N-cadherin peptides modulate integrin-mediated mechanosensing and preserve mesenchymal stem cell phenotype on tunable hydrogels.

Mesenchymal stem cells (MSCs) are a clinically relevant cell source for regenerative therapies, but it is difficult to expand them in vitro without losing stemness. Standard culture on supraphysiologically stiff tissue culture plastic activates mechanosensitive signaling linked to osteogenic differentiation and reduced multipotency. Although very soft materials (∼1 kPa) are known to preserve stemness, proliferation decreases in a stiffness-dependent manner, and hydrogels in the 5-30 kPa range promote early osteogenic signaling and loss of stemness through integrin-driven mechanotransduction. Here, we use norbornene-modified hyaluronic acid hydrogels to tune substrate stiffness and peptide presentation for the culture of human MSCs. Increasing RGD concentration on 5-20 kPa hydrogels elevates cell spreading and nuclear localization of the mechanosensitive regulator Yes-associated protein (YAP), demonstrating increased mechanosensing within this physiological stiffness range. Incorporation of the N-cadherin mimetic peptide HAVDI reduces cell spreading, increases circularity, and lowers nuclear YAP ratios within 24 h across these stiffness conditions, and decreases nuclear Runx2 levels over three days, indicating reduced activation of osteogenic-associated transcriptional signaling. Despite this reduced mechanosensing, MSCs proliferate similarly on HAVDI-containing and control hydrogels over two weeks, and cells expanded on HAVDI substrates exhibit improved retention of MSC surface marker profiles, including higher CD73 positivity and fewer cells expressing non-MSC-associated markers compared to tissue culture plastic. Together, these results identify a peptide-functionalized hydrogel platform that combines integrin and N-cadherin cues to modulate integrin-associated mechanosensing while supporting MSC expansion.