Purpose of Study Preoligodendrocytes (preOLs) are injured in periventricular leukomalacia as shown by histopathological analysis of postmortem brain specimens (Haynes et al, 2003). One difficulty for researchers is the isolation of preOLs from animals. Protocols are now available to culture preOLs from various cell lines including mouse embryonic stem (ES) cells that are extremely labor intensive. The aim of this study was to develop a simple system that would produce preOLs.
Methods Mouse ES cells were ordered from American Tissue Culture Company. The cells were plated at a density of 3×106 cells per T25 flask and allowed to grow to 90% confluence. The cells were then passed 1:3 in Complete Dulbecco's Modified Essential Medium supplemented with 1× β-mercaptoethanol (BME) and 1× leukemia inhibitory factor (LIF). Cytosine arabinofuranoside (Ara-C) was added to the flasks at 5 μM for 48 hours and then the cells were prepared for experimental conditions. The cells derived from this protocol were compared to the standard ES cell 4-/4+ protocol. Standard immunocytochemistry, immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) were performed. Markers used for immunocytochemistry/immunoblot were the oligodendrocyte 1 (O1) and oligodendrocyte 4 (O4) antibodies. Forward and reverse primers for O1, O2, NG2, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were used for RT-PCR.
Results Standard culture techniques were compared to the modified in vitro system demonstrating both preOLs (O4+) and mature oligodendrocytes (O1+ /MBP+) were discovered using the modified technique. Under the experimental conditions some cells were also GFAP+ cells.
Conclusions PreOLs and mature oligodendrocytes are formed using this modified in vitro system. Other neural precursors are also formed as a minor proportion of the cells, which is independent of the standard retinoic acid neural induction pathway. Thus, this technique offers a novel method of producing preoligodendrocytes, which can be used as a tool to help characterize possible protective strategies against injury.
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