Figure 5: β4+/H2-K1^high cells can differentiate into alveolar cells in vivo.

(A) To understand lineage relationship between H2-K1^high progenitors from uninjured lungs and alveolar, club, and expanded H2-K1^high cells post injury, all uninjured club like cells (including H2-K1^high progenitors) were merged with bleomycin injured β4+/CD200+ cells followed by supervised clustering based on progenitor genes as used in Figure 1D. Four cell types are identified: H2-K1^high cells, Foxj1+ ciliated cells, Scgb1a1+ club cells, and Sftpc+ AEC2s (See Figure S5).

(B) UMAP embedded merged objects shows either d0 (uninjured; green) or d9 bleomycin injured cells (red). Uninjured H2-K1^high (d0) progenitors are highlighted by blue circle.

(C) RNA velocity was calculated to predict lineage differentiation of the merged objects and plotted onto the UMAP embedding. The RNA velocity shows three distinct lineages emanating from H2-K1^high cells: H2-K1^high cells to Sftpc+ AEC2s, H2-K1^high cells to Foxj1+ ciliated cells, and H2-K1^high cells into further H2-K1+ cells, some of which express AEC1 markers (Ager, Pdpn; Figure S5).

(D) Sub-setting AEC1s, AEC2s, and Progenitors followed by re-calculation of RNA velocity identified a trajectory from uninjured progenitors to AEC1s and AEC2s. Uninjured H2-K1^high (d0) progenitors are highlighted by blue circle.

(E) 75,000 freshly sorted Sox2-lineage labeled airway cells containing ~4% (or ~3000) H2-K1^high cells or 75,000 freshly sorted Sox2-lineage labeled cells depleted of H2-K1^high cells were transplanted 10 days after mice were injured with bleomycin. Scale bar = 1mm. Dotted line outlines an individual lobe. Sox2-lineage label excludes BASCs and includes H2-K1^high progenitors, P63+ LNEPs, other rare airway progenitors, and mature club cells. Whereas Sox2+/H2-K1^low includes all of those progenitors except the H2-K1^high progenitors. Total fluorescent intensity corresponding to transplanted cells was captured and quantified. 2-3 lobes/mouse were imaged from n=4 mice in each condition. Each data point represents quantification of fluorescent intensity from an individual lobe. Mann-Whitney test was used to determine significance. p-value < 0.05 is considered significant. Data are presented as mean ± SD.

(F) A representative section of a mouse lobe transplanted with either Sox2-labeled or Sox2-labeled/H2-K1^neg cells is visualized showing limited engraftment of Sox2-labeled/H2-K1^neg cells. Image represents a composite of image of multiple images captured at 10X and stitched together (see STAR Methods). Scale bar = 1mm.

(G) Sox2-labeled cells, which include H2-K1^high progenitors, have engrafted in injured lung and express pro-Surfactant Protein C (pro-SPC), indicative of their differentiation towards AEC2 fate. Image represents a composite of image of multiple images captured at 20X and stitched together (see STAR Methods). Scale bar = 200 μm.

See also Figure S5 and S6.