Airway epithelial progenitors are region specific and show differential responses to bleomycin-induced lung injury

Abstract

Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFP(hi) cells were subdivided by CD24 staining into alveolar (CD24(neg)) and conducting airway (CD24(low)) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFP(neg) and GFP(low) progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24(low)GFP(hi) progenitors were present in an overlapping distribution with GFP(low) progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24(low) GFP(hi) progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling.

Figure 1

The airway epithelium is maintained by Scgb1a1-expressing cells. (A): Schematic illustrating Scgb1a1-CreER^T2; Rosa26R-Confetti constructs. Upon Cre activation by treatment of mice with tamoxifen, the Confetti construct randomly recombines for active expression of fluorescent protein reporters. Scgb1a1-expressing cells were efficiently lineage labeled after treatment of Scgb1a1-CreER^T2; Rosa26R-Confetti mice with tamoxifen (B). Naphthalene-induced lung injury led to clonal expansion of lineage-labeled naphthalene-resistant epithelial progenitor cells within either terminal bronchioles (C) or more proximal bronchioles (D). Scale bars = 50 µm. (E): Schematic illustrating Scgb1a1-CreER^T2; Rosa26-mT/mG (Scgb1a1-mT/mG) mice. (F): Lineage labeled cells (GFP⁺) isolated from Scgb1a1-mT/mG mice were sorted by fluorescence-activated cell sorting and placed in 3D MatriGel culture for 14 days (G). Representative image of the culture shows the formation of type A, B, and C colonies.

Figure 2

Segregation of region-specific airway epithelial progenitor cells. (A): Schematic of the SFTPC-GFP transgene. (B): Immunolocalization of Scgb1a1 (red) and GFP (green) in lung tissue of SFTPC-GFP lungs defines three distinct airway locations. (C): Proximal conducting airway is lined by epithelial cells that are Scgb1a1^pos and GFP^neg (arrow). (D): Midlevel bronchioles are lined by epithelium that is Scgb1a1^pos and GFP^low (arrow). (E): Terminal bronchioles and alveoli harbor GFP^hi epithelial cells that are either Scgb1a1^pos (arrows) or Scgb1a1^neg (arrowheads), respectively. Scale bars in (C–E) = 50 µm. (F–K): GFP^neg, GFP^low, and GFP^hi progenitor cells can be fractionated by selection for EpCAM^pos, CD24^low cells that are CD31^neg, CD34^neg, and CD45^neg. Back-gate of these three populations to the CD24 versus Sca-1 dot plot reveals that GFP^neg and GFP^low populations express Sca-1, while GFP^hi cells are predominantly Sca-1^neg. (I): CD24 antigen is broadly expressed in both epithelial lineage and nonepithelial lineages in the lung. (J): CD24^neg Sca-1^neg gate in (H) is largely populated by GFP^hi type 2 cells. (L): Real-time PCR analysis was performed on total RNA isolated from cell fractions to quantify Scgb1a1, Sftpc, Sox2, and Krt5 mRNA’s. Abbreviations: 7-AAD, 7-amino-actinomycin D; FSC, forward scatter; GFP, green fluorescent protein. *, <0.05; **, <0.01.

Figure 3

Segregation of regional progenitor cells based upon SFTPC-GFP expression. (A): Culture of total EpCAM⁺ cells from SFTPC-GFP mice reveals three distinct types of epithelial colonies. (B, C): Two-week cultures of GFP^neg, GFP^low, and GFP^hi cells indicate that type A colonies are enriched within the GFP^neg epithelial fraction. (D, E): Type B colonies are enriched in the GFP^low epithelial fraction. (F, G): Type C colonies are enriched within the GFP^hi epithelial fraction. (H): Quantitative analysis of the colony-forming ability of GFP^neg, GFP^low, and GFP^hi cells. (I): Self-renewal capacity of progenitors yielding type A, B, and C colonies was examined. p0, passage 0; p1, passage 1; p2, passage 2. Data represent combined results from three independent experiments. *, <.05; **, <.01; ***, <.001. Scale bar = 200 µm. Abbreviation: GFP, green fluorescent protein.

Figure 4

In vitro differentiation of airway regional progenitor cells. Green fluorescent protein (GFP^neg), GFP^low, and GFP^hi progenitor cells were cultured in presence of SB431542 for 10 days and switched to SB431542-deficient medium for 4 days. Paraffin sections of each culture type (GFP^neg, A–E; GFP^low, G–K; GFP^hi, M–Q) and normal adult mouse lung tissue (S–Y) were stained with Sftpc, Scgb1a1, FoxJ1, sPlunc1, and Krt5 by dual immunofluorescence for E-caderin, respectively. Rare Krt5-positive cells are indicated in proximal airway by arrows in (X). Dual immunofluorescence stain of GFP and Sox2 on each culture type (F, L, and R) and normal lung section (Z) indicated a variable level of Sox2 among these colonies. Scale bar = 50 µm.

Figure 5

Novel genes that define region-specific progenitor cells. (A): Sorted GFP^neg, GFP^low, and GFP^hi progenitor cells are lyzed to extract RNA, which was then processed for Affymetrix Microarray analysis. The heatmap was made to show distinct gene signatures of these progenitor cells. (B): Venn diagram showing differentially expressed genes in GFP^hi, GFP^low, and GFP^neg progenitor cells with a 1.2-fold as a cutoff using the GeneSpring GX software. (C): Quantitative PCR confirmed that Reg3g, Parm1, and Pard6g are highly enriched in GFP^neg, GFP^low, and GFP^hi airway progenitor cells. Data are presented as mRNA abundance relative to total lung RNA. Data show the average of three samples, representing two independent experiments. The spatial distribution of selected mRNA’s was determined by in situ hybridization of cRNA probes to normal adult mouse lung tissue. (D): An adjacent lung tissue section was stained with H&E to reveal the branching pattern of conducting airways. (E–H): The spatial expression pattern was determined for mRNA’s corresponding to Scgb1a1 (positive control), Reg3g, Parm1, and Pard6g. Abbreviation: GFP, green fluorescent protein.

Figure 6

Response of region-specific progenitor cells to bleomycin injury. (A–C): Trichrome stain of lung sections from control SFTPC-GFP mice and those treated with bleomycin. (D): Immunostain of lung sections from control or bleomycin-treated group (day 20) with Scgb1a1. (E): Percentage of GFP^neg, GFP^low, and GFP^hi fraction relative to Lin^negEpCAM⁺ population. (F): After bleomycin injury, colony-forming efficiency (CFE) of GFP^neg, GFP^low, and GFP^hi progenitor cells was determined. CFEs are shown relative to the control group. Data represent average 6 SEM from three mice in control mice or bleomycin-treated mice. Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; GFP, green fluorescent protein.