Fig. 4.

CD44^high type II cells form alveolar-like organoids in 3-D culture. A: schematic showing the culture conditions used for organoid formation. Five thousand CD44^high or CD44^low type II cells sorted from SpC-CreER/Rosa-mTmG mice were mixed with 100,000 Mlg fibroblast cells and cultured in a 50% Matrigel/collagen mixture inside a 24-well Transwell insert, and 600 μl of culture medium were added outside the Transwell insert. B and C: representative images of GFP⁺ colonies formed with CD44^low (B) or CD44^high (C) type II cells after 14 days of culture. Scale bar = 1,000 μm. D: scatterplot showing the sizes of individual GFP⁺ colonies formed from CD44^low or CD44^high type II cells. More large colonies were formed with CD44^high type II cells. The sizes are presented as areas of the cysts, measured with the ImageJ program using images represented by B and C. Cells from two to four mice were pooled in each experiment, and data are representative of four independent experiments. E–H: immunohistochemistry of sections of GFP⁺ colonies showed that both CD44^low (E and F) and CD44^high (G and H) type II cells formed colonies expressing the type II cell marker Sp-C and the type I cell markers T1α (E and G) and HopX (F and H). CD44^high type II cells formed colonies arranged into alveolar-like structures with a large lumen inside (*; G and H). In contrast, CD44^low type II cells formed colonies that were smaller with no clear identifiable lumen (E and F). I: some cells in the CD44^high type II cell-derived colonies maintained CD44 expression after 2 wk of culture. Scale bar = 50 μm for E–I. J: proportions of type II cells (AT2) or type I cells (AT1) relative to all cells in the colonies were scored on the basis of Sp-C and HopX staining and are plotted for CD44^low or CD44^high cultures. K: CD44^low and CD44^high cells were cultured in Matrigel mixed with or without HMW HA for 11 days. The sizes of individual colonies formed are plotted for these culture conditions.