Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture
Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture
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DOI：
10.3791/61541
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发表时间：
2020-07-01
影响因子：
1.2
通讯作者：
Stripp, Barry R.
中科院分区：
文献类型：
--
作者：
Konda, Bindu;Mulay, Apoorva;Stripp, Barry R.
上皮类器官模型是研究器官系统基本生物学和疾病建模的重要工具。当上皮祖细胞作为类器官生长时，可以自我更新并产生分化的后代，这些后代表现出与体内同类细胞相似的细胞功能。在这里，我们描述了一个循序渐进的方案，从人肺中分离区域特异性祖细胞，并产生3D类器官培养作为实验和验证工具。我们定义肺的近端和远端区域，目的是分离区域特异性祖细胞。我们利用酶解和机械分离相结合的方法从肺和气管中分离出总细胞。然后使用基于细胞类型特异性表面标记的荧光相关细胞分选（FACS）从近端或远端起源细胞中分离特异性祖细胞，如用于基底细胞分选的NGFR和用于肺泡II型细胞分选的HTII-280。分离的基底或肺泡II型祖细胞用于生成三维类器官培养。在第30天培养5000个细胞/孔时，远端祖细胞和近端祖细胞形成类器官的效率分别为9-13%和7-10%。在培养中，远端类器官维持HTII-280+肺泡型细胞，而近端类器官在第30天分化为纤毛细胞和分泌细胞。这些3D类器官培养物可以用作研究肺上皮细胞生物学和上皮间质相互作用的实验工具，也可以用于开发和验证针对疾病中上皮功能障碍的治疗策略。
Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.