Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An In Vitro Axon-Oligodendrocyte Interaction Model

Abstract

Mechanisms that control the differentiation and function of oligodendrocytes in the central nervous system are complex and involve multiple inputs from the surrounding environment, including localized concentrations of growth factors and the extracellular matrix. Dissection and analysis of these inputs are key to understanding the pathology of central nervous system demyelinating diseases such as multiple sclerosis, where the differentiation of myelinating oligodendrocytes from their precursors underlies the remission phase of the disease. In vitro co-culture models provide a mechanism for the study of factors that regulate differentiation of oligodendrocyte precursors but have been difficult to develop due to the complex nature of central nervous system myelination. This study describes development of an in vitro model that merges a defined medium with a chemically modified substrate to study aspects of myelination in the central nervous system. We demonstrate that oligodendrocyte precursors co-cultured with rat embryonic motoneurons on non-biological substrate (diethylenetriamine trimethoxy-silylpropyldiethylenetriamine), can be induced to differentiate into mature oligodendrocytes that express myelin basic protein, using a serum-free medium. This defined and reproducible model of in vitro myelination could be a valuable tool for the development of treatments for demyelinating diseases such as multiple sclerosis.

Keywords: Co-Culture; DETA; In Vitro; MBP; Motoneurons; Oligodendrocytes; Serum-Free.

Fig. 1

Schematic representation of co-cultures of OPCs with EMNs in defined in vitro system. Diagram illustrates the main steps in the OPCs–EMNs co-culture and development of a defined in vitro system. This system involves modification of a glass coverslip with a monolayer of the synthetic, growth promoting substrate DETA. Cells attached to DETA were cultured in the serum free medium shown, containing factors that are optimal for cell survival and the differentiation of OPCs.

Fig. 2

Characterization of newly isolated OPCs from the rat pup cortex. (A) A typical bipolar morphology of OPCs 24 hrs after “shake-off” isolation. (B) Results of immunocytochemical analysis revealed that 78.6±3.1% of cells expressed A2B5 and 53.6±4.7% of cells expressed O4. (C) Immunocytochemistry for A2B5 and O4 expression 24 hrs after shake off. Scale bars 100 µm.

Fig. 3

Expression of markers of oligodendroglial lineage in co-culture. (A) Light microscopy images indicating the morphology of OPCs at day 5, 10 and 20 in co-culture. (B) Immunocytochemical analysis detecting MBP, NF–H and DAPI at day 5, 10 and 20 of co-culture. Images illustrate details of mature ramified oligodendrocytes at day 5 and oligodendrocytes wrapping their processes around axons at day 10 and 20. Scale bars 100 µm.

Fig. 4

Co-culture of OPCs with EMNs and MBP expression on a line pattern. (A) Light microscopy images of co-cultures grown on line patterns composed of DETA/SiPEG SAMs, for 20 days. (B) Immunocytochemical analysis of mature MBP positive oligodendrocyte interacting with multiple NF-positive axons. Scale bars 100 µm.