[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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. · 3.73 Impact Factor
[Show abstract][Hide abstract] 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. · 3.73 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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.).
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations.
[Show abstract][Hide abstract] ABSTRACT: The development of xenobiotics, driven by the demand for therapeutic, domestic and industrial uses continues to grow. However, along with this increasing demand is the risk of xenobiotic-induced toxicity. Currently, safety screening of xenobiotics uses a plethora of animal and in vitro model systems which have over the decades proven useful during compound development and for application in mechanistic studies of xenobiotic-induced toxicity. However, these assessments have proven to be animal-intensive and costly. More importantly, the prevalence of xenobiotic-induced toxicity is still significantly high, causing patient morbidity and mortality, and a costly impediment during drug development. This suggests that the current models for drug safety screening are not reliable in toxicity prediction, and the results not easily translatable to the clinic due to insensitive assays that do not recapitulate fully the complex phenotype of a functional cell type in vivo. Recent advances in the field of stem cell research have potentially allowed for a readily available source of metabolically competent cells for toxicity studies, derived using human pluripotent stem cells harnessed from embryos or reprogrammed from mature somatic cells. Pluripotent stem cell-derived cell types also allow for potential disease modeling in vitro for the purposes of drug toxicology and safety pharmacology, making this model possibly more predictive of drug toxicity compared with existing models. This article will review the advances and challenges of using human pluripotent stem cells for modeling metabolism and toxicity, and offer some perspectives as to where its future may lie.
Advances in pharmacology (San Diego, Calif.) 01/2012; 63:207-56.