Forming control mechanism on pacemaker in the gut differentiated from embryonic stem cells.

利用拟胚体（EB）培养系统，我们从小鼠胚胎干（ES）细胞（ES gut）中培养出了一个功能性器官样细胞簇，即“肠”。每个ES肠表现出自发性收缩，但没有明显的痉挛样运动。在这些自发收缩的ES肠道中，密集分布的Cajal间质细胞（ICC）（c-kit，一种具有酪氨酸激酶活性的跨膜受体，阳性细胞;肠道起搏细胞）和平滑肌细胞被清楚地鉴定;然而，肠神经节在自发分化的ES肠道中不存在。通过仅在EB形成过程中加入脑源性神经营养因子（BDNF），我们首次成功地在体外形成了ES肠中具有连接神经纤维束（ENS）的肠神经节。具有ENS的ES肠表现出强烈的痉挛样运动。在BDNF+培养基中进行EB培养期间，我们检测了与trk原癌基因（trkB; BDNF受体）和神经嵴标志物、原癌基因酪氨酸蛋白激酶受体ret前体（c-ret）、p75或sox 9相关的每种免疫反应性。这些结果表明，本ENS是从肠神经嵴来源的细胞分化。此外，用ENS局灶性电刺激ES肠可引起单个或多个部位细胞内Ca^<2+>浓度（[Ca^<2+]_i）的扩散性增加，阿托品可减弱这种增加，河豚毒素可消除这种增加。这些结果表明，在体外形成的生理功能的肠胆碱能兴奋性神经元。我们第一次成功地在ENS的功能性神经元的分化，通过外源性添加BDNF在ES肠道，导致在生成不同的类神经元的运动。此外，我们发现，自发分化的ICC和BDNF分化的ENS的共存是必不可少的，导致非常相似的水肿样运动在ES肠道在体内肠道。

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not distinct peristalsis-like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICCs)(c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain-derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (ENS) in the ES gut. The ES gut with ENS exhibited strong peristalsis-like movements. During EB culture in BDNF+ medium, we detected each immunoreactivity associated with the trk proto-oncogenes (trkB; BDNF receptors) and neural crest marker, proto-oncogene tyrosine-protein kinase receptor ret precursor (c-ret), p75 or sox9. These results indicated the present ENS is differentiated from enteric neural crest-derived cells. Moreover, focal electrical stimulation of ES guts with ENS elicited propagated increases in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis-like movements. Moreover, we found that the coexistence of spontaneously differentiated ICC and BDNF-differentiated ENS is essential to cause very similar peristalsis-like movements in ES guts to those in in vivo guts.