Characterization of 46C-NS cells.

<p>(A–C) Immunofluorescence staining showed 46C-NS cells expressed nestin, red (A) and 3CB2, red (B) but not GFAP, green (A) or MAP2, green (B). (<b>D–E</b>) Following neuronal or astrocytic differentiation, 46C-NS cells expressed βIII-Tubulin, red and MAP2, green (C) or GFAP (D), respectively.</p

Suppression of <i>in vitro</i> MOG-specific T-cell responses.

<p>(A) Fixed numbers of splenocytes from 2D2 mice stimulated with MOG_35–55 were cultured in the presence of varying numers of GS-N cells, 46C-NS cells or SVZ-NSCs (expressed as NSC: splenocyte ratio). Proliferative responses were measured by ³H-thymidine incorporation and expressed as the mean counts per minute (CPM). (B–G) Supernatants were collected from co-cultures after 48 hrs and the level of pro-inflammatory cytokines was quantified by cytometric bead array. Data represent the mean ± SEM (n = 4 mice). *P&lt;0.05, **P&lt;0.01, ∧P&lt;0.005, ^#P&lt;0.001 vs MOG_35–55 stimulated splenocytes unless otherwise indicated.</p

Differentiation potential of GS-N cells.

<p>(A–C) Immunofluorescence staining showed GS-N cells expressed nestin, red (A), A2B5, red (B) and βIII-Tubulin, red and MAP2, green (C). (D–E) Following neuronal or astrocytic differentiation, GS-N cells expressed MAP2, green and NeuN, red (D) or GFAP (E), respectively. (F–K) Electrophysiological assessment of GS-N cell-derived neurons. Neurons in high density (F) fired spontaneous action potentials (APs), which were blocked by tetrodotoxin (TTX) (G). The depolarization giving rise to the APs (H) was underpinned by an inward current (I). Sparsely growing neurons (J) did not display spontaneous activity but APs could be evoked by depolarising current steps (K).</p

Primer sequences used for gene expression analysis.

<p>Primer sequences used for gene expression analysis.</p

Clinical and pathological outcome of EAE mice.

<p>Data are expressed as mean ± SEM.</p>*<p>p&lt;0.05,</p>**<p>p&lt;0.01 vs PBS.</p

Cell surface expression of homing molecules.

<p>Flow cytometric analysis was used to examine cell surface expression of homing molecules by GS-N cells (A), 46C-NS cells (B) and SVZ-NSCs (C). Representative histograms are shown in grey and respective isotype controls are indicated by the black curve.</p

Effect of intraperitoneal NSC transplantation on the clinical course of EAE.

<p>C57Bl/6 mice were immunized with recombinant myelin oligodendrocyte glycoprotein and treated with intraperitoneal injections of NSCs on days 8, 10 and 12 post immunization. (A) EAE mice received 1×10⁶ SVZ-NSCs or MEFs. Data are expressed as the mean clinical score ± SEM (n = 5 mice). (B) EAE mice received 5×10⁶ GS-N cells or 46C-NS cells. Data are expressed as the mean clinical score ± SEM (n = 9–10 mice). Arrows indicate days of injection. (C–K) Representative spinal cord sections of PBS control mice (C–E), GS-N cell-treated mice (F–H), or 46C-NS cell-treated mice (I–K) were stained with haematoxylin and eosin, luxol fast blue and Bielschowsky silver stain to detect inflammation, demyelination and axonal damage, respectively. Magnification ×400. (L) Proliferative response of splenic T-cells from cell-treated mice and controls to rMOG and non-specific mitogens expressed as the mean counts per minute (CPM). (M–R) Supernatant from rMOG-stimulated splenocyte cultures were collected after 48 hrs and cytokines were quantified by cytometric bead array. (S) Serum rMOG-specific antibody response from NSC-treated mice and controls. Data are expressed as the mean ± SEM (n = 9–10 mice). *P&lt;0.05, **P&lt;0.01, ∧P&lt;0.001 versus PBS controls, ^#P&lt;0.05.</p

Effect of intravenous NSC transplantation on the clinical course of EAE.

<p>(A) C57Bl/6 mice were immunized with recombinant myelin oligodendrocyte glycoprotein and treated with intravenous injections of GS-N cells, 46C-NS cells or SVZ-NSCs on days 8, 10 and 12 post immunization. (B) Proliferative response of splenic T-cells from cell-treated mice and controls to rMOG and non-specific mitogens expressed as the mean counts per minute (CPM). Data are expressed as the mean ± SEM (n = 5–7 mice). (C) Decreasing amounts of DNA from GFP⁺ GS-N cells were titrated with DNA extracted from EAE mice and the minimum amount of GFP⁺ DNA detectable was determined by PCR using primers specific for GFP. (D) GFP⁺ GS-N cells or fibroblasts were injected intravenously into EAE mice at 12 days post disease induction and PCR was used to detect GFP⁺ cells. PCR reactions performed without DNA served as a negative control and DNA from C57Bl/6-GFP chimeric mice served as a positive control.</p

Gene expression profile of GS-N cells.

<p>(A) ES cells aggregated to form round floating EBs. (B) Day 4 neurospheres with neural projections generated from EBs. (C) Day 4 GS-N cells generated by dissociation of EBs. (D–E) Quantitative real time PCR analysis of the expression of neuroectodermal genes (D) and mesodermal and endodermal genes (E) by ES cells, day 7 EBs, day 4 neurospheres and day 4 GS-N cells. cDNA from embryonic heart tissue and Activin A (ActA) treated EBs served as positive controls.</p