Harnessing the murine inner cell mass mechanical environment enhances derivation of in vitro nascent primitive endoderm precursor cells

Stem cell-based models resembling murine blastocysts represent a useful system to investigate subsequent developmental processes. While existing cell lines derived from epiblast and trophectoderm can be aggregated to form 'blastoids', some previously tested in vitro cultured extra-embryonic endoderm cells tended to progress to later stages of development, so integrated inefficiently into blastoids. We attempted to capture the precursor population for extra-embryonic endoderm in vitro by reproducing the mechanical environment of the in vivo peri-implantation embryo as closely as possible. We investigated expression of candidate cell adhesion receptor integrins in the blastocyst inner cell mass, and from this information we assembled an extracellular matrix intended to support primitive endoderm growth by promoting signalling pathways responsible for specification of this lineage. In addition, inner cell mass cells from blastocysts were plated on soft or stiff substrates to investigate whether an appropriate mechano-environment could enhance their self-renewal as primitive endoderm in culture. We could expand nascent primitive endoderm cell lines over several passages, which provided a reproducible, albeit short-term, system sufficient to identify some essential requirements for early primitive endoderm expansion and function.