Figure 4: β4+/H2-K1^high cells expand after injury and acquire senescence-associated markers during fibrotic phase of bleomycin injury.

(A) Fraction of β4+/H2-K1^high cells increases preferentially immediately after injury as determined by flow cytometry at 3 and 6 days after bleomycin injury. These expanded H2-K1^high cells gradually lose their ability to form large colonies in vitro at days 3, 6, and 9 post bleomycin injury. (data representative of n=2 independent experiments from n=3 mice each).

(B, C) Single cell RNA-seq was performed on EpCAM+β4+/CD200+ cell population 9 days after injury followed by supervised clustering using the Sox9-based gene signature of progenitor cells (Figure 1D), which identified H2-K1^high cell cluster (labeled as Progenitors). Cells in this cluster expressed low levels of mature lineage markers such as Surfactant Protein C and secretoglobins (Scgb1a1 and Scgb3a2), as expected. These cells also express senescence associated markers such as Cdkn1a, Cdkn2a, Hif1a, and Trp53.

(D) Injured β4+/H2-K1^high cells (d9 post injury) have increased expression of alveolar genes and decreased expression of airway genes when compared with uninjured β4+/H2-K1^high progenitor cells, suggesting acquisition of alveolar fate.

(E) Pathway analysis of injured β4+/H2-K1^high cells in comparison with uninjured progenitor cells identify upregulation of signaling pathways associated with cellular movement while mature club cells from injured lungs remain unresponsive to the injury when compared to uninjured club cells.

See also Figure S4.