Type 2 alveolar cells are stem cells in adult lung

Abstract

Gas exchange in the lung occurs within alveoli, air-filled sacs composed of type 2 and type 1 epithelial cells (AEC2s and AEC1s), capillaries, and various resident mesenchymal cells. Here, we use a combination of in vivo clonal lineage analysis, different injury/repair systems, and in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair. Genetic lineage-tracing experiments showed that surfactant protein C-positive (SFTPC-positive) AEC2s self renew and differentiate over about a year, consistent with the population containing long-term alveolar stem cells. Moreover, if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal. Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing "alveolospheres," which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s. Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFRα⁺ lung stromal cells. This population included lipofibroblasts that normally reside close to AEC2s and may therefore contribute to a stem cell niche in the murine lung. Results suggest that a similar dynamic exists between AEC2s and mesenchymal cells in the human lung.

Figure 1. Long term self renewal of SFTPC lineage-labeled alveolar cells.

Adult Sftpc-CreER;Rosa-Tm mice were dosed ×4 with Tmx (0.2 mg/g). Tiled confocal images of lung sections 4 dpi show (A) that all Tm⁺ cells express SFTPC. The majority locate in alveoli and resemble AEC2s. No label is seen in bronchi/bronchioles (Br), indicating that the Sftpc-CreER allele is not active in proximal epithelial precursors in the absence of Tmx. (B) Tm⁺ epithelial cells in the BADJ coexpress SFTPC and SCGB1A1. Insets are high magnifications of cell marked with arrow: Left (Tm, red; SFTPC, green); right (SCGB1A1, purple; SFTPC, green). (C) A cohort of 8- to 12-week-old mice was dosed ×2 with 0.25 mg/g Tmx. Controls received vehicle alone. At different dpi, confocal z-stack images of lung sections (n ≥ 3 mice per point, 6 sections/mouse) were analyzed to give the proportion of SFTPC⁺ cells with lineage label. This does not significantly decline over the 48-week chase (red circles). Low recombination was seen without Tmx (black circles). (D) Section from a 24-week-old Tmx^– control mouse from C showing small clusters of Tm⁺ AEC2s (arrows). (E) To confirm proliferation of lineage-labeled (Tm⁺) cells in C, sections of lungs (n = 3 from Tmx⁺ group) were stained for Ki67 and percentage of dual positive cells recorded. Error bars indicate mean ± SEM. Scale bars: 250 μm (A); 100 μm (B); 50 μm (D). See also Supplemental Figure 1.

Figure 2. During repair after bleomycin, SCGB1A1 lineage–labeled cells account for most of the SFTPC lineage-negative AEC2s, and clones can arise in bronchioles.

Sftpc-CreER;Rosa-Tm and Scgb1a1-CreER;Sftpc-CreER;Rosa-Tm mice were dosed with Tmx (0.2 mg/g × 4) and exposed to bleomycin (1.25 U/kg). Controls received Tmx and intratracheal saline. Sections of 21-dpi lungs were stained for SFTPC. Confocal z-stack images (n ≥ 3 mice per point, 6 sections/mouse) were acquired and cells counted from fibrotic areas (high density of DAPI⁺ nuclei) or from random alveolar areas in controls. (A) Fibrotic region of Sftpc-CreER;Rosa-Tm lung showing clusters of SFTPC⁺ AECs that are not lineage labeled. By contrast, in a similar region of Scgb1a1-CreER;Sftpc-CreER;Rosa-Tm lung (B), significantly more AEC2 cells are lineage labeled. This is quantified in C. The smaller decline in value in the Scgb1a1-CreER;Sftpc-CreER;Rosa-Tm group compared with Sftpc-CreER;Rosa-Tm group suggests that there is no other important source of AEC2 precursors besides SCGB1A1⁺ cells. (D–F) Scgb1a1-CreER;Rosa-Confetti mice (n = 3) were dosed with Tmx (0.05 mg/g × 1) and sacrificed 21 days after bleomycin. Uninjured areas show random labeling of single cells in the bronchiolar epithelium (D), while injured areas display clones of labeled cells (E) especially in the BADJ (asterisks). (F) Clones occasionally extend from the BADJ (asterisks) into the alveoli, suggesting that at least some of the SCGB1A1⁺ lineage-labeled cells giving rise to AEC2s and AEC1s originate in the bronchioles. Scale bars: 100 μm. See also Supplemental Figure 2.

Figure 3. Injury/repair model using Rosa-DTA to selectively kill AEC2s in the adult lung.

(A) Sftpc-CreER;Rosa-Tm (left panels) and Sftpc-CreER;Rosa-DTA/Rosa-Tm (right panels) mice (at least 8 weeks of age) were given 1 dose of Tmx (0.05 mg/g) and lungs harvested 2 dpi. TUNEL staining shows significant increase in cell death in lungs with Rosa-DTA (upper right). Staining for SFTPC (lower panels of A) shows that 1 dose of Tmx lineage labels about 52% of the total SFTPC⁺ AEC2s at 2 dpi. The Rosa-DTA allele reduces the number of both SFTPC⁺ and lineage-labeled cells in the alveoli. (B) Loss of AEC2s results in failure of mice to gain weight over a 7-day period after injection. (C–F) Minimal changes in lung architecture at 2 to 4 dpi. (C and D) Histology of matched alveolar regions near mesothelium (Mes). At 4 dpi, the overall architecture of Sftpc-CreER;Rosa-DTA/Rosa-Tm alveoli (D) is very similar to that of controls (C), although abnormal AEC2s can be seen (arrow). (E) By 4 dpi, small groups of healthy AEC2s are seen in injured lung. (F) TEM of lipofibroblast (Lp) adjacent to AEC2 and capillary (cp) at 2 dpi. Scale bars: 100 μm (A); 50 μm (C and D); 2 μm (E and F). See also Supplemental Figure 3.

Figure 4. SFTPC⁺ cells proliferate clonally after targeted AEC2 depletion.

(A) Lungs from SftpcCreER;Rosa-DTA/Rosa-Tm mice (upper panel) were fixed and cleared at different times after Tmx ×1 and 3 hours after EdU. Confocal microscopy shows single-labeled cells at 2 dpi, discrete clones at 7 dpi, and more dispersed clones at 21 dpi. Lineage-labeled AEC1s are present by 21 days (arrow and inset). Controls without Rosa-DTA (lower panels) show no change in Tm⁺ cells and little EdU labeling. (B) Multiphoton images of SftpcCreER;RosaDTA/Rosa-Tm lungs at 2 and 7 dpi. (C) Decline in EdU labeling index as repair proceeds (n = 3 mice each time). (D) To quantify increase in SFTPC⁺ cells during repair, total SFTPC⁺ cells within at least 6 random ×20 fields of view (within 2 lobes/mouse) were counted and scored for lineage label. Without injury, values remained constant. With injury, number of SFTPC⁺ cells increased but proportion of lineage-labeled cells remained the same, suggesting labeled cells do not proliferate preferentially. (E) An Sftpc-CreER;Rosa-DTA/Rosa-Confetti mouse was given Tmx (0.05 mg/g × 1) and 10 weeks later, lungs were fixed, cleared, and viewed by confocal microscopy. Tiled image shows clones labeled with nuclear GFP (nGFP), cytoplasmic YFP (YFP), membrane cyan (mCFP), and cytoplasmic RFP. (F and G) Quantification of clone location (representative boxes in F) 7 days after Tmx in Sftpc-CreER;Rosa-DTA/Rosa-Tm animals suggests that clones do not preferentially arise in or near the BADJ (see text for details). Scale bars: 200 μm (A); 50 μm (B); 100 μm (C). (F) Boxes are 300 μm × 300 μm. See also Supplemental Figures 4 and 5.

Figure 5. Self renewal and differentiation of AEC2 cells in 3D organoid culture.

(A) Sftpc-CreER;Rosa-Tm mice were dosed ×4 with Tmx (0.2 mg/g) and at least 4 dpi lungs were dissociated and sorted by FACS. The Tm⁺ fraction (P3, top left) was seeded at a density of 5,000 cells in 90 μl of 50% Matrigel in a 24-well Transwell insert (top right) together with 1 × 10⁶ PDGFRA-GFP^hi stromal cells freshly sorted from lungs of a Pdgfra-H2B:GFP transgenic mouse (P3, bottom panels). (B and C) After 14 days, spheres of lineage-labeled cells are present in various sizes. (D) CFE is 2.3% ± 0.3% for primary cultures (n = 8 experiments, ≥ 2 replicates per experiment), 6.2% ± 0.7% after passage 1, and 5.1% ± 0.7% after passage 2. (E–I) Histology and immunohistochemistry of sections of spheres after 16–17 days shows (E) alveolus-like areas (asterisk) with more elongated cells, (F) that all cells are lineage labeled and those on the periphery express SFTPC, while cells in the interior express T1a, AQAPORIN 5 (Aqp5) (G and H), and HOPX (I) — markers of AEC1s (see also Supplemental Figure 7). (J and K) TEM of spheres at day 16 shows many cells with lamellar bodies at different stages of maturation and apical membranes with dense microvilli (mv). The cells release surfactant into the interior of the spheres where it accumulates in large amounts. Scale bars: 100 μm (B); 50 μm (C); 50 μm (E); 50 μm (F–I); 2 μm (J and K). See also Supplemental Figures 6 and 7.

Figure 6. Recapitulating the AEC2 stem cell niche.

Sections from adult Pdgfra-H2B:GFP lungs were stained for neutral lipid and/or SFTPC. (A) LipidTOX staining colocalizes with high-intensity GFP⁺ cells in alveoli, but not with lower-intensity GFP⁺ cells (arrows) in the bronchiolar wall. (B) LipidTOX⁺, PDGFRA-GFP⁺ cells are closely apposed to SFTPC⁺ AEC2s (purple). (C) FACS-sorted PDGFRA-GFP^hi cells are also positive for LipidTOX. (D) LipidTox⁺ PDGFRA-GFP⁺ cells express the intermediate filament protein desmin. (E and F) Multiple PDGFRA-GFP⁺;desmin⁺ cells are interspersed throughout an alveolosphere at day 21. (G) TEM of alveolosphere reveals multiple lipid-filled cells (L) adjacent to lamellar body–containing AEC2s. Scale bars: 50 μm (A–F); 2 μm (G).

Figure 7. Human AEC2s form self-renewing colonies in 3D organoid culture.

(A) Immunohistochemistry of human lung sections for SFTPC and HTII-280 shows colocalization of the 2 AEC2 markers. (B and C) Sorting strategy and representative flow plot. (D) Representative sphere at day 14. (E and F) Immunohistochemistry of human spheres at day 14 showing luminal HTII-280 staining and variable SFTPC staining. (G) TEM of a sphere derived from a single HTII-280⁺ cell (passage 1) showing lamellar bodies and microvilli on the luminal surface. Scale bars: 50 μm (A); 100 μm (D); 50 μm (E and F); 2 μm (G).