Heparin-collagen I bilayers stimulate FAK/ERK½ signaling via α2β1 integrin to support the growth and anti-inflammatory potency of mesenchymal stromal cells.

Abstract

Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high-quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin-HEP interactions in the advantages of MSC culture. The layer-by-layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC50 and IC75 levels of BTT-3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow-derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP-ending HEP/COL surfaces significantly increased FAK-ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL-ending HEP/COL and uncoated surfaces. BTT-3033 but not Cilengitide treatment markedly affected FAK-ERK½ activity levels on HEP-ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT-3033 treatment on HEP-ending bilayers reduced MSC-mediated macrophage inhibitory activity and altered the cytokine profile of co-cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1-FAK-ERK½ signaling mechanism.