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Alveolar progenitor and stem cells in lung development, renewal and cancer

Subjects

Abstract

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

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Figure 1: Development of alveolar type 1 (AT1) and AT2 cells from bipotent progenitors.
Figure 2: Mature AT2 cells renew AT1 cells in clonal foci.
Figure 3: Activation of AT2 stem cell function in vivo and proliferation in vitro.
Figure 4: Transformation of mature AT2 cells by activated Kras.
Figure 5: Model of alveolar progenitors and stem cells in development, maintenance, and cancer.

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

Microarray datasets were deposited at Gene Expression Omnibus (accession code GSE49346) and GEXC (https://gexc.stanford.edu/population/detail/998 and https://gexc.stanford.edu/population/detail/999).

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Acknowledgements

We thank A. Andalon for technical assistance; H. Chapman (SftpC–Cre–ER-rtTA), B. Hogan (CCSP–Cre–ER), H. Ueno and I. Weissman (Rainbow), H. Clevers (Confetti), L. Luo (mTmG), and J. Sage (KrasLSL-G12D) for strains; B. Stripp for goat anti-CCSP antibody; F. H. Espinoza for annotated gene lists; R. Metzger, H. Chapman, and members of the Krasnow laboratory for discussions and comments on the manuscript; and Maria Petersen for help preparing figures and the manuscript.

Author information

Affiliations

Authors

Contributions

T.J.D. conducted the experiments except the gene expression profiling and AT2 cell cultures, which were done by D.G.B.; T.J.D., D.G.B. and M.A.K. conceived the experiments, analysed the data and wrote the manuscript. This work was supported by a Parker B. Francis Foundation Fellowship and NIH 5KO8HL084095 Award (T.J.D.), NIH T32HD007249 (D.G.B.), and an NHLBI U01HL099995 Progenitor Cell Biology Consortium grant (M.A.K.). M.A.K. is an investigator of the Howard Hughes Medical Institute.

Corresponding authors

Correspondence to Tushar J. Desai or Mark A. Krasnow.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mature and developing structure of the lung and alveoli.

a, Mature lung showing close up (inset) of transition from bronchial tree to alveoli (from http://www.mayoclinic.com/, with permission). b, Alveoli are surrounded by a dense capillary network in which de-oxygenated blood (blue) from pulmonary arteries is oxygenated (red) and then returned to the heart through pulmonary veins (modified from ref. 51). c, E16.5 Shh–Cre > mTmG mouse lung in which GFP is expressed throughout the epithelium. Lobes are labelled (RAcc, right accessory; RCr, right cranial; RMid, right middle; RCa, right caudal; L, left) and boxed region shows the tip of the accessory lobe that was used for developmental analyses. By E16.5, the bronchial tree has formed and 1 day later sacculation begins as flat (squamous) AT1 and cuboidal AT2 cells mature to generate a functional gas exchange interface. Saccules subsequently undergo subdivision (‘secondary septation’) into mature alveoli that provide an extensive gas-exchange surface. Scale bar, 1 mm; d, Schematic of cell morphogenesis during sacculation (sac) (from ref. 52). Progenitors form flat AT1 cells adjacent to capillaries and AT2 cells specialized to secrete surfactant. e, f, e′, f′, Schematics (e, f) and images (e′, f′) of E-cadherin-stained accessory lobe tips at E18.5 and postnatal day 2 (PN2) showing longitudinal (e) and frontal (f) views of maturing AT1 (1) and AT2 (2) cells. P, proximal; D, distal; red, cell junctions (jxn); yellow, apical surfaces. Note lack of AT1 cells distally in sacculating airway. Scale bar, 10 μm (e′, f′). g, Quantification of ultrastructural classification of cell types in sacculating airways in E18.3 lungs (see Fig. 1r–u). Values shown are the numbers of each progenitor and cell type observed with the indicated ultrastructural features. No cells (n = 36) had features of an AT2> AT1 intermediate (AT1/2) or mature AT2 cell. E, embryonic day; PN, postnatal day.

Extended Data Figure 2 Clonal analysis of alveolar progenitor cells and lineage marking and tracing alveolar type 2 (AT2) cells with LysM-Cre.

a, b, Shh–Cre–ER > mTmG embryos were induced in utero with a limiting dose (2 mg) of tamoxifen (tamox) at E15 to pulse-label epithelial cells at the distal lung tips (alveolar progenitors) with GFP (0.2 labelled cells per embryonic lung lobe) shortly before the onset of differentiation then examined 34 days later at PN30. a, An isolated clone (dashed circle) expressing the GFP lineage tag (green) in a PN30 lung. b, Close up of boxed region showing several flat AT1 cells (open arrows) and a cuboidal AT2 cell (filled arrows) within the alveolar clone, indicating that the tagged progenitor was bipotent. Tagged cells are interspersed with unrecombined cells (tdTomato, red). E, embryonic day; PN, postnatal day; Scale bar, 50 μm. cf, Close-ups of alveoli of 1 (c, e) and 2 month old (d, f) LysM-Cre > mTmG lungs stained for the AT2 lineage tag (GFP, green) and the AT2 (c, d) or AT1 (e, f) markers indicated. Note that at 1 month lineage marked cells (green) express the AT2 (c) but not the AT1 marker (e). At 2 months (d, f), the lineage mark is also observed in some flat AT1 cells. Filled arrows, AT2 cells; open arrows, AT1 cells; E, embryonic day; PN, postnatal day. Scale bar, 20 μm (cf).

Extended Data Figure 3 Proliferation analysis of bipotent progenitors and alveolar epithelial cells.

ad, Late gestational (E17.5, a) and early postnatal (PN7, 14, 21; bd) lungs stained for Nkx2.1 (green) for epithelial and Ki67 (red) for actively cycling cells. Note essentially exclusive labelling, indicating minimal proliferation of bipotent progenitors (a) or AT1 and AT2 cells (bd). Arrow, a rare proliferating AT2 cell. Dashes outline distal epithelial tips; dotted line indicates mesothelium. E, embryonic day; PN, postnatal day; Scale bar, 35 μm.

Extended Data Figure 4 Quantification of cell type labelling and long-term lineage contribution of LysM-Cre and SftpC–Cre–ER marked cells.

a, Alveolar region of a PN 3 month (mo) Shh–Cre> R26EYFP mouse lung co-stained for GFP (green, epithelial cytoplasm) and LysM (red). Inset shows close-up of boxed region. LysM is detected in cytoplasm of many AT2 cells (filled arrow) but not AT1 cells (open arrow). b, Bronchoalveolar lung region of a PN 2 mo LysM reporter mouse expressing GFP from the endogenous locus stained for E-cadherin (red) to mark airway epithelium and GFP (green) to mark LysM-expressing cells. Note AT2 (filled arrows) but not bronchiolar cells (dashes mark bronchoalveolar junction (Badj)) express the LysM reporter. c, PN 17 mo LysM-Cre > mTmG lung stained for E-cadherin (red) and the AT2 lineage marker (green). Note many marked AT2 cells (filled arrows) but absence of lineage-marked cells in the terminal bronchiole (dashes, Badj). d, e, Lungs from LysM-EGFP (d) and LysM-Cre > mTmG (e) mice of the indicated ages stained for ciliated (acetylated tubulin, acTub, red) and neuroendocrine (NE) cell (CGRP, blue) markers and GFP (green) show no co-expressing cells, indicating ciliated (filled arrowhead) and NE (open arrowhead) cell types do not express LysM and do not derive from AT2 cells. Br, bronchus. fh, Lungs from LysM-Cre > mTmG mice of the indicated ages stained for the AT2 lineage tag (green), the AT2 cell marker SftpC (red), and the Clara cell marker CCSP (blue) show CCSP+/SftpC+ (double-positive) cells (*) at the Badj, some of which are tagged. Marked double-positive cells are solitary (g) or in doublets (h). i, Lung from PN 1 mo SftpC–Cre–ER > mTmG mouse (administered 1 mg tamoxifen at PN19) analysed 13 days after pulse-labelling and stained as in d. Note no co-expressing cells, indicating that ciliated (filled arrowhead) and NE (open arrowhead) cell types are not tagged. jl, Lungs from mice labelled as in panel i analysed 13 days (j) and 192 days (k, l) later, stained as in f. Note double-positive CCSP+/SftpC+ cells (*) at the Badj, some of which are pulse-labelled (j). After 192 day chase, marked double-positive cells are solitary (k) or in doublets (l). m, Quantification of lung cell types marked under different labelling and lineage trace conditions. The number of marked and total cells of each type scored (and Badj analysed for CCSP+/SftpC+ cells) is shown for each genotype and age analysed. For the SftpC–Cre–ER line, the dosage of tamoxifen (tamox) and interval time until analysis is also indicated. Because bronchial maintenance involves proliferation without significant cell dispersion50 (as we find for alveolar maintenance), the presence of marked CCSP+/SftpC+ cells primarily in isolation (>90%) at advanced ages suggests they did not contribute significantly to physiological bronchiolar renewal. d, days; PN, postnatal. Scale bar, 50 μm (al), 10 μm (insets in a).

Extended Data Figure 5 Lineage tracing alveolar type 2 (AT2) cells using SftpC–Cre–ER.

SftpC–Cre–ER > mTmG mice were administered 1 mg tamoxifen (Tamox) at PN18 then analysed later by staining for the lineage label (GFP, green) and AT2 cells (SftpC, red). a, b, 13 days later only AT2 cells are marked (a), whereas after 212 days flat AT1 cells also express the AT2 lineage tag (b). A peripheral renewal focus (mesothelium indicated by dotted line) involving multiple alveoli (asterisks) is shown, similar to results using LysM–Cre. Quantification revealed that 94% of AT2 cells were marked 13 days after tamoxifen induction and 97% after 192 days (see Extended Data Fig. 5m), indicating that the SftpC+ population is maintained by self-duplication, and that new AT2 cells do not derive from another cell population during physiological ageing. PN, postnatal day; bar, 100 μm.

Extended Data Figure 6 Alveolar type 2 (AT2) founder cell functional marker expression and self-duplication in vivo, and reprogramming into AT1 cells in vitro.

ac, 16 month LysM-Cre > Confetti mouse lung stained for mCFP lineage tag (green), AT2 cell marker (Nkx2.1, red), and DAPI (blue) to identify clonal renewal foci. a, AT2 founder cell (filled arrow) associated with a daughter AT1 cell (open arrow) shown to express the A2L marker LAMP-1 (white), a protein associated with surfactant-containing lysosomes in mature, secretory AT2 cells. b, c, A clonal focus (b, boxed region) in which an AT2 cell has generated two additional AT2 cells (filled arrows) but no AT1 cells (c, close up of boxed region), demonstrating isolated self-duplication without AT1 cell reprogramming in vivo. Scale bars, 25 μm (a–c). d, Freshly isolated AT2 cells (Fig. 3f) cultured 4 days on glass with 10% serum adopt flat AT1 morphology (E-cadherin, green) and initiate AT1 marker expression (Aqp5, not shown). Scale bar, 10 μm.

Extended Data Figure 7 Clonogenic response to activated Kras using SftpC–Cre–ER and CCSP–Cre–ER.

ac, Lungs of SftpC–Cre–ER (a) and CCSP–Cre–ER (b) mice carrying oncogenic KrasLSL-G12D/+ (Kras*) and Rainbow (Rbw), injected with 3 mg (a) or 1 mg (b) of tamoxifen (tamox) at indicated ages to induce Kras* expression and clonal lineage marking of StfpC- and CCSP-positive cells, respectively. Lungs were analysed after 17 days at PN43 (a) or after 39 days at PN81 (b). Note that multifocal tumours result when SftpC–Cre–ER mice are induced at 25 (a) days of age, whereas induction of Kras* in CCSP–Cre–ER mice at 42 days (b) shows many unresponsive cells and doublets throughout the bronchi (Br) as well as small clonal tumours located at bronchoalveolar duct junctions (Badj). c, An Sftpc–Cre–ER mouse carrying Kras* and mTmG alleles induced at PN116 by injection of 2 mg tamox and analysed after 53 days also demonstrates adenomas. Dotted line indicates mesothelium; PN, postnatal day; scale bar, 100 μm.

Extended Data Figure 8 Marker expression analysis of alveolar type 2 (AT2) cell derived adenomas.

LysM–Cre > mTmG, KrasLSL-G12D/+ (abbreviated Kras*) lungs stained for the AT2-lineage marker (GFP, green), the nuclear stain DAPI (blue), and the indicated AT2, AT1, and Clara cell markers (red). ae, Note tumour cells (green) maintain expression of AT2 markers (a, b) and do not turn on Clara (e) or AT1 markers (c, d), except possibly rare cells (*). c′ and d′ show control (non-tumour) regions with normal AT1 staining (arrows). Scale bars, 10 μm.

Extended Data Table 1 Developmental expression patterns of alveolar epithelial cell markers

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3. Supplementary Table 1 contains validated, robust alveolar epithelial cell markers. Supplementary Table 2 shows percentiles of receptor genes expressed by bipotential progenitors and LysM-lineage alveolar type 2 (AT2) cells; and expression levels of EGF receptor family members with histograms of probe-specific levels. Supplementary Table 3 contains genes highly selectively expressed by bipotential progenitors and LysM-lineage alveolar type 2 (AT2) cells at the 90th percentile or higher; and annotation enrichment analysis for these gene profiles. (PDF 475 kb)

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Desai, T., Brownfield, D. & Krasnow, M. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190–194 (2014). https://doi.org/10.1038/nature12930

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