Role of Ras in regulation of intestinal epithelial cell homeostasis and crosstalk with Wnt signaling.

Cross talk between different signaling pathways is thought to be important for regulation of homeostasis of, as well as oncogenesis of, the intestinal epithelium. Expression of an active form of K-Ras specifically in intestinal epithelial cells (IECs) of mice (IEC-RasDA mice) resulted in the development of hyperplasia in the small intestine and colon of mice. IEC-RasDA mice also manifested the increased proliferation of IECs. In addition, the number of goblet cells markedly increased, while that of Paneth cells decreased in IEC-RasDA mice. Development of intestinal organoids was markedly enhanced for IEC-RasDA mice compared with control mice. Whereas, the expression of Wnt target genes was significantly reduced in the in intestinal crypts from IEC-RasDA mice compared with that apparent for the control. Our results thus suggest that K-Ras promotes the proliferation of IECs as well as generation of goblet cells. By contrast, Ras counter-regulates the Wnt signaling and thereby contribute to the proper regulation of intestinal epithelial cell homeostasis.

RAB11A-mediated YAP localization to adherens and tight junctions is essential for colonic epithelial integrity.

Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells results in increased epithelial proliferation and nuclear accumulation of Yes-associated protein (YAP), a key Hippo-signaling transducer that senses cell-cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, ß-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP-ß-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo-YAP signaling pathway for rapid reparative response after tissue injury.

Berberine augments hypertrophy of colonic patches in mice with intraperitoneal bacterial infection.

Colonic patches, the counterparts of Peyer's patches in the small intestine, are dynamically regulated lymphoid tissues in the colon that have an important role in defensing against microbial infections. Berberine is an isoquinoline alkaloid extracted from medicinal herbs including Rhizoma coptidis and has long been used for the treatment of infectious gastroenteritis, but its impact on the colonic lymphoid tissues (such as colonic patches) is unknown. In this study, we aimed to investigate whether berberine had any influences on the colonic patches in mice with bacterial infection. The results showed that oral berberine administration in bacterial infected mice substantially enhanced the hypertrophy of colonic patches, which usually possessed the features of two large B-cell follicles with a separate T-cell area. Moreover, the colonic patches displayed follicular dendritic cell networks within the B-cell follicles, indicative of mature colonic patches containing germinal centers. Concomitant with enlarged colonic patches, the cultured colon of infected mice treated with berberine secreted significantly higher levels of interleukin-1ß (IL-1ß), IL-6, TNF-α, and CCL-2, while NLRP3 inhibitor MMC950 or knockout of NLRP3 gene abrogated berberine-induced hypertrophy of colonic patches, suggesting the involvement of the NLRP3 signaling pathway in this process. Functionally, oral administration of berberine ameliorated liver inflammation and improved formed feces in the colon. Altogether, these results indicated that berberine was able to augment the hypertrophy of colonic patches in mice with bacterial infection probably through enhancing local inflammatory responses in the colon.

BMP Antagonists Secreted by Mesenchymal Stromal Cells Improve Colonic Organoid Formation: Application for the Treatment of Radiation-induced Injury.

Radiation therapy is crucial in the therapeutic arsenal to cure cancers; however, non-neoplastic tissues around an abdominopelvic tumor can be damaged by ionizing radiation. In particular, the radio-induced death of highly proliferative stem/progenitor cells of the colonic mucosa could induce severe ulcers. The importance of sequelae for patients with gastrointestinal complications after radiotherapy and the absence of satisfactory management has opened the field to the testing of innovative treatments. The aim of this study was to use adult epithelial cells from the colon, to reduce colonic injuries in an animal model reproducing radiation damage observed in patients. We demonstrated that transplanted in vitro-amplified epithelial cells from colonic organoids (ECO) of C57/Bl6 mice expressing green fluorescent protein implant, proliferate, and differentiate in irradiated mucosa and reduce ulcer size. To improve the therapeutic benefit of ECO-based treatment with clinical translatability, we performed co-injection of ECO with mesenchymal stromal cells (MSCs), cells involved in niche function and widely used in clinical trials. We observed in vivo an improvement of the therapeutic benefit and in vitro analysis highlighted that co-culture of MSCs with ECO increases the number, proliferation, and size of colonic organoids. We also demonstrated, using gene expression analysis and siRNA inhibition, the involvement of bone morphogenetic protein antagonists in MSC-induced organoid formation. This study provides evidence of the potential of ECO to limit late radiation effects on the colon and opens perspectives on combined strategies to improve their amplification abilities and therapeutic effects.

Pharmacological activation of TGR5 promotes intestinal growth via a GLP-2-dependent pathway in mice.

Glucagon-like peptide (GLP)-1 and -2-secreting L cells have been shown to express the bile acid receptor Takeda G protein-receptor-5 (TGR5) and increase secretion upon receptor activation. Previous studies have explored GLP-1 secretion following acute TGR5 activation, but chronic activation and GLP-2 responses have not been characterized. In this study, we aimed to investigate the consequences of pharmacological TGR5 receptor activation on L cell hormone production in vivo using the specific TGR5 agonist RO5527239 and the GLP-2 receptor knockout mouse. Here, we show that 1) TGR5 receptor activation led to increased GLP-1 and GLP-2 content in the colon, which 2) was associated with an increased small intestinal weight that 3) was GLP-2 dependent. Additionally, we report that TGR5-mediated gallbladder filling occurred independently of GLP-2 signaling. In conclusion, we demonstrate that pharmacological TGR5 receptor activation stimulates L cells, triggering GLP-2-dependent intestinal adaption in mice.NEW & NOTEWORTHY Using the specific Takeda G protein-receptor-5 (TGR5) agonist RO5527239 and GLP-2 receptor knockout mice, we show that activation of TGR5 led to the increase in colonic GLP-1 and GLP-2 concomitant with a GLP-2 dependent growth response in the proximal portion of the small intestine.

Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling.

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/ß-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

The enteric nervous system undergoes significant chemical and synaptic maturation during adolescence in mice.

Adolescence is a critical period of development. It is very likely that there is significant maturation of the enteric nervous system (ENS) of the gut during this stage of life, especially since there are substantial changes in factors known to influence the ENS including diet and microbiota during this time, but this remains unknown. To examine maturation of the ENS during adolescence, we performed immunohistochemistry using advanced microscopy and analytical methods to compare enteric neurons and glia of the duodenum and colon of mice taken prior to weaning with those of young adult mice. We found significant changes in the architecture of both myenteric and submucosal plexuses and surprisingly found subsets of enteric cells that co-expressed the pan-neuronal marker, Hu, and either glial markers Sox10 or S100ß, not both. About 70% and 35% of all Hu â€‹+ â€‹neurons in the submucous plexus of the young adult duodenum and colon respectively also expressed S100ß. The proportion of Hu+/Sox10 â€‹+ â€‹cells in the duodenal myenteric plexus decreased, while the proportion of Hu+/S100ß+ cells in the colonic submucosal plexus increased during adolescence. In the submucous plexus, there were significant increases in the proportions of vasoactive intestinal peptide+ and choline acetyltransferase â€‹+ â€‹secretomotor neurons, of neurofilament M (NFM)+ neurons in the colon and of calretinin â€‹+ â€‹neurons in the duodenum during adolescence. There were no age-dependent changes in the neurochemistry of various myenteric neuronal subtypes, including those immunoreactive for neuronal nitric oxide synthase (nNOS), Calbindin, Calretinin or NFM. There were significant increases in the somata sizes of Calretinin â€‹+ â€‹submucosal and myenteric neurons, and nNOS â€‹+ â€‹myenteric neurons, and these enteric neurons received significantly more synaptophysin â€‹+ â€‹contacts onto their cell bodies during adolescence. This is the first study showing that enteric neurons and glia in the gut undergo significant changes in their anatomy and chemistry during adolescence. Notably changes in synaptic contacts within the enteric circuitry strongly suggest maturation in gastrointestinal function occurs during this time.

Exploring possible associations of the intestine bacterial microbiome with the pre-weaned weight gaining performance of piglets in intensive pig production.

The pre-weaned weight gain is an important performance trait of pigs in intensive pig production. The bacterial microbiome inside the host is vital to host health and growth performance. The purpose of this study was to explore the possible associations of the intestinal microbiome with the pre-weaned weight gain in intensive pig production. In this study, several anatomical sites (jejunum, ileum, cecum, and colon) were examined for bacterial microbiome structure using 16S rRNA V4-V5 region sequencing with Illumina Miseq. The results showed that the microbial richness (estimated by Chao1 index) in jejunum was positively correlated with the pre-weaned weight gain. This study also revealed that the Firmicutes and Bacteroidetes in colon were the weight gaining-related phyla; while the Selenomonas and Moraxella in ileum and the Lactobacillus in both cecum and colon were the weight gaining-related genera for the pre-weaned piglets in intensive pig prodution. Several intra-microbial interactions within commensal microbiome correlated with the pre-weaned weight gain were excavated, as well. Overall, this study provides an expanded view of the commensal bacterial community inside four anatomical intestinal sites of the commercial piglets and the associations of the intestinal microbiome with the pre-weaned weight gaining performance in intensive pig production.

Recent advances in deriving human endodermal tissues from pluripotent stem cells.

The utilization of directed differentiation of human pluripotent stem cells to generate human tissues is quickly evolving. Here we review recent advances in the derivation and applications of human endodermal tissues, including the esophagus, lung, pancreas, liver, stomach, small intestine, and colon. Improvements in tissue transcriptional and functional maturation, multicellular complexity, and scalability allow better development and disease modeling, large-scale drug and toxicity screening, and potentially cell therapeutic applications.

Developmental and age-dependent plasticity of GABAA receptors in the mouse colon: Implications in colonic motility and inflammation.

Lifelong functional plasticity of the gastrointestinal (GI) tract is essential for health, yet the underlying molecular mechanisms are poorly understood. The enteric nervous system (ENS) regulates all aspects of the gut function, via a range of neurotransmitter pathways, one of which is the GABA-GABAA receptor (GABAAR) system. We have previously shown that GABAA receptor subunits are differentially expressed within the ENS and are involved in regulating various GI functions. We have also shown that these receptors are involved in mediating stress-induced colonic inflammation. However, the expression and function of intestinal GABAARs, at different ages, is largely unexplored and was the focus of this study. Here we show that the impact of GABAAR activation on colonic contractility changes from early postnatal period through to late adulthood, in an age-dependant manner. We also show that the highest levels of expression for all GABAAR subunits is evident at postnatal day (P) 10 apart from the α3 subunit which increased with age. This increase in the α3 subunit expression in late adulthood (18 months old) is accompanied by an increase in the expression of inflammatory markers within the mouse colon. Finally, we demonstrate that the deletion of the α3 subunit prevents the increase in the expression of colonic inflammatory markers associated with healthy ageing. Collectively, the data provide the first demonstration of the molecular and functional plasticity of the GI GABAAR system over the course of a lifetime, and its possible role in mediating the age-induced colonic inflammation associated with healthy ageing.

The production of short chain fatty acid and colonic development in weaning piglets.

Weaning process widely affects the small intestinal structure and function in piglets, while the responses of large intestine to weaning stress are still obscure. The purpose of this study was to determine the developmental changes (i.e., short chain fatty acids (SCFAs) concentrations, growth parameters, crypt-related indices and antioxidant capacity) in colon of piglet during weaning. Forty piglets were weaned at day 21 and euthanized to collect colonic tissues and digesta samples on day 0, 1, 3, 7 and 14 post-weaning (n = 8). Piglet growth performance was improved (p < .001) on day 7 and 14 post-weaning. The concentrations of acetate, propionate, butyrate, valerate, isobutyrate, isovalerate and total SCFAs were higher (p < .001) during the late post-weaning period. The mRNA abundances of SCFAs transporters were greater (p < .001) on day 7 and 14. The absolute and relative weights, absolute length and perimeter of colon were greater (p < .001) on day 7 and 14. Similarly, post-weaning increases (p < .001) in colonic crypt depth and Ki67 positive cells numbers per crypt were observed during the same period. Colonic crypt fission indices decreased (p < .01), while total crypt numbers increased (p < .001) on day 14 after weaning. Moreover, total SCFAs concentration was significantly associated with colonic growth parameters and Ki67 cells/crypt (p < .001). In addition, catalase content was decreased on day 3, 7, and 14, whereas, the concentrations of total superoxide dismutase (T-SOD) and manganese-containing superoxide dismutase (MnSOD) were higher (p < .05) on day 1 and 3 post-weaning. These results showed that weaning process has a significant effect on colonic growth and development, which might be associated with the change of SCFAs concentrations in colon.

Effects of dietary supplementation with yeast glycoprotein on growth performance, intestinal mucosal morphology, immune response and colonic microbiota in weaned piglets.

Antibiotics are commonly provided to weaned piglets; however, this practice has become controversial due to the increased occurrences of microbial resistance, and alternatives are needed. This study aimed to investigate the effects of dietary supplementation with yeast glycoprotein (YG) on growth performance, intestinal mucosal morphology, immune response and colonic microbiota in weaned piglets. A total of 240 weaned piglets (d 23 ± 2) from 16 pens (15 piglets per pen) were randomly allocated to an antibiotics group (25% quinocetone 200 mg kg-1 and 4% enduracidin 800 mg kg-1 of the basal diet) or a YG group (800 mg kg-1 YG of the basal diet), respectively. The trial lasted 14 days, and at the end of the trial, one piglet per pen was chosen to collect plasma, intestinal tissue and colonic digesta samples. The results indicate that piglets fed diets containing YG tended to show increased final body weight (0.05 < P < 0.1), increased average daily gain (P < 0.05) and decreased F/G (P < 0.05) when compared with the antibiotics group. Moreover, intestinal permeability showed that YG led to an improvement in the intestinal development via decreasing serum content of DAO (P < 0.01). Histological evaluations showed that YG contributed to the improvement of the intestinal development via increasing villous height (P < 0.05) and the villous height to crypt depth ratio (P < 0.01), and decreasing crypt depth (P < 0.01) and villous width (P < 0.05) in the ileum. Intestinal integrity also showed that YG was conducive to improvement of the intestinal development via upregulating the m-RNA expression of occludin (P < 0.05) in the duodenal and jejunal mucosa. Interestingly, YG supplementation downregulated the m-RNA expression of IL-12 (P < 0.05), upregulated the m-RNA expression of Hsp-70 (P < 0.05) in the duodenal mucosa, downregulated the m-RNA expression of Hsp-70 (P < 0.05) and IFN-γ (P < 0.05), upregulated the m-RNA expression of Hsp-90 (P < 0.05) in the jejunal mucosa, and upregulated the m-RNA expression of Hsp-70 (P < 0.05) in the ileal mucosa. On the other hand, colonic microbiota results showed that YG supplementation increased the relative abundance of Lactobacillus (P < 0.05) in the genus level. Colonic metabolite results showed that YG supplementation decreased the content of acetate (P < 0.05). Taken together, it is speculated that YG would be a potent alternative to prophylactic antibiotics in improving the gut health in weaned piglets.

The roles of PDGFRα signaling in the postnatal development and functional maintenance of the SMC-ICC-PDGFRα+ cell (SIP) syncytium in the colon.

BACKGROUND: The SIP syncytium in the gut consists of smooth muscle cells, interstitial cells of Cajal, and PDGFRα+ cells. We studied the fate of SIP cells after blocking PDGFRα receptor to explore the roles of PDGFRα signaling in the postnatal development and functional maintenance of the SIP syncytium. METHODS: Crenolanib was administered to mice from P0, P10, or P50. The morphological changes in SIP cells were examined by immunofluorescence. Protein expression in SIP cells was detected by Western blotting. Moreover, colonic transit was analyzed by testing the colonic bead expulsion time. KEY RESULTS: A dose of 5 mg(kg•day)-1 crenolanib administered for 10 days beginning on P0 apparently hindered the development of PDGFRα+ cells in the colonic longitudinal muscularis and myenteric plexus without influencing their proliferative activity and apoptosis, but this result was not seen in the colonic circular muscularis. SMCs were also inhibited by crenolanib. A dose of 7.5 mg(kg•day)-1 crenolanib administered for 15 days beginning on P0 caused reductions in both PDGFRα+ cells and ICC in the longitudinal muscularis, myenteric plexus, and circular muscularis. However, when crenolanib was administered at a dose of 5 mg(kg•day)-1 beginning on P10 or P50, it only noticeably decreased the number of PDGFRα+ cells in the colonic longitudinal muscularis. Crenolanib also caused PDGFRα+ cells to transdifferentiate into SMC in adult mice. Colonic transit was delayed after administration of crenolanib. CONCLUSIONS & INFERENCES: Therefore, PDGFRα signaling is essential for the development and functional maintenance of the SIP cells, especially PDGFRα+ cells.

Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts.

Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFß) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a "just-right" concentration of AG1478 increases colon organoid cryptogenesis.

Intestinal immunity in hypopituitary dwarf mice: effects of age.

Hypopituitary dwarf mice demonstrate advantages of longevity, but little is known of their colon development and intestinal immunity. Herein we found that Ames dwarf mice have shorter colon and colonic crypts, but larger ratio of mesenteric lymph nodes (MLNs) over body weight than age-matched wild type (WT) mice. In the colonic lamina propria (cLP) of juvenile Ames mice, more inflammatory neutrophils (A: 0.15% vs. 0.03% in WT mice) and monocytes (A: 7.97% vs. 5.15%) infiltrated, and antigen presenting cells CD11c+ dendritic cells (A: 1.39% vs. 0.87%), CD11b+ macrophages (A: 3.22% vs. 0.81%) and gamma delta T (γδ T) cells (A: 5.56% vs. 1.35%) were increased. In adult Ames dwarf mice, adaptive immune cells, such as IL-17 producing CD4+ T helper (Th17) cells (A: 8.3% vs. 4.7%) were augmented. In the MLNs of Ames dwarf mice, the antigen presenting and adaptive immune cells also altered when compared to WT mice, such as a decrease of T-regulatory (Treg) cells in juvenile Ames mice (A: 7.7% vs.10.5%), but an increase of Th17 cells (A: 0.627% vs.0.093%). Taken together, these data suggest that somatotropic signaling deficiency influences colon development and intestinal immunity.

Balanced Caloric Restriction Minimizes Changes Caused by Aging on the Colonic Myenteric Plexus.

Aging can promote significant morphofunctional changes in the gastrointestinal tract (GIT). Regulation of GIT motility is mainly controlled by the myenteric neurons of the enteric nervous system. Actions that aim at decreasing the aging effects in the GIT include those related to diet, with caloric restriction (CR). The CR is achieved by controlling the amount of food or by manipulating the components of the diet. Therefore, the objective of this study was to evaluate different levels of CR on the plasticity of nicotinamide adenine dinucleotide phosphate- (NADPH-) reactive myenteric neurons in the colon of Wistar rats during the aging process using ultrastructural (transmission electron microscopy) and morphoquantitative analysis. Wistar male rats (Rattus norvegicus) were distributed into 4 groups (n = 10/group): C, 6-month-old animals; SR, 18-month-old animals fed a normal diet; CRI, 18-month-old animals fed a 12% CR diet; CRII, 18-month-old animals fed a 31% CR diet. At 6 months of age, animals were transferred to the laboratory animal facility, where they remained until 18 months of age. Animals of the CRI and CRII groups were submitted to CR for 6 months. In the ultrastructural analysis, a disorganization of the periganglionar matrix with the aging was observed, and this characteristic was not observed in the animals that received hypocaloric diet. It was observed that the restriction of 12.5% and 31% of calories in the diet minimized the increase in density and cell profile of the reactive NADPH neurons, increased with age. This type of diet may be adapted against gastrointestinal disturbances that commonly affect aging individuals.

Specific protein supplementation using soya, casein or whey differentially affects regional gut growth and luminal growth factor bioactivity in rats; implications for the treatment of gut injury and stimulating repair.

Modulation of regional growth within specific segments of the bowel may have clinical value for several gastrointestinal conditions. We therefore examined the effects of different dietary protein sources on regional gut growth and luminal growth factor bioactivity as potential therapies. Rats were fed for 14 days on isonitrogenous and isocaloric diets comprising elemental diet (ED) alone (which is known to cause gut atrophy), ED supplemented with casein or whey or a soya protein-rich feed. Effects on regional gut growth and intraluminal growth factor activity were then determined. Despite calorie intake being similar in all groups, soya rich feed caused 20% extra total body weight gain. Stomach weight was highest on soya and casein diets. Soya enhanced diet caused greatest increase in small intestinal weight and preserved luminal growth factor activity at levels sufficient to increase proliferation in vitro. Regional small intestinal proliferation was highest in proximal segment in ED fed animals whereas distal small intestine proliferation was greater in soya fed animals. Colonic weight and proliferation throughout the colon was higher in animals receiving soya or whey supplemented feeds. We conclude that specific protein supplementation with either soya, casein or whey may be beneficial to rest or increase growth in different regions of the bowel through mechanisms that include differentially affecting luminal growth factor bioactivity. These results have implications for targeting specific regions of the bowel for conditions such as Crohn's disease and chemotherapy.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

Establishment of a refined culture method for rat colon organoids.

BACKGROUND: Methods for the artificial three-dimensional (3D) culture of mouse and human small-intestinal and large-intestinal stem cells have been established with CD24+ or Paneth cell niches. In contrast, no studies have established stable 3D culture for rat colon stem cells. In this study, we established an advanced method for efficient rat colonic stem cell culture. METHODS: Using various tissue homogenates, we investigated the colonic organoid forming capacity under the TMDU protocol immediately adjacent to Ootani's 3D culture assembly in the same culture dish. Next, we examined whether the supernatant from the colon could be replaced by a colon homogenate. Finally, we identified the bioactive substances that were indispensable for efficient organoid culture using protein purification by three-step column chromatography and proteomic analysis with a quantitative nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By combining Ootani's method with the TMDU protocol, we established a refined culture method for Lewis rat colon organoids, which we refer to as the modified TMDU protocol. Furthermore, we confirmed that PGE2 and galection-4 promoted rat colonic organoid formation. CONCLUSIONS: We established efficient rat colonic stem cell cultures in vitro. This success will contribute to the study of rat intestinal-disease models.

Corrupted Colonic Crypts Bordering Regenerating Mucosal Ulcers in Ulcerative Colitis.

BACKGROUND/AIM: Histology in protracted ulcerative colitis (UC) discloses high numbers of chronic inflammatory cells and crypts with architectural distortions. In severe cases, ulcerations are frequently found. The histogenesis of colonic crypts with architectural distortions in UC remains elusive. A recent review of colectomy specimens from patients with UC revealed crypts surrounding mucosal ulcerations exhibiting severe architectural distortions. They were called corrupted colonic crypts, CCCs. MATERIAL AND METHODS: Archival hematoxylin and eosin (H&E)-stained sections from three colectomies having several mucosal ulcers were selected for the study. The mucosa bordering mucosal ulcers was particularly scrutinized. RESULTS: The review of 49 sections (mean=16.3, range=14-20) in the three colectomies revealed 60 ulcers (mean=20, range=13-27). The following CCC phenotypes were found bordering mucosal ulcers: with asymmetric lateral fission (n=11), with dual or three-foiled corrupted fission (n=19), with cystic dilatations (n=3), L-shaped crypts (n=7), T-inverted crypts (n=6), shoe-shaped crypts (n=3), horizontal crypts (n=14), multi-lobate crypts (n=2), and/or inter-connecting crypts (n=5). CONCLUSION: The regeneration of ulcers in UC seems to proceed with neo-formation of corrupted crypts. In the same colectomies, none to occasional CCCs were found in large areas of the mucosa having severe chronic inflammation. Importantly, none of the occasional CCCs were found in other diseases of the colonic mucosa with chronic inflammation or in unspecific ulcers of the colon. Since neither chronic mucosal inflammation per se, nor unspecific ulcers of the colon are central for the formation of CCCs, it is suggested that crypt distortions of the non-ulcerated colonic mucosa in patients with UC might mirror formerly healed mucosal ulcerations.