Oct4+ stem/progenitor swine lung epithelial cells are targets for influenza virus replication

Abstract

We isolated stem/progenitor epithelial cells from the lungs of 4- to 6-week-old pigs. The epithelial progenitor colony cells were surrounded by mesenchymal stromal cells. The progenitor epithelial colony cells expressed stem cell markers such as octamer binding transcription factor 4 (Oct4) and stage-specific embryonic antigen 1 (SSEA-1), as well as the epithelial markers pancytokeratin, cytokeratin-18, and occludin, but not mesenchymal (CD44, CD29, and CD90) and hematopoietic (CD45) markers. The colony cells had extensive self-renewal potential and had the capacity to undergo differentiation to alveolar type I- and type II-like pneumocytes. Additionally, these cells expressed sialic acid receptors and supported the active replication of influenza virus, which was accompanied by cell lysis. The lysis of progenitor epithelial cells by influenza virus may cause a marked reduction in the potential of progenitor cells for self renewal and for their ability to differentiate into specialized cells of the lung. These observations suggest the possible involvement of lung stem/progenitor cells in influenza virus infection.

Fig 1

Characteristics of lung stem/progenitor epithelial cells. (A) Colony morphology of lung progenitor epithelial cells. The epithelial colony cells were surrounded by mesenchymal cells. Epi, epithelial colony cells; Mes., mesenchymal cells. (B) Colony-forming assay. A single cell can be expanded to generate clones and form colonies (CFU) that are shown by Giemsa staining.

Fig 2

Lung stem/progenitor epithelial cells express stem cell and epithelial cell markers. Shown is the expression of stem cells markers on progenitor epithelial cells. Primary cell cultures were examined for the expression of stem cell markers by using specific antibodies directed against Oct4 (A) and SSEA-1 (B). The expression of epithelial cell markers on lung progenitor epithelial cells also is shown. Colony cells were cultured on coverslips and examined for the expression of specific epithelial cell markers pan-CK (C), CK-18 (D), and occludin (E) by IFA. Staining was specific, as cells incubated with only FITC-labeled anti-rabbit (F) and anti-mouse (G) IgG did not show any staining. Cell nuclei were stained by DAPI.

Fig 3

Progenitor epithelial cells differentiate into type I and II pneumocytes. Lung progenitor cells were subcultured onto collagen I-coated plates and cultured in epithelial cell differentiation medium; on day 5 the cells were examined for (A) the expression of the alveolar cell marker aquaporin 5 (Aqua5; a type I pneumocyte marker) and (B) surfactant protein C (SPC; a type II pneumocyte marker). Primary undifferentiated cells did not express Aqua5 (C) or SPC (D). (E) Quantification of differentiated type I and II pneumocytes. Cells (identified by DAPI staining) were quantified for the expression of Aqua5 or SPC. Data are expressed as means ± SD from two separate experiments using progenitor epithelial cells from two different pigs in each experiment.

Fig 4

Progenitor epithelial cells express influenza virus receptors and support influenza virus replication. (A) Progenitor epithelial cells express α-2,3- and α-2,6-linked sialic acid receptors. The cells were incubated with FITC-labeled Maackia amurensis lectin II (MAA), which is specific for α-2,3-linked sialic acid receptors, and Sambucus niagra agglutinin (SNA), which is specific for α-2,6-linked sialic acid receptors, and they were examined by flow cytometry. Black line, unstained cells; red line, MAA/SNA. (B) Progenitor epithelial cells were infected with SwIV, AvIV, or HuIV at an MOI of 1. All virus types were able to replicate in progenitor cells and induced cytopathic effects. (C) Influenza viral NP was detected in virus-infected progenitor cells by IFA.

Fig 5

Virus replication kinetics in primary cultures of progenitor epithelial cells after virus infection. (A) Progenitor epithelial cells were infected with SwIV, AvIV, or HuIV at an MOI of 1. Virus production in infected culture supernatants at different time points was measured by titration in MDCK cells. *, Significantly different from HuIV; #, significantly different from HuIV (P < 0.05). (B) Virus replication in progenitor epithelial cells in the presence or absence of TPCK-trypsin. Progenitor epithelial cells were infected with SwIV at an MOI of 0.01. TPCK-trypsin (0.5 μg/ml) was added to the culture medium for the duration of the experiment. Virus production in infected culture supernatants at different time points was measured by titration in MDCK cells. The virus titers are expressed as means ± SD from two separate experiments using progenitor epithelial cells from two different pigs in each experiment (n = 4). *, Significantly different (P < 0.05).

Fig 6

Expression of influenza virus receptors on differentiated cells. Lung progenitor epithelial cells were cultured on collagen I-coated 24-well tissue culture plates in epithelial cell differentiation medium. On day 5, differentiated type I- and II-like pneumocytes were examined for the expression of α-2,3- and α-2,6-linked sialic acid receptors. Type I pneumocytes (positive for Aqua5 expression [green]) and type II pneumocytes (positive for SPC marker [green]) express both α-2,3- and α-2,6-linked sialic acid receptors (red).

Fig 7

Replication of influenza virus in differentiated pneumocytes. Lung progenitor epithelial cells were cultured on collagen I-coated 24-well tissue culture plates in epithelial cell differentiation medium; on day 5, differentiated type I- and II-like pneumocytes were infected with SwIV. Type I pneumocytes (green) (A) and type II pneumocytes (green) (B) supported the replication of SwIV virus as indicated by the expression of viral NP protein (red). Cell nuclei were stained with DAPI (blue). (C) Virus replication kinetics in differentiated type I and II pneumocytes. Differentiated type I- and II-like pneumocytes were infected with SwIV at an MOI of 1. At the indicated time intervals, the percentage of viral NP-positive cells was measured. Data are expressed as means ± SD from two separate experiments using progenitor epithelial cells from two different pigs in each experiment. *, Significantly different (P < 0.05).