Mouse embryonic stem cells give rise to gut-like morphogenesis, including intestinal stem cells, in the embryoid body model

Embryonic stem (ES) cells have been proposed as candidates for cell replacement therapy in patients with intestinal failure because these cells can be expanded indefinitely without losing their pluripotent phenotype. We investigated the differentiation capacity of mouse ES cells into gut-like structures, including intestinal stem cells, and defined culture conditions for efficient induction of formation of these structures. ES cell-derived gut-like structures (ES-guts) were reproducibly induced in developing embryoid bodies (EBs) by day 21 of differentiation culture. ES-guts contained an endodermal epithelium, a smooth muscle layer, interstitial cells of Cajal, and enteric neurons and showed spontaneous contraction. Transplantation of ES-guts under the kidney capsules of immunodeficient mice induced formation of highly differentiated epithelium composed of absorptive cells and goblet cells in the grafts. Immunoreactivity for Musashi-1 (Msi-1), a marker of intestinal stem cells, was detected in 1.9% of the columnar epithelial cells in the graft. Culture with 0.1% dimethyl sulfoxide increased the numbers of ES-guts in EBs, and serum-replacement (SR) culture, in comparison to standard ES culture containing 15% serum, increased the area ratio of ES-guts to EBs. SR culture also promoted maturation of epithelium to form a single layer of columnar epithelial cells, including absorptive cells and goblet cells. Expression of Msi-1 mRNA and protein was significantly enhanced when EBs were cultured under SR conditions. In conclusion, SR conditions efficiently induce formation of ES-guts and promote differentiation of epithelium, including intestinal stem cells. These results suggest the feasibility of cell-based therapy for intestinal failure based on ES cell culture systems.     

Interstitial cells of Cajal – their role in pacing and signal transmission in the digestive system

Interstitial cells of Cajal (ICC) are located in most parts of the digestive system. Although they were discovered over 100 years ago, their function began to be unravelled only recently. Morphological observations have led to a number of hypotheses on the possible physiological roles of ICC: (1) these cells may be the source of slow electrical waves recorded in gastrointestinal (GI) muscles; (2) they participate in the conduction of electrical currents, and (3) mediate neural signals between enteric nerves and muscles. These hypotheses were supported by experiments in which the ICC‐containing layer was removed surgically, or when ICC were ablated chemically, and as a consequence the slow waves were absent. Electrophysiological experiments on isolated cells confirmed that ICC can generate rhythmic electrical activity and can also respond to messenger molecules known to be released from enteric nerves. In mice mutants deficient in ICC, or in mice treated with antibody against the protein c‐Kit, slow wave activity was impaired. These results support the role of ICC as pacemaker cells. Physiological studies have shown that ICC in certain GI regions are important for signal transmission between nerves and smooth muscle. There is evidence that pathological changes in ICC may be associated with GI motility disorders. The full interpretation of the role of ICC in disease conditions will require much further study on the physiology and pharmacology of these cells.     

Interstitial cells of Cajal--their role in pacing and signal transmission in the digestive system

Interstitial cells of Cajal (ICC) are located in most parts of the digestive system. Although they were discovered over 100 years ago, their function began to be unravelled only recently. Morphological observations have led to a number of hypotheses on the possible physiological roles of ICC: (1) these cells may be the source of slow electrical waves recorded in gastrointestinal (GI) muscles; (2) they participate in the conduction of electrical currents, and (3) mediate neural signals between enteric nerves and muscles. These hypotheses were supported by experiments in which the ICC-containing layer was removed surgically, or when ICC were ablated chemically, and as a consequence the slow waves were absent. Electrophysiological experiments on isolated cells confirmed that ICC can generate rhythmic electrical activity and can also respond to messenger molecules known to be released from enteric nerves. In mice mutants deficient in ICC, or in mice treated with antibody against the protein c-Kit, slow wave activity was impaired. These results support the role of ICC as pacemaker cells. Physiological studies have shown that ICC in certain GI regions are important for signal transmission between nerves and smooth muscle. There is evidence that pathological changes in ICC may be associated with GI motility disorders. The full interpretation of the role of ICC in disease conditions will require much further study on the physiology and pharmacology of these cells.     

Kit mutants and gastrointestinal physiology

There has been considerable speculation about the function of interstitial cells of Cajal (ICC) since their discovery more than 100 years ago. It has been difficult to study these cells under native conditions, but great insights about the function of ICC have come from studies of genetic models with loss-of function mutations in the Kit signalling pathway. First it was discovered that signalling via Kit (a receptor tyrosine kinase) was vital for the development and maintenance of the ICC phenotype in gastrointestinal (GI) muscles. In compound heterozygotes ( W/W^V and Sl/Sl^d animals), where there are partial loss-of-function mutations in Kit receptors or Kit ligand (stem cell factor), ICC failed to develop in various regions of the GI tract, but no major changes in the smooth muscle layers or enteric nervous system occurred in the absence of these cells. Animals with these mutations provided an unprecedented opportunity to understand the role of ICC in GI motor function, and it is now clear from these studies that ICC serve as: (i) pacemaker cells, generating the spontaneous electrical rhythms of the gut known as slow waves; (ii) a propagation pathway for slow waves so that large areas of the musculature can be entrained to a dominant pacemaker frequency; (iii) mediators of excitatory cholinergic and inhibitory nitrergic neural inputs from the enteric nervous system, and (iv) stretch receptors that modulate membrane potential and electrical slow wave frequency. This review describes the use of genetic models to understand the important physiological role of ICC in the GI tract.     

The interstitial cells of Cajal and a gastroenteric pacemaker system

In spite of a claim by Kobayashi (1990) that they do not correspond to the cells originally depicted by CAJAL, a particular category of fibroblast-like cells have been identified in the gut by electron microscopy (Faussone-Pellegrini, 1977; Thuneberg, 1980) and by immunohistochemistry for Kit protein (Maeda et al., 1992) under the term of the "interstitial cells of Cajal (ICC)". Generating electrical slow waves, the ICC are intercalated between the intramural neurons and the effector smooth muscular cells, to form a gastroenteric pacemaker system. ICC at the level of the myenteric plexus (IC-MY) are multipolar cells forming a reticular network. The network of IC-MY which is believed to be the origin of electrical slow waves is morphologically independent from but associated with the myenteric plexus. On the other hand, intramuscular ICC (IC-IM) usually have spindle-shaped contours arranged in parallel with the bulk smooth muscle cells. Associated with nerve bundles and blood vessels, the IC-IM possess receptors for neurotransmitters and such circulating hormones as cholecystokinin, suggesting their roles in neuromuscular and hormone-muscular transmissions. In addition, gap junctions connect the IC-MY and IC-IM, thereby realizing the electrically synchronized integrity of ICC as a pacemaker system in the gut. The smooth muscle cells are also coupled with ICC via gap junctions, and the functional unit thus formed enables rhythmically synchronized contractions and relaxations. It has recently been found that a lack of Kit-expressing cells may induce hyper-contractility of the tunica muscularis in vitro, whereas a decrease in Kit expression within the muscle wall causes dysmotility-like symptoms in vivo. The pacemaker system in the gut thus seems to play a critical role in the maintenance of both moderate and normal motility of the digestive tract. A loss of Kit positive cells has been detected in several diseases with an impaired motor activity, including diabetic gastroenteropathy. Pathogenesis of these diseases is thought to be accounted for by impaired slow waves and neuromuscular transmissions; a pacemaker disorder may possibly induce a dysmotility-like symptom called 'gastroenteric arrhythmia'. A knowledge of the structure and function of the ICC and the pacemaker system provides a basis for clarifying the normal mechanism and the pathophysiology of motility in the digestive tract.     

Propagation of slow waves requires IP3 receptors and mitochondrial Ca2+ uptake in canine colonic muscles

In the gastrointestinal (GI) tract electrical slow waves yield oscillations in membrane potential that periodically increase the open probability of voltage-dependent Ca²⁺ channels and facilitate phasic contractions. Slow waves are generated by the interstitial cells of Cajal (ICC), and these events actively propagate through ICC networks within the walls of GI organs. The mechanism that entrains spontaneously active pacemaker sites throughout ICC networks to produce regenerative propagation of slow waves is unresolved. Agents that block inositol 1,4,5-trisphosphate (IP₃) receptors and mitochondrial Ca²⁺ uptake were tested on the generation of slow waves in the canine colon. A partitioned chamber apparatus was used to test the effects of blocking slow-wave generation on propagation. We found that active propagation occurred along strips of colonic muscle, but when the pacemaker mechanism was blocked in a portion of the tissue, slow waves decayed exponentially from the point where the pacemaker mechanism was inhibited. An IP₃ receptor inhibitor, mitochondrial inhibitors, low external Ca²⁺, and divalent cations (Mn²⁺ and Ni²⁺) caused exponential decay of the slow waves in regions of muscle exposed to these agents. These data demonstrate that the mechanism that initiates slow waves is reactivated from cell-to-cell during the propagation of slow waves. Voltage-dependent conductances present in smooth muscle cells are incapable of slow-wave regeneration. The data predict that partial loss of or disruptions to ICC networks observed in human motility disorders could lead to incomplete penetration of slow waves through GI organs and, thus, to defects in myogenic regulation.     

Gut pacemaker cells: the interstitial cells of Cajal (ICC).

This review will focus on the pacemaker mechanisms underlying gastrointestinal autonomic rhythmicity in an attempt to elucidate the differences and similarities between the pacemaker mechanisms in the heart and gut. Interstitial cells of Cajal (ICC) form networks that are widely distributed within the submucosal (ICC-SM), intra-muscular (ICC-IM, ICC-DMP) and inter-muscular layers (ICC-MY) of the gastrointestinal tract from the esophagus to the internal anal sphincter. The ICC generate spontaneously active pacemaker currents that may be recorded as plateau and slow potentials. These pacemaker currents drive the spontaneous electrical and mechanical activities of smooth muscle cells. The enteric nervous system, composed of both the myenteric (inter-muscular) plexus and the submucosal plexus, is also distributed in the gastrointestinal tract from the esophagus to the internal anal sphincter. The role of the ICC and the enteric nervous system in the integrative control of gastrointestinal function and especially of spontaneous rhythmic activity, is still unknown. Nevertheless, at least from the results presented in this review of studies of the jejunum, ileum and proximal colon of the mouse, it is convincing that the ICC drive spontaneous rhythmic motility, although a role for the enteric nervous system in the regulation of spontaneous rhythmic motility cannot be overlooked. Furthermore, intracellular Ca2+ handling has a critical role in the generation of pacemaker activity in the gut and heart, although respective players such as the Ca2+-ATPase of the sarcoplasmic reticulum (endoplasmic reticulum), IP3 receptors, ryanodine receptors and plasma membrane ion channels may have divergent roles in the Ca2+-release refilling cycles. In conclusion, intracellular Ca2+ handling plays a key role in the gut pacemaker responsible for spontaneous rhythmicity, as well as in the cardiac pacemaker responsible for spontaneous beating. Pharmacotherapeutic targeting of intracellular Ca2+ handling mechanisms may be a promising approach to the treatment and cure of gut motility dysfunction.     

In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells

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 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.     

Cellular mechanisms of myogenic activity in gastric smooth muscle

In many regions of the intestine, a thin layer of interstitial cells of Cajal (ICC) lie in the myenteric region, between the circular and longitudinal muscle layers. ICC are connected by gap junctions to surrounding ICC and also with circular and longitudinal smooth muscle cells, forming a large electrical syncytium. Damage of the ICC causes a disorder in the patterns of rhythmic activity. Isolated ICC produce a rhythmic oscillation of the membrane potential. All these observations have led to the suggestion that ICC may be the pacemaker cell responsible for intestinal activity. Gastric smooth muscles generate slow oscillatory membrane potential changes (slow waves) and spike potentials. The activity is considered to be linked to the metabolism in the cell. Three types of cells located in the gastric wall (circular and longitudinal smooth muscle cells and ICC) produce synchronized electrical responses with different shapes. The electrical responses appear to originate in ICC and then spread to the smooth muscle layers, indicating that ICC may also be the pacemaker cells responsible for gastric activity. However, isolated circular smooth muscle tissues spontaneously generate regenerative potentials, suggesting that there are at least two sites for the initiation of spontaneous activity in the stomach. Regenerative potentials persist in the presence of Ca-antagonists and are inhibited by agents which disrupt intracellular Ca(2+) homeostasis. Depolarization of the membrane elicits regenerative potentials after a long delay and the potentials have long refractory periods. This suggests that an unidentified 2nd messenger may be formed during the delay between membrane depolarization and the initiation of a regenerative potential. In gastric muscles of mutant mice which do not express inositol trisphosphate (InsP(3)) receptors, spike potentials but not slow waves are generated, suggesting the possible involvement of InsP(3) in the initiation of spontaneous activity.     

新型大麻制剂O-1602和大麻二酚对LPS导致的啮齿动物小肠运动紊乱的影响

目的: 通过离体及在体实验,观测2种新型大麻类制剂O-1602和大麻二酚(CBD)对脂多糖(LPS)诱导的啮齿动物小肠运动紊乱的影响,并探讨其可能的机制。方法: LPS腹腔注射复制小鼠小肠运动紊乱模型,碳末悬液灌胃评估小肠排推功能,Western blotting 测定回肠G蛋白偶联受体55(GPR55)的表达,ELISA法检测血清肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平。LPS与大鼠、小鼠回肠肌条共孵育制备离体动力紊乱模型,应用器官浴槽采集自发性以及10 Hz电刺激时收缩活动的变化;细胞内微电极技术记录平滑肌细胞膜电位变化;定磷法测定平滑肌组织Ca²⁺-ATP酶(Ca²⁺-ATPase)活性变化。结果: 在体实验:LPS引起动物明显的炎症表现及小肠运动紊乱(P<0.01),CBD改善小肠运动(P<0.05),并降低IL-6水平(P<0.01)和GPR55表达(P<0.01)。离体实验:O-1602和CBD在体外选择性逆转LPS诱导的平滑肌自发性及电刺激时的收缩紊乱(P<0.05或P<0.01),但二者不影响生理及病理状态下平滑肌细胞膜电位水平,CBD预处理降低LPS诱导的平滑肌组织Ca²⁺-ATPase活性升高的效应(P<0.05)。结论: 在体内CBD通过减轻炎症程度,降低GPR55表达水平,改善LPS导致的小肠运动紊乱;在体外O-1602和 CBD 可一定程度逆转LPS导致的大、小鼠小肠运动功能紊乱,其机制不是通过改变平滑肌细胞膜电位实现的,而可能与其调节平滑肌组织Ca²⁺-ATPase活性有关。     

Interstitial cells of Cajal: primary targets of enteric motor innervation

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.     

Multiscale modeling of gastrointestinal electrophysiology and experimental validation

Normal gastrointestinal (GI) motility results from the coordinated interplay of multiple cooperating mechanisms, both intrinsic and extrinsic to the GI tract. A fundamental component of this activity is an omnipresent electrical activity termed slow waves, which is generated and propagated by the interstitial cells of Cajal (ICCs). The role of ICC loss and network degradation in GI motility disorders is a significant area of ongoing research. This review examines recent progress in the multiscale modeling framework for effectively integrating a vast range of experimental data in GI electrophysiology, and outlines the prospect of how modeling can provide new insights into GI function in health and disease. The review begins with an overview of the GI tract and its electrophysiology, and then focuses on recent work on modeling GI electrical activity, spanning from cell to body biophysical scales. Mathematical cell models of the ICCs and smooth muscle cell are presented. The continuum framework of monodomain and bidomain models for tissue and organ models are then considered, and the forward techniques used to model the resultant body surface potential and magnetic field are discussed. The review then outlines recent progress in experimental support and validation of modeling, and concludes with a discussion on potential future research directions in this field.     

Organization and function of ICC in the urinary tract

ICC are found in both the upper and lower urinary tract. They are not found in the ureter itself but are confined to the lamina propria of the renal pelvis and pelvi-calyceal junction. They do not appear to have a primary pacemaker role (this is ascribed to atypical smooth muscle cells in the same location) but rather conduct and amplify the pacemaker signals generated by the atypical smooth muscle cells. In the bladder, ICC are widely distributed in the sub-urothelial region, in the lamina propria and at the margins of the detrusor smooth muscle bundles. Again they appear not to have a pacemaking role and such evidence as there is would suggest that they have a role in the modulation of signal transduction. The strongest evidence that ICC in the urinary tract act as pacemakers comes from studies of those in the urethra. Isolated ICC show regular spontaneous depolarizations in current clamp which resemble very closely the slow waves recorded from intact tissue. In voltage clamp they show abundant calcium-activated chloride current and spontaneous transient inward currents which can be blocked by chloride channel blockers. However, their role in the modulation of urethral tone has yet to be fully elucidated.     

Involvement of intramuscular interstitial cells of Cajal in neuroeffector transmission in the gastrointestinal tract

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal (GI) tract. ICC serve as electrical pacemakers, provide pathways for the active propagation of slow waves, are mediators of enteric motor neurotransmission and play a role in afferent neural signalling. Morphological studies have provided evidence that motor neurotransmission in the GI tract does not occur through poorly defined structures between nerves and smooth muscle, but rather via specialized synapses that exist between enteric nerve terminals and intramuscular ICC or ICC-IM. ICC-IM are coupled to smooth muscle cells via gap junctions and post-junctional responses elicited in ICC-IM are conducted to neighbouring smooth muscle cells. Electrophysiological studies from the stomachs and sphincters of wild-type and mutant animals that lack ICC-IM have provided functional evidence for the importance of ICC in cholinergic excitatory and nitrergic inhibitory motor neurotransmission. Intraperitoneal injection of animals with Kit neutralizing antibody or organ culture of gastrointestinal tissues in the presence of neutralizing antibody, which blocks the development and maintenance of ICC, has provided further evidence for the role of ICC in enteric motor transmission. ICC-IM also generate an ongoing discharge of unitary potentials in the gastric fundus and antrum that contributes to the overall excitability of the stomach.