Establishing human lacrimal gland cultures with secretory function

Abstract

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.

Figure 1. Establishment of human lacrimal gland primary cultures.

a) Heterogenous cell population isolated after enzymatic digestion of the lacrimal gland. b) Cell clumps adhere to the substrate as discrete islands and show initiation of proliferation within 1–3 days. c) The islands proliferate and form a confluent monolayer within 15–20 days. d) The cells in the monolayer show thin cytoplasmic border, vesicular nucleus and granularity in the cytoplasm. e–f) Spherules are formed by day 16–18 (arrow head). g–i) Cord-like connections (arrow head) are seen to develop between the spherules.

Figure 2. Other cell types in culture.

a) Spindle shaped cells with slight granularity in their cytoplasm and distinct nucleus, are seen on uncoated culture dishes and these attain confluence within 5–7 days. b) Oval and plump cells that organize themselves in whorls are also seen. These may be myoepithelial in nature.

Figure 3. Immunohistochemistry on normal human lacrimal gland.

H&E staining shows the normal histology of the lacrimal gland. Marker staining pattern shows localization of pan-cytokeratin (AE1/AE3) and lysozyme (Lzy) in the cytoplasm of the acinar cells while c-kit is seen in the plasma membrane of acinar cells. p63, glial fibrillary acidic protein (GFAP), S-100 protein and ∝-SMA localize in the myoepithelial cells enveloping the acinar cells. Vimentin is seen in the myoepithelial cells and also in some of the acinar cells. All images are at 40× magnification except H&E which is at 10×.

Figure 4. Immunocytochemistry on in-vitro cultured human lacrimal gland cells.

Cells with epithelial morphology stain positively with E-cadherin, CK3/12, lysozyme and p63; oval and plump cells stain positive for myoepithelial markers GFAP and S100 protein while the spindle shaped cells are seen to be positive for mesenchymal markers CD90 and vimentin. Some cells also show immunopositivity for ABCG2. Secondary antibody uses is fluoresceine isothiocyanate (green) and the counter-stain is propidium iodide (red).

Figure 5. Flow Cytometry Data.

i) Flow cytometric profile of freshly isolated (FI) cells. ii) Flow cytometric profile of cells 14–18 days post in-vitro culture.

Figure 6. Aldefluor Assay.

i) Flow cytometric data showing ALDH1 high cells in the freshly isolated (FI) cell population. ii) Flow cytometric data showing ALDH1 high cells in day 14–18 in-vitro cultures.

Figure 7. RT-PCR showing appropriate product bands for scIgA, lactoferrin and lysozyme in day 14 in-vitro cultures on all HAM (H), collagen I (C) and Matrigel™(M).

Negative (N) panel shows no amplification product.

Figure 8. Secretome Assessment by ELISA.

a–c): Standard calibration curve for lysozyme, scIgA and lactoferrin. d–f): Plot of mean optical density values for secreted proteins lysozyme, scIgA and lactoferrin on HAM, collagen and Matrigel™ pre and post carbachol stimulation.