In Vitro Developmental Model of the Gastrointestinal Tract from Mouse Embryonic Stem Cells

2007-10Mouse embryonic stem (ES) cells are pluripotent and retain their potential to form cells, tissues and organs originated from three embryonic germ layers. Recently, we developed in vitro organ -gut‐like structures- from mouse ES cells. They had basically similar morphological features to a mouse gastrointestinal tract in vivo composed of three distinct layers (i.e., epithelium, connective tissue and musculature). Gut‐like structures showed spontaneous contractions derived from pacemaker cells (interstitial cells of Cajal) in the musculature. We also examined their formation process and expression pattern of transcription factors crucial for gut organogenesis such as Id2, Sox17, HNF3β/Foxa2 and GATA4. We found that they mimic the development of embryonic gut in vivo and showed a similar expression pattern of common transcription factors. They also maintain their developmental potential after transplantation to a renal capsule. Therefore, gut‐like structures are suitable for in vitro models of gastrointestinal tracts and their development. In addition, we pointed out several unique features different from gut in vivo that provide useful and advantageous tools to investigate the developmental mechanism of the gastrointestinal tract.departmental bulletin pape

ICCの発生

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マウスES細胞を用いた消化管様構造の形成

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues including representatives of all three germ layers in the embryo. We previously reported that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs) (Stem Cells 20:41-49,2002). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e., epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut- like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neuron and ICC development.journal articl

Calcium oscillation linked to pacemaking of interstitial cells of Cajal;Requirement of calcium influx and localisation of TRP4 in caveolae

Interstitial cells of Cajal (ICC) are considered to be pacemaker cells in gastrointestinal tracts. ICC generate rhythmical electronic activity as slow waves, which is dihydropyridine-insensitive, and drive spontaneous contraction of smooth muscles. Although cytosolic Ca2+ has been assumed to play a key role in pacemaking, Ca2+ movements in ICC have not yet been examined in detail. In the present study, using cultured cell clusters isolated from the mouse small intestine, we demonstrated Ca2+ oscillations in ICC. Fluo4 was loaded to the cell cluster, the relative amount of cytosolic Ca2+ was recorded, and ICC were identified by c-Kit immunoreactivity. We specifically detected Ca2+ oscillation in ICC in the presence of dihydropyridine, which abolishes Ca2+ oscillation in smooth muscles. The oscillation was coupled to the electrical activity corresponding to slow waves and depended on Ca2+ influx through a non-selective cation channel, which was SK&F 96365-sensitive and store-operated. We further demonstrated the presence of transient receptor potential-like channel 4 (TRP4) in caveolae of ICC. Taken together, the results infer that the Ca2+ oscillation in ICC is intimately linked to the pacemaker function and depends on Ca2+ influx mediated by TRP4.journal articl

Cajal細胞の形態と機能

The unique cells, interstitial cells of Cajal (ICC) are distributed in the musculature of the gastrointestinal tract and express receptor tyrosine kinase c-Kit. ICC are located in the proximity with enteric neurons and connected with each other to make cellular network. One of their functions is pacemakers of the spontaneous smooth muscle contractions. They generate electrical rhythmicity as slow waves and propagate to smooth muscles. ICC show oscillations of cytosolic calcium linked to slow waves. ICC also mediate neurotransmission from enteric neurons, and regulate excitatory and inhibitory neurotransmissions. They express several receptors of neurotransmitters. Therefore ICC are key cells of gastrointestinal motility.journal articl

2017 HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: executive summary.

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2017 HRS/EHRA/ECAS/APHRS/SOLAECE expert consensus statement on catheter and surgical ablation of atrial fibrillation: executive summary.

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In vitro organogenesis of gut-like structures from mouse embryonic stem cells

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues including representatives of all three germ layers in the embryo. We previously reported that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs) (Stem Cells 20:41-49, 2002). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e., epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neuron and ICC development.journal articl

Resident macrophages activated by lipopolysaccharide (LPS) suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer

A great number of macrophages is found to be evenly distributed in the muscle layer of the gastrointestinal tract. We investigated their effects on smooth muscle contraction and the initiation of immune reactions such as inflammatory responses. Macrophages were demonstrated by uptake of FITC-dextran and their ultrastructural features were elucidated by electronmicroscopy. Muscle layers of rats’ ileums were incubated with lipopolysaccharide (LPS) for 4 to 8 h and the force of smooth muscle contraction was measured. The induction effect of inducible nitric oxide synthase on macrophages was then checked by immunohistochemistry. The expression of Main Histocompatible Complex (MHC) class II was also examined. Macrophages in the muscle layer were confirmed as resident macrophages and were different from a population of dendritic cells. After incubation with LPS, macrophages begun to express iNOS and produced NO, and it reduced smooth muscle contraction. iNOS immunopositive cells increased in a time dependent manner. Macrophages also began to express MHC class II. The total number of macrophages did not alter after incubation. Results indicate that resident macrophages in the muscle layer induced iNOS as an inflammatory reaction,affected smooth muscle contraction, and initiated immune response in the smooth muscle layer of the gastrointestinal tract, when activated by LPS.journal articl