Glycosaminoglycan-Inspired Polymer Brushes Promote Human Mesenchymal Stem Cell Proliferation While Retaining Trilineage Differentiation Potential

Human mesenchymal stem/signaling cells (hMSCs) are promising therapeutics for cancer, autoimmune disorders, and musculoskeletal conditions. Given that the hMSC proliferation capacity and therapeutic potency diminish rapidly over extended passaging, expanding hMSCs into populations sufficient for clinical use remains an outstanding challenge. In this work, we synthesized cell culture substrates that mimicked key structural features of glycosaminoglycans or GAGs (e.g., brush architecture and sulfonation). hMSCs proliferated up to 40% more rapidly on GAG-inspired sulfonated polymer brushes than on currently used tissue culture polystyrene substrates (TCPs). In addition to rapid proliferation, hMSCs expanded on these sulfonated polymer brushes still retained their capacity to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We synthesized 8 polymer brushes with varying sulfonation, hydroxylation, and hydrophobicity. Five of these synthesized polymer brushes outperformed TCPs in hMSC proliferation assays conducted in chemically defined serum-free cell culture media. In comparing the fold change of hMSC numbers over 7 days in culture, brushes with higher sulfonation degrees (88%) and moderate hydrophilicity (37°-48° water contact angles) performed 30-40% better than TCPs. We then selected hMSCs expanded on the polymer brushes with the most consistent proliferation trends (S75H25) for further biological characterization. Genes associated with proliferation (<i>CDK2</i> and <i>KI67</i>), and cell adhesion and stemness (<i>ITGA6</i>) were upregulated in hMSCs cultured on S75H25 compared to TCPs. Interestingly, S75H25 also promoted the expression of basic fibroblast growth factor (bFGF) receptors FGR1. Unlike TCPs, which exhibited zero bFGF adsorption, S75H25 captured significant amounts of bFGF (588 pg/cm²), adsorption levels that are comparable to heparan sulfate. We posit that bFGF molecules sequestered by polymer brushes cluster and activate bFGF receptors to promote hMSC proliferation, and we will explore this in future work. Finally, hMSCs expanded on S75H25 were successfully differentiated into adipogenic, chondrogenic, and osteogenic cell types. We engineered chemically defined and highly economical GAG-mimetic cell culture substrates based on sulfonated polymer brushes that can be readily applied to biomanufacturing platforms such as spinner flasks, bioreactor beads, and microfluidic platforms to boost hMSC throughput under fully defined conditions.