CD44high alveolar type II cells show stem cell properties during steady-state alveolar homeostasis

Abstract

The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here, we have identified a subgroup of type II cells expressing a higher level of cell surface molecule CD44 (CD44^high type II cells) that composed ~3% of total type II cells in 5-10-wk-old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared with CD44^low type II cells. Moreover, in aged mice, 18-24 mo old, the percentage of CD44^high type II cells among all type II cells was increased, but these cells showed decreased progenitor properties. Thus CD44^high type II cells likely represent a type II cell subpopulation important for constitutive regulation of alveolar homeostasis.

Keywords: CD44; alveoli; homeostasis; lung; type II cells.

Fig. 1.

Isolation of CD44^high type II cells from Sp-C lineage-tracing mouse lines. A: to identify type II cells from adult mice, four doses of tamoxifen (TAM; 0.25 mg/g) were given to SpC-CreER/Rosa-Tomato mice, in which TAM-induced Cre activation causes excision of the stop codon upstream of Tomato, resulting in lineage labeling of Sp-C⁺ cells. B and C: alveolar epithelial cells were isolated using a type II cell-enriched protocol and subjected to FACS analysis. Type II cells were selected by gating for the Tomato⁺ population (B) and were further separated into CD44^low and CD44^high populations (C). Approximately 3% of the type II cells were positive for CD44. FS Lin, forward scatter linear; R3, region 3. D: Tomato⁺ cells were gated and analyzed for the expression of EpCAM and CD44. Of the Tomato⁺ cells, 99.8% were EpCAM⁺, and of the Tomato⁺ CD44^high cells, 99.6% were EpCAM⁺. These data are representative of >5 independent experiments. E–G: expression levels of CD44 isoforms in sorted cells were analyzed using qRT-PCR using primers that specifically detect all isoform types (CD44 total; E), the variant isoforms containing variable exons (CD44v; F), or standard isoform (CD44s; G). Compared with CD44^low type II cells, CD44^high type II cells expressed significantly higher levels of total CD44 (E), CD44v (F), and CD44s (G). Data are presented as Tukey box plots. *P < 0.05, **P < 0.01; n = 7 mice.

Fig. 2.

CD44^high type II cells show higher cell proliferation compared with CD44^low type II cells. A and B: relative expression of CDC25C (A) and cyclin B1 (CCNB1; B) in sorted CD44^high and CD44^low type II cells was analyzed using qRT-PCR. Both genes showed significantly higher expression levels in CD44^high type II cells compared with CD44^low type II cells. Data are presented as Tukey box plots. *P < 0.05, **P < 0.01; n = 7 mice. C–G: BrdU-labeled type II cells in C57BL/6 mice. Lungs from BrdU-injected mice were subjected to immunohistological analysis, and cell proliferation rates of CD44^low or CD44^high type II cells were scored as BrdU⁺Sp-C⁺CD44^low cells vs. Sp-C⁺CD44^low cells or BrdU⁺Sp-C⁺CD44^high cells vs. Sp-C⁺CD44^high cells, respectively. C and D show the same section with a BrdU⁺Sp-C⁺CD44^high cell, and E and F show the same section with a BrdU⁺Sp-C⁺CD44^low cell. G: for each mouse, ~600 Sp-C⁺ cells were scored; n = 5 mice. Data are presented as Tukey box plot. ***P < 0.001.

Fig. 3.

CD44^high type II cells differentiate into type I cells in vitro. CD44^high and CD44^low type II cells were grown on 0.2% gelatin-coated plates. A and B: after 48 h of culture, some cells adopted flattened type I cell-like morphology (arrows). C: percentages of the flattened type I-like cells were significantly higher in the CD44^high cell cultures compared with those of CD4^low cells. D and E: higher-magnification images of representative cells derived from CD44^low (D) or CD44^high (E) type II cells after growing 5 days in vitro. Cell diameters were measured on the basis of the longer axis of the cell (pink lines). F: quantification of cell diameters after 5 days of culture showed that average diameter of the flattened cells derived from CD44^high type II cells were significantly larger than those from CD44^low type II cells. G–L: cells were fixed at 72-h postculture and subjected to immunohistochemistry analysis of type I cell markers T1α (G and H), HopX (I and J), and RAGE (K and L). Some of the flattened-shaped cells from both CD44^low (G, I, K) and CD44^high (H, J, and L) cultures expressed these type I cell markers. M: cells expressing any of the above type I cell markers were counted, and the ratio of these type I-like cells to total cells was calculated. N and O: cells were stained for type II cell marker Sp-C at 72-h postculture. CD44^low cells (N) had more Sp-C-expressing cells than CD44^high cells did (O). P: Sp-C⁺ cells were counted, and the ratio of Sp-C⁺ cells to total cells was calculated. Scale bar = 30 µm for A, B, G–L, N, and O. Scale bar = 100 µm for D and E. The data in C, F, M, and P are presented as Tukey box plots. *P < 0.05, ***P < 0.001. Each sample group of the quantification data (C, F, M, and P) was from >5 mice; 50–200 cells randomly selected from each group from each mouse were scored. Because of the slight variation in culture condition of each independent experiment, M and P are plotted as the relative value compared with the average result of CD44^high cells cultured at the same time.

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.

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.

Fig. 6.

CD44^high type II cells are negative for integrin-α6, integrin-β4, and Sca-1 and can be identified from published single-cell RNAseq analysis data. A–C: type II cells were isolated from SpC-CreER/Rosa-Tomato mice that had been given four doses of tamoxifen (0.25 mg/g) and were subjected to FACS analysis. Tomato-positive cells were gated (not shown) and further analyzed for the expression of CD44, integrin-α6, integrin-β4, and Sca-1. Numbers indicate the percentage of cells in the indicated fraction out of the total gated cells. The data are representative of at least two independent experiments. D: FPKM values of CD44 of 46 lineage-labeled adult mouse type II cells (accession no. GSE52583) are plotted. E: TPM values of CD44 from 84, 47, and 77 type II cells from 3 healthy donors (accession no. GSE84147) are plotted. Data are presented as Tukey box plots.

Fig. 7.

Aged mice have more CD44^high type II cells, but these cells exhibit decreased progenitor properties. A and B: type II cells isolated from 6–8-wk-old (young) or 18–24-mo-old (aged) C57BL/6 mice were analyzed for the presence of EpCAM⁺CD44^high cells. C: percentages of EpCAM⁺CD44^high cells among all the EpCAM⁺ type II cells are plotted for young and aged mice; n = 11 mice for each age group. D–F: young (D) or aged (E) C57BL/6 mice were subjected to paraffin sectioning and antibody staining, and the percentages of CD44^highSp-C⁺ vs. all Sp-C⁺ cells were scored and are plotted. Five to eight mice were scored for each age group (F). Arrows in D and E show CD44^highSp-C⁺ cells. Insets in white rectangles are enlarged pictures of blue rectangles. G and H: EpCAM⁺CD44^high cells of the two age groups were isolated by FACS and subjected to qRT-PCR analysis for expression of CDC25C (G) and cyclin B1 (CCNB1; H); n = 7 mice. I–K: EpCAM⁺CD44^high cells from young (I) and aged mice (J) were cultured on gelatin-coated slides for 3 days. Images are representative of similar observations for six to eight mice for each group. The ratios of flat type I-like cells vs. total cells are plotted. For each mouse, 50–200 cells were scored (K). L–O: cultured EpCAM⁺CD44^high cells from young (L and N) and aged (M and O) mice were stained for type I cell markers RAGE (L and M) or T1α (N and O). P: the ratios of cells that expressed either type I cell marker (type I-like cells) vs. total cells were scored; n = 5–7 mice for each group. Q and R: cultured cells from young (Q) and aged (R) mice were stained for type II cell marker Sp-C. S: the ratios of cells that maintained Sp-C expressions were scored; n = 10 mice for each group. Because of the slight variation in culture condition of each independent experiment, P and S are presented as the relative value compared with the average ratios of cells from young mice in the experiment conducted at same time. C, F–H, K, P, and S are Tukey box plots; *P < 0.05, **P < 0.01. Scale bar = 30 μm.