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

Deboux C, Ladraa S, Cazaubon S, Ghribi-Mallah S, Weiss N, Chaverot N, Couraud PO, Baron-Van Evercooren A - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, UMR-S975, Paris, France.

ABSTRACT
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.

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Transduction with lentiviral vector does not affect NPC differentiation in vitro.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.
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pone-0057430-g002: Transduction with lentiviral vector does not affect NPC differentiation in vitro.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.

Mentions: We tested the potential effect of CD44 transduction on NPC phenotype and proliferation. Immunocytochemistry for various markers indicated that control and CD44-NPCs were immature cells with the majority of cells expressing nestin (99.7±0.2% vs. 98.8±0.2%) (Fig. 1E, H, C), and a minority of cells expressing the glial markers GFAP (1.2±0.3% vs. 1.2±0.3%) or A2B5 (8.2±0.2%vs. 6.9±0.1%), or the neuronal marker ß3 tubulin (0.2±0.2% vs. 0.2±0.1%) (Fig. 1C). Moreover CD44 transduction did not modify transduced NPC potential to proliferate since a large proportion of NPCs expressed the proliferation marker Ki67 (57, 9±3% vs. 52,5±6%) (Fig. 1C, F, I). Moreover, lentiviral transduction did not modify NPC multipotency in the major neural cell lineages. After EGF/FGF removal, control and CD44-NPCs differentiated equally in GFAP (45.9±4.3% vs. 45.6±2%), A2B5 (9.7±0.2% vs. 9.8±1.2%) and ß3tubulin (9.3±2% vs. 9.0±0.4%) expressing cells in correlation with an equivalent reduced capacity to proliferate (0.7±0.6% vs. 0.2±0.2%) (Fig. 2).


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

Deboux C, Ladraa S, Cazaubon S, Ghribi-Mallah S, Weiss N, Chaverot N, Couraud PO, Baron-Van Evercooren A - PLoS ONE (2013)

Transduction with lentiviral vector does not affect NPC differentiation in vitro.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585392&req=5

pone-0057430-g002: Transduction with lentiviral vector does not affect NPC differentiation in vitro.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.
Mentions: We tested the potential effect of CD44 transduction on NPC phenotype and proliferation. Immunocytochemistry for various markers indicated that control and CD44-NPCs were immature cells with the majority of cells expressing nestin (99.7±0.2% vs. 98.8±0.2%) (Fig. 1E, H, C), and a minority of cells expressing the glial markers GFAP (1.2±0.3% vs. 1.2±0.3%) or A2B5 (8.2±0.2%vs. 6.9±0.1%), or the neuronal marker ß3 tubulin (0.2±0.2% vs. 0.2±0.1%) (Fig. 1C). Moreover CD44 transduction did not modify transduced NPC potential to proliferate since a large proportion of NPCs expressed the proliferation marker Ki67 (57, 9±3% vs. 52,5±6%) (Fig. 1C, F, I). Moreover, lentiviral transduction did not modify NPC multipotency in the major neural cell lineages. After EGF/FGF removal, control and CD44-NPCs differentiated equally in GFAP (45.9±4.3% vs. 45.6±2%), A2B5 (9.7±0.2% vs. 9.8±1.2%) and ß3tubulin (9.3±2% vs. 9.0±0.4%) expressing cells in correlation with an equivalent reduced capacity to proliferate (0.7±0.6% vs. 0.2±0.2%) (Fig. 2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière, UMR-S975, Paris, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus