Differentiating Human Embryonic Stem Cells in Micropatterns to Study Cell Fate Specification and Morphogenetic Events During Gastrulation
Date of Award
Doctor of Philosophy (PhD)
During mammalian embryogenesis, the first major lineage segregation occurs when embryonic epiblast, and extraembryonic trophectoderm and hypoblast arise in the blastocyst. In the next fundamental and conserved phase of animal embryogenesis known as gastrulation, extraembryonic cells provide signals to epiblast to instruct embryonic patterning, and epiblast gives rise to germ layers ectoderm, mesoderm, and endoderm, that will establish all embryonic tissues. Proper specification and morphogenesis of germ layers during gastrulation is vital for correct embryonic development. Due to ethical and legal restrictions limiting human embryo studies, human gastrulation is poorly understood. Our knowledge of human gastrulation has largely been derived from studies in model organisms, including mouse and more recently, cynomolgus monkey. However, interspecies differences underscore the need for alternative human gastrulation models. In this regard, human and mouse embryonic stem cells have been shown to recapitulate aspects of in vivo gastrulation including germ layer specification, and internalization and elongation morphogenesis. These in vitro systems represent powerful models of gastrulation due to the ease of genetic manipulations and the ability to finely control experimental factors. Human embryonic stem cells, treated with BMP4 for 44 hours in spatially confined micro-discs of extracellular matrix, have been shown to differentiate into 2D micro-colonies termed gastruloids. These gastruloids display highly reproducible differentiation of germ layers and extraembryonic cell types in a radial arrangement. We used combinatorial single-cell RNA sequencing and immunofluorescence imaging to characterize these BMP4-treated 2D gastruloids, and showed the formation in gastruloids of seven cell types, including epiblast, prospective ectoderm, two populations of mesoderm, and endoderm, as well as previously undescribed cell types in 2D gastruloids, primordial germ cell-like cells, and extraembryonic cells that are transcriptionally similar to trophectoderm and amnion. Comparative transcriptomic analyses with human, mouse, and cynomolgus monkey gastrulae support the notion that 2D gastruloid differentiation recapitulates formation of cell types relevant to and models an early-mid stage of in vivo gastrulation. Time course scRNA-seq and immunofluorescence analyses of 2D gastruloid differentiation after 12, 24, and 44 hours of BMP4 treatment showed that germ layer emergence in gastruloids follows the temporal sequence of in vivo gastrulation, with epiblast and ectoderm precursors forming at 12 hour, mesendoderm precursors arising from epiblast at 24 hour to give rise to nascent mesoderm and endoderm at 44 hour, when primordial germ cell-like cells also form. Comparison with human gastrula also showed similarity in transcriptomes and differentiation trajectories of gastruloid cells to their in vivo counterparts. Dynamic changes in transcripts encoding components of key signaling pathways support a BMP, WNT and Nodal hierarchy underlying germ layer specification conserved across mammals, with FGF and HIPPO signaling being active throughout the time course of 2D micropattern gastruloid differentiation. To probe morphogenetic properties of gastruloid cells, differentiated gastruloids treated with BMP4 for 44 hours were dissociated and re-seeded onto extracellular matrix micro-discs. The reseeded cells were highly motile and tended to form aggregates with the same but segregate from or mix with distinct cell types, supporting that 2D gastruloid system exhibits evolutionarily conserved sorting behaviors. In particular, ectodermal cells segregated from endodermal and extraembryonic cells but mixed with mesodermal cells. These results demonstrate that 2D gastruloid system models specification of germ layers and extraembryonic cell types, temporal order and differentiation trajectories of germ layer emergence, and signaling interactions found in early-mid in vivo gastrulation. Dissociated and reseeded gastruloid cells also exhibit conserved cell sorting behaviors. Lastly, this work provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation.
Samantha Morris, Kristen Kroll, Jeffrey Millman, Michael Vahey,
Bioinformatics Commons, Biomedical Engineering and Bioengineering Commons, Developmental Biology Commons