Microglial response to graphene-free or—inclusive PCL scaffold implantation at Week 1 and Week 3.

<p>(A) Image shows an overview of microglial infiltration into the outer shell of a gP6 scaffold at Week 3, and gP6 at high magnification at (B) Week 1 and (C) Week 3. (D) Graphene-free P6 scaffold at Week 3 (Green: Iba1⁺ microglia cells, blue: DAPI stained nucleus). (E) Microglial profile across the tissue/scaffold interfaces (*** p<0.001, n = 4). All brain tissue sections were collected on the transverse plane. Scale bar for (A) represents 100 μm; Scale bar for (B, C and D) represents 20 μm. Error bar in (E) shows standard error of the mean.</p

Astrocyte morphology and infiltration at different time points following H6, P6, gP6, gH6 scaffolds implantation.

<p>Astrocyte/gP6 scaffolds interaction at Week (A) 1, (B) 3 and (C) 7; Astrocytes/P6 scaffolds interaction at Week (E) 3 and (F) 7; Astrocyte process infiltration into (H) gH6 at Week 3 and (I) H6 at Week 7. (D, G) detailed astrocyte morphology of the dash-box indicated area in (B, I) respectively. Green: GFAP positive astrocytes, blue: DAPI stained nucleus, red: surface functionalized scaffolds. * indicates astrocytes that bridge a gap between two scaffold layers in (D). All brain tissue sections were collected on the transverse plane. Scale bar for (D, G) represents 20 μm; for all other images represents 50 μm.</p

GFAP expression at different time points in the outer layer of scaffolds and in adjacent tissue.

<p>(150 μm from tissue/scaffold boundary) in terms of GFAP⁺ astrocytes occupied pixel percentage (pixel%). * (p<0.05, n = 4) indicate significant difference in GFAP expression between Week 3 and 7, within scaffold or tissue for gP6 implants. Error bar shows standard error of the mean.</p

Neuroblast migration/integration along/with gP6 implants at Week 3.

<p>Immunostaining images show DCX⁺ neuroblast (red) and nuclei (DAPI, blue) of tissue sections at the transverse plane. Schematic coronal brain section indicates the gP6 implantation track and locations of A-C, D-E tissue sections respectively. gP6 implant boundary is marked by dotted line. (A) gP6 implant in LV, bottom right part of the outermost scaffold layer is in direct contact with SVZ. Insert enlarges the box region showing neuroblast process across the entire scaffold layer. (B) Neuroblasts migrate along the scaffold surface/inter-layer gap and (C) processes infiltrating into the scaffold layers. Neuroblasts are identified in deeper sections of the gP6 implants either (D) within the scaffold or (E) close to implant in the LSV. LV: lateral ventricle, LSI: lateral septal nucleus, intermediate part, LSV: lateral septal nucleus, ventral part. Scale bars represent 20 μm for all images.</p

Microstructure of electrospun PCL microfibers and polyelectrolyte modified fibers with and without graphene.

<p>SEM images showing the microstructure of (A, B) PCL microfibers, (C) P6 and (D) gP6 modified microfibers. Partially aligned smooth microfiber morphology was revealed in all images at different magnifications.</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

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

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

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