Anne Pryde

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.

Five year prospective study of the incidence, clinical features, and diagnosis of achalasia in Edinburgh.

With the increasing availability of manometry, patients with achalasia are often referred at an early stage when they lack the classic features of established disease. A prospective five year study of the presenting features of untreated achalasia referred to our department was undertaken. Twenty men and 18 women presented throughout adult life, with a mean age at the time of diagnosis of 44 years (range 17 to 76 years). The presenting symptoms were dysphagia: for solids (100%) and for liquids (97%), chest pain (74%), and weight loss (60%). Endoscopy was reported as normal in 15 patients and achalasia was suggested in only 21 of 33 barium examinations. Fourteen had been treated for gastrooesophageal reflux but none had been misdiagnosed as having cardiac or psychiatric disease. The annual incidence of achalasia in the Lothian region is 0.8/100,000 of population. Persistent dysphagia is the cardinal symptom of achalasia which presents throughout adult life. Nevertheless, recent onset achalasia is often misdiagnosed as gastrooesophageal reflux disease. Because endoscopy is frequently normal and the diagnosis is often not made by radiology, manometric investigation is necessary if the condition is to be recognised and treated at an early stage.

Normal pharyngoesophageal motility

Upper esophageal manometry is technically problematic. Published normal values are, therefore, few and wide ranging, reflecting catheter and recording-system variables, while the reproducibility of measurements and the influence of food consistency have been little studied. In this investigation, 50 healthy volunteers were studied with (1) a 2.8-mm-diameter six-sensor catheter-mounted transducer assembly and (2) a 3.2×7.2-mm sleeve device linked to a computerized recorder with a pressure-sample rate of 32/sec. The study protocol included water, bread, and semisolid swallows. Upper esophageal sphincter (UES) tonic pressures measured with the catheter-mounted assembly were lower and more reproducible than pressures measured with the sleeve system. Compared with water, bread swallows showed greater pharnygeal and sphincter after-contraction pressures, while semisolid swallows had less complete sphincter relaxation. Duration of pharyngoesophageal contractions was greater with bread or semisolid than water. The observations have established normal values for measurements of UES function and, in addition, have shown that (1) catheter variables significantly influence the measurement of upper sphincter tonic pressure, (2) pressures recorded with the catheter-mounted transducer are most reproducible, and (3) pharyngoesophageal motility patterns vary significantly according to the substance swallowed.Upper esophageal manometry is technically problematic. Published normal values are, therefore, few and wide ranging, reflecting catheter and recording-system variables, while the reproducibility of measurements and the influence of food consistency have been little studied. In this investigation, 50 healthy volunteers were studied with (1) a 2.8-mm-diameter six-sensor catheter-mounted transducer assembly and (2) a 3.2×7.2-mm sleeve device linked to a computerized recorder with a pressure-sample rate of 32/sec. The study protocol included water, bread, and semisolid swallows. Upper esophageal sphincter (UES) tonic pressures measured with the catheter-mounted assembly were lower and more reproducible than pressures measured with the sleeve system. Compared with water, bread swallows showed greater pharnygeal and sphincter after-contraction pressures, while semisolid swallows had less complete sphincter relaxation. Duration of pharyngoesophageal contractions was greater with bread or semisolid than water. The observations have established normal values for measurements of UES function and, in addition, have shown that (1) catheter variables significantly influence the measurement of upper sphincter tonic pressure, (2) pressures recorded with the catheter-mounted transducer are most reproducible, and (3) pharyngoesophageal motility patterns vary significantly according to the substance swallowed.

The Comparison between Conditioned Media and Serum-Free Media in Human Embryonic Stem Cell Culture and Differentiation

Human embryonic stem cells (hESCs) offer an inexhaustible supply of human somatic cell types through their ability to self-renew while retaining pluripotency. As such, hESC-derived cell types are important for applications ranging from in vitro modeling to therapeutic use. However, for their full potential to be realized, both the growth of the undifferentiated cells and their derivatives must be performed in defined culture conditions. Many research groups maintain hESCs using mouse embryonic fibroblasts (MEF) and MEF conditioned medium (CM). The use of murine systems to support hESCs has been imperative in developing hESC technology; however, they suffer from some major limitations including lack of definition, xenobiotic nature, batch-to-batch variation, and labor-intensive production. Therefore, hESC culture definition is essential if hESC lines, and their derivatives are to be quality assured and manufactured to GMP. We have initiated the process of standardizing hESC tissue culture and have employed two serum-free media: mTeSR (MT) and Stem Pro (SP). hESCs were maintained in a pluripotent state, for over 30 passages using MT and SP. Additionally, we present evidence that hESCs maintained in MT and SP generate equivalent levels of human hepatic endoderm as observed with CM. This data suggests that MT and SP are effective replacements for MEF-CM in hESC culture, contributing to the standardization of hESC in vitro models and ultimately their application.

Bone Marrow Stem Cells Contribute to Alcohol Liver Fibrosis in Humans

Bone marrow-derived stem cell (BMSC) contribution to liver repair varies considerably and recent evidence suggests these cells may contribute to liver fibrosis. We investigated the mobilization and hepatic recruitment of bone marrow (BM) stem cells in patients with alcohol liver injury and their contribution to parenchymal/non-parenchymal liver cell lineages. Liver biopsies from alcoholic hepatitis (AH) patients and male patients, who received a female liver transplant and developed AH, were analyzed for BM stem cell content by fluorescence in situ hybridization and immunostaining. Y chromosome analysis was performed, along with co-staining for hepatocyte, biliary, myofibroblast, and Ki-67 markers. Blood CD34(+) levels were quantified in AH patients by flow cytometry. AH patients had increased CD34(+) cell counts in liver tissue (1.834% +/- 0.605%; P < 0.05) and in blood (0.195% +/- 0.063%; P < 0.05) as compared with matched controls (0.299% + 0.208% and 0.067% +/- 0.01%). A proportion of hepatic myofibroblasts were BM-derived (7.9%-26.8%) as deemed by the co-localization of Y chromosome/alpha-smooth muscle actin (alpha-SMA) staining. In the cross-sex liver grafts with AH, 5.025% of the myofibroblasts were co-staining for CD34, suggesting that a population of CD34(+) cells were contributing to the hepatic myofibroblast population. There was no evidence of BM contribution to hepatocyte or biliary cell differentiation, nor evidence of increased hepatocyte regeneration. Alcohol liver injury mobilizes CD34(+) stem cells into the circulation and recruits them into the liver. These BMSCs contribute to the hepatic myofibroblast population but not to parenchymal lineages and do not promote hepatocyte repair.

Swallowing Performance in Patients with Vocal Fold Motion Impairment

Twenty-seven patients with vocal fold motion impairment underwent detailed pharyngoesophagel manometry with a strain gauge assembly linked to a computer recorder. Nine were known to have lesions of the central vagal trunk or nucleus, 9 had recurrent laryngeal nerve (RLN) palsy, and the remainder were idiopathic. The site of the lesion was a more important determinant of subjective swallowing performance than the position of the involved cord at laryngoscopy. Patients with cental lesions had lower tonic and contraction upper esophageal sphincter (UES) pressures than 25 age-matched controls, suggesting that high cervical branches of the lower cranial nerves are important in UES excitatory innervation. RLN palsy patients showed significantly increased pharyngeal contraction amplitude and reduced pharyngoesophageal wave durations. The results suggest that the dysphagia associated with vocal fold motion impairment is not simply due to the disruption of laryngeal deglutitive kinetics, but to independent effects on pharyngeal function.

Persistence of functional hepatocyte-like cells in immune-compromised mice

Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte‐like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver.

Toxicology of ZnO and TiO2 nanoparticles on hepatocytes: Impact on metabolism and bioenergetics

Abstract Background and aim: Zinc oxide (ZnO) and titanium dioxide (TiO2) nanomaterials (NMs) are used in many consumer products, including foodstuffs. Ingested and inhaled NM can reach the liver. Whilst their effects on inflammation, cytotoxicity, genotoxicity and mitochondrial function have been explored, no work has been reported on their impact on liver intermediary metabolism. Our aim was to assess the effects of sub-lethal doses of these materials on hepatocyte intermediary metabolism. Material and methods: After characterisation, ZnO and TiO2 NM were used to treat C3A cells for 4 hours at concentrations ranging between 0 and 10 μg/cm2, well below their EC50, before the assessment of (i) glucose production and glycolysis from endogenous glycogen and (ii) gluconeogenesis and glycolysis from lactate and pyruvate (LP). Mitochondrial membrane potential was assessed using JC-10 after 0–40 μg/cm2 ZnO. qRT-PCR was used to assess phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression. Dihydroethidium (DHE) staining and FACS were used to assess intracellular reactive oxygen species (ROS) concentration. Results: Treatment of cells with ZnO, but not TiO2, depressed mitochondrial membrane potential, leading to a dose-dependent increase in glycogen breakdown by up to 430%, with an increase of both glycolysis and glucose release. Interestingly, gluconeogenesis from LP was also increased, up to 10-fold and correlated with a 420% increase in the PEPCK mRNA expression, the enzyme controlling gluconeogenesis from LP. An intracellular increase of ROS production after ZnO treatment could explain these effects. Conclusion: At sub-lethal concentrations, ZnO nanoparticles dramatically increased both gluconeogenesis and glycogenolysis, which warrants further in vivo studies.

Systematic comparison of conventional oesophageal manometry with oesophageal motility while eating bread.

Conventional oesophageal manometry is seldom accompanied by symptoms and may indeed be normal in patients with a history of dysphagia. We have recently shown that oesophageal manometry during eating may be helpful in the evaluation of patients with dysphagia but there has been little systematic comparison of fed oesophageal motor patterns with conventional clinical manometry. Oesophageal manometry in response to water swallows and during eating was therefore examined in 58 consecutive patients who had been referred for clinical oesophageal function studies. The patients were divided into three groups according to the percentage of peristaltic activity during conventional manometry: group 1 (n = 21) had 100% peristalsis; group 2 (n = 29) had 1-99% peristalsis and group 3 (n = 8) were aperistaltic. All the patients in group 3 had achalasia and remained aperistaltic during eating, however, was less than with water swallows in both group 1 (53% compared with 100%) and group 2 (49% compared with 82.3%) patients. Synchronous contractions and non-conducted swallows were correspondingly increased during eating. Although there was a significant correlation between the amplitude of peristaltic contractions with water and bread in groups 1 and 2, mean peristaltic amplitudes were less with bread than with water swallows. The data show that there are substantial differences in the distal oesophageal motility patterns produced by water swallows and by eating. Conventional manometry with water swallows does not allow prediction of the fed oesophageal motility pattern, except in patients with achalasia.

Oxidative stress rather than triglyceride accumulation is a determinant of mitochondrial dysfunction in in vitro models of hepatic cellular steatosis

There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question as only a small proportion of individuals with steatosis progress to steatohepatitis. In this study, we focused on defining (i) the effects of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function (ii) the contributions of triglyceride, ROS and subsequent mitochondrial impairment on the metabolism of energy substrates.