Potential of genetically modified ensheathing cells for regeneration after spinal cord injury = Potencial de la glía envolvente genéticamente modificada para la regeneración después de lesión medular

Abstract

Olfactory ensheathing cells (OECs) implantation has emerged as a promising therapy for spinal cord injury (SCI), but their regenerative properties seem to depend on the presence of a substrate permissive to cell migration. Studies demonstrate that when transplanted after SCI, the migratory properties of OECs are reduced compared to transplantation in not-injured spinal cords. As after SCI, inhibitory molecules such as myelin associated inhibitors (MAIs) are overexpressed in the site of lesion. Therefore we thought that these molecules could modulate OECs migration capacity.

We used a rodent clonal OEC line, TEG3, and we demonstrated that these cells express all the components of the NgR-complex. This complex resulted to be active since TEG3s activated RhoA and increased ERK1-2 phosphorylation in response to acute stimuli of myelin. Indeed, myelin inhibited OECs migration over glass surfaces and also over linearly elastic PAA gels. Moreover using traction force microscopy (TFM), we quantitatively demonstrate that TEG3s largely decrease their traction stress over myelin. This decrease in traction force correlated with decreased focal contacts and redistribution the of F-actin cytoskeleton. In fact, OECs cultured on myelin largely reduced the number of protrusions, stress fibers and FAs. Finally we observed that the incubation of TEG3s with the NgR1 blocking peptide NEP1-40 mainly overcame MAIs-mediated migratory inhibition, restoring the traction forces of these cells and their cytoskeletal organization. Altogether these data suggested us that a cell-based strategy, using TEG3s that has the capacity to overcome the inhibitory action of MAIs, is required in other to enhance their migratory properties after SCI transplantation. Indeed in order to overcome the effects of MAIs over TEG3 we tested two strategies.

First we investigated whether a particular type of magnetic nanoparticle (MNP) could be used to control the localization and migration of TEG3s. We confirmed that, in vitro, magnetized TEG3s can survive well without exhibiting stress-associated cellular responses. Moreover, their migration can be modulated by magnetic fields and their transplantation in organotypic culture of spinal cord and peripheral nerve, a model more similar to an in vivo situation, showed positive integration in the model. Second we genetically modified OEC to express the NgR(Ecto)coupled with a GFP using a lentiviral based system in order to visualize them. The NgR(Ecto), contains the amino acids 1–310 of NgR1 and blocks the signaling of myelin and previous studies demonstrated that its presence enhances axonal regeneration after SCI. Engineered OEC were able to express and secrete at high levels the ectodomain in vitro. Moreover, by time lapse analysis we could observe that these cells migrate longer distances than normal OEC over a myelin substrate. To observe the behavior of NgR(Ecto)-OECs in vivo we did a preliminary experiment where cells were implanted in a not-lesioned spinal cord of adult rats. The NgR(Ecto)-OECs migrated more distance both caudal and rostral from the site of injection in comparison with normal OEC.

These data suggest a therapeutic potential of both strategies tested for SCI. NgR(Ecto)-OECs once implanted after SCI would migrate more and this would enhance their regeneration capacity. Moreover the secreted ectodomain would interfere with the myelin present at the lesion site improving intrinsic axonal regeneration. We could combine the use of these modified cells with MNPs. With the design of specific magnetic sources we could concentrate OECs near the lesion site and therefore increase their possible interaction with damaged axons. Indeed, further studies are necessary the behavior of this modified cells in a SCI model.

La implantación de OECs ha surgido como una terapia prometedora para lesiones medulares, pero sus propiedades regenerativas parecen depender de la presencia de un sustrato permisivo para la migración celular. En esta Tesis Doctoral demostramos que los inhibidores asociados a mielina (IAMs) pueden modular la capacitad migración de las OECs través la interacción y activación del complejo receptor de NgR (el receptor común para los IAMs). Con el uso de la microscopia de tracción, demostramos cuantitativamente que las OECs disminuyen en gran medida su fuerza de tracción sobre la mielina. Además, esta disminución se pudo correlacionar con una disminución de las adhesiones focales y la redistribución del citoesqueleto de F-actina. La incubación con el péptido NEP1-40, que bloquea NgR1, restableció in gran parte la capacitad migratoria, las fuerzas de tracción de estas células y su organización citoesqueletal. En base a los resultados obtenidos exploramos la viabilidad de dos estrategias diferentes para mejorar las propiedades migratorias de la OECs. Una de ellas trataba del posible uso de nanoparticulas magnéticas para guiar a las células. Aunque esta técnica no aumenta la velocidad intrínseca de migración de las células, permite la concentración y la guía de las mismas hace a un área específica después de la implantación in vivo. Pudimos comprobar que las OECs marcada con NPM no exhiben respuestas celulares asociadas al estrés, y pueden ser guiadas por un campo magnético in vitro. Además comprobamos que las células se integraban bien después de su trasplante de en un modelo organotípico de regeneración de médula espinal/nervio ciático. La segunda estrategia consistió en modificar genéticamente las OECs para que expresaran un ectodomínio por el NgR1 para bloquear los efectos negativos de los IAMs sobre su migración. Pudimos observar que las células modificadas migran más in vitro sobre un sustrato de mielinas comparadas con OECs normales. Además, una vez implantadas en una médula no lesionada, las células modificadas se integran bien en el modelo y migran más que células normal. Los resultados obtenidos indican un potencial terapéutico de ambas estrategias.