Lacrimal Gland Regeneration:Identification and Isolation of Progenitor Cells

描述(申请人提供)：大约有1000万美国人，主要是女性，患有干眼症，原因多种多样，包括衰老、屈光手术、更年期、自身免疫性疾病和创伤。虽然每种情况的病因不同，但ALL的一个共同特征是泪腺分泌减少和眼表损伤，导致炎症、刺激和疼痛。目前的治疗方法只是针对眼表，而不是导致泪腺分泌减少的原因。大多数干眼症患者可以从改善泪腺的蛋白质、电解质和水分分泌中受益。干眼症患者泪腺细胞因细胞凋亡或坏死而减少，用外源性祖细胞修复病变的泪腺或刺激内源性再生可通过修复丢失的细胞来改善眼表的健康，从而治愈干眼。我们的实验室是第一个开发出一种可重复性的方案，从成年大鼠眼眶外泪腺前体细胞中分离出可用于修复病变泪腺的细胞。这一发现表明，泪腺中存在干细胞或具有再生能力的细胞，可以被激活以修复受损组织。泪腺、腺泡、导管和肌上皮细胞这三种分化细胞类型的再生必须通过三种机制中的一种发生。新形成的分化细胞来自于相同细胞类型的细胞的增殖，来自不同细胞类型的细胞的增殖，或者来自未成熟、未分化的祖细胞。该项目的总体目标是修复泪腺，恢复其分泌功能，并开发一种利用成人泪腺前体细胞治疗干眼的新策略。在初步数据中，我们发现少数泪腺细胞的干细胞标记呈阳性，并且可以重复分离。我们假设成人泪腺含有少量的前体细胞，这些细胞可以在体外或体内被诱导分化为具有功能的泪腺神经、腺泡、导管和肌上皮细胞，从而修复泪腺。为了实现这些目标，我们将使用一组干细胞标记物用于免疫组织化学、免疫印迹和流式细胞术来鉴定培养中的祖细胞，并将这些标记物与它们在体内泪腺中的定位进行比较。祖细胞的生态位将通过选择泪腺主要细胞类型的抗体进行双重标记来确定，以确定形成祖细胞所需的细胞类型。然后，我们将用溴脱氧尿嘧啶核苷标记小鼠泪腺损伤后的增殖细胞，以跟踪再生过程中的细胞谱系。我们还将使用GFAP和Ascl3转基因小鼠进行血统追踪。使用类似于形成神经球的方案，我们将确定培养中的泪腺前体细胞是否形成可用于修复的浮动球。 与公共健康相关：数百万美国人患有干眼病。病因多种多样，包括衰老、屈光手术、更年期、自身免疫性疾病以及导致眼表损伤导致疼痛、炎症和刺激的创伤。干眼无法治愈，由于泪腺是泪膜含水层的主要组成部分，使用干细胞替换患病的泪腺将改善眼表的健康。

DESCRIPTION (provided by applicant): About ten million Americans, predominantly women, suffer from dry eye disease, arising from a variety of causes including aging, refractive surgery, menopause, autoimmune disease, and trauma. Although the etiology of each of these conditions differs, a common feature of all is a decrease in lacrimal gland secretion and damage to the ocular surface resulting in inflammation, irritation, and pain. Current treatments are only palliative as they target the ocular surface, but not the cause of the disease, a decrease in lacrimal gland secretion. Most individuals with dry eye disease could benefit from improved protein, electrolyte, and water secretion from the lacrimal gland. In dry eye lacrimal gland cells are diminished by apoptosis or necrosis, thus restoration of diseased lacrimal gland using exogenous progenitor cells or stimulating endogenous regeneration would improve the health of the ocular surface by replacing lost cells and could cure dry eye. Our laboratory is the first to develop a reproducible protocol to isolate from adult, rat exorbital lacrimal gland progenitor cells that could be used to repair the diseased lacrimal gland. This finding suggests that stem cells or cells capable of regeneration are present in the lacrimal gland and can be activated to repair damaged tissue. The regeneration of the three differentiated cells types of the lacrimal gland, acinar, duct, and myoepithelial cells, must occur via one of three mechanisms. The newly formed differentiated cells are derived from proliferation of cells of the same cell type, derived from proliferation of cells of a different cell type or derived from immature, undifferentiated, progenitor cells. The overall goal of the project is to repair the lacrimal gland, restore its secretory function, and develop a new strategy to cure dry eye by using adult lacrimal gland progenitor cells. In preliminary data we found that a small number of lacrimal gland cells were positive for stem cell markers and could be reproducibly isolated. We hypothesize that the adult lacrimal gland contains a small population of progenitor cells and that these cells can be induced either in vitro or in vivo to differentiate into functioning lacrimal gland nerve, acinar, duct, and myoepithelial cells that can repair the gland. To achieve these goals, we will use a panel of stem cell markers for immunohistochemistry, western blotting, and flow cytometry to identify the progenitor cells in culture and compare these markers with their localization in the in vivo lacrimal gland. The progenitor cell niche will be determined by double labeling with antibodies selective for the major cell types of the lacrimal gland to determine which cell type is needed for progenitor cell formation. We will then label proliferating cells from injured mouse lacrimal glands with bromodeoxyuridine to follow cell lineages during regeneration. We will also use lineage tracing using GFAP and Ascl3 transgenic mice. Using a protocol similar to one that forms neurospheres, we will determine if lacrimal gland progenitor cells in culture form floating spheres that can be used for repair. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from dry eye disease. The causes are varied and include aging, refractive surgery, menopause, autoimmune disease, and trauma causing damage to the ocular surface resulting in pain, inflammation, and irritation. There is no cure for dry eye and as the lacrimal gland is the primary contributor to the aqueous layer of the tear film, replacement of a diseased lacrimal gland using stem cells would improve the health of the ocular surface.