Three-dimensional patterned surface and mechanical stretching effect on human induced pluripotent stem cell differentiation

During embryo development, cell progenitor specification happens via the combined action of morphogens and growth factors and of confinement and mechanical stimulations generated during embryo elongation and tissue morphogenesis. Despite many studies have been dedicated to investigating morphogens and growth factors involved during stem cell commitment, less is known about the role that confinement and mechanical signals have during early stages of development. Advancements in human pluripotent stem cell (hIPSC) technology have recently opened new frontiers for studying the molecular players involved during the early stages of human development by generating complex three-dimensional (3D) in vitro models. However, the investigation of the biophysical signals known to be relevant in vivo is underrepresented in literature when hiPSC-derived in vitro models are considered. In this thesis, we explored how biophysical cues, specifically substrate topography and mechanical stimulation, influence the differentiation process of hiPSC toward cell progenitors able to give rise to the human neuromuscular system. The developed experimental setup combines i) highly reproducible microgrooved PDMS substrates, ii) a custom-built uniaxial bioreactor, which is capable of delivering physiologically relevant strain profiles, iii) hiPSC technology and iv) a custom image analysis pipeline. This allowed us to culture cells into confined patterns that can be subjected to mechanical stretch, thus allowing the qualitative and quantitative investigation of cell progenitors and their spatial organization in presence of these biophysical stimuli. The microgrooved chambers were produced with high structural fidelity, generating an anisotropic environment that confined cells along the channels. The bioreactor demonstrated linear strain delivery, enabling accurate reproduction of human embryonic axial elongation. Cell culture and differentiation was assessed and optimized, allowing the generation of the expected cell progenitors in all the experimental conditions tested. The quantification of cell populations was developed through customized MATLAB scripts, incorporating adjustable thresholding, manual refinement and spatial metrics. From a biological perspective, we found that the provided 3D constraint represented an instructive cue for cell commitment, promoting early differentiation of cells toward mesodermal identity. The application of mechanical stretching further enhanced this effect. Collectively, this work demonstrates that hiPSC commitment can be modulated by biophysical stimuli, such as 3D confinement and mechanical strain. The system and analytical pipeline also provide a foundation for future mechanobiology studies that go also behind the thesis aim, opening new perspectives for modeling and investigating in vitro the early human development.