Establishment of Functional Acinar-like Cultures from Human Salivary Glands

Abstract

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinarspecific markers-namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients.

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells

“…Our previous findings demonstrated that phenotypic modulation of these primary salivary epithelial cells is determined by the ambient [Ca 2ϩ ] of the extracellular growth medium. Although phSG cells could be maintained in a proliferative, non-differentiated state, in culture medium containing low (0.05 mM) [Ca 2ϩ ], increasing the [Ca 2ϩ ] in the medium to a relatively higher but more physiological level (Ͼ0.8 -1.2 mM) induced an acinar cell-like phenotype with up-regulation of acinus-specific proteins like AQP5 (24). In this study, we show that this increase in extracellular [Ca 2ϩ ] induces remodeling of the Ca 2ϩ signaling toolkit, which results in increased SOCE as well as NFAT activation, both of which are critical for the up-regulation of AQP5.…”

“…Cell Culture, Western Blotting, and ImmunofluorescencePrimary human salivary gland epithelial cells were isolated and grown on collagen-coated plates (BioCoat, BD Biosciences) as described previously (24). Briefly, phSG cells were maintained in complete KGM (Lonza) supplemented with bovine pituitary extracts, recombinant human EGF, insulin, hydrocortisone, gentamicin, epinephrine, and transferrin, and the calcium concentration was adjusted to 0.05 mM with CaCl 2 solution.…”

“…The complexes were detected with SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to x-ray films (MR, Kodak). The immunofluorescence staining was performed as described previously (24). The antibodies used were AQP5, cystatin C, and an Alexa Fluor 594-conjugated goat antirabbit antibody (Molecular Probes).…”

“…Signaling Proteins-Primary human salivary gland cells obtained from biopsies of minor salivary glands were maintained in modified KGM medium as described earlier (24 (24). To determine the underlying mechanism involved in this Ca 2ϩ -dependent up-regulation of acinar cell-specific proteins, we examined the transcript levels of various proteins that could contribute to the Ca 2ϩ -dependent switch in cell phenotype and gene expression.…”

“…Although a few studies have described successful cultures of primary epithelial cells from salivary gland explants (19 -22), there has been little success in maintaining the acinar phenotype or preserving the functionality of the cells in culture (23). In our previous study (24), we described optimal conditions for maintaining primary human salivary gland (phSG) cell cultures derived from biopsies of human salivary glands and for promoting an acinar-like phenotype with enhanced expression of acinar-specific proteins (AQP5, NKCC1, and CST3) but not ductal cell markers (KLK1 and KRT19). Importantly, we found that * The authors declare that they have no conflicts of interest with the contents of this article.…”