Identification and Characterization of Adult Salivary Gland Stem Cells

描述(申请人提供)：唾液腺功能丧失影响全球数百万人，由各种疾病引起，包括头颈癌的放射治疗、唾液肿瘤的手术切除、自身免疫性疾病和细胞毒性侮辱。这些患者的唾液分泌显著减少，或者说唾液分泌减少，这是一种严重影响他们生活质量的情况。目前，唯一的治疗选择是药物治疗，但这是姑息性的，往往不太令人满意。由于分泌腺泡细胞因细胞凋亡或坏死而减少，唾液流量的恢复需要通过干细胞/祖细胞移植或内源性干细胞/祖细胞的诱导来替代受损或丢失的细胞。然而，目前唾液腺干细胞还没有被鉴定出来，我们对细胞在正常内稳态和损伤诱导再生过程中的更新机制缺乏基本的了解。从针对唾液腺的再生医学的角度来看，这些都是中心问题。在这方面，我们的实验室首次开发了一种原位谱系追踪方法，我们使用该方法来证明表皮和皮脂腺中存在干细胞。我们还表明，这些细胞的功能独立于位于隆起中的毛囊干细胞。我们建议使用类似的方法来确定唾液腺中细胞维持和再生的机制。唾液腺中分泌腺泡细胞的再生必须通过三种机制中的一种发生：腺体中有功能的腺泡细胞的自我复制，如推测的导管细胞的增殖，或来自未成熟、未分化、分裂缓慢的干细胞群体。为了探索腺体维持和更新的性质和位置，我们将使用两种新的遗传学方法：1)我们将建立针对唾液腺上皮细胞的H2 B-GFP调控表达的双转基因小鼠，并进行长期脉冲追逐标记以鉴定分裂缓慢的细胞。这些绿色荧光蛋白标记的细胞将使用针对唾液腺主要细胞类型的抗体来表征。2)我们将利用慢病毒介导的原位遗传标记和命运图谱来阐明包括导管细胞和腺泡细胞在内的各种细胞类型在唾液腺正常动态期和再生/修复期间的线性关系。成体的鉴定及功能特征 唾液腺干细胞和腺体再生机制的阐明是制定有效的唾液腺损伤再生策略的关键步骤。这项研究中获得的知识将是利用成体祖细胞恢复唾液分泌功能的长期计划的第一步。 与公共卫生相关：数以百万计的美国人患有口干症，原因是唾液腺受到不可逆转的损害。它是由衰老、自身免疫性疾病、辐射损伤、创伤或其他细胞毒性侮辱引起的，导致口腔健康状况不佳，严重影响患者的生活质量。目前，这种情况还没有治愈的方法。成人唾液腺干细胞的鉴定和功能鉴定是制定有效的受损唾液腺再生策略的关键一步，这是治疗大量唾液腺功能低下患者的重要方法。

DESCRIPTION (provided by applicant): Loss of salivary gland function affects millions of people worldwide and is caused by various conditions including radiation therapy for head and neck cancers, surgical excision of salivary tumors, autoimmune diseases and cytotoxic insults. These patients have dramatically reduced salivary secretion, or hyposalivation, a condition that severely affects their quality of life. Currently, the only treatment option is pharmacologic management, but this is palliative and often less than satisfactory. As the secretory acinar cells are diminished by apoptosis or necrosis, restoration of salivary flow requires replacement of damaged or lost cells by either transplantation of stem/progenitor cells or by induction of endogenous stem/progenitor cells. At present, however, salivary gland stem cells have not been identified and we lack essential knowledge about the mechanism of cell renewal during normal homeostasis and injury-induced regeneration. These are central issues from the perspective of regenerative medicine targeted to salivary glands. In this regard, our laboratory was first to develop a method for in situ lineage tracing that we used to demonstrate the presence of stem cells in epidermis and sebaceous glands. We also showed that these cells functioned independently of hair follicle stem cells located in the bulge. We propose using a similar approach to identify the mechanism of cell maintenance and regeneration in salivary glands. Regeneration of secretory acinar cells in the salivary gland must occur via one of three mechanisms: self-duplication of functioning acinar cells in the gland, proliferation of ductal cell, as speculated, or derived from a population of immature, undifferentiated, slow-dividing stem cells. To explore the nature and location of glandular maintenance and renewal, we will use two novel genetic approaches: 1) we will generate bi- transgenic mice with regulated expression of H2B-GFP targeted to epithelial cells in salivary glands and perform long-term pulse-chase labeling to identify slow-dividing cells. These GFP-labeled cells will be characterized using antibodies selective for the major cell types of the salivary gland. We will then recover these slowly-cycling cells by flow cytometry and assess their regenerative abilities; 2) we will utilize lentiviral-mediated in situ genetic marking and fate mapping to elucidate the lineal relationship between various cells types including ductal and acinar cells during normal homeostasis and regeneration/repair in salivary glands. The identification and functional characterization of adult salivary gland stem cells and elucidation of the mechanism of gland renewal are critical steps toward developing effective strategies for regeneration of damaged salivary glands. The knowledge gained in this study will be a first step in a long-range plan to restore salivary secretory function using adult progenitor cells. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from xerostomia caused by irreversible damage to salivary glands. The is caused by aging, autoimmune disease, radiation induced damage, trauma or other cytotoxic insults resulting in poor oral health and severely affecting patient's quality of life. Currently, there is no cure for this condition. The identificaion and functional characterization of adult salivary gland stem cells is a critical step toward developing effective strategies for regeneration of damaged salivary glands, an important therapy for a large patient population suffering from hyposalivation.