Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker
Masafumi Horie,
Alessandra Castaldi,
Mitsuhiro Sunohara,
Hongjun Wang,
Yanbin Ji,
Yixin Liu,
Fan Li,
Thomas A. Wilkinson,
Long Hung,
Hua Shen,
Hidenori Kage,
Ite A. Offringa,
Crystal N. Marconett,
Per Flodby,
Beiyun Zhou,
Zea Borok
Affiliations
Masafumi Horie
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
Alessandra Castaldi
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
Mitsuhiro Sunohara
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
Hongjun Wang
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
Yanbin Ji
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
Yixin Liu
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
Fan Li
Single-Cell, Sequencing, and CyTOF Core (SC2), Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA
Thomas A. Wilkinson
Single-Cell, Sequencing, and CyTOF Core (SC2), Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA
Long Hung
Single-Cell, Sequencing, and CyTOF Core (SC2), Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA
Hua Shen
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
Hidenori Kage
Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Ite A. Offringa
Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
Crystal N. Marconett
Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
Per Flodby
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
Beiyun Zhou
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
Zea Borok
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
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, CD31-CD45-E-cadherin+tdTomato+ 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 (Aqp5, Pdpn, Hopx, Ager), 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, Ager, Rtkn2 and Gprc5a, 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.
