An in vitro model of human yolk sac and epiblast crosstalk
Embryogenesis entails complex signalling interactions between embryonic and extraembryonic lineages. Improved embryo culture techniques have recently allowed human embryos to be cultured in vitro past the point at which they would implant in vivo, until the internationally accepted culture limit of 14 days. Nonetheless, the reliance on surplus in vitro fertilised (IVF) embryos, along with the ethical and legal considerations associated with genetic manipulation of human embryos, present barriers to the study of postimplantation human development. However, human stem cell-based embryo models offer a system that is amenable to genetic and molecular manipulation. Within mouse development, the anterior visceral endoderm (AVE), formed from the extraembryonic endoderm, plays a crucial role in restricting primitive streak formation to the posterior epiblast, thereby establishing the anterior-posterior axis of the mouse embryo. Recent studies of human embryos cultured to postimplantation stages have alluded to the presence of a human equivalent of the mouse AVE in the anterior hypoblast, the hypoblast being the portion of the human yolk sac that lies adjacent to the epiblast. However, the functional role of such a structure within the context of human development is yet to be understood. In vitro stem cell modelling of the human yolk sac- epiblast interaction offers a system through which the role of the yolk sac in postimplantation epiblast morphogenesis can be interrogated. Consequently, the aim of this study was to derive yolk sac-like cells (YSLCs) that could be used to model human epiblast-yolk sac crosstalk in vitro. Following a screen of developmentally relevant signalling pathway activators, this project identified that the coactivation of NODAL, WNT and JAK/STAT signalling via ACTIVIN-A, CHIRON, and leukaemia inhibitory factor (LIF) (ACL) induced an endodermal fate in human pluripotent stem cells (hPSCs). Functional and transcriptional analysis of ACL-treated cell populations revealed that the pluripotent state harboured by hPSCs determines the type of endoderm that ACL treatment induces. Human embryonic stem cells (hESCs), which harbour a primed pluripotent state, were found to respond to ACL by adopting a definitive endoderm-like fate, whereas Rset hPSCs, which harbour a pluripotent state that is an intermediary between naïve and primed, gave rise to a yolk sac-like population. Accordingly, Rset hPSCs treated with ACL were coined YSLCs. YSLCs were found to express inhibitors of BMP, WNT and NODAL signalling, while YSLC conditioned medium was shown to inhibit downstream activity of BMP and WNT signalling in hESCs in both 2D and 3D culture conditions. Similarly, YSLC conditioned medium supported the coexpression of pluripotency and anterior ectoderm markers in hESCs, at the expense of posterior epiblast markers. These results indicated that YSLCs share functional characteristics with the mouse AVE, and also potentially the human anterior hypoblast. Collectively, therefore, this project proposes YSLCs as a tool for studying human epiblast-yolk sac crosstalk.
stem cell, embryology, hESCs, hypoblast, human embryo, epiblast, trophoblast, human development
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