Figure 1.
Heatmap of representative cluster transcripts involved in NGF signaling.
A. OBNS/PC-GFP proliferation (OBNSC prol.), OBNS/PC-hNGF proliferation (NGF prol.), OBNS/PC-GFP differentiation (OBNSC diff.), and OBNS/PC-GFP-hNGF differentiation (NGF diff). Transcripts that are highly up-regulated are red compared with the down-regulated ones which are green. NGF is up-regulated in OBNS/PC-GFP-hNGF respect to OBNS/PC-GFP in proliferation conditions. In differentiation conditions NGF is repressed in both of cell. B. Elisa results showing significant differences between NGF protein levels in OBNS/PC-GFP-hNGF compared to control cells. C. hNGF signal intensity between OBNS/PC-GFP and OBNS/PC-GFP-hNGF. Biological replicates (n=3) of the aforementioned 4 cell classes were compared under proliferation and differentiation conditions.
Figure 2.
Fluorescence image (40X) of wild type OBNS/PC at day 5 post differentiation (passage 15).
The OBNS/PC were stained for the NSC phenotype markers nestin (green) and GFAP (green, b, c, and red, d), MAP (red, c), β-TubulinIII (red, b). Cell nuclei were counterstained with Hoechst (blue, a-f). At the immunohistochemical level, differentiated wild type OBNS/PC exhibited positive immunoreactivity for astrocytes marker (65-75%) (B, C, D), MAP2, mature neuronal marker (8%) (C), β-TubulinIII, early neuronal marker (3%) (B).
Figure 3.
Phase contrast microscope image for OBNS/PC-GFP-hNGF (a,b), and wild type OBNS/PC (c,d) at day four of differentiation.
The OBNS/PC-GFP-hNGF were more confluent and showed extensively branched processes in comparison to the wild type OBNS/PC.
Figure 4.
Between P7 and P10 the OBNS/PC were infected with lentivirus transducing GFP.
Differentiation of OBNS/PC-GPP was applied between passage 12-15. The differentiated OBNS/PC-GFP exhibited positive immunoreactivity for GFAP astrocytes marker (60-70%) (Figure 5, A,B,C,D), MAP2 mature neuronal marker (15%) (C, D), β-TubulinIII immature neuronal marker (8%) (A,B), NG2 positive cells w/o T3 + PDGFAA (E) (7-8%), and with T3 + PDGFAA (F) (16%). The nuclei were stained blue with Hoechst.
Figure 5.
The differentiation potential of OBNS/PC-GFP-hNGF was assessed by examining their reactivity against different neuronal and glial cells molecular markers between passage 12-15.
In comparison to wild type (control) OBNS/PC and OBNS/PC-GFP, differentiated OBNS/PC-GFP-hNGF exhibited positive immunoreactivity for GFAP astrocytes marker (45-55%) (D,E,F), MAP2 mature neuronal marker (25-30%) (C,D,F). β-TubulinIII, immmature neuronal marker (6%) (A,B,E). The nuclei were stained blue with Hoechst.
Figure 6.
Western blot data revealed that the NGF protein levels look similar although the NGF levels were higher in differentiated OBNS/PC-GFP-NGF respect to OBNS/PC-GFP.
Up- regulation of early oligodendroglia markers (PDGFRα, NG2 and CNPase) was observed in OBNS/PC-GFP-hNGF respect to OBNS/PC-GFP. Sox2 and Oct4 were up-regulated in OBNS/PC-GFP vs. OBNS/PC-GFP-hNGF. Nurr1, TH, NeuroD1 and β-TubulinIII expression were similarly expressed in the 4 cell populations. Survival genes (ERK, AKT1, CREB) were differently modulated between the 4 classes of cell populations; high-affinity Trk receptor was down modulated in differentiated cell populations vs proliferated ones. B. Without T3+PDGFAA, 14% of the differentiated OBNS/PC-GFP-hNGF exhibited positive immunoreactivity for NG2 oligodendrocyte marker. C. In the presence of T3+PDGFAA, almost 25% of differentiated OBNS/PC-GFP-hNGF were NG2 positive. The nuclei were stained blue with Hoechst.