Figure 3

(A) Human fetal astrocyte proliferation in response to S1P receptor ligands. Immunocytochemistry stains for astrocytes undergoing cell division (Ki-67, red) and cell nuclei (Hoechst nuclear stain, blue). (i) Untreated cells (basal proliferation). (ii) FTY720 (100 nM) overnight. (iii) S1P (100 nM) overnight. (iv) DMSO control. (v) Methanol control (MeOH). (B) Quantification of human fetal astrocyte proliferation in response to S1P receptor ligands. Proliferation indices were generated by determining the percentage of cells positive for Ki-67 relative to total cell population (Hoechst nuclear stain). Fold changes in proliferation were calculated relative to rate of proliferation under basal conditions. S1P induced a 1.8-fold increase in astrocyte proliferation, whereas FTY720 did not mediate proliferation beyond the basal rate (normalized to 1) (n = 3). (C) FTY720 pre-incubation blocks proliferative response of S1P on human fetal astrocytes. At outset (Day 0), astrocytes were either treated with S1P (S) or FTY720 (F). Following overnight culture, cells were either (i) left untreated (−) or (ii) incubated with S1P for another 24 h (Day 1). Proportion of astrocytes undergoing proliferation (Ki-67+) was then determined. Hoechst nuclear stain was used to determine total cell number. Proliferation fold change was calculated relative to rate of proliferation under basal conditions (normalized to 1). (i) S1P induced a 1.6-fold increase in astrocyte proliferation whereas FTY720 was comparable to basal proliferation rate. (ii) Significant proliferation (1.5-fold increase) was observed in untreated astrocytes (Day 0) when given S1P for 24 h (Day 1). S1P-pre-treated astrocytes on Day 0 continued to proliferate (1.8-fold increase), whereas FTY720 exposure on Day 0 did not result in astrocyte proliferation when exposed to S1P stimulation added on Day 1 (n = 3).