Fast Isolation and Characterisation of Rat Oligodendrocyte Precursor Cells for Differentiation and Proliferation Studies

Dysmyelinating and demyelinating diseases are characterised by impaired production or loss of myelin that ensheathes neuronal axons, respectively. Diseases such as multiple sclerosis and leukodystrophies are increasing in prevalence globally and, with a lack of available treatments to repair or mitigate myelin loss, further efforts are required to develop effective therapeutics.

Mature oligodendrocytes that differentiate from proliferating oligodendrocyte precursor cells (OPCs) are responsible for myelin production. Current in vitro approaches to study myelination often utilise immortalised human cell lines, though the suitability of these cells as functionally relevant in vitro models of oligodendrocytes is debated.

Our method for rapidly isolating primary rat OPCs can be used to study different cell properties, including proliferation. In brief, brain tissue isolated from neonatal rats was digested enzymatically and OPCs were subsequently isolated using O4⁺ magnetic selection, a protein that is expressed on the surface of oligodendrocyte lineage cells. Immunofluorescent staining was performed using antibodies against several oligodendrocyte lineage markers to determine the differentiation and maturity of cells at different stages in culture. Over 11 days, changes in OPC markers were observed, while myelin basic protein levels remained very low, indicating that OPCs had not yet differentiated into mature oligodendrocytes. The proliferation of OPCs was measured using a CellTiter-Glo assay. A slight increase in OPC growth rate was seen when cells were cultured in the presence of growth factors, demonstrating the utility of the assay in measuring the effect of pharmacological agents that may alter OPC growth properties. Additionally, literature precedent indicates that this method can feasibly be adapted to produce mature oligodendrocytes, enabling the investigation of treatments to modulate myelin production.

Our optimised protocol is an effective method for culturing rat OPCs that can be used as a model to investigate cell differentiation and proliferation, serving as a valuable platform for drug discovery studies.