RGDSP-Decorated Hyaluronate Hydrogels Facilitate Rapid 3D Expansion of Amylase-Expressing Salivary Gland Progenitor Cells

Abstract

In vitro engineering of salivary glands relies on the availability of synthetic matrices presenting essential cell-instructive signals to guide tissue growth. Here, we describe a biomimetic, hyaluronic acid (HA)-based hydrogel platform containing covalently immobilized bioactive peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP). The HA network was established by the thiol/acrylate reaction, and bioactive peptides were conjugated to the network with high efficiency without significantly altering the mechanical property of the matrix. When encapsulated as single cells in peptide-modified HA hydrogels, human salivary gland stem/progenitor cells (hS/PCs) spontaneously organized into multicellular spheroids with close cell–cell contacts. Conjugation of RGDSP and TWSKV signals in HA gels significantly accelerated cell proliferation, with the largest spheroids observed in RGDSP-tagged gels. Peptide conjugation did not significantly alter the expression of acinar (AMY1), ductal (TFCP2L1), and progenitor (KRT14) markers at the mRNA level. Characterization of three-dimensional (3D) cultures by immunocytochemistry showed positive staining for keratin-5 (K5), keratin-14 (K14), integrin-β1, and α-amylase under all culture conditions, confirming the maintenance of the secretory progenitor cell population. Two-dimensional (2D) adhesion studies revealed that integrin-β1 played a key role in facilitating cell–matrix interaction in gels with RGDSP, IKVAV, and TWSKV signals. Overall, conjugation of the RGDSP peptide to HA gels improved cell viability, accelerated the formation of epithelial spheroids, and promoted the expansion of the progenitor cell population in 3D. This work represents an essential first step toward the development of an engineered salivary gland.

Perlecan, A Multi-Functional, Cell-Instructive, Matrix-Stabilizing Proteoglycan With Roles in Tissue Development Has Relevance to Connective Tissue Repair and Regeneration

“…From ECM Components in Repair Biology: In Vitro Engineering of Salivary Glands Using Perlecan Domain IV, Laminin-111 and Fibronectin Peptide Scaffolds Peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP) have been incorporated into HA scaffolds with RGDSP and TWSKV peptide HA scaffolds significantly accelerating cell proliferation (Fowler et al, 2021). Perlecan peptide TWSKV triggers differentiation of salivary gland cells into self-assembling acinilike structures expressing salivary gland biomarkers that secrete α-amylase (Pradhan et al, 2009;Pradhan et al, 2010).…”

“…Laminin-111 peptide-HA hydrogels have also been shown to act as a synthetic basement membrane (Yamada et al, 2013). Conjugation of RGDSP peptide to HA gels improves cell viability, accelerates formation of epithelial spheroids, and promotes the expansion of 3D progenitor cell populations, representing the first step toward the development of an engineered salivary gland (Fowler et al, 2021). Primary salivary human progenitor stem cells undergo acinar-like differentiation in HA hydrogel cultures and incorporation of basement membrane peptides derived from perlecan and laminin-111 further directs the development of 3D salivary gland-like spheroids (Srinivasan et al, 2017).…”

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

“…Hydrogel precursors, including thiolated (HA-SH) and acrylated HA (HA-AES), as well as maleimide-functionalized RGDSP peptide (MI-RGDSP), were prepared as previously reported 47 . Cellular constructs were produced by dispersing hS/PCs in a solution containing HA-SH or HA-SH/MI-RGDSP before adding the HA-AES solution, resulting in HA or RGDSP cultures, respectively.…”

“…We have also developed hyaluronic acid (HA)-based hydrogels that support the 3D culture of hS/PCs under defined conditions and identified a mechanical stiffness regime that promotes the development of multicellular spheroids 46 . Recently, we demonstrated that HA matrices with a covalently immobilized RGDSP peptide, a fibronectin-derived integrin-binding ligand, promoted the rapid 3D expansion of amylase-expressing hS/PCs 47 . However, the expression of K7 as the 3D cultures expand remains unexplored.…”

Hydrogels for the treatment of oral and maxillofacial diseases: current research, challenges, and future directions

“…One possible explanation is that L 1p -FH could have activated resident hS/PCs that contributed to gland homeostasis through interactions with specific integrins. Likewise, a fibronectin (RGDSP)-modified HA hydrogel also improved cell viability, accelerated epithelial spheroids formation and promoted the progenitor cell population expansion 225 . Peptide-modified hydrogels probably promote the proliferation and differentiation of salivary gland stem cells by regulating the growth microenvironment of salivary gland stem cells.…”