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

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.基于细胞的治疗（cell-based therapies）可通过直接施用上皮祖细胞，或是借助工程化组织移植物、完整器官，在治疗众多慢性肺部疾病方面展现出应用前景。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物扩增的基底细胞（pharmacologically expanded basal cells，peBC）。在基底标志物表达峰值阶段，我们将peBC同时分为四类体外培养平台——类器官、气液界面（air-liquid interface，ALI）、工程化气管与工程化肺。诱导分化完成后，我们采用单细胞RNA测序（single-cell RNA sequencing，scRNAseq）对这些样本进行评估，并开发了计算分析流程以从全局水平与细胞群体层面比对各组样本。我们还通过scRNAseq对天然大鼠气管上皮样本进行了测序，以作为工程化上皮的对照样本。综上，本研究明确了各平台的特异性效应，佐证了工程化模型在肺部上皮再生相关应用中可实现最具生理相关性的分化结局的实用性。