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

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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.