Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara Shogoin Sakyo, Kyoto, 606-8507, Japan.
Pulmonary Medicine, Kishiwada City Hospital, 1001 Gakuhara Kishiwada, Osaka, 596-8501, Japan.
Respir Res. 2017 Aug 7;18(1):150. doi: 10.1186/s12931-017-0635-5.
Alveolar type 2 (AT2) cells play important roles in maintaining adult lung homeostasis. AT2 cells isolated from the lung have revealed the cell-specific functions of AT2 cells. Comprehensive molecular and transcriptional profiling of purified AT2 cells would be helpful for elucidating the underlying mechanisms of their cell-specific functions. To enable the further purification of AT2 cells, we aimed to discriminate AT2 cells from non-AT2 lung epithelial cells based on surface antigen expression via fluorescence activated cell sorting (FACS).
Single-cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/epithelial cell adhesion molecule (EpCAM)/ major histocompatibility complex class II (MHCII)) and for pro-surfactant protein C (proSP-C), followed by FACS analysis for surface antigen expression on AT2 cells. AT2 cells were sorted, and purity was evaluated by immunofluorescence and FACS. This newly developed strategy for AT2 cell isolation was validated in different strains and ages of mice, as well as in a lung injury model.
FACS analysis revealed that EpCAM epithelial cells existed in 3 subpopulations based on EpCAM and MHCII expression: EpCAMMHCII cells (Population1:P1), EpCAMMHCII cells (P2), and EpCAMMHCII cells (P3). proSP-C cells were enriched in P1 cells, and the purity values of the sorted AT2 cells in P1 were 99.0% by immunofluorescence analysis and 98.0% by FACS analysis. P2 cells were mainly composed of ciliated cells and P3 cells were composed of AT1 cells, respectively, based on the gene expression analysis and immunofluorescence. EpCAM and MHCII expression levels were not significantly altered in different strains or ages of mice or following lipopolysaccharide (LPS)-induced lung injury.
We successfully classified murine distal lung epithelial cells based on EpCAM and MHCII expression. The discrimination of AT2 cells from non-AT2 epithelial cells resulted in the isolation of pure AT2 cells. Highly pure AT2 cells will provide accurate and deeper insights into the cell-specific mechanisms of alveolar homeostasis.
肺泡 II 型（AT2）细胞在维持成人肺部稳态方面发挥着重要作用。从肺部分离出的 AT2 细胞揭示了 AT2 细胞的细胞特异性功能。对纯化的 AT2 细胞进行全面的分子和转录谱分析将有助于阐明其细胞特异性功能的潜在机制。为了进一步纯化 AT2 细胞，我们旨在通过荧光激活细胞分选（FACS）根据表面抗原表达从非 AT2 肺上皮细胞中区分 AT2 细胞。
从酶消化的鼠肺中获得的单细胞悬液被标记为表面抗原（CD45/CD31/上皮细胞黏附分子（EpCAM）/主要组织相容性复合体 II 类（MHCII））和前表面活性蛋白 C（proSP-C），然后进行 FACS 分析以检测 AT2 细胞的表面抗原表达。对 AT2 细胞进行分选，并通过免疫荧光和 FACS 评估纯度。该用于 AT2 细胞分离的新策略在不同品系和年龄的小鼠以及肺损伤模型中得到了验证。
FACS 分析显示，EpCAM 上皮细胞根据 EpCAM 和 MHCII 表达存在 3 个亚群：EpCAMMHCII 细胞（群体 1：P1）、EpCAMMHCII 细胞（P2）和 EpCAMMHCII 细胞（P3）。proSP-C 细胞在 P1 细胞中富集，通过免疫荧光分析和 FACS 分析，P1 中分离的 AT2 细胞的纯度值分别为 99.0%和 98.0%。基于基因表达分析和免疫荧光，P2 细胞主要由纤毛细胞组成，P3 细胞由 AT1 细胞组成。在不同的品系或年龄的小鼠或脂多糖（LPS）诱导的肺损伤后，EpCAM 和 MHCII 的表达水平没有明显改变。
我们成功地根据 EpCAM 和 MHCII 表达对鼠远端肺上皮细胞进行了分类。从非 AT2 上皮细胞中区分 AT2 细胞导致了纯 AT2 细胞的分离。高纯度的 AT2 细胞将为肺泡稳态的细胞特异性机制提供更准确和更深入的见解。
