Overexpression of CD44 in neural precursor cells improves trans-endothelial migration and facilitates their invasion of perivascular tissues in vivo.

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues

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

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

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

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

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