Effects of CD44 overexpression on trans-endothelial migration and spreading <i>in vitro</i>.

<p>(A, B) Trans-endothelial migration of actin eGFP CD44-NPCs (+) and control NPCs (-); quantification was performed using immunofluorescence for (A) eGFP or (B) c-myc. In response to SDF1α cells overexpressing CD44 migrate 2 fold more compared to control cells (experiments made in triplicate). (C, D) Spreading of NPCs <i>in vitro</i> on laminin: the number and length of filopodia are increased. (E, F) CD44 overexpressing cells are more elongated and spread than control NPCs. (* = p<0.5; ** = p<0.01, *** = p<0.01). (G) Immunocytochemistry for actin illustrating filopodia (arrowheads) in control-NPCs (Gi, Gii) and CD44-NPCs (Giii, Giv).</p

Transduction with lentiviral vector does not affect NPC differentiation <i>in vitro</i>.

<p>NPCs were plated in the absence of EGF and FGF for 12 days. Immunocytochemistry for (A, D) GFAP, (B, E) ß3-tubulin, (C, F) c-myc of (A–C), control and (D–F), CD44-c-myc transduced actin eGFP cells (green). (G) Quantitative evaluation of the percentage of cells expressing the different markers over the total cell population identified by Hoechst staining (H+). Arrows in B and E point to cells enlarged in insets.</p

Time-course distribution of NPCs after i.v. delivery in the mouse tail.

<p>(A–F) Detection of GFP(green) to localize grafted cells and of von Willebrand factor (VWF, red) to identify endothelial cells, reveals that NPCs are localized in or around the vascular wall at 6 h (A, B) and 12–24 h (C, D) post injection, but are more distant from the vascular wall at 21 days (E, F). (A, C, E) actin eGFP control-NPCs and (B, D, F) CD44-NPCs. CD44 NPCs become more rapidly elongated and distant from the vascular wall. Moreover, in D and F, GFP+ CD44-NPCs form a double wave, which is not observed in control NPCs. Arrows point to regions enlarged in insets on top of each panel.</p

Semi-quantitative evaluation of the distribution of GFP cells in the vascular wall 6 hours after i.v. delivery.

<p>(A) Schematic representation of the blood vessel structure, The vascular wall is composed of the endothelial layer and vascular mantel. (B) Evaluation of the percentage of animals containing cells present in the vascular wall (intravascular) and beyond the wall (perivascular). (C, D) Schematic representation of the relative distribution of GFP cells in control (C) and CD44 (D) injected animals. (E) Evaluation of the percentage of mice, in which NPC migration occurred beyond the perivascular space (extravascular). (F) Evaluation of the amounts of GFP cells found at the delivery site and expressed in GFP+ surface area (µm2).</p

Illustrations of a focal spinal cord lesion of MOG induced EAE in the DA rat.

<p>LFB staining shows a focal demyelinated lesion in the dorsal funiculus with myelin loss (A–B). ED-1 immunostaining highlights the presence of macrophages/microglia cells within the lesion (C). MBP immunohistochemistry illustrates the loss of myelin and the presence of myelin-laden macrophages (close up in insert) is indicative of recent myelin phagocytosis (D).</p

One route of SC migration: the blood vessels.

<p>SC grafted in the spinal cord parenchyma are often localized in white matter around blood vessels (asterisks), evidenced with anti-laminin antibody (blue). While at 7 days SC are present close to the blood vessel wall (A), at 21 days they are embedded in the perivascular space but remote from the vascular wall (B).</p

Distribution of GFP labeled SC after delivery in the cisterna magna and the spinal cord.

<p>GFP-SC (green) are detected both in the cerebellar parenchyma (A, B) and meninges (C) 7 days (A–C) after cisterna magna delivery as well as 21 days after in the proximal spinal cord (D). GFP-SCs grafted in the spinal cord parenchyma (E, F) are concentrated around blood vessels, some migrate away from the graft toward a lesion (L) identified by MOG immunostaining through white matter (E, arrows). (F) Same field illustrating GFP-SCs, inset is a higher magnification.</p

Effect of NPC delivery on EAE clincial features.

<p>Intravenous delivery of control (light grey) and CD44-NPCs (dark grey) in the tail vein after disease onset significantly improves clinical features over sham (black) in EAE mice. While clinical signs reach a plateau more rapidly after the first peak in CD44-NPC treated animals differences in severity between control and CD44-NPCs are not observed.</p

Average scores, maximal scores, cumulative scores (calculated from day 25 to day 45) and mortality rate in non-operated, CT and SCs grafted animals.

*<p>: is significantly different from control and non-operated animals, p<0.05.</p

EAE score evolution after GFP-SC graft in the spinal cord.

<p>The graph depicts the clinical scores of EAE animals grafted with GFP-SC (white circles, 7 animals) and medium injected animal (black squares, 5 animals). Surgeries were performed 2 days after the first clinical signs (occurring 12 days after the induction of the disease). A difference between the two groups is observed from day 25 and is significant (Mann-Whitney rank test; p<0.05) around 30 days after the first clinical signs.</p