Mechanisms governing the physiologic regulation of cell fate: Hypoxia-induced differentiation of neuroendocrine cells from stem cells

项目摘要 几十年的研究表明，肺神经内分泌细胞具有多种功能，包括 氧气感应，机械转导，调节肺血流，调节呼吸道直径， 化学感觉，以及最近对炎症的调节。此外，NE细胞丰度增加 已经记录了几种呼吸系统疾病，包括肺高压、神经内分泌 婴儿增生症(NEHI)、婴儿猝死综合征、哮喘、支气管肺发育不良、先天性 肺炎、囊性纤维化、慢性阻塞性肺疾病和先天性横隔疝。然而，目前还不清楚是否 这些NE细胞是疾病的原因，或者它们是否代表了对损伤的保护性再生反应。 以前的研究主要是对神经内分泌体(NEB)进行的，神经内分泌体是聚集的NE细胞 发生在呼吸道分支点。在这里，我们关注孤立的神经内分泌细胞的作用，它可能 代表一个不同的NE细胞群体，在与NE细胞相关的疾病中，与NEB一样，NE细胞也会增加 增生症。 我们提供的证据表明，严重肺部疾病中常见的低氧刺激神经内分泌。 分化是一种保护性反应。利用谱系追踪，我们将测试低氧诱导的NE细胞是否 可塑，并能够在受伤后分化为其他类型的细胞。我们还将评估NE细胞是否 使用一种新的偶联的呼吸道外植体系统恢复常氧后，增生是可逆的 长期双光子成像。我们假设低氧依赖的神经内分泌分化是由 通过HIF信号级联定位于基础干细胞室，并将检测是否为呼吸道 干细胞拥有这种氧气感应机制，而氧气感应机制又负责触发NE细胞的启动 差异化。我们将进一步研究正常情况下是否需要独立于低氧的HIF信号 组织驻留NE细胞的维护。利用基因细胞消融，我们将评估细胞的功能相关性 低氧诱导损伤模型的神经内分泌分化。然后我们将测试富含的多肽 降钙素基因相关肽是一种保护性神经肽，通过促进前体细胞减轻缺氧性上皮损伤 促进细胞增殖，防止细胞死亡。为了解决NE细胞的异质性，我们将Tuj1确定为一种 NEBs中不存在的孤立性NE细胞的上皮标记。我们发现，增生性NE细胞在 婴儿期神经内分泌增生症(NEHI)为阳性。最后，我们将建立一个制度， 低氧后人原代呼吸道干细胞向Tuj1孤立NE细胞分化的研究。

Project Summary Decades of studies have suggested that pulmonary neuroendocrine cells perform multiple functions including oxygen sensing, mechanotransduction, regulation of pulmonary blood flow, regulation of airway diameter, chemosensation, and most recently the regulation of inflammation. Additionally, increased NE cell abundance has been documented in several respiratory diseases including pulmonary hypertension, neuroendocrine hyperplasia of infancy (NEHI), sudden infant death syndrome, asthma, bronchopulmonary dysplasia, congenital pneumonia, cystic fibrosis, COPD, and congenital diaphragmatic hernia. However, it remains unclear whether these NE cells are a cause of disease or whether they represent a protective regenerative response to injury. Prior research has largely been conducted on neuroendocrine bodies (NEBs) which are clustered NE cells occurring at airway branch points. Herein, we focus on the role of solitary neuroendocrine cells which may represent a distinct population of NE cells that, like NEBs, are increased in diseases associated with NE cell hyperplasia. We provide evidence that hypoxia, frequently encountered in severe lung disease, stimulates neuroendocrine differentiation as a protective response. Using lineage tracing, we will test whether hypoxia-induced NE cells are plastic and capable of differentiating into other cell types following injury. We will also assess whether NE cell hyperplasia is reversible following the restoration of normoxia using a novel airway explant system coupled to long term 2 photon imaging. We hypothesize that hypoxia-dependent neuroendocrine differentiation is mediated through the HIF signaling cascade localized to the basal stem cell compartment, and will test whether airway stem cells possess this oxygen sensing machinery which in turn is responsible for triggering the onset of NE cell differentiation. We will further examine whether HIF signaling independent of hypoxia is required for the normal maintenance of tissue resident NE cells. Using genetic cell ablation, we will assess the functional relevance of hypoxia-induced neuroendocrine differentiation in injury models. We will then test whether the abundant peptide CGRP is a protective neuropeptide that ameliorates hypoxia-induced epithelial injury by promoting progenitor cell proliferation and preventing cell death. To address NE cell heterogeneity, we have identified Tuj1 as an epithelial marker of solitary NE cells that is absent in NEBs. We show that hyperplastic NE cells in Neuroendocrine Hyperplasia of Infancy (NEHI) are positive for this marker. Lastly, we will establish a system for studying human primary airway stem cell differentiation into Tuj1+ solitary NE cells following hypoxia.