Isolation and propagation of mesenchymal stem cells from the lacrimal gland.

The tear film is the interface between the external environment and the ocular surface.1 It forms a smooth refractive surface over the corneal surface, lubricates the eyelids, and maintains the optimal extracellular environment for the epithelial cells of the cornea and conjunctiva.1 The tear film consists of three interacting layers: an inner mucous layer, a middle aqueous layer, and an outer lipid layer that floats on the middle layer. Different sets of glands are responsible for maintaining each specific layer. The lacrimal gland secretes the proteins, electrolytes, and water that make up the middle aqueous layer.2,3 The lacrimal gland is made up of acini, ducts, nerves, myoepithelial cells, and plasma cells. The majority, approximately 80%, of the gland are the acinar cells, which secrete electrolytes, water, and proteins.2,3 Dry eye syndrome is caused by inadequate quantity or quality of tears. Aqueous-deficient dry eye or keratoconjunctivitis sicca (KCS) is one of two major subtypes of dry eye syndrome, the other being evaporative dry eye.4–7 Although there are tear substitutes that can be used to temporarily lubricate the eye, there is no curative treatment for dry eye syndrome.4–6 Chronic inflammation of the lacrimal gland can lead to insufficient lacrimal gland secretion, leading to symptoms of dry eye.8–10 Pathologies associated with chronic inflammation, such as autoimmune diseases (Sjogren's syndrome, sarcoidosis, rheumatoid arthritis), organ transplantation, or viral infections (hepatitis, HIV), are often accompanied with symptoms of dry eye syndrome. Lacrimal gland inflammation is characterized by the presence of focal lymphocytic infiltrates, increased production of proinflammatory cytokines, and destruction of the tear-producing parenchymal cells.8–10 Development of the lacrimal gland occurs through a process known as branching morphogenesis.11,12 This process of morphogenesis is achieved through epithelial-mesenchymal interactions that include a highly coordinated spatio-temporal release of several growth factors and subsequent activation of critical transcription factors.11,12 Several growth and transcription factors have been shown to be crucial for branching morphogenesis of the lacrimal gland. Bone morphogenetic protein (BMP)7 plays a major role in tissue development and remodeling and was shown to play a pivotal role in murine lacrimal gland development.13 We have recently reported that the BMP7 pathway was not only activated but was upregulated in the lacrimal gland during the repair phase after injury.14 The presence of stem cells in adult tissues is a very active area of research because of the potential clinical benefits.15–18 Several studies have reported the self-regenerating capabilities of salivary glands and the pancreas, liver, intestine, and mammary gland.19–24 In particular, the regenerative capacity of the salivary glands has been well documented.22,23,25–30 Long-term (7–21 days) ligation of the main excretory ducts of the salivary glands leads to atrophy of the glands. The ligation of the salivary gland corresponds with inflammation, edema, and death of acinar cells. After the duct ligation is released, the salivary glands go through a phase of increased cellular proliferation and repair. Nestin-positive stem cells have been isolated and propagated in vitro from injured rat pancreas as well as salivary and mammary glands.22–24,31 We have recently reported that stem/progenitor cells were present in the murine lacrimal gland and that their number increased during the repair phase after experimentally induced inflammation.14 These cells were identified by their ability to express nestin, a stem cell marker. Some of the nestin-positive cells also expressed α-smooth muscle actin, a marker of myoepithelial cells.14 This suggested a common source of stem cells between myoepithelial cells and acinar cells in the lacrimal gland; that is, the same stem cell can give rise to both myoepithelial as well as acinar cells. The purpose of the present studies was to determine whether stem/progenitor cells can be isolated from the murine lacrimal gland undergoing repair and if they can be propagated in vitro.