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Aims: The study aims to explore the effects of the single-nucleotide polymorphism of miR-27a and its expression in Helicobacter pylori (H. pylori)-related diseases and the relationship between gastric pathology and traditional Chinese medicine (TCM). Methods: Subjects were classified into six histopathological groups and five TCM syndrome groups. All specimens underwent H. pylori detection through rapid urease test and methylene blue staining. Histopathological characteristics were observed by hematoxylin-eosin. The expression of miR-27a and its genotype were, respectively, detected by Quantitative Real-Time PCR and direct sequencing. Results: H. pylori promoted the malignant evolution of gastric mucosa and were involved in the formation of TCM syndrome. In H. pylori-positive patients, the frequency of miR-27a CT genotype at the rs895819 locus and its expression in the gastric cancer group were higher than those in other pathological groups. TCM syndrome had a close relationship with histopathological changes, and patients with spleen-qi deficiency syndrome had a higher risk of gastric cancer than other syndromes, regardless of H. pylori infection. Conclusion: The C allele at miR-27a rs895819 locus may be an oncogene in gastric cancer. High levels of miR-27a could play an important role in gastric malignant evolution, especially cancerization. There is a certain connection between TCM syndrome and pathological changes of the gastric mucosa to some extent, where patients with SQD syndrome had a higher risk of GC.
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Background: Helicobacter pylori (Hp) persistent infection is an important pathogenic factor for a series of chronic gastric diseases from chronic gastritis to gastric cancer. Genetic and epigenetic abnormalities of microRNAs may play a vital role in the pathological evolution of gastric mucosa in Helicobacter pylori-related gastric diseases (HPGD). This study aimed to investigate the relationship between miR-146a, miR-196a2, miR-149, miR-499 and miR-27a gene single nucleotide polymorphisms (SNPs) and their expressions with pathological changes in gastric mucosa, and to further analyze the interactions between SNPs and Hp. Methods: Subjects in this study included patients diagnosed with HPGD and healthy controls. MiR-146a rs2910164, miR-196a2 rs11614913, miR-149 rs2292832, miR-499 rs3746444 and miR-27a rs895819 were genotyped by direct sequencing. Fluorescence quantitative PCR was used to detect microRNA expressions. Gene-gene and gene-environment interactions were evaluated by multifactor dimensionality reduction (MDR) method. Results: we found that frequency distribution of miR-196a2 rs11614913 CT genotype in gastric precancerous lesion (GPL) group and gastric cancer (GC) group was significantly higher than normal control (NOR) group [adjusted OR = 6.16, 95%CI (1.46-26.03); adjusted OR = 11.83, 95%CI (1.65-84.72), respectively]. CT genotype and C allele of miR-27a rs895819 were associated with increased risk of GC [adjusted OR = 10.14, 95%CI (2.25-45.77); adjusted OR = 3.71, 95%CI(1.46-9.44), respectively]. The MDR analysis results showed that the interaction between miR-196a2 rs11614913 and Hp was associated with the risk of GPL (p = 0.004). Meanwhile, the expression level of miR-196a2 in GC group was significantly higher than NOR, chronic inflammation (CI) and early precancerous lesion (EPL) groups among Hp-positive subjects. And expressions of miR-499 and miR-27a in GC group were both higher than EPL group. Also, miR-27a expression in GC group was higher than CI and gastric atrophy (GA) groups. Conclusion: miR-196a2 rs11614913 and miR-27a rs895819 may affect the genetic susceptibility to GPL or GC. MiR-196a2 rs11614913 and Hp have a synergistic effect in the occurrence and development of GPL. The up-regulation of miR-499, miR-196a2 and miR-27a expression caused by Hp infection may be an important mechanism of gastric carcinogenesis.
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OBJECTIVE: We aimed to build a ferroptosis-based classifier to characterize the molecular features of gastric cancers (GC) and investigate the relationship between different ferroptosis patterns and GC tumor microenvironment (TME). METHODS: Based on the genomic and clinical information from TCGA portal and GEO database, non-negative matrix factorization (NMF) was used to identify ferroptosis subtypes in GC patients. In order to estimate the ferroptosis levels, we established ferroptosis subtype score (FSS) to quantify ferroptosis patterns and ferroptosis potential index (FPI) by principal component analysis (PCA). The correlations of different ferroptosis patterns with TME cell-infiltrating characteristics (including immune cell infiltration, immune checkpoints expression levels, tumor mutational burden (TMB) and immunotherapy response) were systematically analyzed. RESULTS: Two ferroptosis subtypes, C1 (with lower FSS) and C2 (with higher FSS), were determined. C2 displayed a significantly lower FPI than C1. Besides, C2 was associated with diffuse subtype while C1 with intestinal subtype. As for TME characteristics, C2 was in accordance with the immune-excluded phenotype as it showed more active immune and stromal activities but lower TMB, less probability of immunotherapy response and poorer prognosis. C1 was linked to immune-inflamed phenotype as it had lower stromal activities but increased neoantigen load, enhanced response to immunotherapy and relatively better prognosis. CONCLUSION: The systematic assessment of ferroptosis patterns and ferroptosis levels presented in our study implied that ferroptosis serves as an important factor in the formation of TME, which may expand the understanding of TME and provide a novel perspective for the development of targeted immunotherapeutic strategies for GC patients.
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BACKGROUND: Cassia mimosoides Linn (CMD) is a traditional Chinese herb that clears liver heat and dampness. It has been widely administered in clinical practice to treat jaundice associated with damp-heat pathogen and obesity. Emodin (EMO) is a major bioactive constituent of CMD that has apparent therapeutic efficacy against obesity and fatty liver. Here, we investigated the protective effects and underlying mechanisms of EMO against high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD). OBJECTIVE: We aimed to investigate whether EMO activates farnesoid X receptor (FXR) signaling to alleviate HFD-induced NAFLD. MATERIALS AND METHODS: In vivo assays included serum biochemical indices tests, histopathology, western blotting, and qRT-PCR to evaluate the effects of EMO on glucose and lipid metabolism disorders in wild type (WT) and FXR knockout mice maintained on an HFD. In vitro experiments included intracellular triglyceride (TG) level measurement and Oil Red O staining to assess the capacity of EMO to remove lipids induced by oleic acid and palmitic acid in WT and FXR knockout mouse primary hepatocytes (MPHs). We also detected mRNA expression of FXR signaling genes in MPHs. RESULTS: After HFD administration, body weight and serum lipid and inflammation levels were dramatically increased in the WT mice. The animals also presented with impaired glucose tolerance, insulin resistance, and antioxidant capacity, liver tissue attenuation, and pathological injury. EMO remarkably reversed the foregoing changes in HFD-induced mice. EMO improved HFD-induced lipid accumulation, insulin resistance, inflammation, and oxidative stress in a dose-dependent manner in WT mice by inhibiting FXR expression. EMO also significantly repressed TG hyperaccumulation by upregulating FXR expression in MPHs. However, it did not improve lipid accumulation, insulin sensitivity, or glucose tolerance in HFD-fed FXR knockout mice. CONCLUSIONS: The present study demonstrated that EMO alleviates HFD-induced NAFLD by activating FXR signaling which improves lipid accumulation, insulin resistance, inflammation, and oxidative stress.
Subject(s)
Cassia/chemistry , Emodin/pharmacology , Non-alcoholic Fatty Liver Disease/drug therapy , Receptors, Cytoplasmic and Nuclear/genetics , Animals , Diet, High-Fat/adverse effects , Dose-Response Relationship, Drug , Emodin/administration & dosage , Emodin/isolation & purification , Glucose/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Inflammation/drug therapy , Inflammation/pathology , Insulin Resistance , Lipid Metabolism/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Non-alcoholic Fatty Liver Disease/physiopathology , Oxidative Stress/drug effects , RNA, Messenger/metabolism , Triglycerides/bloodABSTRACT
[This corrects the article DOI: 10.3389/fmed.2021.629080.].
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Background & Aims: Gastric cancer is the common malignancies from cancer worldwide. Endoscopy is currently the most effective method to detect early gastric cancer (EGC). However, endoscopy is not infallible and EGC can be missed during endoscopy. Artificial intelligence (AI)-assisted endoscopic diagnosis is a recent hot spot of research. We aimed to quantify the diagnostic value of AI-assisted endoscopy in diagnosing EGC. Method: The PubMed, MEDLINE, Embase and the Cochrane Library Databases were searched for articles on AI-assisted endoscopy application in EGC diagnosis. The pooled sensitivity, specificity, and area under the curve (AUC) were calculated, and the endoscopists' diagnostic value was evaluated for comparison. The subgroup was set according to endoscopy modality, and number of training images. A funnel plot was delineated to estimate the publication bias. Result: 16 studies were included in this study. We indicated that the application of AI in endoscopic detection of EGC achieved an AUC of 0.96 (95% CI, 0.94-0.97), a sensitivity of 86% (95% CI, 77-92%), and a specificity of 93% (95% CI, 89-96%). In AI-assisted EGC depth diagnosis, the AUC was 0.82(95% CI, 0.78-0.85), and the pooled sensitivity and specificity was 0.72(95% CI, 0.58-0.82) and 0.79(95% CI, 0.56-0.92). The funnel plot showed no publication bias. Conclusion: The AI applications for EGC diagnosis seemed to be more accurate than the endoscopists. AI assisted EGC diagnosis was more accurate than experts. More prospective studies are needed to make AI-aided EGC diagnosis universal in clinical practice.
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AIM: Hepatocellular carcinoma (HCC) is a common malignancy associated with a poor prognosis due to difficulties in reliably estimating overall survival (OS). MicroRNAs (miRNAs) play critical roles in HCC initiation, progression, and metastasis and are highly correlated with patient prognosis. Thus, miRNA-based risk signatures and nomograms are urgently required for predicting OS in patients with HCC. METHODS: We constructed a 13-miRNA-based signature and prognostic nomogram using 408 HCC samples and 58 normal tissues with miRNA sequencing data and clinical data from 323 patients downloaded from The Cancer Genome Atlas. A total of 195 patients were assigned as the internal validation cohort for verification and testing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis was applied to investigate pathway enrichment for the signature. RESULTS: We identified and validated a 13-miRNA risk signature highly associating with the OS of HCC patients. The signature showed good performances by calculating C-index, area under the curve, and calibration curves. After verification and testing using an internal validation cohort, the results yielded a miRNA-based signature and a prognostic nomogram with reliable predictive accuracy. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that various genes and multiple pathways were closely related to the mechanisms of HCC proliferation and metastasis. CONCLUSION: We successfully identified a 13-miRNA-based signature and prognostic nomogram that are capable of predicting OS in patients with HCC.
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H. pylori-related gastric diseases (HPGD) are a series of gastric mucosal benign and malignant lesions associated with H. pylori infection. Exploring the pathogenesis of HPGD will be of great significance to prevent and treat gastric malignancy. Traditional Chinese medicine (TCM) syndrome is the essence of TCM, reflecting the state of whole body. Potential similarities of TCM syndrome may provide a new perspective in understanding development and treatment of diseases. To seek an early warning signal for gastric malignant pathology and similarities of TCM syndrome from the viewpoint of molecular biology, we examined the relationships among H. pylori, gastric pathology, and TCM syndrome and effects of Interleukin-1ß (IL-1ß) gene polymorphisms and expression on gastric pathology and TCM syndrome in HPGD. The results indicated that detection of H. pylori with differentiation of TCM syndrome may have a predictive function to gastric pathology. H. pylori may lead to gastric atrophy via enhancing IL-1ß mRNA expression, and IL-1ß mRNA overexpression in gastric mucosa may be one of the generality characteristics for H. pylori-negative subjects with syndrome of dampness-heat in the spleen and stomach.
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Cyclooxygenase-2 (COX-2) is an inducible enzyme stimulated by various inflammatory factors (IFs). Chronic gastritis is a classic model of "inflammation-cancer transformation" and Helicobacter pylori-related gastric diseases (HPGD) are specific ones of this model. Traditional Chinese Medicine (TCM) syndromes could play a predictive role in gastric histopathological evolution. To search for early warning evidence about "inflammation-cancer transformation," this study is about to explore interaction of COX-2 with Helicobacter pylori (Hp) in HPGD with different TCM syndromes. All included subjects underwent endoscopy and biopsy. Hp infection was detected by rapid urease test and methylene blue staining. Histopathological characteristics and COX-2 expression in gastric mucosa (GM) were, respectively, observed by hematoxylin-eosin and Elivision™ plus. SPSS 18.0 and Stata 11.0 statistical software packages were used for statistical analysis. Results of immunohistochemical staining in this study showed COX-2 expression in Hp-positive patients was stronger than that in Hp-negative ones. Spearman' analysis indicated that degrees of both Hp infection and COX-2 expression were positively correlated with those of gastric inflammation and inflammatory activity. Compared with the relative normal group, both severe dysplasia group and gastric carcinoma group had more severe Hp infection and COX-2 expression. Compared with the nonsyndrome, syndrome of internal block of static blood (IBSB) had higher scores in semiquantitative analysis of COX-2 protein expression among TCM groups. Moreover, multivariate logistics regression analysis suggested that patients with Hp infection could increase the risk of IBSB. These results indicated that COX-2 interacting with Hp could play an important role in transforming gastric chronic nonresolving inflammation into carcinoma in subjects with HPGD, as well as inducing the formation of IBSB. HPGD together with IBSB could be an early warning evidence for GM with histopathological evolution from benign to malignant.
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OBJECTIVE: To evaluate efficacy of traditional Chinese medicine (TCM) for gastric precancerous lesion (GPL). METHOD: Literature retrieval was conducted in seven databases from their inception through Dec. 24th, 2018. The Cochrane collaboration, Review Manager (RevMan5.3) and GRADE profiler software were conducted for this meta-analysis. RESULTS: In primary outcomes, results of meta-analysis showed that TCM had superior to current routine pharmacotherapy (RP) in clinical efficacy, Helicobacter pylori (Hp) eradication rate, efficacy under endoscopy, and TCM syndrome efficacy. Meanwhile, no potential publication bias was detected by Begg's and Egger's tests. In secondary outcomes, compared with control groups, experimental groups were more positive effects on improvement of stomach distention, stomachache, and heartburn. CONCLUSION: Our findings indicated that TCM could have positive effects on GPL. However, further standardized RCTs of rigorous design should be required to obtain more forceful evidence.
Subject(s)
Drugs, Chinese Herbal/therapeutic use , Medicine, Chinese Traditional/methods , Precancerous Conditions/drug therapy , Humans , Randomized Controlled Trials as Topic , Treatment OutcomeABSTRACT
BACKGROUND & AIMS: The protective intestinal mucosal barrier consists of multiple elements including mucus and epithelial layers and immune defense; nonetheless, barrier dysfunction is common in various disorders. The imprinted and developmentally regulated long noncoding RNA H19 is involved in many cell processes and diseases. Here, we investigated the role of H19 in regulating Paneth and goblet cells and autophagy, and its impact on intestinal barrier dysfunction induced by septic stress. METHODS: Studies were conducted in H19-deficient (H19-/-) mice, mucosal tissues from patients with sepsis, primary enterocytes, and Caco-2 cells. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer fluorescein isothiocyanate-dextran assays. The function of Paneth and goblet cells was examined by immunostaining for lysozyme and mucin 2, respectively, and autophagy was examined by microtubule-associated proteins 1A/1B light chain 3 II immunostaining and Western blot analysis. Intestinal organoids were isolated from H19-/- and control littermate mice and treated with lipopolysaccharide (LPS). RESULTS: Intestinal mucosal tissues in mice 24 hours after exposure to CLP and in patients with sepsis showed high H19 levels, associated with intestinal barrier dysfunction. Targeted deletion of the H19 gene in mice enhanced the function of Paneth and goblet cells and promoted autophagy in the small intestinal mucosa. Knockout of H19 protected Paneth and goblet cells against septic stress, preserved autophagy activation, and promoted gut barrier function after exposure to CLP. Compared with organoids from control littermate mice, intestinal organoids isolated from H19-/- mice had increased numbers of lysozyme- and mucin 2-positive cells and showed increased tolerance to LPS. Conversely, ectopic overexpression of H19 in cultured intestinal epithelial cells prevented rapamycin-induced autophagy and abolished the rapamycin-induced protection of the epithelial barrier against LPS. CONCLUSIONS: In investigations of mice, human tissues, primary organoids, and intestinal epithelial cells, we found that increased H19 inhibited the function of Paneth and goblet cells and suppressed autophagy, thus potentially contributing to barrier dysfunction in intestinal pathologies.
Subject(s)
Autophagy/genetics , Goblet Cells/pathology , Paneth Cells/pathology , RNA, Long Noncoding/metabolism , Sepsis/pathology , Animals , Autophagy/immunology , Caco-2 Cells , Disease Models, Animal , Female , Goblet Cells/immunology , Humans , Intestine, Small/cytology , Intestine, Small/immunology , Intestine, Small/pathology , Male , Mice , Mice, Knockout , Organoids , Paneth Cells/immunology , Permeability , RNA, Long Noncoding/genetics , Sepsis/immunologyABSTRACT
The majority of Musashi 1 (Msi1)positive cells derived from mouse embryonic stem cells (mESCs) are prone to differentiate into neural epitheliallike cells, and only a small proportion of Msi1positive cells differentiate into intestinal epitheliallike cells. Whether inhibiting the phosphatidylinositol 3kinase (PI3K) signaling of mESCs can promote the differentiation of Msi1positive cells into intestinal epitheliallike cells remains to be fully elucidated. In the present study, to inhibit PI3K signaling, mESCs were treated with LY294002. A pMsi1green fluorescence protein reporter plasmid was used to sort the Msi1positive cells from mESCs treated and untreated with LY294002 (5 µmol/l). The Msi1positive cells were hypodermically engrafted into the backs of nonobese diabetic/severe combined immunodeficient mice. The presence of neural and intestinal epitheliallike cells in the grafts was detected by reverse transcriptionquantitative polymerase chain reaction analysis and immunohistochemistry. Compared with the Msi1positive cells derived from mESCs without LY294002 treatment, Msi1positive cells derived from mESCs treated with LY294002 expressed higher levels of leucinerich repeatcontaining Gprotein coupled receptor, a marker of intestinal epithelial stem cells, and lower levels of Nestin, a marker of neural epithelial stem cells. The grafts from Msi1positive cells treated with LY294002 contained more intestinal epitheliallike tissues and fewer neural epitheliallike tissues, compared with those from untreated Msi1positive cells. LY294002 had the ability to promote the differentiation of mESCs into intestinal epitheliallike tissues. The Msi1positive cells selected from the cell population derived from mESCs treated with LY294002 exhibited more characteristics of intestinal epithelial stem cells than those from the untreated group.
Subject(s)
Cell Differentiation/drug effects , Chromones/pharmacology , Intestinal Mucosa/cytology , Morpholines/pharmacology , Mouse Embryonic Stem Cells/drug effects , Nerve Tissue Proteins/analysis , Protein Kinase Inhibitors/pharmacology , RNA-Binding Proteins/analysis , Animals , Cell Culture Techniques , Cell Line , Cell Survival/drug effects , Gene Expression Regulation/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nerve Tissue Proteins/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , RNA-Binding Proteins/genetics , Signal Transduction/drug effectsABSTRACT
To investigate the relationship of MUC1, MUC5AC, and the syndrome of spleen and stomach, 109 subjects (34 peptic ulcer (PU), 62 chronic gastritis (CG), and 13 healthy volunteers (CON)) were included. All the subjects included were surveyed with questionnaire to classify them into damp-heat syndrome of spleen and stomach (DHSS), spleen-qi deficiency syndrome (SQD), and CON, examined by gastric endoscope, and biopsied. Rapid urease and methylene blue staining (MBS) were performed on every subject to diagnose for Helicobacter pylori (Hp) infection, and both were defined as Hp-positive. Hematoxylin and eosin (HE) staining was performed on every specimen to explore the histomorphology, inflammatory degree, and inflammatory activity of different groups; then Elivision™ plus kit was used to test the expression of MUC1 and MUC5AC. All the results of digital images were reviewed by two experts blindly. The inflammatory degree with Hp infection was higher than those uninfected or CON, but no significant difference was found between DHSS and SQD. And the expressions of MUC5AC with positive Hp was higher than those with negative Hp or CON regardless of the deficiency and solid syndrome of spleen-stomach but not for MUC1. We speculate that the deficiency and solid syndrome of spleen-stomach is a condition like Tai Ji symbol of dynamic equilibrium, showing the higher expression of MUC5AC but no change of MUC1 in the circumstance of Hp infection.
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INTRODUCTION: Although the efficacy of probiotics has been extensively studied in hepatic encephalopathy (HE), the results remain controversial. The objective of this study is to identify and update the association between probiotics and HE. METHODS: Up to December 2014, we searched Medline, Embase, Web of Science, Cochrane CENTRAL, and SinoMed of China for all relevant articles about probiotics and HE. Jadad score was used to evaluate the quality of studies. Pooled relative risk (RR), publication bias and heterogeneity were assessed. RESULTS: Nine studies met the inclusion criteria. Probiotics was associated with improvement of minimal HE and prophylaxis of overt HE [RR 1.52; 95% confidence intervals (95% CI) 1.00-2.33]. Studies with probiotics showed reduction of ammonia concentration [standard mean difference (SMD) -0.32, 95% CI -0.54 to -0.11]. Probiotics could reduce physical and psychosocial sickness impact profile (SIP) score with weight mean difference (WMD) -3.13 (95% CI -4.10 to -2.17) and WMD -3.50 (95% CI -4.91 to -2.08), respectively. Similar result was obtained with total SIP score (WMD -4.83; 95% CI -6.24 to -3.43). Reduction of severe adverse events, defined as minimal HE developing into overt HE, hospitalizations, infections or unrelated emergency room (ER) visits, was observed in HE with probiotics (RR 0.59; 95% CI 0.39-0.90). CONCLUSION: Our pooled results indicated that probiotics was associated with improvement of minimal HE, prophylaxis of overt HE, and reduction of SIP score and severe adverse events. Large well-designed randomised controlled trials are needed to confirm these results.
Subject(s)
Hepatic Encephalopathy/therapy , Probiotics/therapeutic use , Humans , Treatment OutcomeABSTRACT
BACKGROUND: Purifying stem cells is an inevitable process for further investigation and cell-therapy. Sorting side population (SP) cells is generally regarded as an effective method to enrich for progenitor cells. This study was to explore whether sorting SP could enrich for the Musashi1 (Msi1) positive cells from Msi1 high expression cells (Msi1(high) cells) derived from mouse embryonic stem cells (ESCs) in vitro. RESULTS: In this study, Msi1(high) cell population derived from ESCs were stained by Hoechst 33342, and then the SP and non-SP (NSP) fractions were analyzed and sorted by fluorescence activated cell sorter. Subsequently, the expressions of Msi1 and other markers for neural and intestinal stem cells in SP and NSP were respectively detected. SP and NSP cells were hypodermically engrafted into the backs of NOD/SCID mice to form grafts. The developments of neural and intestinal epithelial cells in these grafts were investigated. SP fraction was identified and isolated from Msi1(high) cell population. The expression of Msi1 in SP fraction was significantly higher than that in NSP fraction and unsorted Msi1(high) cells (P< 0.05). Furthermore, the markers for neural cells and intestinal epithelial cells were more highly expressed in the grafts from SP fraction than those from NSP fraction (P< 0.05). CONCLUSIONS: SP fraction, isolated from Msi1(high) cells, contains almost all the Msi1-positive cells and has the potential to differentiate into neural and intestinal epithelial cells in vivo. Sorting SP fraction could be a convenient and practical method to enrich for Msi1-positive cells from the differentiated cell population derived from ESCs.
Subject(s)
Cell Separation/methods , Embryonic Stem Cells/cytology , Flow Cytometry/methods , Nerve Tissue Proteins/analysis , RNA-Binding Proteins/analysis , Animals , Cell Differentiation , Cells, Cultured , Embryonic Stem Cells/metabolism , Mice , Mice, SCID , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Real-Time Polymerase Chain Reaction/methodsABSTRACT
BACKGROUND: Embryonic stem cells have great plasticity. In this study, we repaired impaired small intestine by transplanting putative intestinal epithelial stem cells (Musashi1 and hairy and enhancer of split 1 high expression cells) derived from embryonic stem cells. METHODS: The differentiation of definitive endoderm in embryoid bodies, derived from male ES-E14TG2a cells by the hanging-drop method, was monitored to define a time point for maximal induction of putative intestinal epithelial stem cells by epidermal growth factor. Furthermore, to evaluate the regenerative potential of intestinal epithelium, these putative stem cells were engrafted into NOD/SCID mice and female mice with enteritis. Donor cells were located by SRY DNA in situ hybridization. RESULTS: The results revealed that definitive endodermal markers were highly expressed in 5-day embryoid bodies. These embryoid body cells were induced into putative intestinal epithelial stem cells on the 5th day of epidermal growth factor administration. Grafts from these cells consisted of adenoid structures and nonspecific structural cells with strong expression of small-intestinal epithelial cell markers. In situ hybridization revealed that the donor cells could specifically locate in damaged intestinal epithelium, contribute to epithelial structures, and enhance regeneration. CONCLUSIONS: In conclusion, the Musashi1 and hairy and enhancer of split 1 high expression cells, derived from mouse embryonic stem cells, locate predominantly in impaired small-intestinal epithelium after transplantation and contribute to epithelial regeneration.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Embryonic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Intestine, Small/injuries , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Stem Cell Transplantation , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cells, Cultured , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Homeodomain Proteins/genetics , Male , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/genetics , RNA-Binding Proteins/genetics , Transcription Factor HES-1ABSTRACT
Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1-positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1-GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1-positive cells out of the differentiated cell population. The GFP-positive cells (i.e. Msi1-positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP-positive cells, but not in the GFP-negative cells. The markers for neural cells (Nestin and Tubulin ß III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1-positive cells than those from Msi1-negative cells (P<0.05). The grafts from the Msi1-negative cells contained more mesodermal-like tissues than those from the Msi1-positive cells. The pMsi1-GFP vector can be used to sort Msi1-positive cells from a cell population derived from mouse embryonic stem cells. The Msi1-positive cells can differentiate into neural and intestinal epithelial-like cells in vivo.