Establishing Human Lacrimal Gland Cultures with Secretory Function

Abstract

PurposeDry 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.MethodsFresh 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.ResultsNative 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).ConclusionThe 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.

Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland

“…Several studies have evaluated adult human lacrimal glands (Paulsen, 2006;Tiwari et al, 2012Tiwari et al, , 2014. Although the morphological development of the early human lacrimal gland has been described (de la Cuadra-Blanco et al, 2003), to our knowledge no study has investigated the molecular characteristics of the human fetal lacrimal gland or whether its cell identities are similar to the mouse.…”

Effect of Subconjunctival Lacrimal Gland Transplantation in a Rabbit Dry Eye Model

“…5,6 However, these drugs cannot promote tear secretion from the lacrimal gland, so the application of lacrimal glandregenerative techniques, such as stem cell transplantation or induction of induced pluripotent stem cells, may be essential to increase tear volume. [7][8][9] Currently, these techniques have not been clinically approved for use.…”

Adult Gland Derived Stem Cells (Gdscs); Potentials, Hurdles and Expectations

“…The established cultures were maintained in vitro for 30-35 days, providing evidence for the presence of putative stem cells in the tissues that express ABCG2 and ALDH1 stem cell markers. In addition, the cultures lead to the formation of spheres similar to the well documented Salispheres [106] and Prostaspheres [107] in their cellular organization [99,108].…”

“…Although there are numerous well-known tear substitutes that can be used to temporarily lubricate the eye, there is actually no curative treatment for the dry eye syndrome [97,98]. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to such patients [99].…”