Stem Cell-Derived Embryo Models in Mouse and Human to Illuminate the “Black Box” of Pre- to Post-Implantation Development

Citation

Jorgensen, Victoria Lynn (2023) Stem Cell-Derived Embryo Models in Mouse and Human to Illuminate the “Black Box” of Pre- to Post-Implantation Development. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/t1fe-3915. https://resolver.caltech.edu/CaltechTHESIS:06112023-211027828

Abstract

Mammalian development is a complex and highly regulated process by which a single cell, the totipotent zygote, gives rise to all lineages of the future organism. While incredible advancements have been made to study and understand the earliest events of our life, many questions are still unanswered. Moreover, the most precarious stage of development, implantation, remains a “black box” to researchers due to inaccessibility of the embryo within the uterus of the mother. In the last decade, however, the emergence of stem cell derived embryos represents an exciting alternative avenue to study these dynamic stages.

During my PhD, I worked to establish two pre-implantation stem cell models, one in human and one in mouse, to better understand the earliest days of mammalian development. These models replicate the blastocyst stage of development; at this point in time the embryo is ready to implant into the uterus and contains all embryonic and extra-embryonic tissues needed to form the future organism: the epiblast, the hypoblast, and the trophectoderm. Beginning with my human model, I demonstrate the ability of a single cell type, expanded potential stem cells (EPSCs), to give rise to structures that replicate the natural blastocyst in size, morphology, and initiation of lineage segregation. Furthermore, these human blastocyst-like structures can undergo the very beginning of post-implantation remodeling by forming an epiblast rosette and initiating lumenogenesis. Nevertheless, single cell RNA-seq (scRNA-seq) analysis reveals that lineages are not fully committed in this model, perhaps explaining why development is limited in these structures up to about Day 7/8. In the context of my mouse model, I combine not one but three distinct cell types to generate blastocyst-like structures: 1) wildtype embryonic stem cells (ESCs) to form the epiblast, 2) trophoblast stem cells (TSCs) to form the trophectoderm, and 3) Gata4-inducible ESCs to form the primitive endoderm. Again, these structures mimic the natural mouse blastocyst in morphology and lineage segregation and demonstrate the ability to transition to post-implantation stages. Development of the three blastocyst lineages was further confirmed via global scRNA-seq analysis comparing our Gata4i-Blastoids to natural embryos; importantly, however, this analysis also showed that differentiation of the mural trophectoderm, the tissue responsible for uterine invasion, is lacking in our stem cell model and likely explains the inability for these blastoids to implant in vivo.

Altogether, this dissertation explains key aspects of pre- to post-implantation development and highlights the incredible power of stem cell-derived embryos to self-organize into structures that closely mimic the natural embryo.

Thesis Files