Huang Sarah X L, Green Michael D, de Carvalho Ana Toste, Mumau Melanie, Chen Ya-Wen, D'Souza Sunita L, Snoeck Hans-Willem
1] Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA. [2] Department of Medicine, Columbia University Medical Center, New York, New York, USA.
Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Experimental Therapeutic Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Nat Protoc. 2015 Mar;10(3):413-25. doi: 10.1038/nprot.2015.023. Epub 2015 Feb 5.
Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.
从人多能干细胞(hPSC)体外生成的肺和气道上皮细胞在再生医学、肺病建模、药物筛选以及人类肺发育研究中具有应用价值。在此,我们描述了一种将hPSC定向分化为发育性肺祖细胞,并随后将其分化为主要是远端肺上皮细胞的策略。该方案包括四个阶段,概括了肺的发育过程,耗时约50天。首先,在高浓度激活素A存在的情况下诱导确定内胚层(DE)。随后,通过依次抑制骨形态发生蛋白(BMP)、转化生长因子-β(TGF-β)和Wnt信号通路来指定肺偏向的前肠内胚层(AFE)。然后通过应用Wnt、BMP、成纤维细胞生长因子(FGF)和视黄酸(RA)信号通路使AFE腹侧化,以获得肺和气道祖细胞。最后,使用Wnt、FGF、cAMP和糖皮质激素激动剂将这些细胞进一步分化为更成熟的上皮细胞类型。该方案在明确的条件下进行,不涉及细胞的基因操作,并且所得到的培养物中大多数细胞表达各种肺和气道上皮细胞的标志物,其中主要是可鉴定为功能性II型肺泡上皮细胞的细胞。