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.

Keywords: Hyaluronic acid; Salivary glands; Stem/progenitor cells; Three-dimensional hydrogel culture; Tissue engineering.

Figure 1

Expanded populations of human salivary gland cells display progenitor properties. (A): Expanded populations of human salivary gland cells cultured in 2D or 3D hyaluronate (HA) hydrogels are enriched for transcripts encoding progenitor markers when compared with whole adult hPG tissue. Error bars represent SEM. Statistical analysis was performed using one‐way analysis of variance followed by the Tukey‐Kramer post hoc test; ∗, p < .05. (B): Protein levels of stem/progenitor markers in hPG tissue, isolated human stem/progenitor cells (hS/PCs) in 2D and in 3D HA hydrogels. K14 (green) and K5 (red) staining is observed in a few cells within the ducts, along with myoepithelial cells in hPG tissue, and in the majority of the population of isolated hS/PCs in 2D as well as those cultured in 3D HA hydrogels. KIT staining (red) was seen in a relatively minor population in hPG tissue, 2D and 3D. Nuclei are stained in blue. Scale bars = 50 μm. Abbreviations: 2D, two‐dimensional; 3D, three‐dimensional; hPG, human parotid gland.

Figure 2

Primary human stem/progenitor cells (hS/PCs) retain transcripts encoding progenitor markers over multiple passages and retain stem/progenitor potential in long‐term culture. (A): hS/PCs retained transcript levels encoding stem/progenitor markers over passages 2 through 15 in two‐dimensional culture. Early: passages 2 and 3; middle: passages 9 and 10; late: passages 13 and 15. Error bars represent SEM. Statistical analysis was performed using one‐way analysis of variance followed by the Tukey‐Kramer test; ∗, p < .05. (B): Stem cell sphere forming assay. hS/PCs form primary and secondary spheroids when grown in stem cell media. Inset shows magnified image of spheroids. (C): A representative confocal image of an approximately 300‐μm three‐dimensional (3D) spheroid at its largest diameter, maintained for 118 days in 3D hyaluronate hydrogel culture. Nuclei are stained in blue. Scale bars = 50 μm. Abbreviations: D, day; hPG, human parotid gland.

Figure 3

Bioactive basement membrane peptides enhance progenitor potential, viability, and proliferation of hS/PC spheroids in three‐dimensional (3D) HA hydrogels. (A): Basement membrane‐derived bioactive peptides from laminin (YIGSR, IKVAV) and perlecan (PlnDIV peptide) increase transcript levels encoding stem/progenitor markers in hS/PCs grown in 3D HA hydrogels. See study text for details. Error bars represent SEM. Statistical analysis was performed using one‐way analysis of variance; ∗, p < .05. (B): Spheroids cultured with basement membrane‐derived peptides gain expression of KIT in a majority of the population. Expression of K14 and K5 persists. Scale bar = 50 μm. (C): Brightfield image of hS/PC spheroids in unmodified 3D HA hydrogels (Ci) and in peptide‐modified HA hydrogels (Cii). A significant increase in the size of spheroids (D) and number of hS/PCs per spheroid (E) is seen in HA hydrogels with basement membrane‐derived peptides. (F): hS/PC spheroids grown in peptide‐modified 3D HA gels display an increase in viability assessed via the PrestoBlue Assay. (G): Ki67 staining (green) indicates increased proliferation in hS/PC spheroids in hydrogels modified with basement membrane‐derived peptides. Nuclei are stained in blue. Scale bar = 20 μm. Abbreviations: HA, hyaluronate; hS/PC, human stem/progenitor cell.

Figure 4

Transcript levels and localization of differentiation markers in tissue, 2D, and peptide‐modified 3D hyaluronate (HA) hydrogels. (A): Levels of acinar and ductal differentiation markers are significantly lower among human stem/progenitor cells grown in 2D and 3D peptide‐modified HA hydrogels compared with the whole hPG tissue. Error bars represent SEM. Statistical analysis was performed using one‐way analysis of variance followed by the Tukey‐Kramer test; ∗, p < .05. (B): Localization of MIST1/BHLHA15 and amylase in hPG tissue and 2D and 3D HA hydrogels with basement membrane‐derived peptides. Scale bar = 20 μm. Abbreviations: 2D, two‐dimensional; 3D, three‐dimensional; hPG, human parotid gland.

Figure 5

Stimulation of human stem/progenitor cells (hS/PCs) with β‐adrenergic and cholinergic agonists leads to differentiation of hS/PC spheroids toward an acinar lineage. Cotreatment with carbachol (25 μM) and isoproterenol (25 μM) increases levels of MIST1/BHLHA15 in hS/PCs cultured in 2D HA hydrogels at 4 hours (A) and 20 hours (B) after treatment. (C): Treatment with neurotransmitter agonists (25 μM carbachol and 25 μM isoproterenol) increases mRNA levels of acinar‐specific differentiation markers in hS/PCs cultured in 3D peptide‐modified hyaluronate hydrogels, 20 hours after treatment. Error bars represent SEM. Statistical analysis was performed using one‐way analysis of variance; ∗, p < .05. (D): Stimulation of hS/PCs in 3D with carbachol and isoproterenol increases acinar markers MIST1/BHLHA15 (green) and amylase (red) after 20 hours. Scale bar = 50 μm. Abbreviation: 2D, two‐dimensional; 3D, three‐dimensional.