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1.
Am J Respir Crit Care Med ; 207(6): 693-703, 2023 03 15.
Article in English | MEDLINE | ID: mdl-36457159

ABSTRACT

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted DlCO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.


Subject(s)
COVID-19 , Lung Diseases, Interstitial , Humans , SARS-CoV-2 , COVID-19/epidemiology , Bayes Theorem , Lung/diagnostic imaging , Hospitalization
2.
Hepatology ; 58(2): 799-809, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23526496

ABSTRACT

UNLABELLED: Failure to predict hepatotoxic drugs in preclinical 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 judgment 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. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained 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. Furthermore, clustering showed that primary adult hepatocytes 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 the hepatocyte proteome in culture, and provide a new protein "toolkit" for determining human hepatocyte maturity in cultured cells.


Subject(s)
Cell Differentiation/genetics , Hepatocytes/metabolism , Hepatocytes/pathology , Proteomics/methods , Alcohol Dehydrogenase/metabolism , Cells, Cultured , Cytochrome P-450 Enzyme System/metabolism , Glutathione Transferase/metabolism , Hep G2 Cells , Humans , Liver/cytology , Liver/embryology , Liver/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology
3.
Hepatology ; 56(3): 1108-16, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22488688

ABSTRACT

UNLABELLED: Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. CONCLUSION: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis.


Subject(s)
Liver Cirrhosis/diagnosis , Liver Cirrhosis/etiology , Osteopontin/physiology , SOX9 Transcription Factor/physiology , Animals , Disease Progression , Humans , Male , Osteopontin/biosynthesis , Rats , Rats, Sprague-Dawley , SOX9 Transcription Factor/biosynthesis
4.
Biomedicines ; 11(5)2023 May 18.
Article in English | MEDLINE | ID: mdl-37239147

ABSTRACT

Differentiation of induced pluripotent stem cells to a range of target cell types is ubiquitous in monolayer culture. To further improve the phenotype of the cells produced, 3D organoid culture is becoming increasingly prevalent. Mature organoids typically require the involvement of cells from multiple germ layers. The aim of this study was to produce pulmonary organoids from defined endodermal and mesodermal progenitors. Endodermal and mesodermal progenitors were differentiated from iPSCs and then combined in 3D Matrigel hydrogels and differentiated for a further 14 days to produce pulmonary organoids. The organoids expressed a range of pulmonary cell markers such as SPA, SPB, SPC, AQP5 and T1α. Furthermore, the organoids expressed ACE2 capable of binding SARS-CoV-2 spike proteins, demonstrating the physiological relevance of the organoids produced. This study presented a rapid production of pulmonary organoids using a multi-germ-layer approach that could be used for studying respiratory-related human conditions.

5.
Sci Rep ; 13(1): 8769, 2023 05 30.
Article in English | MEDLINE | ID: mdl-37253969

ABSTRACT

Rome IV bowel disorders of gut brain interaction (DGBI) and non-alcoholic fatty liver disease (NAFLD) are highly prevalent entities with overlapping pathophysiology and risk factors. We aimed to evaluate the prevalence and burden of Rome IV irritable bowel syndrome (IBS) in patients with NAFLD. Patients diagnosed with NAFLD were recruited from a specialist liver clinic. All participants completed assessments to determine liver fibrosis severity, including liver stiffness measurement (LSM), completed the Rome IV diagnostic questionnaire for bowel disorders of gut brain interaction, the IBS symptom severity score (IBS-SSS), and the EQ-5D-5L to measure of quality-of-life (QoL). 142 patients with NAFLD (71 (50%) female, mean age 53.5 (SD ± 14.9), BMI 35.2 (SD ± 8.1) kg/M2) were recruited. 79 (55.6%) patients met criteria for a Rome IV bowel DGBI, including 50 patients (35.2%) who met the criteria for IBS (mean IBS-SSS 277.2 (SD ± 131.5)). There was no difference in liver fibrosis scores between those with and without Rome IV IBS (FIB-4 scores p = 0.14, LSM p = 0.68). Patients with NAFLD and Rome IV IBS had significantly worse QoL scores (EQ-VAS p = 0.005 and EQ-5D-5L index p = 0.0007), impairment of usual activities of daily living (p = 0.012) and were more likely to report anxiety or depression (p = 0.038). Rome IV bowel DGBI such as IBS are highly prevalent in patients with NAFLD attending liver clinics and are associated with impaired QoL and psychosocial distress.


Subject(s)
Brain Diseases , Irritable Bowel Syndrome , Non-alcoholic Fatty Liver Disease , Humans , Female , Middle Aged , Male , Irritable Bowel Syndrome/complications , Irritable Bowel Syndrome/epidemiology , Irritable Bowel Syndrome/diagnosis , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/epidemiology , Cross-Sectional Studies , Quality of Life/psychology , Prevalence , Activities of Daily Living , Rome , Surveys and Questionnaires , Brain Diseases/complications , Brain , Liver Cirrhosis/complications , Liver Cirrhosis/epidemiology
6.
Drugs ; 83(18): 1699-1707, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37966582

ABSTRACT

BACKGROUND: The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator elexacaftor/tezacaftor/ivacaftor (E/T/I) has been associated with substantial multisystem benefits for people with CF eligible for therapy. In a minority, tolerance has been limited by hepatic toxicity. It is unknown whether there may be particular risk factors for significant drug-induced elevation in transaminases. OBJECTIVE: We aimed to determine the cause of raised transaminases following the introduction of E/T/I, and whether E/T/I can safely be continued in some individuals with elevated transaminases. METHODS: At a large, single, adult CF centre, individuals with transaminases >3 × the upper limit of normal (ULN) since commencing E/T/I underwent clinical assessment to exclude known causes of raised transaminases. Where an alternative cause could not be identified, individuals were discussed with hepatology to advise on further investigations to establish aetiology in addition to calculation of the updated Roussel Uclaf Causality Assessment Method (RUCAM) score to assess causality grading of drug-induced liver injury (DILI) due to E/T/I, and to guide management of ongoing CFTR modulator therapy. RESULTS: Of 337 adults taking E/T/I for a median of 27 months, 19 (5.6%) had transaminases >3 × ULN. In 12 individuals, there was clear evidence of an aetiology unrelated to E/T/I (RUCAM scores -2 to 1 [excluded-unlikely]). Of the remaining cases, two had RUCAM scores in the 'possible' range and one had a RUCAM score in the 'probable' range. Liver biopsy was performed in four individuals, showing hepatic steatosis in one individual, normal histology in one individual, and hepatocyte necrosis suggestive of DILI in two individuals. E/T/I was suspended in those with hepatocyte necrosis, with one permanent discontinuation due to synthetic dysfunction. One individual with hepatocyte necrosis on histology was successfully re-established on E/T/I therapy. CONCLUSIONS: Alternative causes were identified in the majority of patients with clinically significant increases in transaminases following E/T/I, highlighting the importance of thorough investigation. Multidisciplinary assessment involving an experienced hepatologist is crucial in cases of diagnostic uncertainty or suggestion of significant DILI, as discontinuation of therapy can have significant consequences for individuals.


Subject(s)
Chemical and Drug Induced Liver Injury , Cystic Fibrosis , Liver Diseases , Adult , Humans , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Aminophenols/adverse effects , Benzodioxoles/adverse effects , Chemical and Drug Induced Liver Injury/etiology , Transaminases/therapeutic use , Necrosis/chemically induced , Mutation
7.
J Cyst Fibros ; 2023 Sep 19.
Article in English | MEDLINE | ID: mdl-37735009

ABSTRACT

BACKGROUND: There are limited studies to date on the effects of elexacaftor/tezacaftor/ivacaftor (E/T/I) on markers of liver fibrosis in adults with cystic fibrosis (CF). This study aims to analyse changes in makers of liver fibrosis before and after initiation of E/T/I in CF adults. METHODS: Outcome measures of liver fibrosis, including liver stiffness measurement (LSM) using FibroScan, AST-to-platelet-ratio index (APRI) and gamma-GT-to-platelet-ratio (GPR) were available in 74 CF adults following initiation of E/T/I. This was compared to historical data collected in 2018 prior to UK availability of E/T/I. RESULTS: The median duration of E/T/I therapy at the time liver fibrosis markers were repeated was 21 (IQR: 17-25) months. There was an increase in APRI from historical measurement to follow-up but no change in LSM or GPR. There were no differences in change in fibrosis markers according to CF liver disease (CFLD) status, although those with a raised LSM at baseline (>6.8 kPa) (n = 14) had a significant reduction in LSM from historical measurement to follow-up versus those with a normal historical value (-3.3 kPa vs 0.25 kPa, p < 0.01). CONCLUSIONS: Apart from APRI, we found no changes in liver fibrosis outcomes after initiation of E/T/I in adults with CF. Those with a historical diagnosis of CFLD had no significant worsening or improvement of liver fibrosis markers. We did observe a reduction in LSM in those with liver nodularity, with an initial highest result suggesting a potential positive treatment effect of E/T/I in this category of those with severe CFLD.

8.
Eur J Endocrinol ; 188(7): 564-577, 2023 Jul 10.
Article in English | MEDLINE | ID: mdl-37358209

ABSTRACT

BACKGROUND: Growing evidence suggests the key role of ghrelin in the onset and progression of nonalcoholic fatty liver disease (NAFLD). The potential participation of ghrelin and the ghrelin receptor antagonist, LEAP-2, in the onset of liver fibrosis in patients with severe obesity and NAFLD through the regulation of TGF-ß1-induced hepatic stellate cell (HSC) activation was investigated. METHODS: Circulating (n = 179) and hepatic expression (n = 95) of ghrelin and LEAP-2 were measured in patients with severe obesity and available liver pathology analysis undergoing Roux-en-Y gastric bypass (RYGB). The effect of ghrelin isoforms and LEAP-2 on TGF-ß1-induced HSC activation, fibrogenic response, and contractile properties was evaluated in vitro in human LX-2 cells. RESULTS: Plasma and hepatic ghrelin were negatively associated, while LEAP-2 exhibited a positive association with liver fibrosis in patients with obesity and NAFLD. Six months after RYGB, hepatic function was improved and, although acylated ghrelin and LEAP-2 concentrations remained unchanged, both hormones were inversely related to post-surgical levels of profibrogenic factors TGF-ß1 and TIMP-1. Acylated ghrelin treatment reversed TGF-ß1-induced myofibroblast-like phenotype, collagen contractile properties, and the upregulation of factors involved in HSC activation and fibrogenesis via PI3K/Akt/mTOR pathway. Moreover, acylated ghrelin inhibited the mild HSC activation induced by LEAP-2. CONCLUSIONS: Ghrelin is an anti-fibrogenic factor blocking HSC activation induced by the most potent fibrogenic cytokine, TGF-ß1, and LEAP-2. The imbalance between acylated ghrelin and ghrelin receptor antagonist LEAP-2 might contribute to maintain liver fibrosis in patients with obesity and NAFLD.


Subject(s)
Non-alcoholic Fatty Liver Disease , Obesity, Morbid , Humans , Non-alcoholic Fatty Liver Disease/metabolism , Transforming Growth Factor beta1/adverse effects , Transforming Growth Factor beta1/metabolism , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Ghrelin/adverse effects , Receptors, Ghrelin , Liver Cirrhosis/etiology , Liver/metabolism
9.
Dev Biol ; 344(2): 720-30, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20515681

ABSTRACT

The cranial base is essential for integrated craniofacial development and growth. It develops as a cartilaginous template that is replaced by bone through the process of endochondral ossification. Here, we describe a novel and specific role for the homeoprotein Six2 in the growth and elongation of the cranial base. Six2-null newborn mice display premature fusion of the bones in the cranial base. Chondrocyte differentiation is abnormal in the Six2-null cranial base, with reduced proliferation and increased terminal differentiation. Gain-of-function experiments indicate that Six2 promotes cartilage development and growth in other body areas and appears therefore to control general regulators of chondrocyte differentiation. Our data indicate that the main factors restricting Six2 function to the cranial base are tissue-specific transcription of the gene and compensatory effects of other Six family members. The comparable expression during human embryogenesis and the high protein conservation from mouse to human implicate SIX2 loss-of-function as a potential congenital cause of anterior cranial base defects in humans.


Subject(s)
Skull Base/growth & development , Animals , Animals, Newborn , Bone and Bones , Cartilage/growth & development , Cartilage/metabolism , Cell Differentiation , Chondrogenesis , Homeodomain Proteins/metabolism , Humans , Mice , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins , Osteogenesis/physiology , Proteins/metabolism
10.
Nat Commun ; 11(1): 3920, 2020 08 06.
Article in English | MEDLINE | ID: mdl-32764605

ABSTRACT

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.


Subject(s)
Developmental Disabilities/genetics , Epigenesis, Genetic , Organogenesis/genetics , Animals , Animals, Genetically Modified , Databases, Genetic , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Histone Code/genetics , Humans , Models, Genetic , Mutation , Organogenesis/physiology , Promoter Regions, Genetic , Tissue Distribution , Transcription Factors/metabolism , Zebrafish/embryology , Zebrafish/genetics
11.
Mol Cell Endocrinol ; 288(1-2): 79-85, 2008 Jun 25.
Article in English | MEDLINE | ID: mdl-18450368

ABSTRACT

Understanding how beta-cells maintain themselves in the adult pancreas is important for prioritizing strategies aimed at ameliorating or ideally curing different forms of diabetes. There has been much debate over whether beta-cell proliferation, as a means of self-renewal, predominates over the existence and differentiation of a pancreatic stem cell or progenitor cell population. This article describes the two opposing positions based largely on research in laboratory rodents and its extrapolation to humans.


Subject(s)
Insulin-Secreting Cells/cytology , Stem Cells/cytology , Animals , Cell Proliferation , Cell Size , Humans , Mice , Pancreas/growth & development , Species Specificity
12.
Sci Rep ; 8(1): 12866, 2018 08 27.
Article in English | MEDLINE | ID: mdl-30150762

ABSTRACT

The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is thought to contribute to the onset of intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific marker CD24 was used to specifically label and isolate (using FACS) notochordal cells from human embryonic and fetal spines (7.5-14 weeks post-conception). Microarray analysis and qPCR validation identified CD24, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2 as notochord-specific markers. Expression of these markers was confirmed in nucleus pulposus cells from aged and degenerate discs. Ingenuity pathway analysis revealed molecules involved in inhibition of vascularisation (WISP2, Noggin and EDN2) and inflammation (IL1-RN) to be master regulators of notochordal genes. Importantly, this study has, for the first time, defined the human notochordal cell transcriptome and suggests inhibition of inflammation and vascularisation may be key roles for notochordal cells during intervertebral disc development. The molecules and pathways identified in this study have potential for use in developing strategies to retard/prevent disc degeneration, or regenerate tissue.


Subject(s)
Intervertebral Disc/cytology , Intervertebral Disc/embryology , Notochord/cytology , Stem Cells/cytology , Stem Cells/metabolism , Transcriptome , Biomarkers , CD24 Antigen/genetics , CD24 Antigen/metabolism , Computational Biology/methods , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Reproducibility of Results
13.
Brain Res Bull ; 68(1-2): 76-82, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16325007

ABSTRACT

Stem cell therapy offers exciting potential for ambitious cellular replacement to treat human (h) disease, such as Parkinson's disease, Alzheimer's disease or even replacement of the cell death that follows thromboembolic stroke. The realisation of these treatments requires cellular resources possessing three essential characteristics: (i) self-renewal, (ii) the ability to differentiate to physiologically normal cell types and (iii) lack of tumourigenicity. Here, we describe work on human embryonic germ cells (hEGCs), a population of cells alongside human embryonic stem cells (hESCs) with the potential to address these issues.


Subject(s)
Brain Diseases/therapy , Germ Cells/cytology , Neurons/cytology , Pluripotent Stem Cells/cytology , Stem Cell Transplantation/methods , Animals , Gonads/cytology , Gonads/embryology , Humans
14.
Diabetes ; 64(9): 3182-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25931474

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 studied wider abnormalities in the ß-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared with controls. PAX4 and ARX expression was decreased. A tendency toward increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas, and implied immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. In CHI-D, increased proliferation was most elevated in duct (5- to 11-fold) and acinar (7- to 47-fold) lineages. Increased ß-cell proliferation observed in some cases was offset by an increase in apoptosis; this is 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 ß-cells compared with cytoplasmic localization in control cells. These combined data support normal ß-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 ß-cell disorder.


Subject(s)
Congenital Hyperinsulinism/genetics , Glucagon-Secreting Cells/metabolism , Insulin-Secreting Cells/metabolism , Somatostatin-Secreting Cells/metabolism , Case-Control Studies , Cell Lineage , Cell Proliferation , Child , Child, Preschool , Congenital Hyperinsulinism/metabolism , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Fetus/cytology , Glucagon-Secreting Cells/cytology , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/metabolism , Humans , Infant , Infant, Newborn , Insulin/metabolism , Insulin-Secreting Cells/cytology , Mutation , Nuclear Proteins , Paired Box Transcription Factors/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Somatostatin-Secreting Cells/cytology , Sulfonylurea Receptors/genetics , Transcription Factors/metabolism , Zebrafish Proteins
15.
Trends Mol Med ; 17(3): 166-74, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21237710

ABSTRACT

The transcription factor SOX9 is crucial for multiple aspects of development. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder concordant with the expression profile of SOX9 during embryogenesis. The mechanistic understanding of development has revealed roles for SOX9 in regulating cartilage extracellular matrix (ECM) production and cell proliferation, among others. More recently, it transpires that SOX9 becomes expressed and induces destructive ECM components in organ fibrosis and related disorders. Although commonly absent from the parent cell type, SOX9 is expressed in a wide range of cancers, where it regulates cell proliferation. These data have potential diagnostic, prognostic and therapeutic relevance, suggesting that disease mechanisms might result from re-expressing this developmental transcription factor in ectopic locations.


Subject(s)
Disease/genetics , Gene Expression Regulation, Developmental , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Animals , Humans
16.
Stem Cell Res ; 5(1): 4-22, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20483202

ABSTRACT

Hepatotoxicity is an enormous and increasing problem for the pharmaceutical industry. Early detection of problems during the drug discovery pathway is advantageous to minimize costs and improve patient safety. However, current cellular models are sub-optimal. This review addresses the potential use of pluripotent stem cells in the generation of hepatic cell lineages. It begins by highlighting the scale of the problem faced by the pharmaceutical industry, the precise nature of drug-induced liver injury and where in the drug discovery pathway the need for additional cell models arises. Current research is discussed, mainly for generating hepatocyte-like cells rather than other liver cell-types. In addition, an effort is made to identify where some of the major barriers remain in translating what is currently hypothesis-driven laboratory research into meaningful platform technologies for the pharmaceutical industry.


Subject(s)
Cell Culture Techniques/methods , Cell Lineage , Liver/cytology , Liver/drug effects , Pluripotent Stem Cells/cytology , Animals , Drug Evaluation, Preclinical , Drug Industry , Humans
17.
Gene ; 460(1-2): 20-9, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20381594

ABSTRACT

Mutations in the human gene ALMS1 cause Alström syndrome, a disorder characterised by neurosensory degeneration, metabolic defects and cardiomyopathy. ALMS1 encodes a centrosomal protein implicated in the assembly and maintenance of primary cilia. Expression of ALMS1 varies between tissues and recent data suggest that its transcription is modulated during adipogenesis and growth arrest. However the ALMS1 promoter has not been defined. This study focused on identifying and characterising the ALMS1 proximal promoter, initially by using 5' RACE to map transcription start sites. Luciferase reporter assay and EMSA data strongly suggest that ALMS1 transcription is regulated by the ubiquitous factor Sp1. In addition, reporter assay, EMSA, chromatin immunoprecipitation and RNA interference data indicate that ALMS1 transcription is regulated by regulatory factor X (RFX) proteins. These transcription factors are cell-type restricted in their expression profile and known to regulate genes of the ciliogenic pathway. We show binding of RFX proteins to an evolutionarily conserved X-box in the ALMS1 proximal promoter and present evidence that these proteins are responsible for ALMS1 transcription during growth arrest induced by low serum conditions. In summary, this work provides the first data on transcription factors regulating general and context-specific transcription of the disease-associated gene ALMS1.


Subject(s)
Alstrom Syndrome/genetics , DNA-Binding Proteins/physiology , Promoter Regions, Genetic , Proteins/genetics , Sp1 Transcription Factor/physiology , Transcription Factors/physiology , Transcription Initiation Site , Adult , Carrier Proteins/metabolism , Cell Cycle Proteins , Cells, Cultured , Chromosome Mapping , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Mutation , RNA Interference , Regulatory Factor X Transcription Factors , Transcription Factors/metabolism , Transcription, Genetic
18.
J Biol Chem ; 283(20): 14063-71, 2008 May 16.
Article in English | MEDLINE | ID: mdl-18296708

ABSTRACT

Appropriate temporospatial expression of the transcription factor SOX9 is important for normal development of a wide range of organs. Here, we show that when SOX9 is expressed ectopically, target genes become expressed that are associated with disease. Histone deacetylase inhibitors in clinical trials for cancer therapy induced SOX9 expression via enhanced recruitment of nuclear factor Y (NF-Y) to CCAAT elements in the SOX9 proximal promoter. The effect of histone deacetylase inhibitors could be elicited in cells that normally lack SOX9, such as hepatocytes. In human fetal hepatocytes, this aberrant induction of SOX9 protein caused ectopic expression of COL2A1 and COMP1 that encode extracellular matrix (ECM) components normally associated with chondrogenesis. Previously, ectopic expression of this "chondrogenic" profile has been implicated in vascular calcification. More broadly, inappropriate ECM deposition is a hallmark of fibrosis. We demonstrated that induction of SOX9 expression also occurred during activation of fibrogenic cells from the adult liver when the transcription factor was responsible for expression of the major component of fibrotic ECM, type 1 collagen. These combined data identify new aspects in the regulation of SOX9 expression. They support a role for SOX9 beyond normal development as a transcriptional regulator in the pathology of fibrosis.


Subject(s)
CCAAT-Binding Factor/metabolism , Extracellular Matrix/metabolism , Fibrosis/pathology , High Mobility Group Proteins/biosynthesis , Transcription Factors/biosynthesis , Transcription Factors/metabolism , Base Sequence , Cell Line, Tumor , Collagen/chemistry , Gene Silencing , HeLa Cells , Hepatocytes/cytology , Hepatocytes/metabolism , High Mobility Group Proteins/metabolism , Histone Deacetylase Inhibitors , Humans , Models, Biological , Molecular Sequence Data , SOX9 Transcription Factor , Sequence Homology, Nucleic Acid
19.
Diabetes ; 57(6): 1745-52, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18356407

ABSTRACT

OBJECTIVE: Mutations in the alternatively spliced HNF4A gene cause maturity-onset diabetes of the young (MODY). We characterized the spatial and developmental expression patterns of HNF4A transcripts in human tissues and investigated their role as potential moderators of the MODY phenotype. RESEARCH DESIGN AND METHODS: We measured the expression of HNF4A isoforms in human adult tissues and gestationally staged fetal pancreas by isoform-specific real-time PCR. The correlation between mutation position and age of diagnosis or age-related penetrance was assessed in a cohort of 190 patients with HNF4A mutations. RESULTS: HNF4A was expressed exclusively from the P2 promoter in adult pancreas, but from 9 weeks until at least 26 weeks after conception, up to 23% of expression in fetal pancreas was of P1 origin. HNF4A4-6 transcripts were not detected in any tissue. In whole pancreas, HNF4A9 expression was greater than in islets isolated from the endocrine pancreas (relative level 22 vs. 7%). Patients with mutations in exons 9 and 10 (absent from HNF4A3, HNF4A6, and HNF4A9 isoforms) developed diabetes later than those with mutations in exons 2-8, where all isoforms were affected (40 vs. 24 years; P = 0.029). Exon 9/10 mutations were also associated with a reduced age-related penetrance (53 vs. 10% without diabetes at age 55 years; P < 0.00001). CONCLUSIONS: We conclude that isoforms derived from the HNF4A P1 promoter are expressed in human fetal, but not adult, pancreas, and that their presence during pancreatic development may moderate the diabetic phenotype in individuals with mutations in the HNF4A gene.


Subject(s)
Alternative Splicing , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus/genetics , Fetal Development/genetics , Hepatocyte Nuclear Factor 4/genetics , Mutation , Promoter Regions, Genetic , Adult , Aged , Female , Gene Expression Profiling , Humans , Kidney/physiology , Kidney/physiopathology , Male , Middle Aged , Pancreas/physiology , Pancreas/physiopathology , Polymerase Chain Reaction , Protein Isoforms/genetics , White People
20.
Stem Cells ; 24(2): 212-20, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16144875

ABSTRACT

The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations.


Subject(s)
Embryo, Mammalian/cytology , Germ Cells/physiology , Pluripotent Stem Cells/physiology , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Movement , Cell Proliferation , Cell- and Tissue-Based Therapy/methods , Culture Media/chemistry , Embryo, Mammalian/physiology , Gene Expression Profiling , Gene Expression Regulation, Developmental , Germ Cells/chemistry , Growth Substances/physiology , Humans , Mice
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