Establishing Human Lacrimal Gland Cultures with Secretory Function

PURPOSE: Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures. METHODS: Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA. RESULTS: Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons). CONCLUSION: The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future

Phorbol ester-stimulated exocytosis in lacrimal gland: PKC might not be the sole effector

In this work we show that, although both phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PdBu) stimulate the protein discharge in the rat lacrimal gland with the same half-maximal effective concentration (EC50 approximately 2 x 10(-7) M), PdBu is more efficient in eliciting this response compared with PMA. We also show that sphingosine and chelerythrine have no inhibitory effect on the protein discharge stimulated by PMA or PdBu at concentrations up to 2 x 10(-4) and 3 x 10(-5) M, respectively. With staurosporine, a complete inhibition could not be obtained even at 1 microM. However, only with trifluoperazine (TFP) we obtained a complete inhibition of the PMA-induced protein discharge at 10(-4) M TFP. On the other hand, we show that three diacylglycerol-permeant analogues (1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-didecanoyl-sn-glycerol) do not stimulate protein discharge. In a previous report from our laboratory (30), we showed that the rat lacrimal gland expresses the alpha-isoform of protein kinase C (PKC). In this study, using specific antibodies directed against the newly identified isoforms of PKC, we show on a diethylaminoethyl-cellulose fraction that, besides PKC-alpha, the rat lacrimal gland expresses PKC-epsilon, as previously suggested by Dartt et al. (11), and PKC-delta. Our results question the direct implication of PKC activity as a sole effector of the phorbol ester-stimulated protein secretion in the rat lacrimal gland.</jats:p