Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies.
Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies.
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DOI：
10.1016/j.ebiom.2018.05.032
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发表时间：
2018-07
期刊：
影响因子：
11.1
通讯作者：
Meyer TF
中科院分区：
文献类型：
--
作者：
Imai-Matsushima A;Martin-Sancho L;Karlas A;Imai S;Zoranovic T;Hocke AC;Mollenkopf HJ;Berger H;Meyer TF
肺上皮细胞作为多种病原体的靶器官，在健康和疾病中发挥着至关重要的作用。然而，这一领域的研究一直受到标准培养条件下肺泡上皮静止的阻碍。在这里，我们使用人远端呼吸道上皮细胞(DAECs)来产生肺泡上皮细胞。通过与饲养层细胞共培养，并添加表皮生长因子(EGF)、Rho相关蛋白激酶抑制剂Y27632和Notch途径抑制剂二苯扎西平(DBZ)，实现了人DAECs长期、强劲的生长。去除饲养器，并用DBZ和肺成熟因子的混合物预充，阻止了自发分化为气道俱乐部细胞，而是诱导分化为肺泡上皮细胞。我们成功地将这种方法转移到鸡的远端呼吸道细胞，从而建立了一个人畜共患感染模型，使研究甲型流感病毒复制成为可能。这些细胞也可以使用RNAi技术进行基因敲除，这表明该模型适用于肺功能和疾病的机制研究。通过与饲养层细胞共培养和复合补充，实现了DAECs的长期生长。经Notch抑制剂和生长因子处理的DAECs向肺泡上皮细胞分化。人和鸡的DAEC支持复制不同的IAV毒株。Imai-Matsushima等人。描述一种稳定培养人和鸡远端呼吸道上皮细胞(DAECs)的方法。在Notch抑制剂和生长因子的补充下，这些细胞可以产生肺泡上皮细胞。DAEC支持IAV病毒感染，并容易受到基因敲除的影响，从而能够研究肺功能和呼吸道感染。
As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease. Long-term growth of DAECs was achieved using co-culture with feeder cells and compound supplementation. DAECs treated with Notch inhibitors and growth factors differentiated towards alveolar epithelial cells. Human and chicken DAECs support replication of different IAV strains. Imai-Matsushima et al. describe a method for stable culture of human and chicken distal airway epithelial cells (DAECs). Upon Notch inhibitor and growth factor supplementation these cells can generate alveolar epithelial cells. DAECs support IAV virus infection and are amenable to gene knockdown, enabling studies into lung function and respiratory infections.
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