Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
Epithelial Cell Biology in ENT Research (EpiCENTR) Group, Developmental Biology and Cancer Department, Great Ormond Street UCL Institute of Child Health, University College London, London, United Kingdom.
Am J Physiol Lung Cell Mol Physiol. 2024 Oct 1;327(4):L587-L599. doi: 10.1152/ajplung.00047.2024. Epub 2024 Aug 13.
Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the , , and genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures. Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence.
基底细胞是气道上皮的成体干细胞，可再生分化细胞群体，包括执行黏液清除功能的黏液分泌细胞和纤毛细胞。人基底细胞可在体外增殖并产生分化的上皮细胞。然而，气道上皮分化的研究主要依赖于免疫组织化学或免疫荧光染色方法，这意味着缺乏动态方法，且定量数据有限。在这里，我们使用慢病毒报告基因方法将生物发光报告基因构建体转导到原代人基底细胞中，以纵向监测气道上皮分化。我们生成了三个由 、 和 基因启动子序列驱动的构建体，分别定量评估基底细胞、黏液分泌细胞和纤毛细胞的丰度。我们通过跟踪在气液界面和类器官（“支气管球体”）培养物中基底细胞的分化来验证这些构建体。转导细胞还对白细胞介素 13（IL-13；增加黏液分泌细胞分化和黏液产生）和白细胞介素 6（IL-6；增加纤毛细胞分化）的刺激做出适当反应。这些构建体代表了一种新的工具，可用于监测原代上皮细胞和/或诱导多能干细胞（iPSC）衍生细胞培养物中的气道上皮细胞分化。Orr 等人生成并验证了新的慢病毒载体，使用生物发光监测气道基底细胞、杯状细胞或多纤毛细胞的分化。
