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

非神经性胆碱能系统最初由申请者与来自美国和日本的一个研究小组一起描述，同时也被世界科学界接受。乙酰胆碱是由原始生物体(细菌、原生动物、植物)以及几乎所有所谓的非神经细胞(如上皮细胞、内皮细胞、间充质细胞和免疫细胞)合成的。非神经元性乙酰胆碱通过自身和旁分泌机制刺激烟碱和毒扁豆碱受体参与细胞增殖和分化的调节。胚胎干细胞的特点是具有分化为上述体细胞类型的能力。在过去的两年中，申请人证明了乙酰胆碱的合成，并在小鼠干细胞系CGR8中证明了乙酰胆碱的显著高分段释放率(每分钟2%)。除了迄今未知的途径外，该释放还由有机阳离子转运体介导。合成酶胆碱乙酰转移酶定位于胞浆和胞核。去除白血病抑制因子(LIF)后，CGR8细胞失去其多能性，并表现出早期分化。此时，乙酰胆碱的合成、细胞含量和释放显著上调，这可能表明其在早期分化中的功能作用。在目前的应用中，我们将研究上调的细胞机制。因此，与细胞内信号转导通路(STAT3、PI3PK、MAPK、AC、PKA和PKC)的相互作用将被研究。此外，还将测试在诱导干细胞(IPS)和非人类灵长类动物的胚胎干细胞中是否存在类似的机制。在iPS的背景下，必须测试原始分化的体细胞系和诱导的多能细胞系之间的胆碱能系统是否不同，以及是否应该识别修改胆碱能系统的可能的关键信号。此外，还将测试在分化过程中尼古丁和毒碱受体的表达是否会发生变化。总之，目前的“后续应用”应该可以通过研究胆碱能系统来阐明多能性前沿中分化和去分化的调节过程。

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.