Generation of an <em>in vitro</em> embryo model based on cellular reprogramming paradigms in embryonic stem cells

Abstract

This research project focuses on the generation and characterization of a stem cell-based organoid model system, that can model early murine embryonic development <em>in vitro</em> and <em>ex utero</em>. Such embryo-like cell culture models allow for deeper insights into early lineage segregation and cellular reprogramming in complex 3D co-culture environments. The recently emerging field of stem cell-based embryo models aims to recreate hallmark events of embryonic development <em>in vitro</em>. So far, research in the field was based on the co-culture of the three stem cell lines that can be generated from blastocysts at embryonic day (E) 3.5, embryonic stem cells (ESC), trophoblast stem cells (TSC) and extraembryonic endoderm (XEN) stem cells. These stem cell populations were shown to aggregate and self-organize into structures resembling early murine embryos at ~E5.0. However, the generation of these embryo-like structures relies on complex cell culture requirements for maintenance of each of the three stem cell types. In this study, a new strategy for the generation of embryo-like organoids was developed, based on transcription factor mediated reprogramming of a solely ESC-based starting cell population in a 3D cell culture environment. Therefore, three genetically modified ESC lines were used for the inducible system presented in this thesis: Kermit ESCs, carrying an <em>Oct3/4</em> promotor driven GFP expression cassette, 5 Factor ESCs (5F ESCs) carrying doxycycline inducible transgenes of <em>Cdx2, Tfap2c, Eomes, Gata3 and Ets2</em>, and iGATA6 ESCs, carrying doxycycline inducible <em>Gata6</em> transgenes. Upon doxycycline mediated induction of transgene expression, 5F ESCs underwent cell fate conversion towards an induced TSC- (iTSC) identity, while iGATA6 ESCs reprogrammed towards an induced XEN (iXEN) cell fate. Reprogramming of the two ES cell lines in co-culture with a third ES cell line (Kermit ESCs) in a 3D cell culture environment resulted in the generation of embryo-like organoids, that acquire early embryo architecture with an epiblast (Epi), extraembryonic ectoderm (ExE) and visceral endoderm (VE) -like compartment. These embryo-like organoids were shown to induce several hallmarks of embryogenesis, such as induction of rosettes and subsequent lumenogenesis, in Epi- and ExE-like compartments. Additionally, the generation of a distal-/anterior- VE cell lineage was detected, indicating patterning events mediated by interaction of all three induced cellular compartments, as observed during murine embryonic development. Furthermore, single cell RNA-Seq (scRNA-Seq) analysis showed individual, highly specific transcriptional profiles for each of the three embryo-like compartments and revealed high similarity to natural murine embryos between E4.5 – E5.5. Together, the system introduced in this study provides an easy-to-use, inexpensive tool to study specific hallmarks of early embryogenesis and cellular reprogramming in 3D co-culture with cellular signaling interactions comparable to those observed during natural embryonic development.