K20 - Matrix stiffness-induced cytoskeletal remodeling drives fate heterogeneity in induced pluripotent stem cells

Introduction: : Stem cells actively sense and respond to physical cues such as matrix stiffness, which regulates key behaviors including adhesion, migration, and lineage commitment. While multipotent stem cells show stiffness-dependent differentiation toward specific lineages [1], the mechanobiological response of pluripotent stem cells remains poorly defined, especially regarding actin cytoskeletal remodeling and its link to cell fate. Given their high plasticity and therapeutic potential, elucidating how iPSCs interpret mechanical cues is crucial for improving control over their fate and minimizing lineage heterogeneity. In this study, we cultured fibroblast-derived iPSCs on hydrogels with tunable stiffness (150 Pa to 90 kPa) and quantitatively analyzed their morphology, cytoskeletal architecture, traction force generation, and gene expression. We found that increasing matrix stiffness induced cytoskeletal polarization and mechanical asymmetry, accompanied by stiffness-dependent priming of multiple lineage markers. These results suggest that matrix stiffness alone, in the absence of chemical cues, can direct fate bias in iPSCs while partially retaining pluripotency, offering insight into biophysical strategies for guiding stem cell behavior.

Materials and

Methods: : Materials and Methods
Matrix preparation: Polyacrylamide (PA) hydrogels with tunable stiffnesses of 150 Pa, 3 kPa, 11 kPa, 38 kPa, and 90 kPa were prepared on glass-bottom dishes. Surface activation was performed using sulfo-SANPAH crosslinking, followed by coating with Matrigel to promote cell adhesion.
Cell culture: Fibroblast-derived iPSCs from a healthy donor (1×10⁴ cells/cm²) were seeded onto PA hydrogels with stiffness values of 150 Pa, 3 kPa, 11 kPa, 38 kPa, and 90 kPa, and cultured for 96 hours. The PA hydrogels were coated with Matrigel using sulfo-SANPAH crosslinking.
Morphological and cytoskeletal analysis: Cell morphology was monitored using phase-contrast microscopy. Actin cytoskeletal organization was analyzed by live-cell imaging after transfection with a fluorescent actin probe.
Gene expression analysis: Quantitative real-time PCR (RT-qPCR) was conducted at 96 hours using stem cell lineage and pluripotency panels to assess transcript levels of germ layer-specific and pluripotency-associated genes.

Results, Conclusions, and Discussions:: 1. Matrix stiffness-induced morphological and mechanical adaptations (Fig 1)
On soft matrices (150 Pa, 3 kPa), iPSCs formed compact, rounded clusters with homogenous actin organization and uniformly distributed traction forces. In contrast, on stiffer matrices (38 kPa, 90 kPa), iPSCs spread into monolayers, accompanied by actin accumulation at the periphery and traction concentrated along the edges. These findings indicate that increasing matrix stiffness promotes cytoskeletal polarization and mechanical asymmetry in iPSCs.
2. Stiffness-dependent priming of heterogeneous lineage marker expression
RT-qPCR analysis demonstrated that key pluripotency-associated genes were retained across all stiffness conditions, suggesting maintenance of the undifferentiated state at the transcriptional level. However, stiffness-dependent changes in lineage marker expression were observed. Ectodermal markers (PAX6,) were upregulated at intermediate stiffness, while mesodermal (T) and endodermal (SOX17) markers showed increased expression at higher stiffness. These findings indicate that mechanical cues alone, without chemical differentiation factors, can initiate heterogeneous lineage priming in iPSCs in a stiffness-dependent manner.

Matrix stiffness serves as a critical biophysical cue that regulates the cytoskeletal architecture, mechanical force distribution, and ultimately the differentiation potential of iPSCs [2]. In this study, we demonstrated that substrate stiffness modulates iPSC morphology and traction patterns, with soft matrices preserving compact colony organization and uniform mechanical stress, whereas stiff matrices induced cytoskeletal polarization and peripheral force localization. These morphological and mechanical changes suggest activation of mechanotransductive signaling pathways sensitive to extracellular stiffness.
Previous studies have implicated integrin-mediated adhesion complexes, RhoA-ROCK signaling, and YAP/TAZ nuclear localization as key mechanotransduction mechanisms translating mechanical stimuli into transcriptional regulation [3]. Under these stiffness conditions, cytoskeletal remodeling likely enhanced intracellular tension and altered nuclear signaling cascades, leading to the observed upregulation of lineage-specific genes. Importantly, the maintenance of pluripotency-associated gene expression indicates that iPSCs under elevated stiffness conditions were primed toward heterogeneous lineage fates.
These findings suggest that mechanical imbalance between the cell and substrate can bias stem cell fate decisions even in the absence of chemical differentiation cues. Understanding the mechanobiological control of iPSC fate has important implications for regenerative medicine, offering strategies to direct differentiation outcomes through tailored mechanical microenvironments without relying solely on biochemical factors.

Acknowledgements and/or References (Optional):: Funding and financial support - National Research Foundation of Korea (NRF – 2020M3A9E4039658).
Cell source provided by laboratory of Neural Stem Cell Biology, Biological science Engineering, KAIST