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Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells
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Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids.

Haneen S Alsehli1,2, Errin Roy1, Thomas Williams1

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Physical confinement of human stem cell spheroids dramatically reduces germ layer formation. Softening hydrogels rescue this effect, revealing mechanical forces

Keywords:
PEG-based hydrogelsgerm layer differentiationhigh content image analysismorphogenesispluripotent stem cells

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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.