Post Date
Sep 19 2021

Deciphering the Molecular Basis of Trophoblast Stem Cell Differentiation and Function

Dr. Amir Faisal
Rahim Ullah
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Cellular differentiation is one of the fundamental developmental processes through which complex cell types and tissues are formed from a single zygote due to spatiotemporal regulation of gene expression. During mammalian development, differentiation is first marked at the blastocyst stage of the developing embryo when a single outer layer of polarized epithelial cells, called trophectoderm, is differentiated and segregated from the inner cell mass (embryoblast). Trophoblast stem cells (TSCs) within trophectoderm differentiate into various cells of the trophoblast lineage including polyploid trophoblast giant cells (TGCs) that help with implantation of the fetus into the mother’s uterus and the subsequent placenta formation. Mouse TSCs can be derived from the polar trophectoderm of the blastocyst and have been used as an in vitro model to study the regulation of placental development. Similarly, the trophoblast-derived human choriocarcinoma cell line BeWo is functionally equivalent to TSCs and can mimic in vivo syncytialization. Differentiation of both these cell types can be induced in vitro for studying the regulation of the differentiation process and the role these cells play in placental development and function. While several differentially expressed genes, including regulators of TSC differentiation, have been identified, a comprehensive analysis of the global expression of genes and splice variants in TSCs and TGCs has not been reported.

The work described in this thesis profiled the transcriptome of undifferentiated (TSCs) and 4-days differentiated (TGCs) cells through RNA-seq and identified ~7,800 differentially expressed genes which include regulators of the cell cycle, apoptosis, cytoskeleton, cell mobility, embryo implantation, metabolism and various signaling pathways. We revealed that several mitotic proteins, including Aurora A kinase, were downregulated in TGCs and that the activity of Aurora A kinase is required for the maintenance of TSCs. We also identified hitherto undiscovered, cell-type-specific alternative splicing events in 31 genes and expression of 19 novel exons in 12 genes in TSCs and TGCs. Similarly, we discovered that the expression of Cyclin-dependent Kinase 1 (Cdk1) was downregulated in fused BeWo cells and its inhibition through small-molecule inhibitor induced fusion in these cells. The role of Caspase3 downregulation in conferring resistance to genotoxic stress-induced apoptosis in developmentally programmed polyploid cells was also investigated. Finally, we discovered that the polyploid nuclei in fused BeWo cells result from nuclear fusion following the fusion of the cells. Overall, results described in this dissertation uncover several potential regulators of TSC differentiation and TGC function, thereby providing a valuable resource for developmental and molecular biologists interested in the study of stem cell differentiation and embryonic development. Moreover, these results will help in understanding the role and regulation of apoptosis in differentiated TGCs.