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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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Effective CD44 lentiviral transduction of NPCs :(A) FACS analysis and (B) immunoblot of CD44-c-myc transduced cells with an antibody revealing CD44-c-myc and endogenous cm-myc showing c-myc (and thus CD44) overexpression in transduced cells. (C) Quantitative data indicating that lentiviral transduction does not change the cell phenotype. (D–I) Representative illustrations of actin eGFP cells (green) expressing CD44 (red), nestin (red) and Ki67 (red) in response to EGF and FGF (D;E;F: control NPCs; G;H;I: CD44-NPCs). (J–L) Transduced cells represent nearly 70% of the cell population; immunocytochemistry for c-myc (red) in actin eGFP (green) control-(K) and CD44-NPCs (L).
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pone-0057430-g001: Effective CD44 lentiviral transduction of NPCs :(A) FACS analysis and (B) immunoblot of CD44-c-myc transduced cells with an antibody revealing CD44-c-myc and endogenous cm-myc showing c-myc (and thus CD44) overexpression in transduced cells. (C) Quantitative data indicating that lentiviral transduction does not change the cell phenotype. (D–I) Representative illustrations of actin eGFP cells (green) expressing CD44 (red), nestin (red) and Ki67 (red) in response to EGF and FGF (D;E;F: control NPCs; G;H;I: CD44-NPCs). (J–L) Transduced cells represent nearly 70% of the cell population; immunocytochemistry for c-myc (red) in actin eGFP (green) control-(K) and CD44-NPCs (L).

Mentions: Since CD44 controls trans-endothelial migration of NPCs in vitro, we investigated the gain of function of CD44 on NPC in vitro and in vivo. To overexpress CD44 in mouse NPCs, actin-GFP NPCs were transduced with CD44-c-myc lentiviral constructs. FACS analysis using IM7 antibody showed that 47% of NPCs transduced with CD44-c-myc (thereafter named CD44-NPCs) express CD44 compared to only 7.3% in non-transduced NPCs (basal expression of CD44 in cells without infection thereafter named control NPCs) (Fig. 1A). Overexpression of the CD44 protein in the CD44-NPCs was further confirmed by immunoblot using anti-CD44 KM114 antibody or anti-c-myc antibodies (Fig. 1B). Finally, immunocytochemistry for CD44 showed that CD44 was expressed in 60±6% of CD44-NPCs and only in 12±1% of control NPCs (Fig. 1D and G). Quantification of c-myc tag expressing cells showed that CD44-NPCs represented nearly 70±3% of the cell population compared to control NPCs (0%) (Fig. 1J–L).


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)

Effective CD44 lentiviral transduction of NPCs :(A) FACS analysis and (B) immunoblot of CD44-c-myc transduced cells with an antibody revealing CD44-c-myc and endogenous cm-myc showing c-myc (and thus CD44) overexpression in transduced cells. (C) Quantitative data indicating that lentiviral transduction does not change the cell phenotype. (D–I) Representative illustrations of actin eGFP cells (green) expressing CD44 (red), nestin (red) and Ki67 (red) in response to EGF and FGF (D;E;F: control NPCs; G;H;I: CD44-NPCs). (J–L) Transduced cells represent nearly 70% of the cell population; immunocytochemistry for c-myc (red) in actin eGFP (green) control-(K) and CD44-NPCs (L).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057430-g001: Effective CD44 lentiviral transduction of NPCs :(A) FACS analysis and (B) immunoblot of CD44-c-myc transduced cells with an antibody revealing CD44-c-myc and endogenous cm-myc showing c-myc (and thus CD44) overexpression in transduced cells. (C) Quantitative data indicating that lentiviral transduction does not change the cell phenotype. (D–I) Representative illustrations of actin eGFP cells (green) expressing CD44 (red), nestin (red) and Ki67 (red) in response to EGF and FGF (D;E;F: control NPCs; G;H;I: CD44-NPCs). (J–L) Transduced cells represent nearly 70% of the cell population; immunocytochemistry for c-myc (red) in actin eGFP (green) control-(K) and CD44-NPCs (L).
Mentions: Since CD44 controls trans-endothelial migration of NPCs in vitro, we investigated the gain of function of CD44 on NPC in vitro and in vivo. To overexpress CD44 in mouse NPCs, actin-GFP NPCs were transduced with CD44-c-myc lentiviral constructs. FACS analysis using IM7 antibody showed that 47% of NPCs transduced with CD44-c-myc (thereafter named CD44-NPCs) express CD44 compared to only 7.3% in non-transduced NPCs (basal expression of CD44 in cells without infection thereafter named control NPCs) (Fig. 1A). Overexpression of the CD44 protein in the CD44-NPCs was further confirmed by immunoblot using anti-CD44 KM114 antibody or anti-c-myc antibodies (Fig. 1B). Finally, immunocytochemistry for CD44 showed that CD44 was expressed in 60±6% of CD44-NPCs and only in 12±1% of control NPCs (Fig. 1D and G). Quantification of c-myc tag expressing cells showed that CD44-NPCs represented nearly 70±3% of the cell population compared to control NPCs (0%) (Fig. 1J–L).

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