Platform effects on regeneration by pulmonary basal cells as evaluated by single-cell RNA sequencing [array]

Cell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, we isolated and cultured a population of pharmacologically expanded basal cells (peBC) from rat tracheas. At peak basal marker expression we simultaneously split peBC into four in vitro platforms – organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples were evaluated using single-cell RNA sequencing (scRNAseq) and computational pipelines were developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium was also evaluated by scRNAseq as a control for engineered epithelium. Overall this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications.基于细胞的疗法通过直接递送上皮祖细胞（epithelial progenitors），或借助工程化组织移植物（engineered tissue grafts）、完整器官的方式，在多种慢性肺部疾病的治疗中展现出应用前景。为阐明体外环境对上皮再生结局的影响，本研究从大鼠气管中分离并培养了一群经药理学扩增的基底细胞（pharmacologically expanded basal cells，peBC）。在基底标志物表达峰值阶段，我们将peBC同时分配至四种体外培养平台：类器官（organoid）、气液界面（air-liquid interface，ALI）、工程化气管及工程化肺。诱导分化完成后，我们采用单细胞RNA测序（single-cell RNA sequencing，scRNAseq）对上述样本进行检测，并开发了计算分析流程，以从全局及细胞群体层面开展样本间的对比分析。此外，我们还通过scRNAseq检测了天然大鼠气管上皮样本，以作为工程化上皮（engineered epithelium）的对照样本。本研究整体揭示了培养平台特异性效应，证实工程化模型可用于肺部上皮再生相关应用，以实现最贴近生理状态的分化结局。