Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids

Introduction: Cell lineage specification is tightly associated with profound morphological changes in the developing human embryo, particularly during gastrulation. The interplay between mechanical forces and biochemical signals is poorly understood. Methods: Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model based on spheroids formed from human induced pluripotent stem cells (hiPSCs). Results: First, we compare self-renewing versus differentiating media conditions in free-floating cultures and observe the emergence of tri-germ layers. In these unconfined conditions, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Discussion: Our study combines high-content imaging, synthetic hydrogels, and hiPSCs-derived models of early development to define the drivers that cause changes in the shape and the emergence of germ layers.