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

Area of Science:

• Developmental biology
• Stem cell biology
• Biophysics

Background:

• Cell lineage specification during human embryonic development involves significant morphological changes, especially during gastrulation.
• The interaction between mechanical forces and biochemical signals in early human embryogenesis remains largely unexplored.

Purpose of the Study:

• To investigate the impact of biochemical cues and physical confinement on human induced pluripotent stem cells (hiPSCs) in a 3D *in vitro* model.
• To elucidate the role of mechanical forces in regulating germ layer formation and cell shape changes during early human development.

Main Methods:

• Utilized a 3D *in vitro* model using hiPSCs cultured in spheroids.
• Compared self-renewing and differentiating media conditions in both free-floating and physically confined environments.
• Employed PEG-peptide hydrogels for physical confinement, including gels with time-dependent softening properties.
• Integrated high-content imaging for quantitative analysis.

Main Results:

• In unconfined conditions, differentiating media promoted the emergence of tri-germ layers, with BMP4 inducing polarized SOX17 expression and spheroid elongation.
• Physical confinement using hydrogels significantly reduced SOX17 expression, indicating impaired germ layer specification.
• The inhibitory effect of confinement on SOX17 expression was reversible when using hydrogels that softened over time.

Conclusions:

• Mechanical confinement plays a critical role in regulating cell lineage specification and germ layer formation in human embryonic stem cell models.
• The interplay between substrate stiffness and biochemical signaling dictates developmental outcomes.
• This study provides insights into the physical drivers of early human development using advanced *in vitro* models.