Investigating the Specification and Regulation of the Human Trophoblast Lineage Using Cell Culture Models
Date of Award
Doctor of Philosophy (PhD)
The placenta is a complex organ system that mediates the exchange of nutrients, gases, and waste products between the mother and the developing fetus. Multiple pathologies including miscarriage, pre-eclampsia, and intrauterine growth restrictions are caused by deficient placental development and function. Cells in the placenta, which are derived from the trophectoderm (TE) of the blastocyst, are termed trophoblasts. Despite their importance, there is a significant gap in knowledge regarding the mechanisms of human trophoblast specification and regulation, partially due to the historic lack of a faithful human trophoblast model system. Therefore, I first established an in vitro model system of human trophoblast development. I showed that naïve human pluripotent stem cells (hPSCs), which possess morula and inner cell mass characteristics, can directly and efficiently give rise to bona fide human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous trophoblast (EVT) and syncytiotrophoblasts (STB). Global transcriptome and chromatin accessibility analyses confirmed the identity of hTSCs derived from naïve hPSCs, and showed that they acquire features of late post-implantation TE. Next, to systematically explore the molecular regulators of human trophoblast identity, I utilized a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing my data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, I have defined hTSC-specific and -enriched regulators. These include both well-established and novel trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data revealed that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. I also compared hTSC regulators to previously reported mouse placental regulators, and explored the identity of conserved, as well as species-specific, trophoblast regulators. Overall, my study provides a cellular model system of early mechanisms governing human trophoblast specification, as well as a robust methodology for a renewable, patient-specific source of hTSCs. It will also serve as a valuable resource for dissecting the molecular regulation of human placental development and diseases.
Chair and Committee
Thorold W. Theunissen
Thorold W. Theunissen
Dong, Chen, "Investigating the Specification and Regulation of the Human Trophoblast Lineage Using Cell Culture Models" (2022). Arts & Sciences Electronic Theses and Dissertations. 2716.
Available for download on Friday, May 20, 2112