Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.
and kc370@cam.ac.uk 2 AUTHOR CONTRIBUTIONS KJC and JCRS designed the project. CCA and KJC developed the hydrogel technology. CL, CCA, BXT, MH, AW, CMV, and HTS performed the experiments and analysis. GGS analysed RNA sequencing data. WM performed the blastocyst injections. JCRS and KJC supervised ES cell experiments and analysis. PB supervised analysis of RNA sequencing data. KF supervised AFM analysis. CL and KJC wrote the paper. ACKNOWLEDGEMENTSWe are grateful to Peter Humphreys for assistance with imaging and imaging analysis, Sally Lees and staff for tissue culture work, Maike Paramor and staff for preparation of sequencing libraries, Michael A. Barber for alignment of sequencing data, G. Chu and staff for animal husbandry, CarlaMulas for comments on the manuscript, Ivan B. Dimov and Amelia Joy Thompson for help with AFM measurements. Rosa26-CreERT2+/+ cells were a kind gift from the lab of Bon-Kyoung Koo. Illumina RNA sequencing was performed at the CIGC, Cambridge. ABSTRACTStudies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness.We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.
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