Expression and function of the non-neuronal cholinergic system in murine embryonic stem cells

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The non-neuronal cholinergic system, which has been described at first by the applicants together with a research group from the States and Japan, is meanwhile accepted in the scientific community worldwide. Acetylcholine is synthesized by primitive organisms (bacteria, protozoa, plants) as well as by more or less all so called non-neuronal cells including the human organism (for example epithelial, endothelial, mesenchymal and immune cells). Non-neuronal acetylcholine is involved via auto- and paracrine mechanisms stimulating nicotinic and muscarinic receptors in the regulation of cellular proliferation and differentiation. Embryonic stem are characterized by their potency to differentiate into the somatic cell types indicated above. In the last 2 years the applicants have demonstrated the synthesis of acetylcholine and a remarkable high fractional release rate (2% per min) of acetylcholine in the murine stem cell line CGR8. The release is mediated by organic cation transporters in addition to so far unknown pathways. The synthesizing enzyme choline acetyltransferase is localized within the cytosol and the cell nucleus. Upon removal of leukemia inhibitory factor (LIF) CGR8 cells lose their pluripotency and show early differentiation. At this time the synthesis, cell content and release of acetylcholine is substantially up-regulated, which may indicate its functional role in early differentiation. In the present application the cellular mechanism of the upregulation will be investigated. Therefore, the interaction with intracellular signal transduction pathways known to interfer with the state of pluripotency will be examined (STAT3, PI3PK, MAPK, AC, PKA and PKC). Moreover it will be tested, whether comparable mechanisms are active in induced stem cells (iPS) and in embryonic stem cell of non-human primates.In context with iPS it has to be tested, whether the cholinergic system differs between the original differentiated somtic cell line and the induced pluripotent cell line and possible key signals modifying the cholinergic system should be identified. Moreover it will be tested whether the expression of nicotinic and muscarinic receptors will be changed during differentiation. In conclusion, the present "follow-application" should allow, to illuminate regulation processes of differentiation and de-differentiation within the cutting edge of pluripotency by investigating ther cholinergic system.