Neil A Hanley

Central Manchester University Hospitals NHS Foundation Trust, Manchester, England, United Kingdom

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Publications (83)510.76 Total impact

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    ABSTRACT: Background & aims Hepatocyte-like cells (HLCs) differentiated from pluripotent stem cells by the use of soluble factors can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages and two different protocols for direct comparison with fresh fetal and adult hepatocytes. Methods Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. Expression of 81% of phase 1 enzymes in HLCs was significantly upregulated and half were statistically no different from in fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests devised by principal components analysis to distinguish fetal from adult hepatocytes HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.
    Journal of Hepatology. 10/2014;
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    ABSTRACT: Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), IκB kinase-α (IKKα), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome.
    The Journal of clinical investigation 08/2014; · 15.39 Impact Factor
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    ABSTRACT: The basic helix-loop-helix transcription factor, NEUROG3, is critical in causing endocrine commitment from a progenitor cell population in the developing pancreas. In human, NEUROG3 has been detected from 8 weeks post-conception (wpc). However, the profile of its production and when it ceases to be detected is unknown. In this study we have defined the profile of NEUROG3 detection in the developing pancreas to give insight into when NEUROG3-dependent endocrine commitment is possible in the human fetus. Immunohistochemistry allowed counting of cells with positively stained nuclei from 7 wpc through to term. mRNA was also isolated from sections of human fetal pancreas and NEUROG3 transcription analyzed by quantitative reverse transcription and polymerase chain reaction. NEUROG3 was detected as expected at 8 wpc. The number of NEUROG3-positive cells increased to peak levels between 10 wpc and 14 wpc. It declined at and after 18 wpc such that it was not detected in human fetal pancreas at 35-41 wpc. Analysis of NEUROG3 transcription corroborated this profile by demonstrating very low levels of transcript at 35-41 wpc, more than 10-fold lower than levels at 12-16 wpc. These data define the appearance, peak and subsequent disappearance of the critical transcription factor, NEUROG3, in human fetal pancreas for the first time. By inference, the window for pancreatic endocrine differentiation via NEUROG3 action opens at 8 wpc and closes between 21 and 35 wpc.
    Islets 05/2014; 6(3):e954436. · 1.55 Impact Factor
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    ABSTRACT: Glucocorticoids (Gc) regulate cell fate and immune function. We identified the metasasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator, relevant to human disease. Merm1 binds the GR co-activator GRIP1, but not GR. Loss of Merm1 impaired both GR transactivation, and transrepression, by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterised promoter. Inflammation promotes Gc resistance, in part through the actions of TNF α and IFN γ. These cytokines suppressed Merm1 protein expression, by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine-suppression of Merm1, and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of Gc sensitivity in inflammation, with suppressed expression in pulmonary disease.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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    ABSTRACT: Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.
    PLoS ONE 01/2014; 9(1):e86372. · 3.53 Impact Factor
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    ABSTRACT: Liver fibrosis is a major cause of morbidity and mortality. It is characterised by excessive extracellular matrix (ECM) deposition from activated hepatic stellate cells (HSCs). Although potentially reversible, treatment remains limited. Understanding how ECM influences the pathogenesis of the disease may provide insight into novel therapeutic targets for the disease. The extracellular protein Epimorphin (EPIM) has been implicated in tissue repair mechanisms in several tissues, partially, through its ability to manipulate proteases. In this study, we have identified that EPIM modulates the ECM environment produced by activated hepatic stellate cells (HSCs), in part, through down-regulation of pro-fibrotic Sex-determining region Y-box 9 (SOX9).
    PLoS ONE 01/2014; 9(6):e100091. · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: -Hypertension or aortic stenosis causes pressure overload, which evokes hypertrophic myocardial growth. Sustained cardiac hypertrophy eventually progresses to heart failure. Growing evidence indicates that restraining hypertrophy could be beneficial; here we discovered that FTY-720, an immuno-modulator for treating multiple sclerosis, can reverse existing cardiac hypertrophy/fibrosis. METHODS AND RESULTS: -Male C57/Bl6 mice underwent transverse aortic constriction (TAC) for 1 week followed by FTY-720 treatment for 2 weeks under continuing TAC. Compared to vehicle-treated TAC hearts, FTY-720 significantly reduced ventricular mass, ameliorated fibrosis and improved cardiac performance. Mechanistic studies led us to discover that FTY-720 appreciably inhibited NFAT activity. Moreover, we found that in primary cardiomyocytes (rat and human) pertussis toxin (PTX, Gi-coupled receptor inhibitor) substantially blocked the anti-hypertrophic effect of FTY-720. This observation was confirmed in a mouse model of pressure overload. Interestingly, gene array analysis of TAC-hearts revealed that FTY-720 profoundly decreased gene expression of a group of matricellular proteins, of which periostin was prominent. Analysis of periostin protein expression in TAC-myocardium, as well as in rat and human cardiac fibroblasts confirmed the array data. Moreover, we found that FTY-720 treatment or knockdown of periostin protein was able to inhibit TGF-β responsiveness and decrease collagen expression. CONCLUSIONS: -FTY-720 alleviates existing cardiac hypertrophy/fibrosis through mechanisms involving negative regulation of NFAT activity in cardiomyocytes and reduction of periostin expression allowing for a more homeostatic extracellular compartment milieu. Together, FTY-720 or its analogues could be a promising new approach for treating hypertrophic/fibrotic heart disease.
    Circulation Heart Failure 06/2013; · 6.68 Impact Factor
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    ABSTRACT: Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation towards a β-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development prior to PDX1 detection. In contrast to the published data from mouse embryos, during human pancreas development we detected only a single phase of NEUROG3 expression and endocrine differentiation from approximately 8 weeks, prior to which NKX2.2 was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell-types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro towards the pancreatic β-cell so as to model human development, or enable drug discovery and potential cell therapy.
    Diabetes 04/2013; · 7.90 Impact Factor
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    ABSTRACT: Optical approaches have been explored to enable undifferentiated to be distinguished from differentiated stem cells. Optical measurements combined with theoretical and statistical analysis provide a novel approach with the potential for identification and sorting.
    Optical Trapping Applications; 04/2013
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    ABSTRACT: Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.
    The American Journal of Human Genetics 03/2013; · 11.20 Impact Factor
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    ABSTRACT: Failure to predict hepatotoxic drugs in pre-clinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise with differentiated hepatocyte-like cells currently considered to be 'fetal-like' in their maturity. However, this judgement is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. Typical biomarkers showed that adult hepatocytes functioned equally well in sandwich or air-liquid interface culture. Fetal cells, however, were viable over longer culture periods and their function was enhanced over time in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. The clustering also shows that primary cells cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including Cytochrome P450 2A6, Glutathione S transferase P and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. Conclusion: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of hepatocytes in culture and provide a new protein 'toolkit' for determining human hepatocyte maturity in cultured cells. (HEPATOLOGY 2013.).
    Hepatology 03/2013; · 12.00 Impact Factor
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    ABSTRACT: Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.
    The American Journal of Human Genetics 01/2013; · 11.20 Impact Factor
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    ABSTRACT: Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.
    Stem cell reports. 01/2013; 1(4):293-306.
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    ABSTRACT: The molecular basis of type 2 diabetes predisposition at most established susceptibility loci remains poorly understood. KCNQ1 maps within the 11p15.5 imprinted domain, a region with an established role in congenital growth phenotypes. Variants intronic to KCNQ1 influence diabetes susceptibility when maternally inherited. By use of quantitative PCR and pyrosequencing of human adult islet and fetal pancreas samples, we investigated the imprinting status of regional transcripts and aimed to determine whether type 2 diabetes risk alleles influence regional DNA methylation and gene expression. The results demonstrate that gene expression patterns differ by developmental stage. CDKN1C showed monoallelic expression in both adult and fetal tissue, whereas PHLDA2, SLC22A18, and SLC22A18AS were biallelically expressed in both tissues. Temporal changes in imprinting were observed for KCNQ1 and KCNQ1OT1, with monoallelic expression in fetal tissues and biallelic expression in adult samples. Genotype at the type 2 diabetes risk variant rs2237895 influenced methylation levels of regulatory sequence in fetal pancreas but without demonstrable effects on gene expression. We demonstrate that CDKN1C, KCNQ1, and KCNQ1OT1 are most likely to mediate diabetes susceptibility at the KCNQ1 locus and identify temporal differences in imprinting status and methylation effects, suggesting that diabetes risk effects may be mediated in early development.
    Diabetes 11/2012; · 7.90 Impact Factor
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    ABSTRACT: A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.
    Cell metabolism 10/2012; 16(4):435-48. · 17.35 Impact Factor
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    ABSTRACT: Amongst the different types of adverse drug reactions, drug-induced liver injury is the most prominent cause of patient morbidity and mortality. However, the current available hepatic model systems developed for evaluating safety have limited utility and relevance as they do not fully recapitulate a fully functional hepatocyte, and do not sufficiently represent the genetic polymorphisms present in the population. The rapidly advancing research in stem cells raises the possibility of using human pluripotent stem cells in bridging this gap. The generation of human induced pluripotent stem cells via reprogramming of mature human somatic cells may also allow for disease modelling in vitro for the purposes of assessing drug safety and toxicology. This would also allow for better understanding of disease processes and thus facilitate in the potential identification of novel therapeutic targets. This review will focus on the current state in efforts to derive hepatocytes from human pluripotent stem cells for potential use in hepatotoxicity evaluation, and aims to provide an insight as to where the future of the field may lie. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.
    British Journal of Clinical Pharmacology 06/2012; · 3.69 Impact Factor

Publication Stats

2k Citations
510.76 Total Impact Points

Institutions

  • 2014
    • Central Manchester University Hospitals NHS Foundation Trust
      Manchester, England, United Kingdom
  • 1999–2012
    • Newcastle University
      • Institute of Cellular Medicine
      Newcastle upon Tyne, ENG, United Kingdom
  • 2010–2011
    • The University of Manchester
      • School of Biomedicine
      Manchester, ENG, United Kingdom
  • 2002–2010
    • University of Southampton
      • Developmental Origins of Health and Disease
      Southampton, England, United Kingdom
    • University of Toronto
      Toronto, Ontario, Canada
  • 2009
    • Radboud University Medical Centre (Radboudumc)
      Nymegen, Gelderland, Netherlands
  • 2008
    • Centre for Stem Cells Sciences
      Bhaganagar, Andhra Pradesh, India
  • 2007
    • University of Birmingham
      • Institute for Biomedical Research
      Birmingham, ENG, United Kingdom
  • 2006
    • New York University
      • Department of Pediatrics
      New York City, NY, United States
  • 1999–2004
    • University of Texas Southwestern Medical Center
      • Department of Internal Medicine
      Dallas, TX, United States
  • 2000
    • NYU Langone Medical Center
      • Department of Pediatrics
      New York City, NY, United States