Table 1.
RT-PCR primer sequences.
Figure 1.
Differentiation-dependent changes in type I IFN pathway expression in human BE(2)-C neuronal cells.
(A) Immunoblot analysis of the indicated type I IFN pathway component or GAPDH as a loading control. Lysate from IFNα-A/D-treated BE(2)-C/m cells was used as a positive control for the known IFN-inducible components IRF-9, STAT2, and STAT1. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of untreated differentiated over undifferentiated cells. *p<0.05, **p<0.005, relative to GAPDH. (B) Flow cytometry analysis of basal surface IFNAR2 expression in undifferentiated BE(2)-C cells (grey lines) and differentiated BE(2)-C/m cells (black lines). Dashed lines indicate background fluorescence levels obtained with isotype-matched control antibodies, and MHC class I surface expression was examined to assess potential global changes in cell surface protein expression. Representative overlaid histograms from one of five independent experiments are shown.
Figure 2.
IFNAR mRNA analysis in undifferentiated BE(2)-C and differentiated BE(2)-C/m human neuronal cells.
(A) Semi-quantitative RT-PCR analysis of total NGFR, IFNAR1, and IFNAR2 mRNA transcript expression. Ribosomal RNA (rRNA) was analyzed as a loading control and ten-fold serial dilutions of cDNA were used for PCR. (B) Quantitative RT-PCR analysis of NGFR, IFNAR1, and IFNAR2 mRNA transcript expression. Results are presented as inverse rRNA-normalized threshold cycle (1/ΔCt) values so that increased mRNA levels are associated with increased bar height. n.s., not significant; *p<0.05. (C) Schematic of IFNAR2 total (white and black arrows) and isoform-specific (white and grey arrows) primer design. (D) Semi-quantitative RT-PCR analysis of isoform-specific IFNAR2 mRNA transcript expression. Ten-fold serial dilutions of cDNA from the indicated cell line were used for PCR. Representative results from one of four independent experiments are shown.
Figure 3.
Differentiation-dependent changes in type I IFN pathway function in human BE(2)-C neuronal cells.
(A) Immunoblot analysis of type I IFN-induced STAT phosphorylation. Cells were treated with 5, 50, or 500 U/mL IFNα-A/D and lysates were harvested and analyzed 30 min after stimulation for phosphorylated STAT1 (p-STAT1), total STAT1, phosphorylated STAT2 (p-STAT2), and total STAT2. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of differentiated over undifferentiated cells treated with 500 U/ml IFNα-A/D. *p<0.05. (B) Immunoblot analysis of type I IFN-induced MxA and IRF-7 expression. Cells were treated with 500 U/mL IFNα-A/D and harvested at the indicated times post-stimulation. Representative blots from one of three independent experiments are shown. (C) Flow cytometry analysis of type I IFN-induced surface MHC class I expression. Cells were unstimulated (−) or stimulated with 50 U/mL IFNα-A/D for 48 h (+). Representative overlaid histograms from one of four independent experiments are shown.
Figure 4.
Overexpression of STAT2 and IFNAR2 is sufficient to recapitulate differentiation-dependent changes in neuronal type I IFN-stimulated gene expression.
(A) Type I IFN dose-response curves were analyzed in undifferentiated BE(2)-C (closed circles), differentiated BE(2)-C/m (closed squares), and undifferentiated cells transfected with either empty vector (open circles, solid line) or IRF-9-, IFNAR2-, and STAT2-overexpression vectors (open circles, dashed line). Normalized SEAP responses 24 h after stimulation with IFNα-A/D were fit using a variable slope nonlinear regression to calculate EC50 and Hill slope values. (B) BE(2)-C cells were transfected with the indicated expression plasmid combinations, and EC50 and Hill slope values were calculated from independently fit type I IFN dose-response curves as noted above in (A). *p<0.05, **p<0.005, relative to vector control. Upper and lower dashed reference lines indicate calculated response values for untransfected undifferentiated and differentiated cells, respectively. Representative immunoblots from one of four independent experiments indicate equivalent overexpression of IRF-9, IFNAR2, and STAT2 in the appropriate samples. A GFP-overexpression plasmid was co-transfected as a control for transfection efficiency, and GAPDH was analyzed as a loading control.
Figure 5.
Overexpression of type I IFN signaling components selectively modulates antiviral responses in human neuronal cells.
Undifferentiated BE(2)-C cells were transfected with the indicated expression vector combinations and either unstimulated (open bars) or stimulated with 500 U/mL IFNα-A/D for 24 h (closed bars), infected with the indicated virus at an MOI of 0.01, and infectious virus titers in tissue culture supernatants were measured by plaque assay at 72 hpi for FMV (A) or 36 hpi for WEEV (B). Results represent mean ± SEM from at least four independent experiments, and the change in virus titer with IFNα-A/D stimulation was used for statistical analyses. ND, not detected. *p<0.05, relative to the IFN-stimulated reduction in vector control-transfected cells.
Figure 6.
Differentiation of hESC to NPCs and mature neurons.
Phase-contrast micrographs of a pluripotent hESC colony growing on a layer of irradiated mouse embryonic fibroblasts on day 0 (top image), subsequent differentiation in the absence of noggin into embryoid bodies on day 4 and neuroepithelial rosettes on day 21 (left images) or in the presence of noggin into neurospheres in suspension culture on day 21 (right image), and final differentiation into adherent cultures enriched in NPCs on day 28 and mature neurons by day 42 (bottom images). Arrows in day 21 noggin-independent cultures indicate neuroepithelial rosettes. Open arrowheads in day 28 NPC cultures indicate characteristic cells with large perikaryon and minimal processes, whereas closed arrowhead in day 42 mature neuron cultures indicate characteristic cells with small perikaryon and extensive processes. Scale bars = 200 μm.
Figure 7.
hESC-derived cultures are neural-lineage pure and enriched in NPCs at day 28 or mature neurons at day 42.
Immunocytochemical analysis of day 28 differentiated (A) and day 42 differentiated (B) cultures generated using the noggin-dependent protocol outlined in Fig. 6. Expression of the indicated marker is shown as an overlay image (top panels), and nuclear DAPI staining identifies all cells in the field (bottom panels). Scale bars = 200 μm. Higher magnification inset images more clearly depict cell morphology and intracellular staining pattern. (C) Flow cytometry analysis of day 28 differentiated (green lines) and day 42 differentiated (blue lines) neuronal cultures derived using the noggin-dependent protocol outlined in Fig. 6. Surface PSA-NCAM expression assessed neural-lineage purity. Intracellular SOX3 and NeuN expression were used to approximate NPC and mature neuronal percentages, respectively. Representative overlaid histograms from one of two independent experiments are shown, where quantitative values represent mean ± SD percentage positive relative to isotype control. The noggin-dependent differentiation protocol produced less pure populations of NPCs and mature neurons, where NPC cultures were ∼43% SOX3+ and mature neuronal cultures were ∼58% NeuN+ (data not shown).
Figure 8.
Differentiation of hESC-derived neurons enhances type I IFN pathway component expression and function.
(A) Lysates from hESC-derived NPCs and mature neurons were immunoblotted for basal IRF-9, STAT1, and STAT2 expression. GAPDH was analyzed as loading control. Representative blots from one of three independent experiments are shown, and quantitative immunoblot results shown on the graph represent mean ± SEM densitometry ratios of mature versus immature cells. *p<0.05, relative to GAPDH. (B) Flow cytometry analysis of basal surface IFNAR2 expression on hESC-derived neurons. Solid grey and black lines represent IFNAR2 levels on immature and mature cells, respectively. Dashed lines indicate background fluorescence levels obtained with isotype-matched control antibodies. Representative overlaid histograms from one of four independent experiments are shown. (C) Cell viability analysis of type I IFN-mediated protection from WEEV-induced cell death. hESC-derived NPCs or mature neurons were either unprimed (closed bars) or primed with 50 U/mL IFNα-A/D (open bars) for 24 h prior to infection with WEEV at an MOI of 0.1, and viability was analyzed at 72 hpi. Results represent mean ± SEM from four independent experiments (*p<0.05; n = 2 each for noggin-dependent and noggin-independent protocols).