Impact of Substrate Stiffness on Physical Traits and Pluripotency in iPSCs

Introduction: Induced pluripotent stem cells (iPSCs) have significant potential for personalized medicine, disease modeling, and regenerative therapies due to their ability to differentiate into various cell types. Mechanical cues, particularly the stiffness of the extracellular matrix (ECM), play a crucial role in regulating iPSC fate and behavior. These mechanical interactions are essential for guiding cell behavior and function.
Mechanical forces influence iPSCs at various levels, from subcellular mechanisms like actomyosin motors to larger-scale responses to external forces, including shear, tensile, and compressive stresses. These interactions with the cells’ biochemical and epigenetic landscapes broadly affect iPSC function, including self-renewal and differentiation.
In vitro, iPSC cultures benefit from controlled models that allow precise manipulation of ECM stiffness. These models facilitate detailed studies on how different stiffness levels direct iPSC differentiation and functionality. Insights into the mechanobiology of iPSCs, particularly how ECM stiffness influences them, are essential for improving both their cultivation and their application in various therapies.
This study investigates how ECM stiffness modulates iPSC behavior, aiming to develop improved stem cell cultivation methods and enhance their therapeutic applications. By optimizing conditions for maintaining iPSC pluripotency and differentiation potential, these findings could significantly advance the field of regenerative medicine.

Materials and

Methods: iPSCs were derived from fibroblasts from a healthy donor's arm and characterized via RNA sequencing to identify groups with high and low pluripotency. The iPSCs were cultured for 48 hours at 37°C and 5% CO₂ in E8 medium with 1% penicillin-streptomycin on polyacrylamide (PA) gel substrates with Young’s moduli of 3 kPa, 11 kPa, and 90 kPa, and a thickness of 100 μm. To prepare PA-gel surfaces, 24 μl of PA-gel mixed with fluorescent beads (diameter = 0.5 μm) was polymerized in a silane-treated glass bottom dish under a cover slip (diameter = 18 mm). The gel surfaces were functionalized with 1 mg/ml of Sulfo-SANPAH in warm 50 mM HEPES buffer to immobilize 50 μg/ml Matrigel. Traction force microscopy (TFM) was used to measure traction forces exerted by the cells using unconstrained Fourier transform traction microscopy (FTTM)[1]. Cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with Triton-X, blocked in bovine serum albumin (BSA), and incubated with primary antibodies for OCT4 and Nanog overnight. Secondary antibodies (Alexa Fluor 488 and 568) and DAPI were then applied for nuclear staining. Cells were imaged using a multichannel fluorescence microscope. Pluripotency marker expression levels were quantified using ImageJ software. Total RNA was isolated using RNAiso reagent, reverse transcribed to cDNA, and analyzed by qPCR, with GAPDH as the reference gene. The -ΔΔCt method was used to express log2(Fold Change) relative to the control group, providing a detailed assessment of gene expression changes in response to substrate stiffness.

Results, Conclusions, and Discussions:

Results: Immunofluorescence staining revealed that iPSCs with high pluripotency maintained consistent expression levels of pluripotency markers OCT4 and Nanog across different substrate stiffness levels (3 kPa, 11 kPa, and 90 kPa). Conversely, iPSCs with low pluripotency showed a significant increase in the expression of these markers as substrate stiffness increased, peaking at 90 kPa (Fig. 1 a, b). Quantitative PCR analysis corroborated these findings, showing elevated mRNA levels of OCT4 and Nanog on stiffer substrates, indicating enhanced gene expression linked to increased substrate stiffness (Fig. 2).Traction force microscopy (TFM) data demonstrated that high pluripotency iPSCs exerted strong traction forces consistently across all stiffness levels, while low pluripotency iPSCs exhibited significantly greater traction forces at 90 kPa. This suggests a shift in cellular responsiveness to mechanical cues, where higher stiffness promotes enhanced traction forces and pluripotency marker expression in low pluripotency iPSCs.

Conclusions: The study highlights that substrate stiffness plays a critical role in modulating the physical traits and pluripotency of iPSCs. High pluripotency iPSCs exhibit robust differentiation potential and consistent behavior across various stiffness levels, whereas low pluripotency iPSCs are more sensitive to mechanical cues, showing enhanced pluripotency marker expression and traction forces at higher stiffness levels. These findings suggest that manipulating substrate stiffness can be a strategic approach to optimizing iPSC cultivation conditions.

Discussions: Understanding the influence of substrate stiffness on iPSCs provides valuable insights into the mechanobiology of stem cells. The differential response of iPSCs based on their pluripotency levels to substrate stiffness underscores the importance of mechanical cues in stem cell regulation. For high pluripotency iPSCs, maintaining consistent behavior across different stiffness levels suggests their robust nature and potential for various therapeutic applications without the need for specific mechanical conditioning.

Acknowledgements (Optional): This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (No. NRF-2020M3A9E4039658)