Abstract
Oligodendrocytes are well known as myelin-forming cells in the central nervous system (CNS). However, detailed mechanisms of oligodendrocyte differentiation and myelination are poorly understood, particularly due to the difficulty of the purification of murine oligodendrocyte precursor cells (OPCs). We have recently established a transgenic mouse line that expresses a fluorescent protein Venus under the promoter of Sox10, whose expression is restricted to OPCs and oligodendrocytes in the CNS. Here, we have characterized Venus-positive cells from the Sox10-Venus mouse brain for analyzing oligodendrocyte differentiation. We first purified Venus-positive cells from the postnatal day 0–2 brain by flow cytometry. Most of the Venus-positive cells expressed NG2, an OPC marker. After induction of differentiation, an increased population of galactocerebroside-positive oligodendrocytes and decrease of OPCs were observed in the Venus-positive culture. Furthermore, a time-lapse analysis showed that Venus-positive oligodendrocytes dynamically changed their morphology with highly branched cell processes during differentiation. In addition, we found that Venus-positive OPCs were able to differentiate to type II astrocytes. In vivo, OPCs and oligodendrocytes express Venus, and some of astrocytes were positive for Venus in the ventral cortex. Taken together, the Sox10-Venus mouse system is useful for analyzing differentiation and multipotency of murine OPCs.
Highlights
Isolation of rat oligodendrocyte precursor cells (OPCs) from the central nervous system (CNS) has been previously established, it is still less efficient to obtain sufficient quantity and purity of mouse OPCs5
Quantitative RT-PCR using cells after the cell sorting revealed that Venus (+) cells highly expressed an OPC marker neural/glial antigen 2 (NG2), but not markers for oligodendrocytes: myelin associated glycoprotein (MAG), astrocytes: glial fibrillary acidic protein (GFAP) and glutamate/aspartate transporter (GLAST), microglia: ionized calcium-binding adapter molecule 1 (Iba1), or neurons: neuronal nuclei (NeuN), compared with control whole cells without cell sorting (Fig. 1b)
We found that Venus (+) cells were positive for Adenomatous Polyposis Coli (APC), in addition to NG2, but negative for GFAP, Iba[1], or NeuN (Fig. 6a)
Summary
Isolation of rat OPCs from the CNS has been previously established, it is still less efficient to obtain sufficient quantity and purity of mouse OPCs5. Another marker PDGFRα is available for immunopanning of OPCs from mouse cortices[10] This is a useful and established method, but the possibility exists that in general, antibodies used for sorting may affect the cells during culture or analysis[11]. This problem can be overcome by using a fluorescent protein expression system under an OPC/oligodendrocyte-specific promoter. Several transgenic mouse lines that express a fluorescence protein DsRed or GFP under the regulation of OPC genes Cspg[4] and Pdgfra, respectively, have been developed to analyze the fate of oligodendrocyte lineage cells[12,13,14]. We here propose that Sox10-Venus mice could be a useful tool for purifying and analyzing oligodendrocyte lineage cells
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