Studies on gut differentiated from mouse embryonic stem cells


（Misawa, Matsuyoshi, Takaki）

　Recently, embryonic stem (ES) cells gave rise to a functional organ-like unit, the "gut" (ES gut), which was manifested with spontaneous motility and topographically well-organized by the enteric derivatives of all three embryonic germ layers: epithelial cells (endoderm), smooth muscle cells and interstitial cells of Cajal (ICC) (mesoderm), and enteric neurons (ectoderm). On approximately day-21 outgrowth culture, the ES gut showed distinct and highly coordinated contraction patterns with regular rhythms. This mechanical activity was composed of periodic contraction and relaxation. It was very similar to gastrointestinal (GI) motilities (Fig. 1, 2) .

ES Cell Culture.
Undifferentiated ES cells (EB3) were maintained on gelatin-coated dishes without feeder cells in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; GIBCO/BRL, Grand Island, NY), 0.1 mM 2-mercaptoethanol (Wako, Tokyo, Japan), 0.1 mM non-essential amino acids (GIBCO/BRL), 1 mM sodium pyruvate (Bio-WHITTAKER,), and 1000 U/ml of LIF (CHEMICON, Temecula, CA). The EB3 cells (a kind gift from Dr. Hitoshi Niwa, CDB of RIKEN, Kobe) carried the blasticidin S-resistant selection marker gene driven by the Oct-3/4 promoter (active in undifferentiated cells) and were maintained in medium containing 10 ?g/ml blasticidin S to eliminate differentiated cells. To induce EB formation, dissociated ES cells were cultured in hanging drops as previously described with minor modifications. The cell density of one drop was 500 cells per 15 ?l of ES cell-medium in the absence of LIF.
In vitro formation of enteric neural network structure
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 (not peristalsis observed in in vivo gut). 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 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 (Fig. 3).

　　
Fig.1 Fig.2
Fig.3

References

１. Yamada, T., Yoshikawa, M., Takaki, M., Torihashi, S., Kato, Y., Nakajima, Y., Ishizaka, S. and Tsunoda, Y. (2002). In vitro function gut-like organ formation from mouse embryonic stem cells. Stem Cells 20, 41-49.

２. Ishikawa, T., Nakayama, S., Nakagawa, T., Horiguchi K., Misawa, H., Kadowaki, M., Nakao, A., Inoue, S., Komuro, T., and Takaki, M.: Characterization of in vitro gutlike organ formed from mouse embryonic stem cells. Am J Physiol Cell Physiol 286, C1344-C1352, 2004.

３．Takaki M, Nakayama S, Misawa H, Nakagawa T, Kuniyasu H: In vitro Formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells. Stem Cells 24: 1414-1422, 2006.

４．Takaki M, Misawa H, Shimizu J, Kuniyasu H, Horiguchi K: Inhibition of gut pacemaker cell formation from mouse ES cells by the c-kit inhibitor. Biochemical and Biophysical Research Communications 359: 354-359, 2007.

５． Miyako Takaki, Hikaru Suzuki, Shinsuke Nakayama: Recent advances in studies of spontaneous activity in smooth muscle: ubiquitous pacemaker cells. Prog. Biophys. Mol. Biol.　102: 129-135, 2010.

６．Yi Luo, Miyako Takaki, Hiromi Misawa, Hiroko Matsuyoshi, Tomonori Sasahira, Yoshitomo Chihara, Kiyomu Fujii, Hitoshi Ohmori, Hiroki Kuniyasu:　Determinants of the epithelial-muscle axis on embryonic stem cell-derived gut-like structures.　Pathobiology （in press）, 2010.