Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells.

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity.

Development of a cost-effective automated platform to produce human liver spheroids for basic and applied research.

Liver disease represents an increasing cause of global morbidity and mortality. Currently, liver transplant is the only treatment curative for end-stage liver disease. Donor organs cannot meet the demand and therefore scalable treatments and new disease models are required to improve clinical intervention. Pluripotent stem cells represent a renewable source of human tissue. Recent advances in three-dimensional cell culture have provided the field with more complex systems that better mimic liver physiology and function. Despite these improvements, current cell-based models are variable in performance and expensive to manufacture at scale. This is due, in part, to the use of poorly defined or cross-species materials within the process, severely affecting technology translation. To address this issue, we have developed an automated and economical platform to produce liver tissue at scale for modelling disease and small molecule screening. Stem cell derived liver spheres were formed by combining hepatic progenitors with endothelial cells and stellate cells, in the ratios found within the liver. The resulting tissue permitted the study of human liver biology 'in the dish' and could be scaled for screening. In summary, we have developed an automated differentiation system that permits reliable self-assembly of human liver tissue for biomedical application. Going forward we believe that this technology will not only serve as anin vitroresource, and may have an important role to play in supporting failing liver function in humans.

Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering.

Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions.

Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue: process optimization and cell population metrics across a large cohort of diverse demographics.

BACKGROUND: Adipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction. Until recently MSC could only be isolated in a process involving ex-vivo culture and their in-vivo identity, location and frequency remained elusive. We have documented that pericytes (CD45-, CD146+, and CD34-) and adventitial cells (CD45-, CD146-, CD34+) (collectively termed perivascular stem cells or PSC) represent native ancestors of the MSC, and can be prospectively purified using fluorescence activated cell sorting (FACS). In this study we describe an optimized protocol that aims to deliver pure, viable and consistent yields of PSC from adipose tissue. We analysed the frequency of PSC within adipose tissue, and the effect of patient and procedure based variables on this yield. METHODS: Within this twin centre study we analysed the adipose tissue of n = 131 donors using flow cytometry to determine the frequency of PSC and correlate this with demographic and processing data such as age, sex, BMI and cold storage time of the tissue. RESULTS: The mean number of stromal vascular fraction (SVF) cells from 100 ml of lipoaspirate was 34.4 million. Within the SVF, mean cell viability was 83 %, with 31.6 % of cells being haematopoietic (CD45+). Adventitial cells and pericytes represented 33.0 % and 8 % of SVF cells respectively. Therefore, a 200 ml lipoaspirate would theoretically yield 23.2 million viable prospectively purified PSC - sufficient for many reconstructive and regenerative applications. Minimal changes were observed in respect to age, sex and BMI suggesting universal potential application. CONCLUSIONS: Adipose tissue contains two anatomically and phenotypically discreet populations of MSC precursors - adventitial cells and pericytes - together referred to as perivascular stem cells (PSC). More than 9 million PSC per 100 ml of lipoaspirate can be rapidly purified to homogeneity using flow cytometry in clinically relevant numbers potentially circumventing the need for purification and expansion by culture prior to clinical use. The number and viability of PSC are minimally affected by patient age, sex, BMI or the storage time of the tissue, but the quality and consistency of yield can be significantly influenced by procedure based variables.

Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

Ethical, legal and practical issues of establishing an adipose stem cell bank for research.

Access to human tissue is critical to medical research, however the laws and regulations surrounding gaining ethical and legal access to tissue are often poorly understood. Recently, there has been a huge increase in the interest surrounding the therapeutic application of adipose tissue, and adipose-derived stem cells. To facilitate our own research interests and possibly assist our local colleagues and collaborators, we established a Research Tissue Bank (RTB) to collect, store and distribute human adipose tissue derived cells with all the appropriate ethical approval for subsequent downstream research. Here we examine the legal, ethical and practical issues relating to the banking of adipose tissue for research in the UK, and discuss relevant international guidelines and policies. We also share our experiences of establishing an RTB including the necessary infrastructure and the submission of an application to a Research Ethics Committee (REC).

Surgical findings in the treatment of Dupuytren's disease after initial treatment with clostridial collagenase (Xiaflex).

We investigated the difficulty of surgical fasciectomy after previous treatment with clostridial collagenase injection. The 35 clinicians who had participated in the initial trials of this injection were contacted via email. Twenty-eight responded, nine of whom reported on 15 patients. Most (seven of nine) felt there was no significant distortion of anatomy and rated the level of technical difficulty as equivalent to a primary Dupuytren's fasciectomy at the observed degree of contracture (nine of 15 cases). One respondent (four of 15 cases) reported significantly more difficulty and grossly distorted anatomy. One surgical complication, a wound dehiscence, was reported.

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription.

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.

Testing instance models of face repetition priming.

Two experiments examining repetition priming in face recognition are reported. They employed eight rather than the more usual two presentation trials so that the prediction made by Logan's (1988) instance model of power function speedup of response time (RT) distributions could be examined. In Experiment 1, we presented the same photograph on each trial; in Experiment 2, we presented photographs of varying poses. Both experiments showed repetition priming effects for familiar and unfamiliar faces, power function speedup for both the mean and the standard deviation of RT and the power function speedup of the quanties of the RT distributions. We argue that our findings are consistent with the predictions made by the instance model and provide an explanatory challenge for alternative theoretical approaches.

Movement, face processing and schizophrenia: evidence of a differential deficit in expression analysis.

Three dynamic face-processing tasks based on the Bruce & Young (1986) functional model of face processing were presented to 10 schizophrenic and 10 depressed inpatients and to 10 non-patient subjects. Familiar face recognition, facial expression recognition and unfamiliar face matching were examined. Schizophrenic patients' performance was significantly poorer than that of depressed patients and non-patient controls. Significantly lower scores were obtained on the facial expression recognition task than on the familiar face recognition task. There was a differential pattern of group performance on each of the three tasks: schizophrenic and depressed patients were as accurate as non-patient controls on the familiar face recognition task, but significantly less accurate than non-patient controls on the unfamiliar face-matching task. Schizophrenic patients were significantly less accurate than depressed patients and non-patient controls on the facial expression recognition task. The results are contrasted with an analogous static face-processing study.

Face perception after brain injury. Selective impairments affecting identity and expression.

Current theoretical models of face perception postulate separate routes for processing information needed in the recognition of a familiar face, for matching photographs of unfamiliar faces and for the analysis of facial expressions. The present study investigated this claim in a group of ex-servicemen who had sustained unilateral brain injuries affecting posterior areas of the left or right cerebral hemisphere. Care was taken to confirm the nature of impairment by using two different tasks to assess each of the three theoretically defined abilities (leading to a total of six tasks). We adopted a stringent application of the double dissociation methodology to investigate the pattern of performance across tasks of individual ex-servicemen. A selective impairment was defined as a significantly impoverished performance on both tests of a specific ability, while all other tasks were performed within normal limits. In addition, we used both accuracy and response latency measures to substantiate evidence for spared or defective abilities. The results showed selective impairments of all three abilities on accuracy scores. Response latency data confirmed the finding of a selective deficit in the processing of facial expressions, but produced evidence suggesting that impairments affecting familiar face recognition and unfamiliar face matching were not completely independent from each other in this group of ex-servicemen.

Impaired discrimination of familiar from unfamiliar faces.

We report investigations of the face processing abilities of J.T., a man who had suffered a right hemisphere stroke. J.T. showed a marked problem in discriminating between familiar and unfamiliar faces, with no corresponding problem in discriminating familiar from unfamiliar names. The problem with faces was still found 2 years after the stroke, but had resolved at long-term follow-up (9 years post stroke). When given unlimited time to respond, J.T. did not show any problem in recognising familiar faces, but tended to think that he recognised unfamiliar faces. When under time pressure, however, J.T. also failed to recognise a number of familiar faces which he could readily identify when the time pressure was removed. J.T.'s ability to remember a face and to think of other people who might be similar in appearance was affected by whether or not the face seemed familiar or unfamiliar to him. Hence, whatever underlay the spurious sense of familiarity was sufficient to produce real differences between the way in which genuinely unfamiliar and spuriously familiar faces were seen, leading us to suggest that his impaired discrimination of unfamiliar from familiar faces reflected a malfunction of face recognition units.

Face processing in psychiatric conditions.

Functional models of face processing have indicated that dissociations exist between the various processes involved, e.g. between familiar face recognition and matching of unfamiliar faces, and between familiar face recognition and facial expression analysis. These models have been successfully applied to the understanding of the different types of impairment that can exist in neuropsychological patients. In the present study, aspects of face processing in psychiatric patients were investigated in relation to Bruce & Young's (1986) model. Based on this functional model different predictions can be made. We contrast here the impaired expression analysis hypothesis, which is that psychiatric patients would show a deficit in facial expression recognition, but not in facial identity recognition or unfamiliar face matching, with the generalized deficit hypothesis, that patients would be impaired on all tasks. These hypotheses were examined using three forced-choice tasks (facial recognition, facial expression recognition, and unfamiliar face matching) which were presented to schizophrenic and depressed patients, and to non-patient controls. Results showed that schizophrenic patients performed at a significantly lower level than non-patient controls on all three tasks, supporting the generalized deficit hypothesis.

Routes through the face recognition system.

Two experiments are reported which seek to examine the proposition first put forward by Hay and Young (1982), that recognition of a known person after seeing his or her face proceeds through a series of sequentially organized stages. In both experiments subjects were shown a selection of famous and unfamiliar faces and required to state whether each face was familiar. They were then asked to recall semantic information and the person's name. Of all the possible response types, only some are predicted by models derived from Hay and Young (1982), and only these responses were observed in Experiment 1. In order to give as complete an account as possible of the slips and errors made by subjects, they were interrogated some days after completing the testing phase in Experiment 2. As in the first experiment, the results supported the view that distinct but successive stages are involved in everyday face recognition. The method developed here provides an extension of the "dairy" type of study of everyday recognition errors into laboratory conditions, which confirms the findings of studies of everyday errors and provides strong support for sequential models.

Facial neglect.

Following a stroke, a retired industrial chemist, K.L., complained that faces looked "different" and had become difficult to recognize. Investigation of this problem revealed that it particularly affected the left half of a face as seen by K.L. Defective recognition of this (left) half was found for normal faces, chimaeric faces, and for half-faces presented in isolation, whether upright or inverted. The problem was apparent for both internal and external facial features. Further studies with chimaeric faces demonstrated inattention to left-side features in K.L.'s judgements of facial expression and of resemblance between faces. Moreover, the left-half of a chimaeric face was affected even when it was itself forming part of the face positioned on the right in a display of two horizontally aligned chimaerics. K.L.'s spatial contrast sensitivity function was within normal limits for his age. He did not experience differential difficulty in recognizing the left side of everyday objects or of car-fronts (another stimulus class demanding within-category discrimination between visually similar items that display approximate left-right symmetry). He was also able to sort left or right half-stimuli correctly into the categories "human face", "dog face", or "tree". Although K.L. had a left visual field defect, the problem with faces occurred within otherwise intact parts of his field of vision. We suggest that his disorder can be considered a domain-specific form of unilateral neglect.

Configurational information in face perception.

A new facial composites technique is demonstrated, in which photographs of the top and bottom halves of different familiar faces fuse to form unfamiliar faces when aligned with each other. The perception of a novel configuration in such composite stimuli is sufficiently convincing to interfere with identification of the constituent parts (experiment 1), but this effect disappears when stimuli are inverted (experiment 2). Difficulty in identifying the parts of upright composites is found even for stimuli made from parts of unfamiliar faces that have only ever been encountered as face fragments (experiment 3). An equivalent effect is found for composites made from internal and external facial features of well-known people (experiment 4). These findings demonstrate the importance of configurational information in face perception, and that configurations are only properly perceived in upright faces.

Face-name interference.

Interference effects between the processing of simultaneously presented photographs of faces of familiar people and printed names of familiar people were investigated. Printed names interfered with identifying faces, whereas faces did not interfere with saying printed names (Experiments 1 and 3). In contrast, faces interfered more with name categorization than names interfered with face categorization (Experiments 2 and 4). Despite a priori reasons as to why faces might be thought to possess functional properties different from those of other visual objects, the observed effects are comparable to those found in object-word interference studies, with photographs of faces behaving like pictures of objects and printed people's names behaving like printed names of objects. In face naming tasks, the presence of related names produced more interference than did the presence of unrelated names (Experiment 1). This effect was examined in greater detail in Experiment 3, where we found that the effect arises when the face and the name belong to people of similar appearance. An effect of common category membership was not found in Experiment 3. Experiment 5, however, showed that names of people highly associated with the person whose face is presented also produce more interference than do names of unrelated people.