A TGFβR inhibitor represses keratin-7 expression in 3D cultures of human salivary gland progenitor cells

Abstract

Salivary gland tissue engineering offers an attractive alternative for the treatment of radiation-induced xerostomia. Key to the success of this approach is the maintenance and expansion of secretory acinar cells in vitro. However, recent studies revealed that in vitro culture of primary salivary gland epithelial cells led to undesirable upregulation of the expression of keratin-7 (K7), a marker of ductal phenotype and frequently associated with cellular stress. We have previously shown that hyaluronic acid (HA)-based, RGDSP-decorated hydrogels support the 3D growth and assembly of primary human salivary gland stem/progenitor cells (hS/PCs). Here, we investigate whether the RGDSP culture also promotes K7 expression, and if so, what factors govern the K7 expression. Compared to hS/PCs maintained in blank HA gels, those grown in RGDSP cultures expressed a significantly higher level of K7. In other tissues, various transforming growth factor-β (TGF-β) superfamily members are reported to regulate K7 expression. Similarly, our immunoblot array and ELISA experiments confirmed the increased expression of TGF-β1 and growth/differentiation factor-15 (GDF-15) in RGDSP cultures. However, 2D model studies show that only TGF-β1 is required to induce K7 expression in hS/PCs. Immunocytochemical analysis of the intracellular effectors of TGF-β signaling, SMAD 2/3, further confirmed the elevated TGF-β signaling in RGDSP cultures. To maximize the regenerative potential of h/SPCs, cultures were treated with a pharmacological inhibitor of TGF-β receptor, A83-01. Our results show that A83-01 treatment can repress K7 expression not only in 3D RGDSP cultures but also under 2D conditions with exogenous TGF-β1. Collectively, we provide a link between TGF-β signaling and K7 expression in hS/PC cultures and demonstrate the effectiveness of TGF-β inhibition to repress K7 expression while maintaining the ability of RGDSP-conjugated HA gels to facilitate the rapid development of amylase expressing spheroids. These findings represent an important step towards regenerating salivary function with a tissue-engineered salivary gland.

Design of dual peptide-conjugated hydrogels for proliferation and differentiation of human pluripotent stem cells

“…Most researchers have used peptides derived from vitronectin (PQVTRGDVFTMP or KGGPQVTRGDVFTMP) for the preparation of peptide-conjugated surfaces [ [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] ], where hPSCs can proliferate. The peptide derived from fibronectin (RGDSP) is typically used to enhance the adhesion of any type of cell [ [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] ] but cannot effectively support hPSC proliferation on RGDSP-conjugated surfaces. The peptide derived from the laminin β4 chain (PMQJMRGDVFSP) was originally developed by Jia et al for the adhesion of hPSC-induced cardiomyocytes [ 24 ]; this peptide was effective for hPSC-derived cardiomyocyte adhesion (but not pluripotent hPSC adhesion) and was shown to be useful for hPSC proliferation on PMQJMRGDVFSP-conjugated surfaces in our previous studies [ 8 , 28 ].…”

Matrix Degradability Contributes to the Development of Salivary Gland Progenitor Cells with Secretory Functions

“…Cell adhesion molecules regulate the growth and differentiation of epithelial cells and play a key role in maintaining their structural integrity. , The predominant way in which cell–cell adhesion is realized is through cadherin/catenin adhesion complexes. Here, we characterized the cell–cell interactions by immunofluorescence targeting β-catenin (Figure ), which localizes to epithelial adherens junctions .…”

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

“…We have developed hyaluronic acid (HA)‐based hydrogels that are conducive to the establishment of multicellular hS/PC spheroids, and identified the optimal matrix stiffness, protease degradability, and peptide ligands that promote the rapid development of spheroids with enhanced expression of progenitor and acinar markers. [ 14–20 ] Although our work represents a significant step forward toward the creation of tissue‐engineered human salivary glands, a functional gland consisting of polarized acini remains to be realized.…”

“…enhanced expression of progenitor and acinar markers. [14][15][16][17][18][19][20] Although our work represents a significant step forward toward the creation of tissue-engineered human salivary glands, a functional gland consisting of polarized acini remains to be realized.…”