Genetically unmodified neural precursor cells (NPCs) derived from rate embryonic cortex were stimulated with Long-epidermal growth factor in vitro to form multicellular neurospheres. In our study, histology and transmission electron microscopy was for the first time utilized for analysis of three-dimensional neurospheres. Immunohistochemical phenotypization carried out on paraffin-embedded section revealed that most cells in the neurosphere expressed high levels of vimentin that is normally found in immature neural cells. Majority of glial cells expressed glial fibrillary acidic protein (GFAP) that is a specific marker for differentiated astrocytes. On the other hand, myelin basic protein (MBP), a marker of oligodendrocytes, was found only in a discrete subpopulation of glial cells. Similarly, electron microscopy recognized only rate myelin sheaths surrounding axons. Some cells displayed neuron-like morphology and were labelled with antibodies recognizing the neuronal antigens: 210 kDa neurofilaments (NF 210) and microtubule associated protein-2 (MAP-2). Surprisingly well-developed interneuronal synapses were found electronmicroscopically. The presence of synaptic vesicles was also confirmed by immunohistochemical detection of synaptophysin. Our results show that NPCs in neurospheres gradually differentiate into glial and neuronal cells.

Department of Histology and Embryology Charles University Faculty of Medicine