Neil A Hanley

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

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Publications (92)634.5 Total impact

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    ABSTRACT: Diffuse congenital hyperinsulinism in infancy (CHI-D) arises from mutations inactivating the KATP channel, however, the phenotype is difficult to explain from electrophysiology alone. Here we have studied wider abnormalities in the β-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared to control. PAX4 and ARX expression was decreased. A tendency to increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas and implying immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. Increased proliferation in CHI-D was most elevated in duct (5-11 fold) and acinar (7-47 fold) lineages. Increased β-cell proliferation observed in some cases was offset by an increase in apoptosis; in keeping with no difference in INSULIN expression or surface area stained for insulin between CHI-D and control pancreas. However, nuclear localization of CDK6 and P27 was markedly enhanced in CHI-D β-cells compared to cytoplasmic localization in control cells. These combined data support normal β-cell mass in CHI-D, but with G1/S molecules positioned in favor of cell cycle progression. New molecular abnormalities in δ-cells and marked proliferative increases in other pancreatic lineages indicate CHI-D is not solely a β-cell disorder.
    Diabetes 04/2015; DOI:10.2337/db14-1202 · 8.47 Impact Factor
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    ABSTRACT: The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.
    Nature Cell Biology 04/2015; 17(5). DOI:10.1038/ncb3160 · 20.06 Impact Factor
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    ABSTRACT: Collectively lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell based therapy, disease modelling and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells co-expressesing NKX2.1 and FOXP2. These progenitors are then able to form cells representative of distal airway epithelium that express NKX2.1, GATA6, CFTR and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508 enabling the development of in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can not only differentiate into both liver and pancreatic cells but also into lung endoderm. Furthermore our study establishes a new approach for the generation of functional lung cells that can be used for disease modelling as well as drug screening and the study of lung development.
    Stem Cells and Development 03/2015; 24(14). DOI:10.1089/scd.2014.0512 · 4.20 Impact Factor
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    ABSTRACT: Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular co-culture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology.
    Toxicological Sciences 12/2014; 144(1). DOI:10.1093/toxsci/kfu269 · 4.48 Impact Factor
  • Neil Hanley
    Nature Biotechnology 11/2014; 32(11):1100-1102. DOI:10.1038/nbt.3064 · 39.08 Impact Factor
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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; 62(3). DOI:10.1016/j.jhep.2014.10.016 · 10.40 Impact Factor
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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; 124(9). DOI:10.1172/JCI71946 · 13.77 Impact Factor
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    ABSTRACT: Background and Aims 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). Methods Influence of EPIM on ECM was investigated in cultured primary rat HSCs. Activated HSCs were treated with recombinant EPIM or SOX9 siRNA. Core fibrotic factors were evaluated by immunoblotting, qPCR and chromatin immunoprecipitation (ChIP). Results During HSC activation EPIM became significantly decreased in contrast to pro-fibrotic markers SOX9, Collagen type 1 (COL1), and α- Smooth muscle actin (α-SMA). Treatment of activated HSCs with recombinant EPIM caused a reduction in α-SMA, SOX9, COL1 and Osteopontin (OPN), while increasing expression of the collagenase matrix metalloproteinase 13 (MMP13). Sox9 abrogation in activated HSCs increased EPIM and MMP13 expression. Conclusion These data provide evidence for EPIM and SOX9 functioning by mutual negative feedback to regulate attributes of the quiescent or activated state of HSCs. Further understanding of EPIM's role may lead to opportunities to modulate SOX9 as a therapeutic avenue for liver fibrosis.
    PLoS ONE 06/2014; 9(6):e100091. DOI:10.1371/journal.pone.0100091 · 3.23 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. DOI:10.4161/19382014.2014.954436 · 1.59 Impact Factor
  • Gastroenterology 05/2014; 146(5):S-919. DOI:10.1016/S0016-5085(14)63338-2 · 13.93 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; 289(13). DOI:10.1074/jbc.M113.540906 · 4.57 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. DOI:10.1371/journal.pone.0086372 · 3.23 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 10/2013; 1(4):293-306. DOI:10.1016/j.stemcr.2013.09.003 · 5.64 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 08/2013; 58(2). DOI:10.1002/hep.26414 · 11.19 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(4). DOI:10.1161/CIRCHEARTFAILURE.112.000123 · 5.95 Impact Factor

Publication Stats

3k Citations
634.50 Total Impact Points

Institutions

  • 2014
    • Central Manchester University Hospitals NHS Foundation Trust
      Manchester, England, United Kingdom
  • 2008–2014
    • The University of Manchester
      • School of Biomedicine
      Manchester, England, United Kingdom
  • 2012–2013
    • University of Liverpool
      • Department of Molecular and Clinical Pharmacology
      Liverpool, England, United Kingdom
  • 2002–2010
    • University of Southampton
      • Developmental Origins of Health and Disease
      Southampton, England, United Kingdom
    • University of Toronto
      Toronto, Ontario, Canada
  • 2008–2009
    • Centre for Stem Cells Sciences
      Bhaganagar, Telangana, India
  • 2000–2004
    • University of Texas Southwestern Medical Center
      • • Division of Endocrinology
      • • Department of Internal Medicine
      Dallas, Texas, United States
  • 1999–2002
    • Newcastle University
      Newcastle-on-Tyne, England, United Kingdom