Differentiation of mature oligodendrocytes from small biopsy of adult rat spinal cord meninges

In most diseases of the spinal cord, demyelination plays an important role in the generation and progression of the neurodegenerative lesion. Regeneration of oligodendrocytes is apparently insufficient for repair and attempts with regenerative transplantation approaches have been so far limited by lack of adequate sources of oligodendrocytes. Here we report results obtained with a new protocol for production of oligodendrocyte precursors (OPCs ) from LeSCs, a population of neural stem cells of the leptomeninges. Starting from a small (approx 3x10 mm strip) 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. NG2 was expressed at high level at step 1 and almost disappeared at step 3. O4 expression was highest at step 2. At step 3, 100% of the cells expressed MBP at levels up to 30 folds higher than in neurosphere stage. Mature cells also expressed plp1, cnp, mag and mog, markers of mature oligodendrocytes. Efficiency was high and usually 1.8-3.6 x 1012 OPCs were obtained from 1x 105 cells from neurospheres (n=7). In conclusion, we have established an efficient and reproducible method to generate large numbers of cells of oligodendrocyte-lineage at different and controlled levels of differentiation. This observation paves the way for exploitation of LeSCs in regenerative therapies of demyelinating disorders.