Embryoid bodies (EBs) resemble self-organizing aggregates of pluripotent stem cells that recapitulate some aspects of early embryogenesis. Within few days, the cells undergo a transition from rather homogeneous epithelial-like pluripotent stem cell colonies into a three-dimensional organization of various cell types with multifaceted cell–cell interactions and lumen formation—a process associated with repetitive epithelial-mesenchymal transitions. In the last few years, culture methods have further evolved to better control EB size, growth, cellular composition, and organization—e.g., by the addition of morphogens or different extracellular matrix molecules. There is a growing perception that the mechanical properties, cell mechanics, and cell signaling during EB development are also influenced by physical cues to better guide lineage specification; substrate elasticity and topography are relevant, as well as shear stress and mechanical strain. Epithelial structures outside and inside EBs support the integrity of the cell aggregates and counteract mechanical stress. Furthermore, hydrogels can be used to better control the organization and lineage-specific differentiation of EBs. In this review, we summarize how EB formation is accompanied by a variety of biomechanical parameters that need to be considered for the directed and reproducible self-organization of early cell fate decisions.

Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP^-/-) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP^-/- iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP^-/- lines. Notably, in undirected self-organized YAP^-/- EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.

Epigenetic regulatory mechanisms, which include histone modifications and DNA methylation, play a central role in development and aging. Dimethylation of H3K36, deposited mainly by the histone methyltransferase NSD1, occurs predominantly in intergenic regions, and recruits the DNA methyltransferase DNMT3A to facilitate DNA methylation. Haploinsufficiency of NSD1 results in the overgrowth disorder Sotos syndrome in vivo, which is associated with aberrant DNA methylation signatures and an enhanced epigenetic age. To understand the mechanisms by which NSD1 may regulate development, differentiation, and diseases, we generated human iPSC lines deficient in functional NSD1 (NSD1-KO). NSD1-KO cells exhibit a substrate-specific decrease in proliferation, reduced H3K36me2 levels, and extensive DNA hypomethylation. Notably, the loss of functional NSD1 altered the differentiation potential of iPSCs, with aberrant endodermal and mesodermal lineage commitment. Further analysis revealed that NSD1 might drive endodermal differentiation by regulating the expression of an endodermal lncRNA ‘HIDEN’ by mechanisms independent of the regulation of DNA methylation. Our NSD1-KO iPSC lines partially recapitulate the DNA methylation defects associated with NSD1-related developmental disorders. Additionally, we uncovered a novel mechanism by which NSD1 may regulate endodermal differentiation of human iPSCs.