Figure 1.
Characterization of progenitor epithelial cells isolated from pig bone marrow.
(A) Colony morphology of progenitor epithelial cells derived from bone marrow. (B) Expression of stem cells markers on progenitor epithelial cells. Epithelial colony cells from primary cell cultures were examined for the expression of lung and other stem cell markers by using specific antibodies directed against Ccsp, Oct4 and SSEA-1. (C) Expression of epithelial cell markers on progenitor epithelial cells. Primary cell cultures were examined for the expression of specific epithelial cell markers: Pan-K, K-18, and occludin. (D–F) Absence of non-specific binding by progenitor epithelial cells. The cells were incubated with FITC-labeled secondary antibodies (D) goat anti-rabbit (E) goat anti-mouse (F) donkey anti-goat antibodies. Nuclei were stained by DAPI.
Figure 2.
Differentiation potential of progenitor epithelial cells.
Epithelial colony cells were sub-cultured onto collagen I-coated plates and cultured in epithelial cell differentiation medium; on day 5 the cells were examined for (A) morphology of control and differentiated cells and (B) the expression of alveolar cell markers, Aqua5 (a type I pneumocyte marker) and SPC (a type II pneumocyte marker).
Figure 3.
Replication of influenza virus in progenitor epithelial cells.
(A) Progenitor epithelial cells express α-2,3- and α-2,6-linked sialic acid receptors. Progenitor epithelial cells were examined by flow cytometry for the expression of α-2,3- and α-2,6-linked sialic acid receptors. The cells were stained with FITC-labelled Maackia amurensis lectin II (MAA) specific for α-2,3-linked sialic acid receptors and Sambucus niagra agglutinin (SNA), specific for α-2,6-linked sialic acid receptors. MDCK cells were included as positive controls. Compared to unstained cells (black), cells stained with lectins (red) showed right shift indicating positive staining. BM cells (progenitor bone marrow epithelial cells), MDCK (MDCK cells) (B) Progenitor epithelial cells were infected with SwIV, AvIV or HuIV at a MOI of 1. All virus types tested induced cell cytotoxicity. (C) Virus replication kinetics in primary culture of progenitor epithelial cells after virus infection at 1 MOI. Virus production in infected culture supernatants was measured by titration in MDCK cells. The values are averages ± S.D. of two separate experiments using progenitor epithelial cells from two different pigs in each experiment (n = 4).
Figure 4.
Replication of influenza virus in differentiated pneumocytes:
Primary cells were subcultured onto collagen I-coated plates in epithelial cell differentiation medium; on day 5 differentiated cells were infected with SwIV and examined for the presence of viral proteins at 24 h after infection. Type II pneumocytes, positive for SPC marker (green) supported the replication of SwIV virus as indicated by expression of viral NP protein (red). Cell nuclei were stained with DAPI (blue). No viral proteins were detected in Aqua5 expressing (green) type I pneumocytes.