Synchronization, Neuro-differentiation And Amyloid-β Peptides Inducing NFκB Activation During Differentiation From Mouse Embryonic Stem Cells To Neuronal Cells In Vitro

Mouse ESC (embryonic stem cells) are cell lines derived from ICM (inner cell mass) of the 3.5-4 days blastocysts, and can be differentiated into neuronal cells in vitro by RA or lineage selection. In these protocols, the differentiational capacity of EBs (embryoid bodies) plays an important role in the follow differentiation. The status of EBs development will decide whether this ESC can successfully grow and further differentiate into functional neuronal cells. ESC differentiated neuronal cells could act as powerful models of neural development in neuroscience experiment in vitro. It is unknown that the states of cell cycle effect on the differentiation from ESC to neuronal cells. At early stages during the differentiation of EBs, bFGF (basic fibroblast growth factor) is required for differentiation of neuronal cells from ES cells by applying lineage selection. In addition, ESC differentiated neuronal cells could act as a neural development model to search the pathogenesis of AD (Alzheimer's disease). The neurotoxic Aβ (β-amyloid peptide) is a potent inducer of NFkB (nuclear factor kB) in primary neurons. It is unknown whether NFkB pathway could be activated by A|3 from the neuronal cells which ESC differentiated into just.First, an optimal protocol was proved in the differentiation from ESC into neuronal cells. ES derived neuronal cells were identified by the assay of specific neuronal antigen of Nestin, MAP2 and NF-M. NMDA acceptor subunits of R2A and R2B expressed in differentiated neuronal cells by assay of RT-PCR. The free cytoplasmic calcium was labeled by fluorescence indicator Fluo-3-AM, and was measured by CLSM (confocal laser scanning microscopy) after Glu stimulation. The change of [Ca~2+] could be blocked by MK801 (0.6 mM) in the differentiated neuronal cells.Second, two ways of serum removing and the cell cycle inhibitor, thymidine and colchicines were used, to synchronize ESC before differentiation. The parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen (SSEA)-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher thanthat in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully. EBs formed from synchronized ES cells has higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells.Third, the bFGF-induced release of glutamate was measured over four successive minutes in 4, 8 and 12 days differentiated neuronal cells. Changes of Ca2+ concentration labeled by fluorescence intensity occurred in response to minimal concentrations of glutamate. The Ca2+ concentration was changed more rapidly by bFGF than by glutamate during the early stages of differentiation of ES-derived neurons. High concentrations of the inhibitor MK801 applied before bFGF stimulation caused a dramatic decrease of fluorescence intensity in neurons derived from both hippocampal and ES cells. These results showed that bFGF serves not only as a neurotrophin but also as a stimulator of excitability during the differentiation of ES cells into neurons.At last, ESC was first differentiated into functional neuron using an improved RA-induced method. Exposure of differentiated neurons to aggregated Api-42 induced the activation of NFkB. NFkB was only activated in the neuron after differentiation for 12 days, suggesting a different effect at different differentiated stages. Further analysis of the molecular mechanisms revealed the subunit p65 and p50 participated in activation of NFkB in neuron derived from ESC after differentiation of 12 days. These results strongly suggested that ApM2 selectively affected the cell activity of aged neuron derived from ESC via NFkB activation pathway.