Gap Junctional Intercellular Communication during Neuronal Differentiation of Mouse Embryonic Stem Cells under Dispersed Culture in vitro

Mammalian brains consist of a variety of cells such as neurons,astrocytes,oligodendrocytes and vascular endothelia. It is believed that appropriate cell- cell interaction is essential for the development of the mammalian brain. To clarify changes in gap junctional intercel-lular communication during the early stages of neural differentiation,we established an in vitro system for neural induction from mouse embryonic stem (ES) cells and analyzed their capability of gap junctional intercellular communication by microinjection tracer coupling with neurobiotin.Undifferentiated ES cells were culturedirL SuSpenSion for8days and treated with all-trans retinoic acid(500 nM) during the latter half of tlle period. Thereafter,Cells were transferred to adhesion culture. Neurites were observed in more than 90% of embryoid bodies at day 6 after the beginning of adhesion culture. Concerning neural markers, in small clusters at day 1 of adhesion culture cells were positive for A2B5, which is known to be expressed from the neuroepithelial stage in vivo. Cells with neurites showed positive signals of microtuble-associated protein 2(MAP2)and neurofilament M subunit, markers for more differentiated neurons on and after day 3 of adhesion culture. Utilizing this in vitro neural-differentiation system from ES cells, We dispersed embryoid bodies by treatment with trypsin and developed a dispersed cell culture system for early neuronal cells. In this system,cells positive for neuronal markers formed small colonies after one day of culture. Neurobiotin microinjected into a single cell was found to spread into all cells constituting colonies that expressed A2B5 at day 1 of adhesion culture after dispersion, but the dye did not spread into flat cells when there was no A2B5 expression around the colony. On the other hand, after day 3 of adhesion culture after dispersion, neurobiotin injected into a single cell in the colony was restricted to small portions of the colony. Connlexin26 was expressed in neural cells within the colony. These results suggest that compartmentalization of gap junctional communication was stabiished between neuron cells and other cell lineages and that the capability of communication within neural cells decreases rapidly during the process of neural differentiation