Identification and Characterization of Adult Salivary Gland Stem Cells

描述（由申请人提供）：唾液腺功能丧失影响全世界数百万人，由各种疾病引起，包括头颈癌的放射治疗、唾液腺肿瘤的手术切除、自身免疫性疾病和细胞毒性损伤。这些患者的唾液分泌显著减少，或唾液过少，严重影响他们的生活质量。目前，唯一的治疗选择是药物管理，但这是姑息性的，往往不太令人满意。由于分泌腺泡细胞因凋亡或坏死而减少，唾液流的恢复需要通过干/祖细胞移植或通过诱导内源性干/祖细胞来替换受损或丢失的细胞。然而，目前，唾液腺干细胞尚未被确定，我们缺乏必要的知识，在正常的稳态和损伤诱导再生的细胞更新的机制。从再生医学的角度来看，这些都是针对唾液腺的中心问题。在这方面，我们的实验室首先开发了一种原位谱系追踪的方法，我们用来证明表皮和皮脂腺中干细胞的存在。我们还发现，这些细胞的功能独立于位于隆突部的毛囊干细胞。我们建议使用类似的方法来确定细胞的维护和再生的机制，在唾液腺。唾液腺中分泌腺泡细胞的再生必须通过以下三种机制之一发生：腺体中功能腺泡细胞的自我复制，导管细胞的增殖，如推测的那样，或来源于未成熟、未分化、缓慢分裂的干细胞群体。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方法：1）我们将产生具有靶向唾液腺中的上皮细胞的H2 B-GFP的调节表达的双转基因小鼠，并进行长期脉冲追踪标记以鉴定缓慢分裂的细胞。将使用对唾液腺的主要细胞类型具有选择性的抗体来表征这些GFP标记的细胞。然后，我们将通过流式细胞术恢复这些缓慢循环的细胞，并评估其再生能力; 2）我们将利用慢病毒介导的原位遗传标记和命运作图来阐明各种细胞类型之间的线性关系，包括导管和腺泡细胞在唾液腺的正常稳态和再生/修复过程中。成年人的鉴定和功能特征 唾液腺干细胞和阐明腺体再生机制是发展有效的受损唾液腺再生策略的关键步骤。这项研究中获得的知识将是使用成体祖细胞恢复唾液分泌功能的长期计划的第一步。 公共卫生相关性：数百万美国人患有唾液腺不可逆损伤引起的口干症。它是由老化、自身免疫性疾病、辐射引起的损伤、创伤或其他细胞毒性损伤引起的，导致口腔健康不良，严重影响患者的生活质量。目前，没有治愈这种情况的方法。成人唾液腺干细胞的鉴定和功能表征是开发用于受损唾液腺再生的有效策略的关键一步，这是患有唾液过少的大患者群体的重要疗法。

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.