Abstract
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.
Highlights
The lung alveolar epithelium comprises two morphologically and functionally distinct cell types: type I (AT1) and type II (AT2) cells
fluorescence-activated cell sorting (FACS) analysis of cells isolated from three lungs showed that 31 ± 10% of epithelial cells were positive for tdTomato
ACID;R26tdTomato reporter mice and integrated these results with publicly available mouse and human datasets, with the goal of identifying and further characterizing novel AT1 cell genes that could be used as surface markers to facilitate cell sorting
Summary
The lung alveolar epithelium comprises two morphologically and functionally distinct cell types: type I (AT1) and type II (AT2) cells. AT2 cells cover ~5% of the alveolar surface and have important functions including the production of surfactant proteins and both self-renewal and differentiation to AT1 cells during homeostasis and repair following injury [1,2,3,4]. In contrast to AT2 cells, which have been extensively studied, far less is known about AT1 cell contributions to alveolar homeostasis due to difficulty in isolating viable AT1 cell populations of sufficient yield and purity for detailed molecular and functional characterization. An in vitro culture model which recapitulates aspects of AT2 to AT1 cell transdifferentiation has provided important insights into functional and phenotypic properties of AT1 cells [7,8,9,10].
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