FIGURE 1.

PMN-CM promotes astroglial differentiation in vitro. (A and B) PMN or Mϕ retain viability after 24 h of culture in DM. (A) The number of PI⁺ PMN and Mϕ was quantified using flow cytometry, as illustrated for PMN by scatterplot. Dead cells were quantified by gating to viable cells that did not receive PI. (B) Quantification of PI⁺ PMN (orange bars) and Mϕ (yellow bars); PMN = 6.04% ± 0.30, Mϕ = 5.27% ± 0.86. Mean percentage ± SEM relative to control IgG or IgM isotype-labeled cells (Student t test, p > 0.05, N.S.). n = 3 technical replicates and n = 3 biological replicates/condition. (C and D) GFAP is a reliable marker for hNSC astroglial lineage commitment under differentiation conditions in vitro. (C) hNSC at passages 9–11 were dissociated into single cells, plated with DM onto eight-well chamber slides (30,000 cells/ml), and maintained at 37°C under standard 5% CO₂ 20% O₂ culture conditions for 2 DIV (black bars) or 14 DIV (gray bars) and mRNA collected for quantitative RT-PCR. GFAP mRNA exhibited a 2-fold increase between 2 and 14 DIV under these differentiation conditions (Student t test, ***p ≤ 0.0001). n = 3 biological replicates/condition. (D) In contrast, Nestin mRNA remained unchanged between 2 and 14 DIV (Student t test, p > 0.05, N.S.). n = 3 biological replicates/condition. (E–G) hNSC were exposed to either DM (control), or PMN-CM or Mϕ-CM. PMN-CM (orange bars) or Mϕ-CM (yellow bars) versus DM control (dashed line) comparisons were conducted using one-sample t tests (^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001). Comparisons between groups were performed using one-tailed Student t tests (****p < 0.0001). (E) PMN-CM increased the percentage of hNSC expressing GFAP compared with control and Mϕ-CM at 14 DIV. (F) PMN-CM and Mϕ-CM reduced the percentage of Olig2⁺ cells compared with control at 14 DIV, but were not significantly different from each other. (G) PMN-CM reduced the percentage of β-tubulin III⁺ cells compared with control and Mϕ-CM at 14 DIV. Mϕ-CM increased the percentage of β-tubulin III expression. n = 2 technical replicates and n = 2–3 biological replicates/condition. (H) PMN-CM derived from immune-sufficient rats promotes astroglial differentiation in vitro. Immunocytochemical staining for GFAP (red), β-tubulin III (green), and Hoechst counterstain (blue) in hNSC exposed to DM control or PMN-CM isolated from the peritoneal cavity of immune-sufficient Sprague–Dawley rats. hNSC treated with PMN-CM exhibited increased GFAP⁺ cell number in comparison with DM control. Mean ± SEM. Scale bars, 25 μm (E–G) and 150 μm (H).