Basement Membrane Mimetic Hydrogel Cooperates with Rho-Associated Protein Kinase Inhibitor to Promote the Development of Acini-Like Salivary Gland Spheroids

Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels are prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce “hyperGel+” or “hyperGel”, respectively. Differential scanning fluorescence experiments confirm the ability of the sulfated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels are nanoporous, cytocompatible, and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. The incorporation of sulfated HPG species in the hydrogel enhances cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor Y-27632 (Y-27) leads to the development of spheroids with a central lumen, increases the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreases the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 is also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the Rho-associated protein kinase inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.