Identification of the central intermediate in the extra-embryonic to embryonic endoderm transition through single-cell transcriptomics.

High-resolution maps of embryonic development suggest that acquisition of cell identity is not limited to canonical germ layers but proceeds via alternative routes. Despite evidence that visceral organs are formed via embryonic and extra-embryonic trajectories, the production of organ-specific cell types in vitro focuses on the embryonic one. Here we resolve these differentiation routes using massively parallel single-cell RNA sequencing to generate datasets from FOXA2Venus reporter mouse embryos and embryonic stem cell differentiation towards endoderm. To relate cell types in these datasets, we develop a single-parameter computational approach and identify an intermediate en route from extra-embryonic identity to embryonic endoderm, which we localize spatially in embryos at embryonic day 7.5. While there is little evidence for this cell type in embryonic stem cell differentiation, by following the extra-embryonic trajectory starting with naïve extra-embryonic endoderm stem cells we can generate embryonic gut spheroids. Exploiting developmental plasticity therefore offers alternatives to pluripotent cells and opens alternative avenues for in vitro differentiation.

Differentiation and Expansion of Human Extra-Embryonic Endoderm Cell Lines from Naïve Pluripotent Stem Cells.

In human, endoderm is induced in two waves, with the first being the extra-embryonic primitive endoderm (PrE), otherwise known as hypoblast, induced during blastocyst development, and the second being gastrulation-stage definitive endoderm (DE). The PrE gives rise to the primary and secondary yolk sac, and has supportive functions during pregnancy for nutrient provision, with descendants of this extra-embryonic lineage also playing a role in embryonic patterning. As in DE specification, we recently found that PrE could be induced in vitro by Wnt and Nodal-related signaling, but that the critical difference was in the pluripotent starting point for differentiation. Thus, blastocyst-like naïve human pluripotent stem cells retain the unique capacity to differentiate into PrE cultures, a cell type resembling the pre-implantation hypoblast. The PrE cells could then be expanded as stable naïve extra-embryonic endoderm (nEnd) cell lines, capable of indefinite self-renewal. Here, we describe detailed protocols to differentiate naïve pluripotent stem cells into PrE and then expand the cultures as nEnd, including descriptions of morphology, passaging technique, and troubleshooting.

From pluripotency to totipotency: an experimentalist's guide to cellular potency.

Embryonic stem cells (ESCs) are derived from the pre-implantation mammalian blastocyst. At this point in time, the newly formed embryo is concerned with the generation and expansion of both the embryonic lineages required to build the embryo and the extra-embryonic lineages that support development. When used in grafting experiments, embryonic cells from early developmental stages can contribute to both embryonic and extra-embryonic lineages, but it is generally accepted that ESCs can give rise to only embryonic lineages. As a result, they are referred to as pluripotent, rather than totipotent. Here, we consider the experimental potential of various ESC populations and a number of recently identified in vitro culture systems producing states beyond pluripotency and reminiscent of those observed during pre-implantation development. We also consider the nature of totipotency and the extent to which cell populations in these culture systems exhibit this property.

Inhibition of cortical neuron differentiation by Groucho/TLE1 requires interaction with WRPW, but not Eh1, repressor peptides.

In both invertebrates and vertebrates, transcriptional co-repressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental mechanisms, including neuronal differentiation. The pleiotropic activity of Gro/TLE depends on context-specific interactions with a variety of DNA-binding proteins. Most of those factors engage Gro/TLE through two different types of short peptide motifs, the WRP(W/Y) tetrapeptide and the Engrailed homology 1 (Eh1) sequence (FXIXXIL). The aim of this study was to elucidate the contribution of WRP(W/Y) and Eh1 motifs to mammalian Gro/TLE anti-neurogenic activity. Here we describe point mutations within the C-terminal WD40 repeat domain of Gro/TLE1 that do not perturb protein folding but disrupt the ability of Gro/TLE1 to inhibit the differentiation of cerebral cortex neural progenitor cells into neurons. One of those mutations, L743F, selectively blocks binding to Hes1, an anti-neurogenic basic helix-loop-helix protein that harbors a WRPW motif. In contrast, the L743F mutation does not disrupt binding to Engrailed1 and FoxG1, which both contain Eh1 motifs, nor to Tcf3, which binds to the Gro/TLE N terminus. These results demonstrate that the recruitment of transcription factors harboring WRP(W/Y) tetrapeptides is essential to the anti-neurogenic function of Gro/TLE1.