Neuron‐oligodendrocyte myelination co‐culture derived from embryonic rat spinal cord and cerebral cortex

Abstract

An in vitro myelination model derived from rat central nervous system (CNS) remains to be established. Here, we describe a simple and reproducible myelination culture method using dissociated neuron-oligodendrocyte (OL) co-cultures from either the embryonic day 16 (E16) rat spinal cord or cerebral cortex. The dissociated cells are plated directly on poly-L-lysine-coated cover slips and maintained in a modified myelination medium that supports both OL and neuron differentiation. The spinal cord derived OL progenitor cells develop quickly into myelin basic protein (MBP)+ mature OLs and start to myelinate axons around 17 days in vitro (DIV17). Myelination reaches its peak around six weeks (DIV40) and the typical nodes of Ranvier are revealed by paranodal proteins Caspr and juxaparanodal protein Kv1.2 immunoreactivity. Electron microscopy (EM) shows typical myelination cytoarchitecture and synaptic organization. In contrast, the cortical-derived co-culture requires triiodothyronine (T3) in the culture medium for myelination. Finally, either hypomyelination and/or demyelination can be induced by exposing proinflammatory cytokines or demyelinating agents to the co-culture, suggesting the feasibility of this modified in vitro myelination model for myelin-deficit investigation.

Metabolomics-based discovery of a metabolite that enhances oligodendrocyte maturation

“…Cells were allowed to attach for 2 h and then coverslips were rinsed with PBS before being transferred to a 24-well plate containing 0.5 mL growth media consisting of: Neurobasal medium, B27 supplement (Gibco), 0.5× Glutamax (Thermo), 5 µg/mL gentamycin and 5 µM glutamic acid. Half of the cell media was replaced after 3 days in culture with supplemental growth media not containing glutamic acid 46 . After 5 days in culture, rat-derived cortical OPCs were added to the neuronal culture at a density of 5,000 cells/cm 2 .…”

Axonal abnormalities in vanishing white matter

“…Astrocytes or forebrain mixed cells were isolated from cerebral hemispheres of E18 mice and cultured following previously described methods with minor modifications 27, 28, 29, 30, 31. Cultures were kept in a humidified incubator at 37°C and 7.5% CO 2 at all times.…”

Live-imaging in the CNS: New insights on oligodendrocytes, myelination, and their responses to inflammation

“…Despite this drawback, a simple model for autoimmune mediated OL and myelin injury suggests that they still hold value for future studies examining OL-axon interactions within the injury context (Pang et al, 2012). In this model, coapplications of MOG antibody and complement produced a robust demyelination in co-cultures of CNS-derived primary OPC and neurons.…”

“…In this model, coapplications of MOG antibody and complement produced a robust demyelination in co-cultures of CNS-derived primary OPC and neurons. Additionally, the commonly used membranesolubilizing agent lysolecithin can also be used to demyelinate these cultures (Pang et al, 2012). Complex cultures containing multiple neural cell types dissociated from the CNS can also provide a myelinating culture suitable for the study of myelin injury and repair (Boomkamp et al, 2012).…”