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bioRxiv · 10.1101/2024.05.09.593319

Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid development

Blotenburg, M.·Suurenbroek, L.·Bhardwaj, V.·Zeller, P.
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Abstract

By aggregating low numbers of mouse embryonic stem cells (mESCs) followed by exposure to Wnt activation, structures termed gastruloids are produced that display an anteroposterior organisation of cell types derived from all three germ layers. Since current gastruloid protocols display considerable heterogeneity between experiments in terms of morphology, elongation efficiency, and cell type composition, we investigated whether an optimised mESC pluripotency state would provide more consistent results. By growing mESCs in different intervals of ESLIF and 2i medium the pluripotency state of cells was modulated, and mESC culture as well as the resulting gastruloids were analysed. Microscopy analysis showed a pre-culture-specific effect on gastruloid formation, in terms of efficiency, elongation index and reproducibility. RNA-seq analysis of the mESC start population confirmed that short-term pulses of 2i and ESLIF modulate the pluripotency state, and result in different cellular states. With multiple epigenetic regulators among the top differentially expressed genes we further analysed genome-wide DNA methylation and H3K27me3 distributions. We observed the same divergence between conditions, most dominantly displayed in the promoter regions of developmental regulators. Lastly, when we investigated the cell type composition of gastruloids grown from these different pre-cultures, we observed that mESCs subjected to 2i-ESLIF preceding aggregation generated gastruloids more consistently, including more complex mesodermal contributions as compared to the ESLIF-only control. This indicates that optimisation of the mESCs pluripotency state allows the modulation of cell differentiation during gastruloid formation.

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Blotenburg, M., Suurenbroek, L., Bhardwaj, V., Zeller, P.. 2024-05-09. Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid development. https://doi.org/10.1101/2024.05.09.593319

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