Temporospatial relationships between macroglia and microglia during in vitro differentiation of murine stem cells

Embryonic stem (ES) cells of the permanent line BLC6 derived from a 129/Sv Gat mouse blastocyst were differentiated as spheroid aggregates (embryoid bodies, EBs) in the presence of retionic acid. After 2 days in suspension, EBs were plated on gelatine-coated glass coverslips and cultivated for 5, 9, and 16 days post plating (DPP) in normal medium. In this study we investigated whether the well-known retinoic acid-induced differentiation of ES cells into neurons (identified by immunostaining for neuron-specific enolase and synaptophysin) was accompanied by cells expressing astroglial (GFAP), oligodendroglial (O4), and microglial(5C6, galectin-3) markers. Whereas differentiation of neurons was closely related to their centrifugal migration towards the periphery of the EBs, the maturation of neuroglia followed a strict time-dependent manner. At 5 DPP, only neurons but no cells expressing glia-specific markers, were observed. At 9 DPP, GFAP-positive and O4-positive macroglial cells appeared. At 16 DPP, microglial cells (5C6-positive and galectin-3-positive) occurred. The established dynamic of relationships between neuronal and nonneuronal cells shows that the model of EBs is similar to the sequence differentiation of the nervous tissue. Thus, enabling in vivo observation of neurons, astrocytes, oligodendrocytes, and microglia, the model of EBs provides a basis for further investigations on the relationships between neurons and neuroglia under various experimental conditions.