StemBond hydrogels optimise the mechanical microenvironment for embryonic stem cells

Abstract

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.

Coculture techniques for modeling retinal development and disease, and enabling regenerative medicine

“…107,110,112,113 The presence of specific ECM proteins facilitates tethering to biological or synthetic substrates and thereby facilitates efficient mechano-transduction of substrate stiffness to intracellular signals. 109,111,112,114 Oligodendrocyte precursor cells are multipotent cells responsible for regeneration in the central nervous system. It has recently been shown that age-dependent loss of function in oligodendrocyte precursor cells can be recapitulated by culture on stiff hydrogels which mimic the aged connective tissue and this loss of function can be restored on soft hydrogels which mimic young brains.…”

Mechanoresponse of epithelial monolayers to in-plane and out-of-plane curvatures imposed by 3D microwells

“…Indeed, cells reside in native tissues in a complex microenvironment that guides and regulates their functions [2] . For instance, numerous studies have shown that the mechanical stiffness of the cellular microenvironment has a fundamental effect on cell migration [3] , stem cell differentiation [4] and can impact tissue regeneration [5] . Recently, evidence is rising that the geometrical properties of the cell's microenvironment also play an important role in the regulation of cellular functions, such as the intestinal crypt-villus architecture where dividing stem cells reside exclusively in the concave zone [6] .…”