Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture

Abstract

Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three‐dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long‐term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate‐based hydrogel system modified with bioactive basement membrane‐derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with β‐adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar‐like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human‐compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications. Stem Cells Translational Medicine 2017;6:110–120

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function

“…Towards bioengineering complex glandular models[11, 23], we previously reported the isolation and phenotypic characterization of hS/PCs, as confirmed by the expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) and the ability of hS/PCs in hydrogels to differentiate into a specialized acini-like cell type upon neurotransmitter stimulation. [12] Our custom-designed HA hydrogels promoted spheroid assembly from dispersed hS/PCs. [14] We further discovered that hS/PCs cultured in HA gels decorated with bioactive peptides expressed a significantly higher levels of progenitor markers as compared to the unmodified HA gels.…”

“…[14] We further discovered that hS/PCs cultured in HA gels decorated with bioactive peptides expressed a significantly higher levels of progenitor markers as compared to the unmodified HA gels. [12] The current study aimed to create physiologically relevant microtissues composed of myoepithelial cells and organized, acini-like spheroids to replicate the function of intact acini-hSMEC assemblies in tissue. At present, there is no similar physiologically relevant in vitro model for providing myoepithelial cell functions, in terms of phenotype, contractility and cell-cell interactions, to engineered acini-like spheroids.…”

“…[12] Briefly, human parotid gland tissue was disinfected using 1% (v/v) Betadine solution in DMEM/F12, followed by mincing into a slurry in complete HepatoSTIM media (Corning Inc., Corning, NY). After reaching 70–80 % confluence, cells were trypsinized using 0.05% trypsin/EDTA, neutralized by equal volume of Soybean Trypsin Inhibitor (ATCC, Manassas, VA) and subcultured into a new flask in complete HepatoSTIM media.…”

“…We have previously isolated and expanded hS/PCs,[11] confirmed their expression of stem/progenitor markers,[12] encapsulated them as dispersed single cells hyaluronic acid (HA)-based hydrogels[13], investigated the role of hydrogel properties on the formation of hS/PC spheroid,[14] and differentiated them to acinar cells using neurotransmitters. [12] Separately, consistent isolation, immunophenotypic characterization and functional analyses of hSMECs are essential for the generation of in vitro models with lobular cells. With the two types of cells in hand, a flexible, high-throughput 3D culture platform is desirable for the organization of the respective cell types in a well-defined spatial manner.…”

Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D

“…13 These hydrogels were prepared using thiolated HA (HA-SH) and acrylated HA (synthesized via reaction with mono-2-( a cryloyloxy) e thyl s uccinate, HA-AES) with a molecular weight and stoichiometry mismatch. We altered the hydrogel properties by varying HA concentration and thiol/acrylate ratio.…”