Hastings Center for Pulmonary Research and Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Cells. 2020 Nov 11;9(11):2460. doi: 10.3390/cells9112460.
Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31CD45E-cadherintdTomato cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed 'conventional' AT1 cell markers (, , , ), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, , and , that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.
由于难以分离足够数量的活细胞，肺泡上皮细胞 I 型（AT1）的分子和功能特征一直具有挑战性。在这里，我们对 AT1 细胞特异性 Aqp5-Cre-IRES-DsRed（ACID）；R26tdTomato 报告小鼠肺部的 tdTomato 细胞进行了单细胞 RNA 测序（scRNA-seq）。在进行酶消化后，使用 CD31CD45E-cadherintdTomato 细胞进行荧光激活细胞分选（FACS），然后进行 scRNA-seq。使用细胞类型特异性抗体通过免疫荧光确认细胞身份。经过质量控制后，分析了 92 个细胞。大多数细胞表达“传统”AT1 细胞标志物（，，，），在该群体中具有异质性表达。其余细胞表达 AT2、club、基底或纤毛细胞标志物。与公共数据集的整合确定了三个稳健的 AT1 细胞和肺富集基因，，和，这些基因在物种间是保守的。GPRC5A 与 HOPX 共定位，在小鼠、大鼠和人肺中的 AT2 或气道细胞中不表达。GPRC5A 与 AQP5 共定位，但与小鼠肺上皮细胞涂片中的 pro-SPC 或 CC10 不共定位。我们富集了小鼠 AT1 细胞，以使用 scRNA-seq 进行分子表型分析。对假定的 AT1 细胞富集基因的进一步表征揭示了 GPRC5A 作为一种保守的 AT1 细胞表面标志物，可能有助于 AT1 细胞的分离。
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