Intersectional genetic tracing reveals the origin of alveolar stem cells during lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Thus, the exact contribution of various epithelial cells to the pools of AT2 cells under different conditions remains unclear. Here, we used dual recombinases-mediated intersectional genetic lineage tracing to investigate the cellular origins of AT2 cells during lung homeostasis, injury and repair. In contrast to previous studies, we found AT1 cells were terminally differentiated cells and did not contribute to AT2 cells after lung injury and repair. Distinctive, but simultaneous, labeling of club cells, bronchioalveolar stem cells (BASCs), and AT2 cells revealed the exact contribution of each to AT2 cells after lung injury. Moreover, we found that club cells have the potential to rebuild virtually all alveoli in some severely injured lung regions. Mechanistically, Notch signaling promotes a BASCs-to-AT2 cell transition, but it inhibits club cell-to-AT2 cell conversion during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of AT1, club, BASCs, and AT2 cells to alveolar regeneration after injury. Overall design: tdT+, ZsG+, tdT+ZsG+ lung epithelial cell of the Scgb1a1-CreER;Sftpc-DreER;R26-TLR mice at 4 weeks after bleomycin injury were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.

肺泡II型细胞（Alveolar type 2 cells, AT2）是肺泡上皮的干细胞。既往遗传谱系示踪（genetic lineage tracing）研究曾报道，损伤后AT2细胞存在多种细胞起源。然而，基于Cre-loxP系统的传统谱系示踪技术存在非特异性标记的局限，因此不同条件下各类上皮细胞对AT2细胞池的实际贡献仍不明确。本研究采用双重组酶介导的交叉遗传谱系示踪技术，探究了肺稳态、损伤及修复过程中AT2细胞的细胞起源。与既往研究不同，我们发现AT1细胞属于终末分化细胞，在肺损伤修复后并不会向AT2细胞池贡献细胞。通过对克拉拉细胞（club cells）、细支气管肺泡干细胞（bronchioalveolar stem cells, BASCs）及AT2细胞进行同步特异性标记，我们明确了各类细胞在肺损伤后对AT2细胞的实际贡献。此外，我们发现克拉拉细胞在部分重度损伤的肺区域中，具备重建几乎全部肺泡的潜能。机制层面，Notch信号通路（Notch signaling）可促进BASCs向AT2细胞的转分化，但在肺修复过程中会抑制克拉拉细胞向AT2细胞的转化。这种精度得以提升的交叉遗传谱系示踪策略，帮助我们阐明了AT1细胞、克拉拉细胞、BASCs及AT2细胞在损伤后肺泡再生中的生理功能。总体实验设计：博莱霉素损伤4周后，通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）分离Scgb1a1-CreER;Sftpc-DreER;R26-TLR小鼠的tdT+、ZsG+及tdT+ZsG+肺上皮细胞，并利用单细胞RNA测序（single-cell RNA sequencing, scRNAseq）进行分析。