Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.
Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.
复制标题
来自胚胎小鼠肺分离尖端上皮的肺泡样器官。
DOI：
10.1007/s13577-019-00236-6
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发表时间：
2019
期刊：
影响因子：
4.3
通讯作者：
Nogawa H
中科院分区：
文献类型：
--
作者：
Seiji Y;Ito T;Nakamura Y;Nakaishi-Fukuchi Y;Matsuo A;Sato N;Nogawa H
胚胎期13.5天的ICR小鼠获得胚肺。将经疾病处理分离的肺尖上皮包埋于低生长因子Matrigel中，在含有0.1%牛血清白蛋白的DMEM/F12培养基中培养，添加胰岛素、转铁蛋白和硒（ITS），添加或不添加成纤维细胞生长因子7 (FGF7)，观察14天。添加FGF7后，培养第7天顶端上皮生长形成囊肿。然后，囊肿表面出现管状簇状肺泡。逆转录聚合酶链反应显示，加入FGF7后，培养的肺外植体表达肺泡1型细胞标记物，如hopxandaquaporin5，以及2型细胞标记物，如lamp3和表面活性剂载脂蛋白（Sftp）CandD。石蜡包埋切片用苏木精和伊红染色，培养第14天肺泡结构由鳞状和立方状上皮细胞组成。免疫组化研究显示鳞状上皮细胞HopX阳性，立方上皮细胞pro-SftpC阳性。透射电镜观察证实鳞状上皮细胞为1型肺泡细胞，立方体细胞为2型细胞，因为鳞状上皮细胞有许多片层包涵体。胚胎肺尖上皮在Matrigel、ITS和FGF7的作用下通过自组织形成肺泡样器官。该方法体外制备肺泡样器官简便、可重复性好、经济。这种方法有可能解决肺泡上皮细胞在正常和病理条件下的许多问题。
Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such asHopXandAquaporin5, and type 2 cell markers, such asLamp3andSurfactant apoproteins(Sftp)CandD. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.