Investigation of astrocyte - oligodendrocyte interactions in human cultures

Abstract

Multiple sclerosis (MS) is characterized by CNS demyelination and oligodendrocyte depletion, axonal loss, and reactive astrogliosis. Myelin loss causes conduction block, while remyelination is associated with recovery of conduction and return of function. Reactive astrocytes are a prominent feature of MS plaques, and have been implicated as producing factors regulating oligodendrocyte progenitor differentiation and myelin formation. Understanding their impact on these events may lead to new approaches for oligodendrocyte protection and/or remyelination in MS. Here, we outline protocols for the establishment and analysis of primary monocultures and cocultures of human astrocytes and oligodendrocytes. These approaches are designed to facilitate analysis of mechanisms underlying astrocytic regulation of progenitor survival and myelin repair.

Fig. 1

Coculture of human astrocytes and human oligodendrocytes. Primary cultures of human fetal astrocytes were established from cortical samples as described in the text, and cells at passage G3 are allowed to reach 70% confluence. Primary human oligodendrocytes grown in parallel from fetal spinal cord samples were then plated onto the astrocyte culture, and resulting cocultures propagated in serum-free medium for 72 h. Cells were fixed with 4% paraformaldehyde, stained sequentially for the oligodendrocyte marker O4 and the astrocytic marker GFAP as described in the text, and counterstained with DAPI. Cocultures were imaged using confocal microscopy, and images captured at 63× magnification. The panel shows a large GFAP⁺ astrocyte (left) and two arborized O4⁺ oligodendrocytes (bottom and right). Scalebar, 10 µm.