Presentation Information
[P03-428]Substrate Stiffness Regulates Endocytosis-Related Pathways and Molecular Uptake in Human Induced Pluripotent Stem Cells
○Xi Yuan1, Masanobu Horie1, Yuichi Tsunoyama1 (1. Kyoto University (Japan))
Keywords:
human induced pluripotent stem cell,Endocytosis,Substrate Stiffness
[Purpose]
The fate and function of human induced pluripotent stem cells (hiPSCs) are governed by a complex interplay of biochemical signals and mechanical cues from the microenvironment. While the effects of mechanotransduction on hiPSC differentiation are well-documented, the impact of physical niche properties on fundamental membrane dynamics remains poorly understood. This study investigates how substrate stiffness modulates endocytic pathways and the resulting cellular response to exogenous molecules.
[Method]
hiPSCs were cultured on polyacrylamide gel-based functionalized substrates with tunable stiffness. Morphological changes and cytoskeletal organization were characterized using high-resolution imaging. To elucidate the molecular mechanisms, RNA sequencing (RNA-seq) was performed, and pharmacological inhibitors, including the ROCK inhibitor Y-27632, were utilized to assess the role of actomyosin contractility.
[Results]
hiPSCs cultured on soft substrates exhibited significant morphological remodeling and altered cytoskeletal tension compared to those on a rigid substrate. Inhibition of ROCK kinase reversed these phenotypic changes, suggesting that the response is driven by Rho/ROCK-mediated mechanosensing. RNA-seq analysis revealed a distinct transcriptomic profile on soft substrates, with significant enrichment in genes associated with vesicle trafficking, membrane dynamics, and caveolae-mediated endocytosis. Furthermore, cells on soft substrates demonstrated altered sensitivity to streptomycin, indicating functional changes in molecular uptake.
[Consideration]
These findings suggest that the mechanical context of the extracellular environment directly influences endocytic activity and membrane trafficking in hiPSCs, potentially through cytoskeleton-dependent regulation of vesicle dynamics. The observed shift in uptake sensitivity implies that mechanotransduction pathways may modulate how cells internalize external compounds.
[Conclusion]
Our findings demonstrate that substrate stiffness is a critical regulator of endocytosis-related pathways in hiPSCs. This linkage between mechanical stimuli and membrane trafficking not only advances our understanding of hiPSC cell biology but also has significant implications for drug delivery, toxicology screening, and the optimization of culture protocols for regenerative medicine.
The fate and function of human induced pluripotent stem cells (hiPSCs) are governed by a complex interplay of biochemical signals and mechanical cues from the microenvironment. While the effects of mechanotransduction on hiPSC differentiation are well-documented, the impact of physical niche properties on fundamental membrane dynamics remains poorly understood. This study investigates how substrate stiffness modulates endocytic pathways and the resulting cellular response to exogenous molecules.
[Method]
hiPSCs were cultured on polyacrylamide gel-based functionalized substrates with tunable stiffness. Morphological changes and cytoskeletal organization were characterized using high-resolution imaging. To elucidate the molecular mechanisms, RNA sequencing (RNA-seq) was performed, and pharmacological inhibitors, including the ROCK inhibitor Y-27632, were utilized to assess the role of actomyosin contractility.
[Results]
hiPSCs cultured on soft substrates exhibited significant morphological remodeling and altered cytoskeletal tension compared to those on a rigid substrate. Inhibition of ROCK kinase reversed these phenotypic changes, suggesting that the response is driven by Rho/ROCK-mediated mechanosensing. RNA-seq analysis revealed a distinct transcriptomic profile on soft substrates, with significant enrichment in genes associated with vesicle trafficking, membrane dynamics, and caveolae-mediated endocytosis. Furthermore, cells on soft substrates demonstrated altered sensitivity to streptomycin, indicating functional changes in molecular uptake.
[Consideration]
These findings suggest that the mechanical context of the extracellular environment directly influences endocytic activity and membrane trafficking in hiPSCs, potentially through cytoskeleton-dependent regulation of vesicle dynamics. The observed shift in uptake sensitivity implies that mechanotransduction pathways may modulate how cells internalize external compounds.
[Conclusion]
Our findings demonstrate that substrate stiffness is a critical regulator of endocytosis-related pathways in hiPSCs. This linkage between mechanical stimuli and membrane trafficking not only advances our understanding of hiPSC cell biology but also has significant implications for drug delivery, toxicology screening, and the optimization of culture protocols for regenerative medicine.
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