Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome
Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome
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DOI：
10.1165/rcmb.2015-0274ma
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发表时间：
2016-09-01
影响因子：
6.4
通讯作者：
Seibold, Max A.
中科院分区：
文献类型：
--
作者：
Reynolds, Susan D.;Rios, Cydney;Seibold, Max A.
条件重编程培养（CRC）方法的应用，鼻气道上皮细胞将允许更广泛地纳入原代气道上皮细胞培养模型到复杂的肺部疾病的研究。在这项研究中，我们采用CRC方法鼻气道上皮细胞，研究了该技术提供的生长优势，标准的培养方法，并确定CRC细胞培养效果的细胞和分子基础。我们发现，CRC方法允许在4次传代后产生7.1 × 10（10）个细胞，比标准支气管上皮生长培养基（BEGM）方法产生的细胞多约379倍。这些鼻气道上皮细胞表达正常的基底细胞标志物，并可被诱导形成粘膜纤毛上皮。与标准培养方法相比，使用CRC方法的祖细胞频率显著更高，并且祖细胞维持依赖于Rho激酶抑制剂Y-27632的添加。全转录组测序分析表明Y-27632处理的基底细胞中广泛的基因表达变化。我们发现，Y-27632治疗改变了基因的表达，这些基因对基底细胞骨架、细胞-细胞连接和细胞-细胞外基质（ECM）相互作用的形成至关重要。重要的是，我们发现Y-27632处理上调了独特的基底细胞中间丝和桥粒基因的表达。相反，Y-27632下调参与ECM重塑的蛋白酶/抗蛋白酶基因的多个家族。我们得出结论，Y-27632从根本上改变了细胞-细胞和细胞-ECM的相互作用，从而保留了基底祖细胞并允许更大的细胞扩增。
The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more widespread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 x 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.