Expansion of Airway Basal Cells and Generation of Polarized Epithelium
Expansion of Airway Basal Cells and Generation of Polarized Epithelium
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DOI：
10.21769/bioprotoc.2877
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发表时间：
2018-06-05
期刊：
影响因子：
0.8
通讯作者：
Mou, Hongmei
中科院分区：
文献类型：
--
作者：
Levardon, Hannah;Yonker, Lael M.;Mou, Hongmei
气道基底干细胞是气道内的祖细胞，具有自我更新能力并产生多种类型的分化气道上皮细胞。这种干细胞衍生的上皮显示出具有气道粘膜功能属性的组织结构。已经开发出一种方案来培养和扩增人类气道基底干细胞，同时保留其干细胞特性和随后粘液纤毛分化的能力。这一成就提供了一个以前未实现的机会，可以维持来自健康捐赠者的祖细胞的持久供应，以分化为人类初级气道上皮，用于基于细胞和分子的研究。此外，可以从患有特定气道疾病（例如囊性纤维化）的患者中采集基底干细胞，从而能够研究气道粘膜适当环境中疾病特异性细胞的潜在行为改变。在这里，我们详细描述了气道基底干细胞连续扩增的方案，以生成几乎无限的气道基底细胞，这些细胞可以储存并随时用于后续培养和分化。此外，我们描述了气道基底干细胞在气液界面的可渗透 Transwell 过滤器上的培养和分化，以创建功能性粘液纤毛假复层极化气道上皮粘膜。
Airway basal stem cells are the progenitor cells within the airway that exhibit the capacity to self-renew and give rise to multiple types of differentiated airway epithelial cells. This stem cell-derived epithelium displays organized architecture with functional attributes of the airway mucosa. A protocol has been developed to culture and expand human airway basal stem cells while preserving their stem cell properties and capacity for subsequent mucociliary differentiation. This achievement presents a previously unrealized opportunity to maintain a durable supply of progenitor cells derived from healthy donors to differentiate into human primary airway epithelium for cellular and molecular-based studies. Further, basal stem cells can be harvested from patients with a specific airway disease, such as cystic fibrosis, enabling investigation of potentially altered behavior of disease-specific cells in the appropriate context of the airway mucosa. Here we describe, in detail, a protocol for the serial expansion of airway basal stem cells to enable the generation of nearly unlimited airway basal cells that can be stored and readily available for subsequent culturing and differentiation. In addition, we describe culturing and differentiation of airway basal stem cells on permeable transwell filters at air-liquid interface to create functional mucociliary pseudostratified polarized airway epithelial mucosa.