Quantitation and functional characterization of neural cells derived from ES cells using nestin enhancer-mediated...

To gain insight into early events of neurogenesis, transgenic embryonic stem (ES) cells were generated using the enhanced green fluorescence protein (EGFP) reporter gene under the regulatory control of the neural stem cell marker, nestin. The expression of EGFP in undifferentiated ES cells suggested that the onset of endogenous nestin occurred before neurulation. Upon differentiation of ES cells, the EGFP expression became confined to the neural lineage and asynchrony in ES-cell-derived neural differentiation was evident. The EGFP intensity was prominent in the proliferative progenitors and unipolar neurons, whereas downregulation occurred in differentiating bi- and multipolar neurons. This was corroborated quantitatively using flow cytometry where maximal generation of neural progenitors was observed 4-12 days post-plating. The proliferative potential of neural progenitors as well as glia, in contrast to post-mitotic neurons, was also evident by time-lapse microscopy. The functional characterization of progenitors revealed an absence of voltage-activated inward currents, whereas the Na+ current (INa) was detected prior to Ca2+ currents (ICa) in differentiating neurons. Additionally, inhibitory receptor-operated channels could be detected at these early stages of development in bi- and multipolar neurons suggesting that the pre-committed progenitors had retained their intrinsic ability to give rise to functional neurons. This study sheds new light on early events of neurogenesis defining the quantitative and qualitative aspects and demarcating the functional neural cell types from ES cells in vitro.