Oral 2 - 2.4
1Ursula Nosi, 2Fredrik Lanner, 1Brian Cox
1 Physiology, University of Toronto, Toronto, ON, Canada; 2 Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden
Stem cells representative of the trophectoderm (TE; TSCs) and inner cell mass have made it possible to investigate processes governing embryogenesis in vitro. We previously used these models to investigate the role of microRNAs in cell fate specification resulting in the derivation of a new, mural TE-like cell type (mTELCs, Nosi et al., 2017). These cells exhibit a reduced differentiation capacity in vitro, yet are able to follow their expected developmental trajectory in vivo. Chimeric embryos were generated by injecting fluorescently labeled TSCs or mTELCs into morulae. A subset of embryos were screened at the blastocyst-stage to assess cell contribution potential. Remaining embryos were transferred into pseudo-pregnant female mice to allow further development in vivo, and dissected at 6.5dpc. Both TSC and mTELCs contribute exclusively to the TE monolayer of blastocysts. In post-implantation embryos, TSCs predominantly contribute to the extra-embryonic ectoderm (ExE) compartment with low-frequency contribution to the Reichert’s membrane (RM)-comprised of trophoblast giant cells deriving from the mural TE, whereas mTELCs only localized to the RM. The RM-containing epiblast (Epi) was micro-dissected away from the ExE of 6.5dpc embryos, generating two fractions. Single cell suspensions were made from these fractions which were further analyzed by flow cytometry. In TSC-injected embryos, fluorescent cells were enriched in the ExE fraction and not the Epi, whereas embryos injected with mTELCs revealed a higher proportion of fluorescent cells in the Epi compared to the ExE fraction. We are currently performing single-cell transcriptomic analysis of TSC and mTELCs “re-isolated” from embryos. The profile of these embryo-isolated cells will be compared to those of TSCs and mTELCs cultured in stem cell promoting or differentiating conditions prior to embryo injections. This in-depth interrogation of mTELCs aims to unravel fundamental mechanisms of embryo implantation mediated by the complex interaction between the mural TE and endometrium. Further, we aim to understand how the microenvironment drives differentiation of TSCs and mTELCs down their appropriate developmental trajectories. These results will have wider implications on how closely in vitro conditions accurately mimic in vivo environments.