Differentiation of Salivary Human Stem/Progenitor Cell Pedigrees in Bioactive Hydrogels

Objectives: The salivary gland is a complex tissue containing acinar, ductal and myoepithelial cells. We aimed to reveal the multipotency of primary salivary human stem/progenitor cell (hS/PC) pedigrees cultured in customized 3D hydrogel culture systems to determine if these cells could provide all lineages for tissue replacement. We hypothesize that a combination of growth factor and matrix cues can drive differentiation of hS/PCs and phenotypically distinct stem cell pedigrees derived from the parent population into distinct cell lineages.
Methods: Total hS/PC populations from salivary tissues from patient donors were used to create morphologically and phenotypically different pedigree subcultures. Two stable pedigrees were obtained: B3 and C8. Stem/progenitor status was assessed and the parent population and derived pedigrees were encapsulated (2x10⁶ cells/mL) into hyaluronic-acid (HA) hydrogels, some of which incorporated MMP-cleavable peptide sequences and integrin binding motifs. Cells in each hydrogel-type were treated with 200 ng/mL FGF-10 between day 5-30, after which all cultures were assessed for biomarkers K14/p63 (stem), K19 (ductal), α-amylase (acinar), α-smooth muscle actin/α-SMA (myoepithelial), and Cx45 (connectivity) by immunocytochemistry.
Results: Primary salivary cell pedigrees were viable, grew and reorganized in HA-hydrogels with a subset retaining stem cell markers in culture. hS/PCs were amylase positive, but the A2 pedigree initially amylase positive failed to grow without other cell types. In the migration permissive hydrogels, parental hS/PCs exhibited a 23% increase in levels of K19 after FGF-10 treatment. In contrast, salivary pedigree B3 treated with FGF-10 exhibited a 22% increase in levels of α-SMA when encapsulated in MMP-sensitive hydrogels, but not in unmodified hydrogels. Salivary pedigrees B3 and C8 encapsulated in unmodified hydrogels exhibited cell junctions expressing Cx45.
Conclusions: hS/PCs can differentiate into acinar, ductal, and myoepithelial phenotypes when encapsulated in customized HA-based hydrogels. The multipotency of these cells provides a mechanism to differentiate primary hS/PCs for gland restoration.