment at STB-specific open chromatin. In summary, this project will provide conceptual and experimental advances in understanding the genetic and epigenetic mechanisms regulating specification of the human trophoblast lineage. Our work will also define the action and significance of CREs and TFs in regulating the differentiation of hTSCs into specialized trophoblast cell types.
