Immunogenicity of hAEC and effects on T cell proliferation.

<p>Primary (P0) and passage 5 (P5) hAEC expressed HLA-A-B-C but lacked HLA-DP-DQ-DR. Co-stimulatory molecules CD40, CD80 and CD86 remained unaltered at P5. Representative flow plots for HLA antigens and co-stimulatory molecules are shown (<b>A</b>). P0 and P5 hAEC suppressed Concanavelin A stimulated splenocytes from C57/BL6 mice, however P5 cells showed reduced suppression at higher splenocyte ratios (<b>B</b>). P0 and expanded cells produced the immunosuppressive factors TGF-β and IL-6. HLA-G that was abundant in P0 cells declined significantly in P5 grown in DMEM/F12+10%FCS (DF), while hAEC expanded in Epilife lacked HLA-G. Open bars = P0, black shaded bars = P5 cells expanded in DF and grey bars = P5 cells expanded in Epilife. Scale bars = 100 µm. *p&lt;0.05; **p&lt;0.01 and ***p&lt;0.001.</p

Expansion of hAEC.

<p>The hAEC grew well in the xenobiotic-free media Epilife until passage 5 (P5) and plateaued thereafter. A similar trend was seen in control cultures grown in DMEM/F12+10% FCS but the cumulative cell number in Epilife was lower than controls (*p = 0.002; <b>A</b>). Cumulative population doubling of hAEC cultured in Epilife media was lower than DMEM/F12+10% FCS (*p = 0.0026; <b>B</b>). hAEC appeared stromal-like at P5 in Epilife and DMEM/F12+10% FCS (<b>C</b>). Stromal-like cells at P4 retained the CFSE label suggesting the stromal cells arose from the labeled P2 epithelial cells (<b>D</b>). Scale bars = 100 µm.</p

Antibodies Used to Phenotype hAEC by Flow Cytometry.

<p>Antibodies Used to Phenotype hAEC by Flow Cytometry.</p

Phenotype of primary (P0) and passage 5 (P5) hAEC.

<p>Cells expressing epithelial markers E-cadherin, CD49f and CK7 declined with expansion while percentage of cells with MSC associated markers CD90, CD146 and the stromal marker vimentin were elevated in P5 cells grown in Epilife and DMEM/F12+10% FCS (DF). However, notable differences were also found between Epilife and DF expanded cells. Representative flow cytometry plots of markers that differed are shown. Open bars = P0, black shaded bars = P5 DF and grey bars = P5 Epilife. *p&lt;0.05; **p&lt;0.01; ***p&lt;0.0001 by ANOVA and Tukey's post hoc test.</p

Features of cultured primary (P0) and cells expanded in Epilife medium to passage 5 (P5).

<p>Normal karyotype of P0 hAEC was retained at P5 (<b>A</b>). Transmission electron micrograph of P0 hAEC showed intercellular junctions (IJ), a multiloculated peripheral appearance compared to P5 hAEC. P5 cells had extensive rough endoplasmic reticulum (rER) and fewer cell surface projections. P0 and P5 cells showed high nuclear (N) to cytoplasmic ratio (<b>B</b>). P0 hAEC seeded at low density formed clonal colonies unlike P5 hAEC (<b>C</b>). hAEC lacked alkaline phosphatase activity unlike human embryonal carcinoma (hEC) cell line used as a positive control (<b>D</b>). Scale bars = 100 µm (<b>A–B</b>); 5 µm and 1 µm (<b>C</b>).</p

Cell migration assay.

<p>Primary, passage 0 (P0) hAEC migrated more rapidly compared with expanded passage 5 (P5) cells in a scratch wound assay (<b>A</b>). Cultured P0 and P5 cells lacked the chemokine receptor CXCR4 that has been implicated in cell migration (data not shown), however hAEC lining term delivered amnion membranes were immunopositive for CXCR4 (white arrows head = hAEC; <b>B</b>). Insert within panel shows staining of isotype control. Scale bars = 500 µm (<b>A</b>) and 100 µm (<b>B</b>).</p

Primers used to analyze expression of M1 and M2 macrophage genes and MMP.

<p>Primers used to analyze expression of M1 and M2 macrophage genes and MMP.</p

Differentiation of hAEC and their characterization.

<p>Primary (P0) and Epilife expanded passage 5 (P5) hAEC grown in SAGM produced prosurfactant protein-C characteristic of type 2 alveolar epithelial cells. Control P5 cultures maintained in Epilife lacked staining. Stimulated P0 hAEC contained glucagon (GCG), found in alpha pancreatic cells (arrow heads). Glucagon was absent in control cultures grown in DMEM/F12 medium with 10% FCS (DF). Inserts within panels show isotype controls (upper panel). P0 hAEC induced with supplements expressed hepatocyte nuclear factor-4α (HNF-4α) and albumin, unlike control cultures grown in DF. Cell nuclei stained with DAPI are shown in the inserts (middle panel). Alizarin red staining indicating calcium deposition characteristic of osteocytes in stimulated P0 and P5 cultures. Cartilage proteoglycans stained with Alcian blue in stimulated P5 DF expanded hAEC. Non-stimulated control cultures maintained in basal medium lacked evidence of differentiation into osteocyte and chondrocyte-like cells (lower panel). Scale bars = 100 µm.</p

A comparison of a single versus two doses of bleomycin on lung injury in C57BL/6 mice.

<p>There was more inflammatory infiltration at day 10 and fibrosis at day 21 post-bleomycin for double versus a single dose of bleomycin (<b>A</b>). Semi-quantitative measures of inflammatory cell infiltration and fibrosis showed significant elevation of inflammation at day 10 and fibrosis at day 21, following two bleomycin doses compared to a single dose (<b>B</b>). * p&lt;0.05.</p

Human amniotic epithelial cell transplantation induces expression of genes associated with alternately activated (M2) macrophages.

<p>mRNA expression of classically (M1) and alternately activated M2 macrophage genes were analyzed by real time quantitative PCR and the fold change calculated by the ^ΔΔCT method. Expression of M2 associated YM-1, CD206 and IL-10 was significantly elevated in livers of mice treated with hAEC compared to animals given carbon tetrachloride (CCl₄) alone (A). M1 associated genes CCL17 and CCL5 were expressed but levels did not alter with hAEC treatment (B). IL-12b:IL-10 ratio was skewed towards an M2 phenotype. MMP-9 increased and MMP-12 expression decreased with hAEC treatment (C). *, ** and *** P&lt;0.05, 0.01 and 0.001, respectively.</p