Pathogenesis of Airway Stem Cell Abnormalities in Obliterative Bronchiolitis

项目总结： 终末期肺部疾病是全世界发病率和死亡率的主要原因。肺移植是一种 对于患有这种疾病的患者来说，这是一个很好的治疗选择，但50%的受者在五年内死于 同种异体移植物发生闭塞性毛细支气管炎。建议上皮干细胞枯竭 有助于OB的发展；然而，很少有研究来检验这一假说， 主要是由于缺乏OB的动物模型，这种动物模型会发展成类似于 人类。我们最近在雪貂身上开发了一种新的原位肺移植模型，以模拟人类OB 很好。使用这个模型，我们首次在雪貂和人类同种异体移植中证明了 同种异体移植物近端和远端的克隆性K5+p63+基础干细胞(BSCs)进行性下降 随着OB严重程度的增加。此外，我们的研究首次证明了近端的呼吸道 粘膜下腺(SMG)是表面气道上皮(SAE)中BSCs的兼性小生境，是早期的靶标。 人类和雪貂同种异体移植呼吸道的免疫破坏。在小鼠中，SMG干细胞利基只服务于 然而，在较大的哺乳动物中，如人和雪貂，SMGs存在于整个 软骨性呼吸道。通过谱系追踪，我们发现SMG的肌上皮细胞(MECs)是 SAE中多能K5+p63+BSCs的前体细胞。在OB的发展过程中，SMG的毁灭 同种异体移植物中的干细胞生境同时发生表型和功能的多能性变化。 SAE中K5+p63+BSCs。我们假设同种异体移植物中失神经SMG的破坏，因此 MECs的耗竭导致SAE中多能K5+p63+BSCs的减少和承诺的增加 多能(K5+P63+K14+)、双能(K5+K14+、P63+K14+)和单能(K14+)基本细胞，所有这些细胞都有 自我更新能力降低。拟议研究的目标是确定功能 微血管内皮细胞和谱系决定的基底细胞的表型变化的意义以及如何去神经 改变维持SMG干细胞生态位所需的Wnt信号(Lef-1/TCF1)和 损伤背景下腺体微血管内皮细胞向SAE骨髓间充质干细胞的谱系定位。此外，一项重要的临床前研究 这项建议的目的是阐明干细胞移植到移植肺和修复的能力。 损伤，从而为干细胞疗法的发展奠定了基础，以延缓或预防肺内OB 异体移植。我们将通过解决以下具体目标来实现这些目标：1)确定如何 SMG干细胞生态位的破坏导致K5+p63+BSCs的枯竭。2)确定以下情况的后果 肺去神经对呼吸道干细胞及其壁龛的影响。3)确定呼吸道干细胞的潜能 移植在预防或延缓OB中的作用。我们希望描绘出干细胞耗尽的机制 这将是理解OB发病机制的重要一步，并将 告知使用干细胞作为预防或延迟OB的治疗方法。

Project Summary: End-stage lung diseases are a major cause of morbidity and mortality worldwide. Lung transplantation is an excellent treatment option for patients with this condition, yet 50% of recipients die within five years due to the development of obliterative bronchiolitis (OB) in the allograft. Epithelial stem cell depletion is suggested to contribute to the development of OB; however, there is little research being performed to test this hypothesis, primarily due to the lack of animal models for OB that develop allograft pathology resembling that seen in humans. We recently developed a novel orthotopic lung transplant model in the ferret that models human OB very well. Using this model, we have shown for the first time in ferret and human allografts that the number of clonogenic K5+p63+ basal stem cells (BSCs) progressively declines in proximal and distal airways of the allograft as the severity of OB increases. Additionally, our research is the first to demonstrate that the proximal airway submucosal gland (SMG), a facultative niche for BSCs in the surface airway epithelium (SAE), is an early target of immune destruction in human and ferret allograft airways. In mice, the SMG stem cell niche serves only the trachea; however, in larger mammals such as humans and ferrets, SMGs are present throughout the cartilaginous airways. Using lineage tracing, we have shown that the myoepithelial cells (MECs) of SMGs are precursors of multipotent K5+p63+ BSCs in the SAE. During the development of OB, the destruction of SMG stem cell niches in the allograft occurs simultaneously with phenotypic and functional changes to multipotent K5+p63+ BSCs in the SAE. We hypothesize that destruction of the denervated SMG in the allograft, and thus depletion of MECs, leads to a decline in multipotent K5+p63+ BSCs in the SAE, and to increases in committed multipotent (K5+p63+K14+), bipotent (K5+K14+, p63+K14+) and unipotent (K14+) basal cells, all of which have a reduced capacity for self-renewal. The objective of the proposed research is to determine the functional significance of the phenotypic changes in the MECS and the lineage-committed basal cells and how denervation of the SMG alters Wnt signaling (Lef-1/TCF1) required for both the maintenance of the SMG stem cell niche and lineage commitment of glandular MECs to SAE BSCs in the setting of injury. Additionally, a major preclinical objective of this proposal is to elucidate the ability of stem cells to engraft into a transplanted lung and repair injury, thus laying the foundation for the development of stem cell therapy to delay or prevent OB in lung allografts. We will achieve these objectives by addressing the following specific aims: 1) Determine how destruction of the SMG stem cell niche contributes to depletion of K5+p63+ BSCs. 2) Identify consequences of lung denervation on airway stem cells and their niches. 3) Determine the potential for airway stem cell transplantation in preventing or delaying OB. We expect that delineating the mechanisms of stem cell depletion in the airways of transplanted lungs will be a major step forward in understanding OB pathogenesis and will inform the use of stem cells as a therapeutic approach to prevent or delay OB.