In Vitro Formation of Enteric Neural Network Structure in a Gut‐Like Organ Differentiated from Mouse Embryonic Stem Cells
In Vitro Formation of Enteric Neural Network Structure in a Gut‐Like Organ Differentiated from Mouse Embryonic Stem Cells
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DOI：
10.1634/stemcells.2005-0394
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发表时间：
2006-06
期刊：
影响因子：
5.2
通讯作者：
M. Takaki;S. Nakayama;H. Misawa;T. Nakagawa;H. Kuniyasu
中科院分区：
文献类型：
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作者：
M. Takaki;S. Nakayama;H. Misawa;T. Nakagawa;H. Kuniyasu
使用胚状体（EB）培养系统，我们从小鼠胚胎干（ES）细胞（ES gut）中开发了一种功能性器官样簇-“肠”。每个ES肠均表现出自发收缩，但未表现出明显的痉挛样运动。在这些自发收缩的ES肠道中，明确鉴定了Cajal间质细胞（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）和平滑肌细胞的密集分布;然而，在自发分化的ES肠道中不存在肠神经节。通过仅在EB形成期间添加脑源性神经营养因子（BDNF），我们首次成功地在ES肠道中体外形成具有连接神经纤维束（肠神经系统[ENS]）的肠神经节。带ENS的ES肠表现出强烈的肠蠕动样运动。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是从肠神经嵴衍生细胞分化而来的。此外，局灶性刺激ES肠ENS引起细胞内Ca 2+浓度（[Ca 2 +]i）在单个或多个网站的衰减阿托品或消除河豚毒素的传播增加。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。我们首次通过在ES肠道中外源性添加BDNF成功地分化了ENS中的功能性神经元，导致产生了明显的类神经元运动。
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 exhibit distinct peristalsis‐like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (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 (enteric nervous system [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 that the present ENS is differentiated from enteric neural crest‐derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca2+ concentration ([Ca2+]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.