Thank you! So nice to hear :)

Thank you Vlad! ✨️

Most importantly - thank you to Lianne, Peter, and @vivekbhr.bsky.social for kickstarting this project, and Danique, Hendrik, Joëlle and Thijs for the wonderful collaboration and adding crucial experiments to our study

We emphasise that the effects of pre-culture conditions are cell line-dependent, necessitating optimisation for each cell line (more info in our discussion!). Even so, this study helped us a lot in increasing reproducibility and recovery of complex cell types. We hope it is helpful to you too! [7/7]

Lastly, we asked what the effect of these pre-cultures is on cell fate specification. We chose the ESLIF conditions and sequenced 5 individual gastruloids for each. This showed that ESLIF-only skewed gastruloids towards a neural fate, whereas short pulses of 2i increase the mesoderm fraction. [6/7]

We profiled H3K27me3 and DNA methylation, and found differential coverage of both modifications in a subset of developmentally associated genes across pre-culture conditions. These genes were not yet transcribed, indicating that epigenetic priming plays a role. [5/7]

RNA sequencing revealed distinct transcriptome profiles between pre-cultures ending with ESLIF vs 2i incubation, with differential expression of epigenetic factors such as Polycomb subunits and DNA methyltransferases. [4/7]

Changing the pre-culture condition, as we call it, affects not only the mESC morphology but also gastruloid elongation, time point of symmetry breaking and reproducibility between experiments, and different cell lines display different optimal pre-culture conditions. But what determines this? [3/7]

This project started with observations of considerable variability in our gastruloid experiments, coupled to variable morphology in ESCs at time point of aggregation. We decided to systematically test this by applying different pulses of 2i and ESLIF medium, followed by gastruloid aggregation. [2/7]