Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro [scRNA-seq]

Rationale: Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. Objectives: To elucidate the role of EMT in the development of aberrant basaloid cells from human bronchial epithelium by mapping EMT-induced transcriptional changes at the population and single-cell level. Methods: Human bronchial epithelial cells (HBECs) grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Measurements and Main Results: Comparison of submerged and ALI cultures revealed differential expression of 9,868—protein coding (PC) and long non-coding RNA (lncRNA)—genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,927—PC and lncRNA—genes. While there was no evidence for fibroblast/myofibroblast conversion, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. Conclusions: The substantial transcriptional differences between submerged and ALI cultures highlights that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and a data visualization package for further exploration for potential roles in the lung epithelium in health and lung disease. 3 individual healthy donor bronchial epithelial tissue samples were used for single-cell RNA sequencing analysis. The single-cell RNA-seq data were generated in two ALI libraries, AW20003 and AW21001, and one submerged cultures library, SC2100310. (1) AW20003 contained the data of donor ID 34 (11 years old Caucasian male). Hashtag-oligos (HTOs) 5 and 6 labelled the Day 0 cells, HTOs 3 and 4 the Day 1 cells and HTOs 1 and 2 the Day 5 cells. (2) AW21001 combined the data of donor IDs 27 (60 years old black male) and 54 (19 years old black female) in different HTOs. HTOs 1 and 4 labelled the Day 0 cells of each donor respectively. Similarly, HTOs 2 and 5 and HTOs 3 and 6 held the data of Days 1 and 5. (3) SC2100310 combined the data of all three donors with HTO 1 marking the cells of donor IDs 27, HTO 2 the cells of donor ID 34 and HTO 3 the cells of donor ID 54.

研究背景：尽管上皮间质转化（epithelial-mesenchymal transition, EMT）是肺纤维化疾病的常见特征，但其在肺纤维化发生中的作用尚存争议。近期，研究人员在肺纤维化组织中鉴定出一类新型上皮细胞——异常基底样细胞，其呈现部分EMT表型。这类细胞的发育起源目前仍未知。
研究目的：通过在群体及单细胞水平绘制EMT诱导的转录变化图谱，阐明EMT在人支气管上皮来源的异常基底样细胞发生中的作用。
研究方法：将人支气管上皮细胞（HBECs）以浸没培养或气液界面（ALI）培养的方式培养，并分别添加或不添加EMT诱导剂，通过批量RNA测序与单细胞RNA测序进行分析。
测量与主要结果：比较浸没培养与ALI培养体系，发现9868个蛋白编码（PC）与长链非编码RNA（lncRNA）基因存在差异表达，并鉴定出上皮细胞类型特异性lncRNA。同样，在ALI培养体系中诱导EMT可导致6927个蛋白编码及lncRNA基因发生显著转录重编程。尽管未观察到成纤维细胞/肌成纤维细胞转化，但细胞呈现出部分EMT基因特征及异常基底样细胞表型。
研究结论：浸没培养与ALI培养体系间存在显著转录差异，这提示在解读浸没培养的实验数据时需格外谨慎。本研究证实肺上皮EMT并不会生成成纤维细胞/肌成纤维细胞，并验证了ALI培养体系是研究异常基底样细胞及EMT机制的生理相关模型。我们提供了蛋白编码及lncRNA基因的转录谱目录与一款数据可视化工具包，以供进一步探索其在健康肺上皮及肺部疾病中的潜在作用。
本研究共使用3份来自健康供体的支气管上皮组织样本开展单细胞RNA测序分析。单细胞RNA测序数据构建于2个ALI文库（AW20003与AW21001）及1份浸没培养文库（SC2100310）：
1. AW20003包含供体ID34（11岁高加索男性）的测序数据：标签寡核苷酸（hashtag-oligos, HTO）5和6标记第0天细胞，HTO3和4标记第1天细胞，HTO1和2标记第5天细胞。
2. AW21001整合了供体ID27（60岁黑人男性）与供体ID54（19岁黑人女性）的测序数据，通过不同HTO进行区分：HTO1和4分别标记两名供体的第0天细胞；同理，HTO2和5、HTO3和6分别对应第1天与第5天的细胞样本。
3. SC2100310整合了全部3名供体的测序数据：HTO1标记供体ID27的细胞，HTO2标记供体ID34的细胞，HTO3标记供体ID54的细胞。