N-cadherin–mimetic 3D hydrogels program pro-regenerative and immunomodulatory states in human adipose-derived mesenchymal stem cells

Mesenchymal stromal cell (MSC) therapies critically depend on culture systems that preserve stem cell identity while appropriately configuring immunomodulatory functions. Here, we investigated how a biomimetic three-dimensional (3D) hydrogel that presents the N-cadherin-derived HAVDI motif shapes the phenotype and inflammatory programming of human adipose-derived MSCs (ADMSCs). Self-assembled HAVDI-functionalized peptide amphiphile hydrogels formed nanofibrous, mechanically stable networks that supported high cell viability and sustained encapsulation. Across all conditions-including tissue culture plastic, micromass aggregates, and 2D/3D peptide formulations-flow cytometry showed that ADMSCs remained > 90% positive for canonical MSC markers CD73, CD90, and CD105, indicating global preservation of MSC surface phenotype, with peptide-modified environments modestly stabilizing marker expression relative to uncoated plastic. In 3D HAVDI hydrogels, gene expression profiling revealed robust upregulation of p120-catenin and β-catenin, together with increased transcription of matrix-remodeling and angiogenesis-related genes (MMP2, PLAU, VEGFR2), consistent with a pro-regenerative program. Notably, 3D HAVDI cultures displayed markedly elevated basal expression of multiple immunoregulatory cytokine genes (IL-1α, IL-1β, IL-8, IFN-γ, TNF-α, GM-CSF) under LPS-negative conditions, followed by a pronounced downregulation upon LPS challenge, suggestive of a tolerance-like, controlled inflammatory response rather than unchecked activation. Collectively, these findings show that HAVDI-functionalized 3D hydrogels provide a bioinstructive niche that maintains MSC phenotype while priming ADMSCs into a regenerative and tightly regulated immunomodulatory state, highlighting their potential as an advanced platform for stem cell-based tissue repair and immune modulation.