The tumor microenvironment offers favorable conditions for tumor progression, and activated fibroblasts, known as cancer‐associated fibroblasts, play a pivotal role. TP53‐deficient cancer cells are ...known to induce strong fibroblast activation. We aimed to elucidate the oncogenic role of exosomes derived from TP53‐deficient colon cancer cells in fibroblast proliferation and tumor growth. Cancer cell‐derived exosomes (CDEs) were isolated from the conditioned media of cancer cells using a sequential ultracentrifugation method. The effects of exosomes on tumor growth were evaluated using human cell lines (TP53‐WT colon cancer, HCT116; TP53‐mutant colon cancer, HT29; and fibroblasts, CCD‐18Co and WI‐38) and an immune‐deficient nude mouse xenograft model. HCT116 (HCT116sh p53) cells deficient in TP53 accelerated cocultured fibroblast proliferation compared to TP53‐WT HCT116 (HCT116sh control) cells in vitro. Exosomes from HCT116sh p53 cells suppressed TP53 expression of fibroblasts and promoted their proliferation. Xenografts of HCT116sh p53 cells grew significantly faster than those of HCT116sh control cells in the presence of co‐injected fibroblasts, but this difference was diminished by CDE inhibition. Microarray analysis identified upregulation of several microRNAs (miR‐1249‐5p, miR‐6737‐5p, and miR‐6819‐5p) in TP53‐deficient CDEs, which were functionally proven to suppress TP53 expression in fibroblasts. Exosomes derived from TP53‐mutant HT29 cells also suppressed TP53 expression in fibroblasts and accelerated their growth. The proliferative effect of HT29 on cocultured fibroblasts was diminished by inhibition of these miRNAs in fibroblasts. Our results suggest that CDEs play a pivotal role in tumor progression by fibroblast modification. Cancer cell‐derived exosomes might, therefore, represent a novel therapeutic target in colon cancer.
Exosomes derived from cancer cells play a pivotal role in tumor progression through fibroblast modification and might represent a novel therapeutic target in colon cancer.
Deficiency of p53 in cancer cells activates the transformation of normal tissue fibroblasts into carcinoma-associated fibroblasts; this promotes tumor progression through a variety of mechanisms in ...the tumor microenvironment. The role of autophagy in carcinoma-associated fibroblasts in tumor progression has not been elucidated. We aimed to clarify the significance of autophagy in fibroblasts, focusing on the TP53 status in co-cultured human colorectal cancer cell lines (TP53-wild-type colon cancer, HCT116; TP53-mutant colon cancer, HT29; fibroblast, CCD-18Co) in vitro. Autophagy in fibroblasts was significantly suppressed in association with ACTA2, CXCL12, TGFβ1, VEGFA, FGF2, and PDGFRA mRNA levels, when co-cultured with p53-deficient HCT116
cells. Exosomes isolated from the culture media of HCT116
cells significantly suppressed autophagy in fibroblasts via inhibition of ATG2B. Exosomes derived from TP53-mutant HT29 cells also suppressed autophagy in fibroblasts. miR-4534, extracted from the exosomes of HCT116
cells, suppressed ATG2B in fibroblasts. In conclusion, a loss of p53 function in colon cancer cells promotes the activation of surrounding fibroblasts through the suppression of autophagy. Exosomal miRNAs derived from cancer cells may play a pivotal role in the suppression of autophagy.
Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. ...Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.
Although oxygen is a vital nutrient for the hepatocytes
, few reports have focused on its effect during hepatic differentiation of induced pluripotent stem cells (iPSCs). In this report, we performed ...the hepatic differentiation of human iPSCs (hiPSCs) under different atmospheric oxygen concentrations and oxygen supply fluxes to investigate the effects of oxygen in terms of both the concentration and the supply flux. Results demonstrate that direct oxygenation through a polydimethylsiloxane (PDMS) membrane enhances the maturation and efficient production of hiPSC-derived hepatocyte-like cells (iHeps). Thus, direct oxygenation through a PDMS membrane is a better alternative culture method over conventional tissue culture-treated polystyrene (TCPS) plates for the maturation of hiPSC-derived hepatocytes
.
In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for ...complete and partial on‐chip differentiation were tested. Unlike full on‐chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin‐1 or albumin, while no stabilin‐1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha (
ESRRA), hepatic nuclear factor 1 (
HNF1A), hepatic nuclear factor 4 (
HNF4A), transcription factor 4 (
TCF4), and CCAAT enhancer binding protein alpha (
CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia‐inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB‐positive cells and the
ɑ‐fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on‐chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte‐like cells in microfluidic biochips with significant improvements over Petri dish cultures.
In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) in microfluidic biochips.
Suspension culture of three-dimensional (3D) spheroid of human induced pluripotent stem cells (hiPSCs) has been known as a potential method to enhance the scalability of hepatic differentiation of ...hiPSCs. However, the impact of size-related factor of initial formed spheroid were not largely considered. To address this problem, we evaluate the impact of different specific spheroid size of hiPSCs by forming the individual spheroid from different number of hiPSCs and differentiated into hiPSCs-derived hepatocytes (iHeps). The results showed that larger spheroid exhibit enhanced capability to differentiated into hepatic lineage by increasing the expression marker albumin, CYP3A4 and lower expression of fetal hepatic marker AFP. Several factor such as the tendency of cystic like structure forming, the necrotic area of the large dense spheroid, and interference of WNT/β-catenin signaling was significantly affecting the resulted iHeps. In this study, we suggest that the optimal spheroid size for hepatic differentiation can be attained from 500 to 600 μm diameter spheroid formed from 12,500–25,000 hiPSCs. This size can be potentially applied for various practical use of hepatic differentiation in scalable suspension culture.
Spontaneous isolated visceral artery dissection (SIVAD) involving the celiac artery or superior mesenteric artery is rare, but it can be fatal. Given its rare incidence, the clinical characteristics ...of SIVAD are not fully understood. Therefore, the aim of this study was to investigate the clinical characteristics and prognosis of SIVAD.
We retrospectively reviewed 39 consecutive patients diagnosed with SIVAD from January 2007 to December 2016. Demographic characteristics, symptoms, vital signs, blood examination results, and computed tomography findings were retrieved through medical record review.
The median age of the patients was 52 years; 94.9% were male, and 64.1% were symptomatic. Median follow-up duration was 11 months. Overall, hypertension (48.7%) and smoking (79.5%) were frequently observed. There were significant differences between symptomatic and asymptomatic patients in white blood cell count and creatine kinase level but not in fibrin degradation products or D-dimer level. There was a significant correlation between symptoms and length of dissection on computed tomography (P < .01). Conservative treatment was performed in 32 patients (82.1%), and only 7 patients required open surgery or intravascular intervention. Notably, the diameter of affected vessels decreased spontaneously with no rupture or symptom recurrence during follow-up, and mortality was 0% at both 30 days and 1 year.
The utility of blood examination, especially for fibrin degradation products and D-dimer levels, for diagnosis of SIVAD is limited. A high index of suspicion is warranted in patients presenting with persistent severe abdominal pain. Conservative treatment should be considered first-line therapy in patients without any signs of bowel ischemia or rupture.
Shrub species are used in restoration projects on dryland for their facilitation effects, which include environmental improvements and protection from herbivore feeding. Facilitation effects on ...forage grasses are potentially important in improving grazing capacity on rangelands. However, the morphology-dependent performance of benefactor plants in facilitating forage species growth and supplementation under moderate grazing intensity remains unclear. Here, our main purpose was to measure facilitation performance in terms of the survival of a native forage grass,
Agropyron cristatum
(L.) Gaertn. (Gramineae)., in accordance with the growth conditions of a sand-fixing benefactor shrub,
Caragana microphylla
Lam., in the Hulun Buir Grassland, northern China. Six study sites with patches of
A. cristatum
and
C. microphylla
were established at the foot of fixed sand dunes. At each site, five quadrats were set in places where
C. microphylla
coverage was 100% and
A. cristatum
grew among the shrubs (shrub quadrats), and another five were set where
A. cristatum
grew alone without
C. microphylla
(grass quadrats). We measured the morphological traits of
C. microphylla
and
A. cristatum
in all 60 quadrats, along with the soil water content and soil temperature. The data were compared between the shrub and grass quadrats by generalized linear mixed-effect models to assess the shrub’s facilitation effects. We also used such models to elucidate the relationship between the average height of
C. microphylla
and the morphological traits of
A. cristatum
in the shrub quadrats. The maximum height, average grazed height, and the number of seed heads of
A. cristatum
were greater in the shrub quadrats than in the grass quadrats. The soil surface temperature was lower in the shrub quadrats. The maximum height and seed head number of
A. cristatum
were positively associated with the average height of
C. microphylla.
These results suggest that the grazing impact and heat stress were smaller in shrub quadrats than in grass quadrats, and that the degree of this protective effect depended on the shrub height. The shrub canopy seemed to reduce the increase in soil temperature and keep the grass vigorous. Livestock likely avoided grazing grasses in the
C. microphylla
patches because of the shrub’s spiny leaves; only the upper parts of the grass stems (including the seed heads) protruding from the shrub canopy were grazed. The sand-fixing shrub thus moderates the grazing impact and soil temperature, and contributes to vegetation restoration and grazing system sustainability.
Inflammatory abdominal aortic aneurysm (AAA) is a member of a family of disorders referred to as "chronic periaortitis" together with retroperitoneal fibrosis. Retroperitoneal fibrosis is included in ...IgG4-related disease, which is characterized by numerous infiltrating IgG4-positive plasma cells and high serum IgG4 concentrations. However, the relationship between IgG4-related disease and inflammatory AAA has not been documented. In this study, we examined the clinicopathologic characteristics of inflammatory (10 cases) and atherosclerotic (22 cases) AAAs, based on the hypothesis that inflammatory AAA might be related to IgG4-related disease. Cases of inflammatory AAA could be classified into 2 groups based on immunostaining of IgG4. Four patients showed diffuse infiltration of abundant IgG4-positive plasma cells (IgG4-related cases), whereas the remaining 6 cases of inflammatory AAA and all cases of atherosclerotic AAA had only a few IgG4-positive plasma cells (non-IgG4-related cases). IgG4-related inflammatory AAA was pathologically characterized by the frequent infiltration of eosinophils, lymph follicle formation, perineural inflammatory extension, and inconspicuous infiltration of neutrophils compared with non-IgG4-related inflammatory AAA. Obliterative phlebitis, which is venous occlusion with inflammatory cell infiltration, is observed in all IgG4-related cases. In addition, serum IgG4 concentrations were significantly higher in IgG4-related inflammatory AAA (109 to 559 mg/dL, normal range: 4 to 110 mg/dL) than non-IgG4-related inflammatory AAA (32 to 59 mg/dL) and all atherosclerotic AAA (12 to 83 mg/dL). In conclusion, inflammatory AAAs might be classified into 2 groups: IgG4-related or nonrelated. The former might be one of the IgG4-related diseases, and could be included in IgG4-related periaortitis together with retroperitoneal fibrosis.
Background and Aim
The morphological diagnosis of microvessels on the surface of superficial esophageal squamous cell carcinomas using magnifying endoscopy with narrow‐band imaging is widely used in ...clinical practice. Nevertheless, inconsistency, even among experts, remains a problem. We constructed a convolutional neural network‐based computer‐aided diagnosis system to classify the microvessels of superficial esophageal squamous cell carcinomas and evaluated its diagnostic performance.
Methods
In this retrospective study, a cropped magnifying endoscopy with narrow‐band images from superficial esophageal squamous cell carcinoma lesions was used as the dataset. All images were assessed by three experts, and classified into three classes, Type B1, B2, and B3, based on the Japan Esophagus Society classification. The dataset was divided into training and validation datasets. A convolutional neural network model (ResNeXt‐101) was trained and tuned with the training dataset. To evaluate diagnostic accuracy, the validation dataset was assessed by the computer‐aided diagnosis system and eight endoscopists.
Results
In total, 1777 and 747 cropped images (total, 393 lesions) were included in the training and validation datasets, respectively. The diagnosis system took 20.3 s to evaluate the 747 images in the validation dataset. The microvessel classification accuracy of the computer‐aided diagnosis system was 84.2%, which was higher than the average of the eight endoscopists (77.8%, P < 0.001). The area under the receiver operating characteristic curves for diagnosing Type B1, B2, and B3 vessels were 0.969, 0.948, and 0.973, respectively.
Conclusions
The computer‐aided diagnosis system showed remarkable performance in the classification of microvessels on superficial esophageal squamous cell carcinomas.
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