Dynamic Assembly of Human Salivary Stem/Progenitor Microstructures Requires Coordinated α1β1 Integrin-Mediated Motility

Abstract

A tissue engineering approach can provide replacement salivary gland structures to patients with hyposalivation disorders and xerostomia. Salivary human stem/progenitor cells (hS/PCs) were isolated from healthy regions of parotid glands of head and neck surgery patients, expanded, then encapsulated in biocompatible hyaluronate (HA)-based hydrogels. These bioactive hydrogels provide a surrogate territorial matrix suitable for the dynamic assembly, growth and reorganization of salivary gland components. This study examined the dynamics of salivary microstructure formation, growth, and reorganization using time-lapse imaging over 15 h. Immunofluorescence detection monitored production of individual basement membrane components forming around developing microstructures, and Ki67 assessed proliferation. Dynamic movements in hydrogels were quantified by measuring angular velocity (ω) of rotating salivary microstructures and changes in basement membrane architecture during microstructure growth. Integrin involvement in the dynamic reassembly was assessed using knockdown and inhibitor approaches. Single hS/PCs expanded over 5 days into spherical microstructures typically containing 3–10 cells. In larger macrostructures, proliferation occurred near the peripheral basement membrane that underwent growth-associated cycles of thinning and collapse. De novo secretion of laminin/collagen IV from reorganizing hS/PCs preceded that of perlecan/HSPG2. Microstructures routinely expressed β1 integrin-containing complexes at basement membrane-associated regions and exhibited spontaneous and coordinated rotation during basement membrane maturation. β1 integrin siRNA knockdown at the single-cell state prevented hS/PC microstructure growth. After microstructure formation, β1 integrin knockdown reduced rotation and mean ω by 84%. Blockade of the α1 integrin subunit (CD49a) that associates with β1 reduced mean ω by 66%. Studies presented here show that initial hS/PC structure growth and basement membrane maturation depends on α1β1-integrin mediated signaling. Coordinated cellular motility during neotissue reorganization reminiscent of salivary gland acini was critically dependent both on hS/PC-secretion of laminin,collagen type-IV, and perlecan/HSPG2 and the force-driven interactions of α1β1-integrin activation. We conclude that α1β1-integrin plays a critical role in establishing human salivary gland coordinated structure and function, and that its activation in tissue engineered systems is essential to tissue assembly.

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

“…Ki67 and PCNA proteins are markers of cell proliferation, and CK7 is the marker of salivary ductal cells, respectively. 27,28 In the non-treated group, CK7 was localized to the intercalated duct, striated duct, granular and excretory duct cells. Ki67 was mainly expressed in these CK7-positive (CK7+) ductal cells, and a small number of Ki67-positive cells (Ki67+) were located in non-ductal tissues.…”

Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures

“…The importance of β1 integrin in salivary epithelial branching morphogenesis has been established via antibody blocking experiments (Wei et al 2007). β1 integrin has also been shown to mediate the assembly of human salivary stem/progenitor microstructures by siRNA-mediated knock down (Wu et al 2019). Our results in this study provide genetic perturbation evidence that strengthens the finding that β1 integrin is required for branching morphogenesis.…”

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

“…Because cellular secretion of ECM proteins is minimal, hS/PCs engage with the synthetic matrix via immobilized cell-adhesive peptides. It has been shown that integrin-β1 is important for salivary gland development and the assembly of multicellular salivary gland spheroids. − While receptors such as CD44, RHAMM, and ICAM can bind the HA matrix, the bioactive peptides are reported to target multiple isoforms of integrin-β1 . The RGDSP peptide predominantly ligates α 5 β 1 and α v β 3 integrin subunits. , The laminin-derived peptides, YIGSR and IKVAV, interact to varying extents with α 3 β 1 , α 4 β 1 , and α 6 β 1 . ,, YIGSR also has an affinity for a 67 kDa laminin receptor, to which much of its biological activity has been attributed. , Derived from perlecan domain IV, TWSKV is reported to primarily target a nonintegrin receptor, with limited integrin-β1 affinity. , While all 3D cultures were stained positive for integrin-β1, more pronounced staining was found on the larger spheroids localized to the basal domains.…”