Spinal cord injury is characterized by multiple pathological reactions resulting in loss of neurons and oligodendrocytes, demyelination of spared axons and formation of scar tissue at the lesion site. Regeneration of oligodendrocytes to prevent or limit demyelination is apparently insufficient for repair and attempts with regenerative transplantation approaches have been so far limited also because of lack of adequate sources of oligodendrocytes. Here we report results obtained with a new protocol for production of oligodendrocyte precursors (OPCs ) from LeSCs [1], a population of neural stem cells of the leptomeninges. Starting from a small biopsy of adult rat spinal cord meninges, neurospheres were produced. By adjusting culture conditions, we developed a three-step protocol (1-induction, 2-differentiation, 3-maturation) to produce oligodendrocytes. Confocal immunofluorescence and Real time-RT PCR were used to monitor cell changes. During the progression of oligodendrocytes lineage we observed different expression of maturation stage-specific markers; at the end of the maturation progression, expression of typical adult olygodendorcyte markers plp1, cnp, mag and mog was revealed by Real time-RT PCR. With this study we want to asses regenerative and reparative potential of transplanted LeSCs-derived OPCs in adult rats exposed to a moderate traumatic lesion at T8 using Impactor as described in ref. 1. Six days after injury (dpi), 600,000 GFP-LeSC-OPCs were transplanted at the site of contusion. For control, lesioned animals were injected with culture medium and no cells. Locomotor evaluation was assessed by the Basso, Beattie, and Bresnahan (BBB) rating scale and subscale, Catwalk analysis and electrophysiology. Measurements were taken 1 and 6 dpi, and at several time points between 1 and 56 days after transplantation (dpt). From 1 week after GFP-LeSC-OPCs transplantation (n=11), rats showed significant improvement in locomotion subparameters compared to controls (n=12) in BBB score. Catwalk analysis showed significant differences at 56 dpt whereas significant differences were not observed by electrophysiological evaluation. Preliminary data indicate that 63 dpt transplanted cells are present in the parenchyma of the spinal cord with tendency to accumulate in the distal regions of the lesioned tissue. Temporal analysis of the fate of transplanted cells is in progress. These findings pave the way for exploitation of LeSCs in regenerative therapies of demyelinating disorders.

Regenerative potential of leptomeninges-derived oligodendrocytes in a rat model of spinal cord injury.

BRAGA, ALICE;Berton, Valeria;MALPELI, Giorgio;Pino, Annachiara;KRAMPERA, Mauro;DECIMO, Ilaria;BIFARI, Francesco;FUMAGALLI, Guido Francesco
2014-01-01

Abstract

Spinal cord injury is characterized by multiple pathological reactions resulting in loss of neurons and oligodendrocytes, demyelination of spared axons and formation of scar tissue at the lesion site. Regeneration of oligodendrocytes to prevent or limit demyelination is apparently insufficient for repair and attempts with regenerative transplantation approaches have been so far limited also because of lack of adequate sources of oligodendrocytes. Here we report results obtained with a new protocol for production of oligodendrocyte precursors (OPCs ) from LeSCs [1], a population of neural stem cells of the leptomeninges. Starting from a small biopsy of adult rat spinal cord meninges, neurospheres were produced. By adjusting culture conditions, we developed a three-step protocol (1-induction, 2-differentiation, 3-maturation) to produce oligodendrocytes. Confocal immunofluorescence and Real time-RT PCR were used to monitor cell changes. During the progression of oligodendrocytes lineage we observed different expression of maturation stage-specific markers; at the end of the maturation progression, expression of typical adult olygodendorcyte markers plp1, cnp, mag and mog was revealed by Real time-RT PCR. With this study we want to asses regenerative and reparative potential of transplanted LeSCs-derived OPCs in adult rats exposed to a moderate traumatic lesion at T8 using Impactor as described in ref. 1. Six days after injury (dpi), 600,000 GFP-LeSC-OPCs were transplanted at the site of contusion. For control, lesioned animals were injected with culture medium and no cells. Locomotor evaluation was assessed by the Basso, Beattie, and Bresnahan (BBB) rating scale and subscale, Catwalk analysis and electrophysiology. Measurements were taken 1 and 6 dpi, and at several time points between 1 and 56 days after transplantation (dpt). From 1 week after GFP-LeSC-OPCs transplantation (n=11), rats showed significant improvement in locomotion subparameters compared to controls (n=12) in BBB score. Catwalk analysis showed significant differences at 56 dpt whereas significant differences were not observed by electrophysiological evaluation. Preliminary data indicate that 63 dpt transplanted cells are present in the parenchyma of the spinal cord with tendency to accumulate in the distal regions of the lesioned tissue. Temporal analysis of the fate of transplanted cells is in progress. These findings pave the way for exploitation of LeSCs in regenerative therapies of demyelinating disorders.
spinal cord injury; neural stem cells, oligodendrocyte differentiation; regenerative medicine
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/866376
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