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

首先由申请人与来自美国和日本的研究小组一起描述的非神经元胆碱能系统同时在全世界科学界被接受。乙酰胆碱由原始生物体（细菌、原生动物、植物）以及或多或少所有所谓的非神经元细胞（包括人类生物体（例如上皮细胞、内皮细胞、间充质细胞和免疫细胞））合成。非神经元乙酰胆碱通过刺激烟碱和毒蕈碱受体的自分泌和旁分泌机制参与细胞增殖和分化的调节。胚胎干的特征在于它们分化成上述体细胞类型的能力。 在过去的2年中，申请人已经证明了在鼠干细胞系CGR 8中乙酰胆碱的合成和乙酰胆碱的显著高的分数释放速率（2%/min）。除了迄今未知的途径外，释放还由有机阳离子转运蛋白介导。合成酶胆碱乙酰转移酶位于细胞质和细胞核内。在去除白血病抑制因子（LIF）后，CGR 8细胞失去其多能性并显示早期分化。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。在本申请中，将研究上调的细胞机制。因此，将检查与已知干扰多能性状态的细胞内信号转导途径的相互作用（STAT 3、PI 3 PK、MAPK、AC、PKA和PKC）。此外，它将被测试，是否类似的机制是积极的诱导干细胞（iPS）和非人灵长类动物的胚胎干细胞。在与iPS的背景下，它必须被测试，是否胆碱能系统之间的原始分化的体细胞系和诱导多能细胞系和可能的关键信号修改胆碱能系统的不同，应确定。此外，将测试烟碱和毒蕈碱受体的表达在分化期间是否会改变。总之，目前的“后续应用”应允许，阐明分化和去分化的调节过程中的多能性的前沿通过研究胆碱能系统。

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.