Effects of acetylcholinesterase overexpression in murine embryonic stem cells and on their differentiation

It has long been acknowledged that acetylcholinesterase (AChE) also displays a large set of non-cholinergic functions. These functions have been attributed to both developmental and degenerative processes. Our previous gene and protein expression studies demonstrated that AChE and other components of the cholinergic system are expressed in murine embryonic stem cells (ESCs). To investigate the function of AChE during early embryonic neurogenesis, we examined the expression of this protein and its role in proliferation and differentiation of murine ESC cells into neural precursor cells (NPC). We stably transfected the D3-stem cell line with a plasmid encoding the synaptic form of mouse AChE. Cells from the same passage were also transfected with a mutated form of AChE (which leads to the ER retention of the cho-linesterases) or GFP and used as experimental control. We examined at the stem cell stage parameters as cell vitality and proliferation. AChE overexpression led to a significant decrease of cell proliferation, but didn't affect cell vitality. Stably transfected cells were differentiated toward neural precursor cells following an ITSF protocol and their differentiation was compared with differentiation of control cells. Expression of stem cell and neuronal markers was examined by RT-PCR, qPCR and immunohistochemistry. Interestingly, the AChE stem cells expressed all tested stem cell markers for an extended period during the differentiation protocol. We also found that AChE overexpression led to a more efficient differentiation of stem cells into neuronal cell type, by comparing expression of nestin, beta3-tubulin, N-CAM.