Fig. 5.

CD44^high type II cells are preferentially located in pericapillary niche. A: schematic showing the labeling strategy for type II cells in SpC-CreER/Rosa-tTA/tetO-H2B-GFP mice. Adult mice were given four doses of tamoxifen (TAM; 0.25 mg/g) 2 wk before lung isolation. Sp-C⁺ cell-specific Cre then excises the stop codon upstream of tTA, leaving it to activate the tetO promoter, which drives the expression of H2B-GFP in Sp-C⁺ cells. B: immunohistochemistry analysis of lung sections from these mice showed that only a small fraction of GFP⁺ cells expressed CD44 at the cell membrane (arrow). C and D: the same lung sections showed staining with GFP and CD44 (C) and GFP and Sp-C (D). The arrows show that GFP⁺CD44^high cells were also positive for Sp-C. Arrowheads indicate cells that were GFP⁺Sp-C⁺ type II cells but had no CD44 expression. E: 6 × 6 tiled confocal image showing the distribution of CD44^high cells in a larger area of the lung section. Blood vessels (bv) are outlined by white circles, pink arrows show GFP⁺CD44^high cells that are adjacent to bv, and tan arrows indicate those cells that are farther away from bv. F: blood vessels in the lung were labeled with vWF. Arrows show GFP⁺CD44^high cells located adjacent to bv. G–I: GFP⁺CD44^high cells were more frequently detected at perivascular regions of the lung. Sixty-two GFP⁺CD44^high type II cells from five 6 × 6 tiled pictures captured at random locations in lung sections (H) and 204 random GFP⁺ type II cells in those pictures (I) were analyzed for their distance to the nearest bv; the percentage of the cells vs. the distance to bv is plotted. The mean distance to nearest bv is significantly less for the GFP⁺CD44^high type II cells compared with the random GFP⁺ type II cells (G); data are presented as Tukey box plot. J–L: areas of nuclei were measured using ImageJ for CD44^high (arrows and insets) and CD44^low (arrowheads) type II cells and are plotted (L). K is the same image as J but only shows DAPI. **P < 0.01, ***P < 0.001. Scale bar = 30 μm for B, C, D, and F. Scale bar = 150 μm for E. Scale bar = 10 μm for J and K.