Embryonic stem cells (ESCs) exist in at least two states that transcriptionally resemble different stages of embryonic development. Naïve ESCs resemble peri-implantation stages and primed ESCs the pre-gastrulation epiblast. In mouse, primed ESCs give rise to definitive endoderm in response to the pathways downstream of Nodal and Wnt signalling. However, when these pathways are activated in naïve ESCs, they differentiate to a cell type resembling early primitive endoderm (PrE), the blastocyst stage progenitor of the extra-embryonic endoderm. Here, we apply this context dependency to human ESCs, showing that activation of Nodal and Wnt signalling drives the differentiation of naïve pluripotent cells toward extra-embryonic PrE, or hypoblast, and these can be expanded as an in vitro model for naïve extra-embryonic endoderm (nEnd). Consistent with observations made in mouse, human PrE differentiation is dependent on FGF signalling in vitro, and we show, that by inhibiting FGF receptor signalling, we can simplify naïve pluripotent culture conditions, such that the inhibitor requirements closer resemble those used in mouse. The expandable nEnd cultures reported here represent stable extra-embryonic endoderm, or human hypoblast, cell lines.

No abstract

SummaryAntisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.

No abstract