ORCID Profile
0000-0002-2146-6614
Current Organisation
University of Queensland
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biomaterials | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) | Synthesis of Materials | Biomedical Engineering | Biomaterials | Fluidization And Fluid Mechanics | Chemical Engineering | Chemical Engineering Not Elsewhere Classified | Medical Biochemistry and Metabolomics not elsewhere classified | Agricultural Biotechnology | Animal Cell and Molecular Biology | Medical Biotechnology | Medical Biochemistry and Metabolomics | Central Nervous System | Industrial Molecular Engineering of Nucleic Acids and Proteins | Transgenesis | Nanobiotechnology | Cell Development, Proliferation and Death
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Nervous System and Disorders | Cancer and related disorders | Plastic Products (incl. Construction Materials) | Animal Production and Animal Primary Products not elsewhere classified | Cardiovascular System and Diseases | Clinical health not specific to particular organs, diseases and conditions | Health Related to Ageing | Expanding Knowledge in Engineering | Expanding Knowledge in the Agricultural and Veterinary Sciences | Blood Disorders | Manufactured products not elsewhere classified |
Related Links
Publications
Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-6955-5_29
Abstract: Reprogramming of cells enables generation of pluripotent stem cells and resulting progeny through directed differentiation, making this technology an invaluable tool for the study of human development and disease. Reprogramming occurs with a wide range of efficiency, a culmination of intrinsic and extrinsic factors including the tissue of origin, the passage number and culture history of the target cells. Another major factor affecting reprogramming is the methodology used and the quality of the reprogramming process itself, including for conventional viral-based approaches viral titer and subsequent viral transduction efficiency, including downstream transgene insertion and stoichiometry. Genetic background is an important parameter affecting the efficiency of the reprogramming process with reports that cells from in iduals harboring specific mutations are more difficult to reprogram than control counterparts.Ataxia-Telangiectasia (A-T) fibroblasts underwent reprogramming at reduced efficiency in contrast to their controls. To optimize reprogramming of fibroblasts from patients with A-T, we examined the response of A-T cells to various cell culture conditions after lentiviral transduction with reprogramming factors Oc4/Sox2 (pSIN4-EF2-O2S) and Klf4/c-Myc (pSIN4-CMV-K2M). Parameters included media type (KSR or serum-containing DMEM), treatment with a p53 inhibitor (small-molecule cyclic pifithrin-α), and either a low or high concentration of bFGF. Post-transduction, equivalent numbers of cells from heterozygote and homozygote patients were plated and assessed at regular intervals for survival and proliferation. Our findings indicate that A-T cells responded favorably to the addition of FCS and gradual weaning away from their native media into KSR-containing stem cell media that produced suitable conditions for their reprogramming. We examined a range of properties to identify and isolate good quality iPSCs including the expression status of important stem cell transcription factors/surface proteins, methylation levels at stem cell associated regulatory loci, persistence of transgenes, karyotype status, and teratoma-forming ability.
A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells
Publisher: Elsevier BV
Date: 08-2018
Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency
Publisher: Hindawi Limited
Date: 25-11-2015
DOI: 10.1002/TERM.1960
Abstract: Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation.
Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development
Publisher: Public Library of Science (PLoS)
Date: 19-08-2015
BMP-11 and Myostatin Support Undifferentiated Growth of Human Embryonic Stem Cells in Feeder-Free Cultures
Publisher: Mary Ann Liebert Inc
Date: 09-2009
Abstract: BMP-11/GDF-11 and Myostatin/GDF-8 are both members of the TGF-beta superfamily that can activate SMAD2/3 phosphorylation via the type I receptors ALK4, ALK5, or ALK7. We tested the ability of BMP-11 and Myostatin to promote self-renewal of human embryonic stem cells (hESC) under feeder-free and serum-free culture conditions in short term (1 week) and medium term cultures (10 weeks). We show that hESC cultured in serum-free medium supplemented with either 20 ng/mL Myostatin or 20 ng/mL BMP-11 maintain the colony and cellular morphology of undifferentiated hESC, maintain POU5f1, NANOG, TRA-1-60, and SSEA4 expression, and display increased SMAD2/3 phosphorylation, similar to hESC cultured in mouse embryonic fibroblast feeder-CM or 20 ng/mL Activin-A. The type I TGF-beta receptor inhibitor SB431542 totally inhibited the maintenance activity of both Myostatin or BMP-11 supplemented medium. Our data show that members of the TGF-beta superfamily, other than Activin-A and GDF3, are able to maintain hES cells in an undifferentiated state under feeder free conditions.
Serotonin Syndrome After Methylene Blue Administration During Cardiac Surgery: A Case Report and Review
Publisher: Elsevier BV
Date: 08-2016
Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body
Publisher: Public Library of Science (PLoS)
Date: 25-03-2015
Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage
Publisher: Public Library of Science (PLoS)
Date: 14-12-2015
hESC Adaptation, Selection and Stability
Publisher: Springer Science and Business Media LLC
Date: 22-05-2007
Author response: Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells
Publisher: eLife Sciences Publications, Ltd
Date: 06-06-2017
Identification of on-target mutagenesis during correction of a beta-thalassemia splice mutation in iPS cells with optimised CRISPR/Cas9-double nickase reveals potential safety concerns
Publisher: AIP Publishing
Date: 12-2018
DOI: 10.1063/1.5048625
Abstract: Precise and accurate gene correction is crucial for enabling iPSC-based therapies, and Cas9-Nickase based approaches are increasingly considered for in vivo correction of diseases such as beta-thalassemia. Here, we generate footprint-free induced pluripotent stem cells from a patient with a beta-thalassemia mutation (IVSII-1 G & A) and employ a double Cas9nickase-mediated correction strategy combined with a piggyBac transposon-modified donor vector for gene correction. Our approach further aimed to minimize the formation of adjacent single-strand breaks at the targeted allele through the destruction of the binding site for one guide and the use of a synonymous protospacer adjacent motif blocking mutation (canonical PAM sequence 5'-NGG-3' is changed to 5'-NCG-3', where N indicates any nucleobase) for the other guide. We show that this strategy indeed not only permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele but also results in unexpected on-target mutagenesis with some guide RNAs (gRNAs) in several targeted clones. This study thus not only validates a framework for seamless gene correction with enhanced specificity and accuracy but also highlights potential safety concerns associated with Cas9-nickase based gene correction.
Gene Expression Variability as a Unifying Element of the Pluripotency Network
Publisher: Elsevier BV
Date: 08-2014
High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4LC01176G
Abstract: In this paper, the design, development and validation of a novel high throughput microfluidic device enabling both the robust and rapid trapping of 100's to 1000's of single cells and their in situ clonal growth is described.
Ascorbate Promotes Epigenetic Activation of CD30 in Human Embryonic Stem Cells
Publisher: Oxford University Press (OUP)
Date: 10-2010
DOI: 10.1002/STEM.500
Abstract: Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions.
Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation
Publisher: Proceedings of the National Academy of Sciences
Date: 27-03-2014
Abstract: Naturally occurring regulatory T (Treg) cells, which specifically express the transcription factor forkhead box P3 (Foxp3), are engaged in the maintenance of immunological self-tolerance and homeostasis. By transcriptional start site cluster analysis, we assessed here how genome-wide patterns of DNA methylation or Foxp3 binding sites were associated with Treg-specific gene expression. We found that Treg-specific DNA hypomethylated regions were closely associated with Treg up-regulated transcriptional start site clusters, whereas Foxp3 binding regions had no significant correlation with either up- or down-regulated clusters in nonactivated Treg cells. However, in activated Treg cells, Foxp3 binding regions showed a strong correlation with down-regulated clusters. In accordance with these findings, the above two features of activation-dependent gene regulation in Treg cells tend to occur at different locations in the genome. The results collectively indicate that Treg-specific DNA hypomethylation is instrumental in gene up-regulation in steady state Treg cells, whereas Foxp3 down-regulates the expression of its target genes in activated Treg cells. Thus, the two events seem to play distinct but complementary roles in Treg-specific gene expression.
Will Brain Cells Derived From Induced Pluripotent Stem Cells or Directly Converted From Somatic Cells (iNs) Be Useful for Schizophrenia Research?
Publisher: Oxford University Press (OUP)
Date: 24-07-2013
Differential mesengenic potential and expression of stem cell‐fate modulators in mesenchymal stromal cells from human‐term placenta and bone marrow
Publisher: Wiley
Date: 17-05-2012
DOI: 10.1002/JCP.24014
Abstract: Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbor populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full-term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs, and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28, and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell-fate mediators and mesodermal differentiation capacity.
A defined medium and substrate for expansion of human mesenchymal stromal cell progenitors that enriches for osteo- and chondrogenic precursors.
Publisher: Mary Ann Liebert Inc
Date: 2010
Abstract: Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29(+), CD44(+), CD49a(+), CD73(+), CD90(+), CD105(+), CD146(+), CD166(+), CD34(-), and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.
Latency of the peroxisomal enzyme acyl-coA:dihydroxyacetonephosphate acyltransferase in digitonin-permeabilized fibroblasts: the effect of ATP and ATPase inhibitors
Publisher: Elsevier BV
Date: 08-1990
DOI: 10.1016/0006-291X(90)90511-K
Abstract: We have studied the activity of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAP-AT) in fibroblasts treated with low concentrations of digitonin so that the cytoplasmic compartment was freely accessible to the substrates of DHAP-AT while intracellular membranes remained intact. DHAP-AT activity exhibited 70% latency under these conditions. This latency could be overcome by addition of ATP, resulting in a four-fold stimulation of DHAP-AT activity. Virtually no stimulatory effect of ATP on DHAP-AT activity was observed in sonicated fibroblasts or when a non-hydrolyzable ATP analogue was used. Furthermore the stimulatory effect of ATP was prevented in part by DCCD. N-ethylmaleimide and high concentrations of oligomycin bafilomycin had no effect. This pattern of inhibitor sensitivity is similar to that of the ATPase activity in peroxisomal fractions from rat liver. We conclude that peroxisomes in situ exhibit structure linked latency and that ATP is required for the transport of at least one of the substrates of DHAP-AT.
Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling
Publisher: Oxford University Press (OUP)
Date: 14-02-2019
DOI: 10.1002/STEM.2966
Abstract: When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019 :476–488
S3_Fig.eps
Publisher: Public Library of Science (PLoS)
Date: 2015
S2_Fig.eps
Publisher: Public Library of Science (PLoS)
Date: 2015
S1_Fig.eps
Publisher: Public Library of Science (PLoS)
Date: 2015
Targeted, Stimuli-Responsive Delivery of Plasmid DNA and miRNAs Using a Facile Self-Assembled Supramolecular Nanoparticle System
Publisher: American Chemical Society (ACS)
Date: 18-01-2018
Overexpression of the chromosome 21 transcription factor Ets2 induces neuronal apoptosis
Publisher: Elsevier BV
Date: 12-2003
DOI: 10.1016/S0969-9961(03)00107-4
Abstract: Down syndrome (trisomy 21) neurons display an increased rate of apoptosis in vitro. The genes on chromosome 21 that mediate this increased cell death remain to be elucidated. Here we show that the chromosome 21 transcription factor Ets2, a gene that is overexpressed in Down syndrome, is expressed in neurons, and that moderate overexpression of Ets2 leads to increased apoptosis of primary neuronal cultures from Ets2 tg mice that involves activation of caspase-3. Our data therefore suggest that overexpression of ETS2 may contribute to the increased rate of apoptosis of neurons in Down syndrome.
S4_Fig.eps
Publisher: Public Library of Science (PLoS)
Date: 2015
Bone disease - Current knowledge and future prospects
Publisher: Bentham Science Publishers Ltd.
Date: 06-2001
Autophagy in stem cells
Publisher: Informa UK Limited
Date: 06-06-2013
DOI: 10.4161/AUTO.24132
Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells
Publisher: Oxford University Press (OUP)
Date: 11-2005
DOI: 10.1634/STEMCELLS.2004-0338
Abstract: Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.
Erratum: Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis
Publisher: Springer Science and Business Media LLC
Date: 27-04-2016
DOI: 10.1038/NATURE17982
S2_Text.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Genetic and Epigenetic Instability of Human Pluripotent Stem Cells
Publisher: Bentham Science Publishers Ltd.
Date: 17-03-2011
Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry
Publisher: Baishideng Publishing Group Inc.
Date: 2012
S1_Text.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Stimulation of autophagy suppresses the intracellular survival ofBurkholderia pseudomalleiin mammalian cell lines
Publisher: Informa UK Limited
Date: 16-08-2008
DOI: 10.4161/AUTO.6246
Abstract: Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chlor henicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.
Reactive Oxygen Species and Their Contribution to Pathology in Down Syndrome
Publisher: Elsevier
Date: 1996
DOI: 10.1016/S1054-3589(08)60992-8
Abstract: Plasma lipid profile and anthropometric variables are known to be under strong genetic control and the identification of genetic variants associated with bioclinical parameters is of considerable public health importance. In this study, a young cohort of healthy in iduals was genotyped for genes related to health and pathological conditions, to analyze the association of single nucleotide polymorphisms (SNPs) with different bioclinical parameters, adherence to the Mediterranean Diet (MD) and physical activity, studying the role of lifestyle and body composition parameters on biochemical metabolic profile. Association analysis of single variants in the genes of lipoprotein lipase (LPL), fibronectin type III domain containing protein 5 (FNDC5), and peroxisome proliferator-activated receptor-gamma (PPARγ) and haplotype analyses were performed. Multiple (n = 14) common variants in the three genes demonstrated a significant effect on plasma lipoprotein-lipid levels and/or on biochemical parameters in our s le. Specifically, SNPs were related to lipid metabolism (rs3866471, rs4922115, rs11570892, rs248, rs316, rs1059507, rs1801282) or glycemic profile (rs3208305) or anthropometric parameters (rs3480, rs726344, rs1570569) for a total of 26 significant associations (P < 0.01 and/or P < 0.05) and two haplotypes, for the first time, were strongly associated with lipid and body composition parameters. Interestingly, we identified twenty-four new variants not previously described in the literature and a novel significant association between rs80143795 and body composition. In this study we confirm the association between these SNPs on lipid metabolism and body parameters also in a young cohort, indicating the important role of these genetic factors as determinants of health.
Energy dependence of autophagic protein degradation in isolated rat hepatocytes
Publisher: Wiley
Date: 04-1987
DOI: 10.1111/J.1432-1033.1987.TB11011.X
Abstract: The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.
Determinants of Zika virus host tropism uncovered by deep mutational scanning
Publisher: Springer Science and Business Media LLC
Date: 18-03-2019
CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes
Publisher: Springer Science and Business Media LLC
Date: 24-06-2014
DOI: 10.1038/SREP05228
Inflammatory responses to a pathogenic West Nile virus strain
Publisher: Springer Science and Business Media LLC
Date: 29-10-2019
DOI: 10.1186/S12879-019-4471-8
Abstract: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNV KUN ). WNV KUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.
Primitive Cardiac Cells from Human Embryonic Stem Cells
Publisher: Mary Ann Liebert Inc
Date: 10-06-2012
Abstract: Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.
A rapid, cost-effective method for counting human embryonic stem cell numbers as clumps
Publisher: Future Science Ltd
Date: 07-2009
DOI: 10.2144/000113151
Abstract: Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ s les. We have developed a reproducible, cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each s le to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05–2 × 10 5 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy.
Ceruloplasmin Is a Novel Adipokine Which Is Overexpressed in Adipose Tissue of Obese Subjects and in Obesity-Associated Cancer Cells
Publisher: Public Library of Science (PLoS)
Date: 27-03-2014
Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density Microbioreactor Arrays
Publisher: Springer Science and Business Media LLC
Date: 21-04-2016
DOI: 10.1038/SREP24637
Abstract: Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either in idually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro , but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.
Multivariate patterning of human pluripotent cells under perfusion reveals critical roles of induced paracrine factors in kidney organoid development
Publisher: American Association for the Advancement of Science (AAAS)
Date: 10-01-2020
Abstract: Multivariate patterning of perfused pluripotent cells reveals critical roles of induced paracrine factors in kidney organoids.
An atlas of active enhancers across human cell types and tissues
Publisher: Springer Science and Business Media LLC
Date: 03-2014
DOI: 10.1038/NATURE12787
Induced pluripotent stem cells from ataxia-telangiectasia recapitulate the cellular phenotype
Publisher: Oxford University Press (OUP)
Date: 28-06-2012
Abstract: Pluripotent stem cells can differentiate into every cell type of the human body. Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) therefore provides an opportunity to gain insight into the molecular and cellular basis of disease. Because the cellular DNA damage response poses a barrier to reprogramming, generation of iPSCs from patients with chromosomal instability syndromes has thus far proven to be difficult. Here we demonstrate that fibroblasts from patients with ataxia-telangiectasia (A-T), a disorder characterized by chromosomal instability, progressive neurodegeneration, high risk of cancer, and immunodeficiency, can be reprogrammed to bona fide iPSCs, albeit at a reduced efficiency. A-T iPSCs display defective radiation-induced signaling, radiosensitivity, and cell cycle checkpoint defects. Bioinformatic analysis of gene expression in the A-T iPSCs identifies abnormalities in DNA damage signaling pathways, as well as changes in mitochondrial and pentose phosphate pathways. A-T iPSCs can be differentiated into functional neurons and thus represent a suitable model system to investigate A-T-associated neurodegeneration. Collectively, our data show that iPSCs can be generated from a chromosomal instability syndrome and that these cells can be used to discover early developmental consequences of ATM deficiency, such as altered mitochondrial function, that may be relevant to A-T pathogenesis and amenable to therapeutic intervention.
Increase of plasma membrane oxidoreductase activity is not correlated with the production of extracellular Superoxide radicals in human Namalwa cells
Publisher: Springer Science and Business Media LLC
Date: 03-1995
DOI: 10.1007/BF01276917
Mitochondrial respiratory chain inhibitors induce apoptosis
Publisher: Wiley
Date: 14-02-1994
DOI: 10.1016/0014-5793(94)80380-3
Abstract: In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.
Corrigendum to “p53 is required for etoposide-induced apoptosis of human embryonic stem cells” [Stem Cell Research 1 (2008) 116–128]
Publisher: Elsevier BV
Date: 09-2009
Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors
Publisher: Wiley
Date: 22-07-2011
DOI: 10.1002/BIT.23260
Abstract: Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number 95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture.
Modelling ischemia-reperfusion injury (IRI) in vitro using metabolically matured induced pluripotent stem cell-derived cardiomyocytes
Publisher: AIP Publishing
Date: 20-03-2018
DOI: 10.1063/1.5000746
Abstract: Coronary intervention following ST-segment elevation myocardial infarction (STEMI) is the treatment of choice for reducing cardiomyocyte death but paradoxically leads to reperfusion injury. Pharmacological post-conditioning is an attractive approach to minimize Ischemia-Reperfusion Injury (IRI), but candidate drugs identified in IRI animal models have performed poorly in human clinical trials, highlighting the need for a human cell-based model of IRI. In this work, we show that when we imposed sequential hypoxia and reoxygenation episodes [mimicking the ischemia (I) and reperfusion (R) events] to immature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), they display significant hypoxia resistance and minimal cell death (∼5%). Metabolic maturation of hPSC-CMs for 8 days substantially increased their sensitivity to changes in oxygen concentration and led to up to ∼30% cell death post-hypoxia and reoxygenation. To mimic the known transient changes in the interstitial tissue microenvironment during an IRI event in vivo, we tested a new in vitro IRI model protocol that required glucose availability and lowering of media pH during the ischemic episode, resulting in a significant increase in cell death in vitro (∼60%). Finally, we confirm that in this new physiologically matched IRI in vitro model, pharmacological post-conditioning reduces reperfusion-induced hPSC-CM cell death by 50%. Our results indicate that in recapitulating key aspects of an in vivo IRI event, our in vitro model can serve as a useful method for the study of IRI and the validation and screening of human specific pharmacological post-conditioning drug candidates.
Gap junction mediated transport of shRNA between human embryonic stem cells
Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.BBRC.2007.09.035
Abstract: Gap junction intracellular communication (GJIC) allows the direct transport of small molecules between adjacent cells. We hypothesized that siRNAs in one hESC could inhibit target RNA expression in another hESC via GJIC. We co-cultured green fluorescent protein (GFP)-expressing ENVY hESC with non-GFP-expressing hESC, which had been transduced to stably express shRNA directed against GFP. We discovered that the GFP shRNA expressing hESC inhibited GFP expression in the adjacent GFP-expressing hESC in a dose-dependent manner. This downregulation of GFP expression in ENVY cells was not observed when the co-cultured cells had been transduced with a non-functional GFP shRNA that was mutated at two nucleotides or when the cells were incubated with the GJIC inhibitor, alpha-glycyrrhetinic acid (alpha-GA). We conclude that 21-23 bp double-stranded shRNA/siRNA oligonucleotides are able to move through gap junctions between hESCs and thus can affect gene expression in neighbouring hESC. This novel intercellular gene expression regulatory mechanism may offer new approaches to manipulation of hESC.
Expression Pattern of the Aspartyl-tRNA Synthetase DARS in the Human Brain
Publisher: Frontiers Media SA
Date: 20-03-2018
Transcriptional evaluation of the developmental accuracy, reproducibility and robustness of kidney organoids derived from human pluripotent stem cells
Publisher: Cold Spring Harbor Laboratory
Date: 22-12-2017
DOI: 10.1101/238428
Abstract: We have previously reported a protocol for the directed differentiation of human induced pluripotent stem cells to kidney organoids comprised of nephrons, proximal and distal epithelium, vasculature and surrounding interstitial elements. The utility of this protocol for applications such as disease modelling will rely implicitly on the developmental accuracy of the model, technical robustness of the protocol and transferability between iPSC lines. Here we report extensive transcriptional analyses of the sources of variation across the timecourse of differentiation from pluripotency to complete kidney organoid, focussing on repeated differentiations to day 18 organoid. In idual organoids generated within the same differentiation experiment show Spearman’s correlation coefficients of .99. The greatest source of variation was seen between experimental batch, with the enrichment for genes that also varied temporally between day 10 and day 25 organoids implicating nephron maturation as contributing to transcriptional variance between in idual differentiation experiments. A morphological analysis revealed a transition from renal vesicle to capillary loop stage nephrons across the same time period. Distinct iPSC clones were also shown to display congruent transcriptional programs with inter-experimental and inter-clonal variation most strongly associated with nephron patterning. Even epithelial cells isolated from organoids showed transcriptional alignment with total organoids of the same day of differentiation. This data provides a framework for managing experimental variation, thereby increasing the utility of this approach for personalised medicine and functional genomics.
Chondro-inductive injectable hydrogels for stem cell-based intervertebral disc tissue engineering
Publisher: Frontiers Media SA
Date: 2016
Arrayed cellular environments for stem cells and regenerative medicine
Publisher: Wiley
Date: 14-08-2012
Abstract: The behavior and composition of both multipotent and pluripotent stem cell populations are exquisitely controlled by a complex, spatiotemporally variable interplay of physico-chemical, extracellular matrix, cell-cell interaction, and soluble factor cues that collectively define the stem cell niche. The push for stem cell-based regenerative medicine models and therapies has fuelled demands for increasingly accurate cellular environmental control and enhanced experimental throughput, driving an evolution of cell culture platforms away from conventional culture formats toward integrated systems. Arrayed cellular environments typically provide a set of discrete experimental elements with variation of one or several classes of stimuli across elements of the array. These are based on high-content/high-throughput detection, small s le volumes, and multiplexing of environments to increase experimental parameter space, and can be used to address a range of biological processes at the cell population, single-cell, or subcellular level. Arrayed cellular environments have the capability to provide an unprecedented understanding of the molecular and cellular events that underlie expansion and specification of stem cell and therapeutic cell populations, and thus generate successful regenerative medicine outcomes. This review focuses on recent key developments of arrayed cellular environments and their contribution and potential in stem cells and regenerative medicine.
Alcohol and DNA Methylation
Publisher: CRC Press
Date: 16-10-2014
DOI: 10.1201/B17530-11
Platypus Induced Pluripotent Stem Cells: the Unique Pluripotency Signature of a Monotreme
Publisher: Cold Spring Harbor Laboratory
Date: 25-07-2018
DOI: 10.1101/377010
Abstract: The generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals. The mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG . Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X 1-5 X 1-5 :AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).
DNA methylation in schizophrenia in different patient-derived cell types
Publisher: Springer Science and Business Media LLC
Date: 23-01-2017
DOI: 10.1038/S41537-016-0006-0
Abstract: DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in in iduals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a “ground state” upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same in iduals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.
Induced Pluripotent Stem Cells from a Marsupial, the Tasmanian Devil (Sarcophilus harrisii): Insight into the Evolution of Mammalian Pluripotency
Publisher: Mary Ann Liebert Inc
Date: 15-01-2018
Abstract: We demonstrate the generation of Tasmanian devil (Sarcophilus harrisii) induced pluripotent stem cells (DeviPSCs) from dermal fibroblasts by lentiviral delivery of human transcription factors. DeviPSCs display characteristic pluripotent stem cell colony morphology, with in idual cells having a high nuclear-to-cytoplasmic ratio and alkaline phosphatase activity. DeviPSCs are leukemia inhibitory factor dependent and have reactivated endogenous octamer-binding transcription factor 4 [OCT4, POU domain, class 5, transcription factor 1 (POU5F1)], POU2 [POU domain, class 5, transcription factor 3 (POU5F3)], sex determining region Y-box 2 (SOX2), Nanog homeobox (NANOG) and dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) genes, retained a normal karyotype, and concurrently silenced exogenous human transgenes. Notably, co-expression of both OCT4 and POU2 suggests that they are representative of cells of the epiblast, the marsupial equivalent of the inner cell mass. DeviPSCs readily form embryoid bodies and in vitro teratomas containing derivatives of all three embryonic germ layers. To date, DeviPSCs have been stably maintained for more than 45 passages. Our DeviPSCs provide an invaluable resource for studies into marsupial pluripotency and development, and they may also serve as an important tool in efforts to combat the threat of devil facial tumor disease.
Cortical Neurons Derived from Equine Induced Pluripotent Stem Cells Are Susceptible to Neurotropic Flavivirus Infection and Replication: An In Vitro Model for Equine Neuropathic Diseases
Publisher: Mary Ann Liebert Inc
Date: 15-05-2018
Abstract: Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this, we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses, such as Hendra virus, enter mammalian neurons. EquiPSC-derived neurons spontaneously depolarize with waves of depolarization conducted unidirectionally to adjacent neurons. We sought to confirm that equiPSC-derived neurons are a possible in vitro model for viral neuropathic diseases in the horse by examining their susceptibility to infection with flaviviruses that are known to be neurotropic in horses, including WNV and Murray Valley encephalitis virus (MVEV), and to compare these to nonpathogenic flaviviruses such as Fitzroy River virus (FRV) and Bamaga virus (BgV). All three strains of WNV tested in this study grew to high titres in the equiPSC-derived neurons, inducing a strong cytopathic effect (cpe), as did MVEV. In contrast, FRV showed restricted replication, and no cpe, which is consistent with the observation that FRV infects, but does not cause disease, in horses. BgV, which is thought to infect only marsupials, did not replicate in the equiPSC-derived neurons. Hence, our equiPSC-derived neurons display virus-specific differences in terms of viral titre and cpe that are similar to observations made in vivo, thus supporting their use as an in vitro model for neurotropic viral infection in horses.
Evaluation of variability in human kidney organoids.
Publisher: Springer Science and Business Media LLC
Date: 20-12-2018
Generation of footprint-free induced pluripotent stem cells from human fibroblasts using episomal plasmid vectors
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2848-4_4
Abstract: Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying in iduals' fibroblasts using episomal plasmids.
Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators
Publisher: Springer Science and Business Media LLC
Date: 12-1996
DOI: 10.1007/BF02110443
Abstract: Neutrophils are polymorphonuclear leukocytes recruited to sites of acute inflammation, in response to pathogen invasion and tissue injury. The modulation of their activity, especially oxidative burst, may be important to control the inflammatory process. 2-Styrylchromones (2-SC) are derived from chromones and despite their recognized multiple biological activities, their anti-inflammatory and antioxidant properties are still poorly explored. Therefore, in this study, 43 structurally related 2-SC were evaluated concerning their effects on freshly isolated human neutrophils' viability and oxidative burst. The studied 2-SC were ided into eight groups according to their substitution at C-4' on B-ring (none, -OH, -OCH
Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage
Publisher: Oxford University Press (OUP)
Date: 28-11-2017
DOI: 10.1189/JLB.4VMA0716-316R
Abstract: Mutations in the ataxia-telangiectasia (A-T)-mutated (ATM) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation of an Atm missense mutant [amino acid change of leucine (L) to proline (P) at position 2262 (L2262P)] rat by intracytoplasmic injection (ICSI) of mutant sperm into oocytes. Atm-mutant rats (AtmL2262P/L2262P) expressed low levels of ATM protein, suggesting a destabilizing effect of the mutation, and had a significantly reduced lifespan compared with Atm+/+. Whereas these rats did not show cerebellar atrophy, they succumbed to hind-limb paralysis (45%), and the remainder developed tumors. Closer examination revealed the presence of both dsDNA and ssDNA in the cytoplasm of cells in the hippoc us, cerebellum, and spinal cord of AtmL2262P/L2262P rats. Significantly increased levels of IFN-β and IL-1β in all 3 tissues were indicative of DNA damage induction of the type 1 IFN response. This was further supported by NF-κB activation, as evidenced by p65 phosphorylation (P65) and translocation to the nucleus in the spinal cord and parahippoc us. Other evidence of neuroinflammation in the brain and spinal cord was the loss of motor neurons and the presence of increased activation of microglia. These data provide support for a proinflammatory phenotype that is manifested in the Atm mutant rat as hind-limb paralysis. This mutant represents a useful model to investigate the importance of neuroinflammation in A-T.
Correction: AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation.
Publisher: Public Library of Science (PLoS)
Date: 21-07-2016
Characterization of monoclonal antibodies specific to the transcription factor ETS-2 protein
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0165-2478(02)00258-4
Abstract: ETS-2 is a member of the ETS family of transcription factors. ETS-2 was initially characterized as a nuclear oncogene and has been shown to play a role in regulation of apoptosis and cell cycle progression. Members of the ETS family display high sequence homology, thus, there is considerable controversy concerning the specificity of existing ETS-2 polyclonal antibodies that have been used to define ETS-2 function. We therefore embarked on the production of ETS-2 specific monoclonal antibodies. In this report, we describe the production and characterization of six antibodies and the localization of their target epitopes to distinct domains of the ETS-2 protein. Four antibodies are ETS-2 specific and two antibodies cross-react with ETS-1, an ETS family member with the highest amino acid sequence homology to ETS-2. This report provides a comprehensive evaluation of ETS-2 specific monoclonal antibodies verified using ETS-2 null cells. These antibodies can be used for EMSA, Western blotting, immunoprecipitation and immunofluorescence staining experiments. Collectively, these reagents are invaluable molecular tools that should help better understand the biological function of ETS-2.
A rat model of ataxia-telangiectasia: evidence for a neurodegenerative phenotype.
Publisher: Oxford University Press (OUP)
Date: 22-12-2017
DOI: 10.1093/HMG/DDW371
Abstract: Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.
Neuronal Lipid Metabolism: Multiple Pathways Driving Functional Outcomes in Health and Disease
Publisher: Frontiers Media SA
Date: 23-01-2018
Platypus Induced Pluripotent Stem Cells: The Unique Pluripotency Signature of a Monotreme
Publisher: Mary Ann Liebert Inc
Date: 02-2019
Abstract: The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors erged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X
Inhibition of the cGAS‐STING pathway ameliorates the premature senescence hallmarks of Ataxia‐Telangiectasia brain organoids
Publisher: Wiley
Date: 30-08-2021
DOI: 10.1111/ACEL.13468
Abstract: Ataxia‐telangiectasia (A‐T) is a genetic disorder caused by the lack of functional ATM kinase. A‐T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A‐T remains elusive. Here, we utilize human pluripotent stem cell‐derived cortical brain organoids to study A‐T neuropathology. Mechanistically, we show that the cGAS‐STING pathway is required for the recognition of micronuclei and induction of a senescence‐associated secretory phenotype (SASP) in A‐T olfactory neurosphere‐derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self‐DNA‐triggered SASP expression in A‐T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A‐T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A‐T and constitutes a novel therapeutic target for treating neuropathology in A‐T patients.
Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology
Publisher: Oxford University Press (OUP)
Date: 25-02-2013
DOI: 10.1002/STEM.1297
Abstract: Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at & % efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.
SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein
Publisher: Springer Science and Business Media LLC
Date: 11-2022
DOI: 10.1038/S41380-022-01831-0
Abstract: Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson’s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in COVID-19 infected in iduals, and a potential therapeutic avenue for intervention.
Proceedings of the Frontiers of Retrovirology Conference 2016
Publisher: Springer Science and Business Media LLC
Date: 09-2016
Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.BIOMATERIALS.2010.07.037
Abstract: Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
Publisher: Informa UK Limited
Date: 02-01-2016
Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a HumanNCX1-EGFPReporter
Publisher: Mary Ann Liebert Inc
Date: 2015
Abstract: The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-cl recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.
ETS2 overexpression in transgenic models and in Down sydrome predisposes to apoptosis via the p53 pathway
Publisher: Oxford University Press (OUP)
Date: 02-2003
DOI: 10.1093/HMG/DDG015
Abstract: ETS2 is a transcription factor encoded by a gene on human chromosome 21 and alterations in its expression have been implicated in the pathophysiological features of Down syndrome (DS). This study demonstrates that overexpression of ETS2 results in apoptosis. This is shown in a number of circumstances, including ETS2-overexpressing transgenic mice and cell lines and in cells from subjects with DS. Indeed we report for the first time that the ETS2 overexpression transgenic mouse develops a smaller thymus and lymphocyte abnormalities similar to that observed in DS. In all circumstances of ETS2 overexpression, the increased apoptosis correlated with increased p53 and alterations in downstream factors in the p53 pathway. In the human HeLa cancer cell line, transfection with functional p53 enables ETS2 overexpression to induce apoptosis. Furthermore, crossing the ETS2 transgenic mice with p53(-/-) mice genetically rescued the thymic apoptosis phenotype. Therefore, we conclude that overexpression of human chromosome 21-encoded ETS2 induces apoptosis that is dependent on p53. These results have important consequences for understanding DS and oncogenesis and may provide new insights into therapeutic interventions.
A protocol for the identification and validation of novel genetic causes of kidney disease.
Publisher: Springer Science and Business Media LLC
Date: 15-09-2015
Testing the Correlates of Consciousness in Brain Organoids: How Do We Know and What Do We Do?
Publisher: Informa UK Limited
Date: 29-12-2020
Substrate stiffness facilitates improved induced pluripotent stem cell production through modulation of both early and late phases of cell reprogramming
Publisher: Cold Spring Harbor Laboratory
Date: 03-2023
DOI: 10.1101/2023.02.27.530366
Abstract: Cell reprogramming involves time-intensive, costly processes that ultimately produce low numbers of reprogrammed cells of variable quality. By screening a range of polyacrylamide hydrogels (pAAm gels) of varying stiffness (1 kPA – 1.3 MPa) we found that a gel of medium stiffness significantly increases the overall number of reprogrammed cells by up to ten-fold with accelerated reprogramming kinetics, as compared to the standard Tissue Culture PolyStyrene (TCPS)-based protocol. We observe that though the gel improves both early and late phases of reprogramming, improvement in the late (reprogramming prone population maturation) phase is more pronounced and produces iPSCs having different characteristics and lower remnant transgene expression than those produced on TCPS. Comparative RNA-Seq analyses coupled with experimental validation reveals that modulation of Bone Morphogenic Protein (BMP) signalling by a novel reprogramming regulator, Phactr3, upregulated in the gel at an earliest time-point without the influence of transcription factors used for reprogramming, plays a crucial role in the improvement in the early reprogramming kinetics and overall reprogramming outcomes. This study provides new insights into the mechanism via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.
Variability in the Generation of Induced Pluripotent Stem Cells: Importance for Disease Modeling
Publisher: Oxford University Press (OUP)
Date: 09-2012
Abstract: In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different in iduals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interin idual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage-specific embryonic antigen-4. Our study shows that it is possible to define a similar “ground state” for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease-associated variability above in idual and cell line variability.
Advances in Reprogramming to Pluripotency
Publisher: Bentham Science Publishers Ltd.
Date: 10-04-2015
DOI: 10.2174/1574888X10666150220154820
Abstract: Pluripotent stem cells (PSCs) derived from somatic cells represent a powerful experimental tool for investigating the molecular mechanisms underlying the disease phenotype with prospects to advance medical therapies. They also have significant potential as a renewable source of autologous cells for cellular therapy. Various approaches for PSC derivation from somatic cells have been reported in the literature. The method used for reprogramming is particularly relevant as it may affect the characteristics and quality of PSCs. This review will present an overview of the basic strategies and methods for reprogramming to pluripotency. These strategies will be briefly discussed in the context of how the mechanism of reprogramming could influence PSC characteristics with respect to safety and quality. Aspects of the reprogramming approach that can influence PSC properties, such as culture conditions and donor cell source, are also discussed.
The chromosome 21 transcription factor ETS2 transactivates the β-APP promoter: Implications for Down syndrome
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0167-4781(03)00121-0
Abstract: The gene that codes for beta-amyloid precursor protein (beta-APP), a protein centrally involved in senile plaque formation in Down syndrome (DS) and Alzheimer's disease (AD), is located on chromosome 21. In DS beta-APP expression is three- to fourfold higher than what is expected from the 1.5-fold increased gene load, suggesting that other genes on chromosome 21 directly or indirectly can further up-regulate beta-APP. Here we show that the chromosome 21 transcription factor ETS2 transactivates the beta-APP gene via specific Ets binding sites in the beta-APP promoter and, in this respect, cooperates with the transcription factor complex AP1. We further show that brains and primary neuronal cultures from Ets2 transgenic mice, as well as 3T3 fibroblasts that overexpress ETS2, display molecular abnormalities also seen in DS, such as elevated expression of beta-APP protein, an increase in presenilin-1 and increased beta-amyloid production. We conclude that ETS2 is a transcriptional regulator of beta-APP and that overexpression of ETS2 in DS may play a role in the pathogenesis of the brain abnormalities in DS and possibly AD.
Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-12-2022
Abstract: All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.
Putative variability markers identified for each stage based on changes in gene expression variability
Publisher: Public Library of Science (PLoS)
Date: 2015
adck3 mutant cell lines used in this study
Publisher: Public Library of Science (PLoS)
Date: 2016
A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells
Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2017
DOI: 10.1101/237644
Abstract: Kidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble ‘capillary loop’ stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.
Factors influencing the latency of the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in permeabilized human skin fibroblasts
Publisher: Elsevier BV
Date: 10-1991
DOI: 10.1016/0167-4889(91)90074-8
Abstract: In selectively permeabilized fibroblasts suspended in a medium mimicking the composition of the cytosol the peroxisomal enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) was found to exhibit about 80% latency (Wolvetang, E.J., Tager, J.M. and Wanders, R.J.A. (1990) Biochem. Biophys. Res. Commun. 1035, 6-11). We investigated which components of the cytosol mimicking medium are important for latency of DHAP-AT and unmasking of latent DHAP-AT activity by ATP. We show that the latency of DHAP-AT is critically dependent upon the presence of reduced glutathione in the medium and that the in vivo prevailing GSH/GSSG ratio is sufficient to maintain DHAP-AT latency. Although thiol-groups in the peroxisomal membrane seem to be essential for the integrity of peroxisomes in selectively permeabilized fibroblasts no latency of DHAP-AT is observed in buffered sucrose media or in cell homogenates, irrespective of the presence of GSH in the medium used. We suggest that during homogenization irreversible damage is inflicted upon the peroxisomal membrane and/or that more factors than at present investigated are involved in maintaining peroxisomal integrity. Furthermore, we demonstrate that cations play a role in the stimulatory effect of ATP on latent DHAP-AT activity while a proton gradient is not directly involved in the stimulatory effect of ATP on latent DHAP-AT activity.
Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1593/TLO.12232
A new model to study neurodegeneration in ataxia oculomotor apraxia type 2
Publisher: Oxford University Press (OUP)
Date: 30-07-2015
DOI: 10.1093/HMG/DDV296
Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.SCR.2014.05.006
Abstract: Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.
Tailorable cell culture platforms from enzymatically cross-linked multifunctional poly(ethylene glycol)-based hydrogels
Publisher: American Chemical Society (ACS)
Date: 15-01-2013
DOI: 10.1021/BM301652Q
Abstract: As stem-cell-based therapies rapidly advance toward clinical applications, there is a need for cheap, easily manufactured, injectable gels that can be tailored to carry stem cells and impart function to such cells. Herein we describe a process for making hydrogels composed of hydroxyphenyl propionic acid (HPA) conjugated, branched poly(ethylene glycol) (PEG) via an enzyme mediated, oxidative cross-linking method. Functionalization of the branched PEG with HPA at varying degrees of substitution was confirmed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. The versatility of this hydrogel system was exemplified through variations in the degree of HPA substitution, polymer concentration, and the concentration of cross-linking reagents (horseradish peroxidase and H(2)O(2)), which resulted in a range of mechanical properties and gelation kinetics for these gels. Cross-linking of the PEG-HPA conjugate with a recombinantly produced Fibronectin fragment (Type III domains 7-10) encouraged attachment and spreading of human mesenchymal stem cells (hMSCs) when assessed in both two-dimensional and three-dimensional formats. Interestingly, when encapsulated in both nonfunctionalized and functionalized cross-linked PEG-HPA gels, MSCs showed good viability over all time periods assessed. With tunable gelation kinetics and mechanical properties, these hydrogels provide a flexible in vitro cell culture platform that will likely have significant utility in tissue engineering as an injectable delivery platform for cells to sites of tissue damage.
Activity-dependent transcriptional dynamics in mouse primary cortical and human iPS-derived neurons
Publisher: No publisher found
Date: 2014
Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2
Publisher: Cold Spring Harbor Laboratory
Date: 12-06-2023
DOI: 10.1101/2023.06.12.544552
Abstract: Why in iduals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.
3-Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism
Publisher: Wiley
Date: 08-1988
DOI: 10.1111/J.1432-1033.1988.TB14200.X
Abstract: 3-Methyladenine is generally used as an inhibitor of autophagy [P. O. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889-1892]. Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well. 1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine. 2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.
AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation.
Publisher: Public Library of Science (PLoS)
Date: 11-02-2016
RELB Alters Proliferation of Human Pluripotent Stem Cells via IMP3- and LIN28-Mediated Modulation of the Expression of IGF2 and Other Cell-Cycle Regulators
Publisher: Mary Ann Liebert Inc
Date: 15-08-2015
Abstract: The molecular mechanisms that orchestrate the exit from pluripotency, cell cycle progression, and lineage-specific differentiation in human pluripotent stem cells (hPSCs) are poorly understood. RELB, a key protein in the noncanonical nuclear factor-kappaB (NFκB) signaling pathway, was previously implicated in controlling the switch between human embryonic stem cell (hESC) proliferation and differentiation. Here, we show that RELB enhances the proliferation of hESCs and human-induced pluripotent stem cells (hiPSCs) without affecting their pluripotency. We demonstrate that RELB does this by interacting with two RNA-binding proteins LIN28A and IMP3 (IGF2 mRNA-binding protein 3) further, these interactions control mRNA levels and protein expression of insulin-like growth factor 2 (IGF2) and key cell-cycle genes. Finally, after stress, these proteins co-localize in stress granules in hESCs and iPSCs. Our data identify RELB as a novel regulator of hPSC proliferation, and suggest a new function for RELB, in addition to its widely accepted role as a transcription factor, that involves recruitment of IMP3 and LIN28 to the cytosolic mRNA translation-control domains for post-transcriptional modulation of IGF2 and cell-cycle gene expression.
Dynamic methylation of an L1 transduction family during reprogramming and neurodifferentiation
Publisher: Informa UK Limited
Date: 04-2019
DOI: 10.1128/MCB.00499-18
Klotho inhibits neuronal senescence in human brain organoids
Publisher: Springer Science and Business Media LLC
Date: 02-08-2021
DOI: 10.1038/S41514-021-00070-X
Abstract: Aging is a major risk factor for many neurodegenerative diseases. Klotho (KL) is a glycosylated transmembrane protein that is expressed in the choroid plexus and neurons of the brain. KL exerts potent anti-aging effects on multiple cell types in the body but its role in human brain cells remains largely unclear. Here we show that human cortical neurons, derived from human pluripotent stem cells in 2D cultures or in cortical organoids, develop the typical hallmarks of senescent cells when maintained in vitro for prolonged periods of time, and that moderate upregulation or repression of endogenous KL expression in cortical organoids inhibits and accelerates senescence, respectively. We further demonstrate that KL expression alters the expression of senescence-associated genes including, extracellular matrix genes, and proteoglycans, and can act in a paracrine fashion to inhibit neuronal senescence. In summary, our results establish an important role for KL in the regulation of human neuronal senescence and offer new mechanistic insight into its role in human brain aging.
Opportunities and Limitations of Modelling Alzheimer’s Disease with Induced Pluripotent Stem Cells
Publisher: MDPI AG
Date: 05-12-2014
DOI: 10.3390/JCM3041357
The universality of bioenergetic disease: The role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase
Publisher: Elsevier BV
Date: 1994
GRGD‐decorated three‐dimensional nanoworm hydrogels for culturing human embryonic stem cells
Publisher: Wiley
Date: 20-02-2019
DOI: 10.1002/POLA.29342
Ets-2 is induced by oxidative stress and sensitizes cells to H(2)O(2)-induced apoptosis: implications for Down's syndrome
Publisher: Elsevier BV
Date: 10-2001
Development of Myocardial Constructs Using Modulus-Matched Acrylated Polypropylene Glycol Triol Substrate and Different Nonmyocyte Cell Populations
Publisher: Mary Ann Liebert Inc
Date: 09-2011
Generation and characterization of LIF-dependent canine induced pluripotent stem cells from adult dermal fibroblasts
Publisher: Mary Ann Liebert Inc
Date: 10-08-2012
Abstract: Dogs provide a more clinically relevant model of human disease than rodents, particularly with respect to hereditary diseases. Thus, the availability of canine stem cells will greatly facilitate the use of the dog in the development of stem cell-based gene therapies and regenerative medicine. In this study we describe the production of canine induced pluripotent stem cells (ciPSCs) from adult dermal fibroblasts. These cells have a morphology resembling previously described canine embryonic stem cells, a normal karyotype, and express pluripotency markers including alkaline phosphatase, Nanog, Oct4, Telomerase, SSEA1, SSEA4, TRA1-60, TRA1-81, and Rex1. Furthermore, the inactive X chromosome is reactivated indicating a ground-state pluripotency. In culture they readily form embryoid bodies, which in turn give rise to cell types from all 3 embryonic germ layers, as indicated by expression of the definitive endoderm markers Cxcr4 and α-fetoprotein, mesoderm markers Collagen IIA and Gata2, and ectoderm markers βIII-tubulin, Enolase, and Nestin. Of particular significance is the observation that these ciPSCs are dependent only on leukemia inhibitory factor (LIF), making them similar to mouse and canine embryonic stem cells, but strikingly unlike the ciPSCs recently described in two other studies, which were dependent on both basic fibroblast growth factor and LIF in order to maintain their pluripotency. Thus, our ciPSCs closely resemble mouse ESCs derived from the inner cell mass of preimplantation embryos, while the previously described ciPSCs appear to be more representative of cells from the epiblast of mouse postimplantation embryos.
Mitochondrial Dysfunction in Mouse Oocytes Results in Preimplantation Embryo Arrest in Vitro
Publisher: Oxford University Press (OUP)
Date: 12-2004
p53 is required for etoposide-induced apoptosis of human embryonic stem cells
Publisher: Elsevier BV
Date: 06-2008
DOI: 10.1016/J.SCR.2007.10.003
Abstract: The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.
Erratum: Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro (Biology of Reproduction (July 30, 2004) 71 (1936-1942) DOI: 10.1095/biolreprod.104.033589)
Publisher: Oxford University Press (OUP)
Date: 12-2005
Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons
Publisher: Cold Spring Harbor Laboratory
Date: 09-01-2020
DOI: 10.1101/2020.01.08.898429
Abstract: DNA methylation functions in genome regulation and is implicated in neuronal maturation. Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of mice and humans, but its precise function remains unknown. Here we investigate mCH deposition in neurons derived from mouse ES-cells in vitro and in cultured primary mouse neurons. We find that both acquire comparable levels of mCH over a similar period as in vivo. In vitro mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, is preceded by expression of the post-mitotic neuronal marker Rbfox3 (NeuN) and is enriched at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals that mCH patterning in mESC-derived neurons partially differs from in vivo . mESC-derived neurons therefore represent a valuable model system for analyzing the mechanisms and functional consequences of correct and aberrantly deposited CG and non-CG methylation in neuronal maturation.
Full factorial screening of human embryonic stem cell maintenance with multiplexed microbioreactor arrays
Publisher: Wiley
Date: 07-2013
Abstract: Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine aracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.
A promoter-level mammalian expression atlas
Publisher: Springer Science and Business Media LLC
Date: 03-2014
DOI: 10.1038/NATURE13182
Adipocyte Differentiation in Human Embryonic Stem Cells Transduced With Oct4 shRNA Lentivirus
Publisher: Mary Ann Liebert Inc
Date: 05-2009
Abstract: Oct4 is one of the master pluripotency genes that controls differentiation of human embryonic stem cells (hESCs). We generated HES2 and HES3 hESC lines stably transduced with lentivirus carrying Oct4 short hairpin RNA (shRNA) that display 80-90% reduction of Oct4 expression. Analysis of pluripotency marker expression shows that these Oct4 shRNA-transduced hESCs display normal wild-type expression levels of the pluripotency marker CD9 but an absence of GCTM2 expression. These hESC-derived adipocyte precursor cells display a characteristic morphology and can be propagated and cryopreserved as a standard stem cell line. Interestingly, Oct4 shRNA-transduced hESCs display a remarkably high lineage-specific spontaneous differentiation toward adipocytes. After two weeks of spontaneous differentiation under feeder-free conditions, 60-70% of cells display a mature adipocyte morphology as well as the expression of multiple adipocyte-specific mRNAs as assessed by RT-PCR. The upregulation of trophoblast, mesoderm, and endoderm transcripts is, however, also detected in these spontaneously differentiating cultures. These Oct4 shRNA hESCs will be an interesting model system to study human fetal adipogenesis and constitutes a renewable resource for obesity drug screening purposes.
TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation
Publisher: American Society for Cell Biology (ASCB)
Date: 03-2015
Abstract: CD30 activates NFκB signaling in human embryonic stem cells. A single threonine residue in the CD30v protein is critical for this and recruitment of TRAF2. The data reveal the importance of this interaction for hESC survival and proliferation.
Pushing the Limits of High Throughput PET-RAFT Polymerization
Publisher: American Chemical Society (ACS)
Date: 20-09-2018
Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells
Publisher: American Association for the Advancement of Science (AAAS)
Date: 27-02-2015
Abstract: In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010
Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells
Publisher: eLife Sciences Publications, Ltd
Date: 08-09-2017
DOI: 10.7554/ELIFE.24502
Abstract: Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells.
Self-Organizing 3D Human Choroid Plexus-Ventricle-Cortical Organoids
Publisher: Cold Spring Harbor Laboratory
Date: 10-2020
DOI: 10.1101/2020.09.30.321554
Abstract: Both the choroid plexus (CP) and the cortex are derived from the rostral neural tube during early embryonic development. In addition to producing CSF, the CP secretes essential factors that orchestrate cortical development and later neurogenesis. Previous brain modeling efforts with human pluripotent stem cells (hPSCs) generated either cortical or CP tissues in 3D culture. Here, we used hPSC-derived neuroectodermal cells, the building blocks of the anterior body, to simultaneously generate CP that forms ventricles and cortical cells in organoids (CVCOs), which can be maintained as 3D organoid cultures. Large scale culture revealed reproducibility of the protocol independent of cell lines, clones or batches. CVCOs contain mature and functional CP that projects multiple cilia into the ventricle-like fluid filled cysts and is in direct contact with appropriately patterned cortical cells. CVCOs thus recapitulate key features of developing forebrain structures observed in in vivo and constitute a useful for dissecting the role of CP in human forebrain development in health and disease.
Use of human iPSCs and kidney organoids to develop a cysteamine/mTOR inhibition combination therapy to treat cystinosis
Publisher: Cold Spring Harbor Laboratory
Date: 04-2019
DOI: 10.1101/595264
Abstract: Cystinosis is a lysosomal storage disease caused by mutations in CTNS , encoding a cystine transporter, and in its severest form is characterized by cystine accumulation, renal proximal tubule dysfunction and kidney failure. Cystinosis is treated with the cystine-depleting drug cysteamine, however this only slows progression of the disease and there is an urgent need for better treatments. Here, we have generated and characterized the first human induced pluripotent stem cell (iPSC) and kidney organoid models of cystinosis. These models exhibit elevated cystine and cysteine levels, enlarged lysosomes and a block in basal autophagy flux. Cysteamine treatment ameliorates this phenotype except for the basal autophagy flux defect. We found that treatment with Everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes and activates autophagy but does not rescue the cystine/cysteine loading defect. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and Everolimus corrects all of the observed phenotypes indicating that a combination therapy has therapeutic potential to improve the treatment of cystinosis.
FANTOM5 CAGE profiles of human and mouse samples
Publisher: Springer Science and Business Media LLC
Date: 29-08-2017
Abstract: In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of s les, consisting of a variety of primary cells, tissues, cell lines, and time series s les during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.
Vitamin C Promotes Widespread Yet Specific DNA Demethylation of the Epigenome in Human Embryonic Stem Cells
Publisher: Oxford University Press (OUP)
Date: 10-2010
DOI: 10.1002/STEM.493
Abstract: Vitamin C (ascorbate) is a widely used medium supplement in embryonic stem cell culture. Here, we show that ascorbate causes widespread, consistent, and remarkably specific DNA demethylation of 1,847 genes in human embryonic stem cells (hESCs), including important stem cell genes, with a clear bias toward demethylation at CpG island boundaries. We show that a subset of these DNA demethylated genes displays concomitant gene expression changes and that the position of the demethylated CpGs relative to the transcription start site is correlated to such changes. We further show that the ascorbate-demethylated gene set not only overlaps with gene sets that have bivalent marks, but also with the gene sets that are demethylated during differentiation of hESCs and during reprogramming of fibroblasts to induced pluritotent stem cells (iPSCs). Our data thus identify a novel link between ascorbate-mediated signaling and specific epigenetic changes in hESCs that might impact on pluripotency and reprogramming pathways.
Fabrication of nanopatterned, porous microspheres using a glass capillary microfluidic device
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2SM25950H
CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination
Publisher: MDPI AG
Date: 28-09-2023
Human iPSC-derived cerebellar neurons from a patient with ataxia-telangiectasia reveal disrupted gene regulatory networks
Publisher: Frontiers Media SA
Date: 13-10-2017
Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology
Publisher: Research Square Platform LLC
Date: 16-03-2023
DOI: 10.21203/RS.3.RS-2675698/V1
Abstract: Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cells in vivo strongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected in iduals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2. In vivo treatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.
The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.TAAP.2012.03.009
Abstract: The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chlor henicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure,membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chlor henicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chlor henicol toxicity in humans.
Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis
Publisher: Wiley
Date: 07-2003
DOI: 10.1002/ART.11165
Abstract: To study the capacity of macrophage migration inhibitory factor (MIF) to regulate proliferation, apoptosis, and p53 in an animal model of rheumatoid arthritis (RA) and in fibroblast-like synoviocytes (FLS) from humans with RA. Antigen-induced arthritis (AIA) was induced in MIF(-/-) mice and littermate controls. FLS were obtained from patients with RA. Western blotting and immunohistochemistry were used to measure p53 in cells and tissues. Apoptosis was detected in cells by flow cytometry using TUNEL and annexin V ropidium iodide labeling. Apoptosis in tissue was detected using TUNEL. Proliferation was assessed in cultured cells and tissue by (3)H-thymidine incorporation and Ki-67 immunostaining, respectively. MIF inhibited p53 expression in human RA FLS. Levels of p53 were correspondingly increased in MIF(-/-) mouse tissues and cells. Spontaneous and sodium nitroprusside-induced apoptosis were significantly increased in MIF(-/-) cells. In vitro exposure of FLS to MIF reduced apoptosis and significantly induced FLS proliferation. Synoviocyte proliferation in MIF(-/-) mice was correspondingly reduced. A decrease in the severity of AIA in MIF(-/-) mice was associated with an increase in p53 and apoptosis in synovium. Evidence of in situ proliferation was scant in this model, and no difference in in situ proliferation was detectable in MIF(-/-) mice compared with wild-type mice. These results indicate a role for MIF in the regulation of p53 expression and p53-mediated events in the inflamed synovium and support the hypothesis that MIF is of critical importance in the pathogenesis of RA.
The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome
Publisher: Public Library of Science (PLoS)
Date: 18-12-2015
Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.
Publisher: Public Library of Science (PLoS)
Date: 20-11-2014
S3_Text.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation
Publisher: Public Library of Science (PLoS)
Date: 26-12-2012
Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency
Publisher: Oxford University Press (OUP)
Date: 08-2013
DOI: 10.1002/STEM.1425
Abstract: Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify “unsafe” hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these “unsafe” hiPSC lines should be interpreted carefully.
PCDH19 regulation of neural progenitor cell differentiation suggests asynchrony of neurogenesis as a mechanism contributing to PCDH19 Girls Clustering Epilepsy.
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.NBD.2018.05.004
Abstract: PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) is a childhood epileptic encephalopathy characterised by a spectrum of neurodevelopmental problems. PCDH19-GCE is caused by heterozygous loss-of-function mutations in the X-chromosome gene, Protocadherin 19 (PCDH19) encoding a cell-cell adhesion molecule. Intriguingly, hemizygous males are generally unaffected. As PCDH19 is subjected to random X-inactivation, heterozygous females are comprised of a mosaic of cells expressing either the normal or mutant allele, which is thought to drive pathology. Despite being the second most prevalent monogeneic cause of epilepsy, little is known about the role of PCDH19 in brain development. In this study we show that PCDH19 is highly expressed in human neural stem and progenitor cells (NSPCs) and investigate its function in vitro in these cells of both mouse and human origin. Transcriptomic analysis of mouse NSPCs lacking Pcdh19 revealed changes to genes involved in regulation of neuronal differentiation, and we subsequently show that loss of Pcdh19 causes increased NSPC neurogenesis. We reprogramed human fibroblast cells harbouring a pathogenic PCDH19 mutation into human induced pluripotent stem cells (hiPSC) and employed neural differentiation of these to extend our studies into human NSPCs. As in mouse, loss of PCDH19 function caused increased neurogenesis, and furthermore, we show this is associated with a loss of human NSPC polarity. Overall our data suggests a conserved role for PCDH19 in regulating mammalian cortical neurogenesis and has implications for the pathogenesis of PCDH19-GCE. We propose that the difference in timing or "heterochrony" of neuronal cell production originating from PCDH19 wildtype and mutant NSPCs within the same in idual may lead to downstream asynchronies and abnormalities in neuronal network formation, which in-part predispose the in idual to network dysfunction and epileptic activity.
Interactive visualization and analysis of large-scale sequencing datasets using ZENBU
Publisher: Springer Science and Business Media LLC
Date: 03-2014
DOI: 10.1038/NBT.2840
S3_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Derivation of mesenchymal stromal cells from canine induced pluripotent stem cells by inhibition of the tgfβ/activin signaling pathway
Publisher: Mary Ann Liebert Inc
Date: 15-12-2014
S2_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
CD146-assisted isolation and multi-omics characterisation of human pluripotent stem cell derived cranial Schwann cell precursor-like cells.
Publisher: Research Square Platform LLC
Date: 19-09-2023
S1_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
sample information of the mouse samples
Publisher: No publisher found
Date: 2017
ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1
Publisher: Oxford University Press (OUP)
Date: 03-08-2015
DOI: 10.1093/NAR/GKV754
Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase
Publisher: Elsevier BV
Date: 07-1990
DOI: 10.1016/0304-4165(90)90166-T
Abstract: Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 +/- 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the beta-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F1F0-ATPase and the lysosomal V-type ATPase.
sample information of the human samples
Publisher: No publisher found
Date: 2017
Transcriptome of the developing Ataxia-Telangiectasia cerebellum: RNA Sequencing of human iPSC-derived cerebellar progenitors
Publisher: Unpublished
Date: 2015
A synthetic elastomer based on acrylated polypropylene glycol triol with tunable modulus for tissue engineering applications.
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.BIOMATERIALS.2010.07.007
Abstract: As strategies for manipulating cellular behaviour in vitro and in vivo become more sophisticated, synthetic biomaterial substrates capable of reproducing critical biochemical and biophysical properties (or cues) of tissue micro-environments will be required. Cytoskeletal tension has been shown to be highly deterministic of cell fate decisions, yet few synthetic biomaterials are capable of modulating cytoskeletal tension of adhered cells through variations in stiffness, at least in the ranges applicable to tissue properties (e.g., 1-100 kPa), whilst also possessing other required properties, such as biodegradability, biocompatibility and processability. In this paper we describe a non-cytotoxic polymer system based on acrylated polypropylene glycol triol (aPPGT). This new elastomer system has tunable elastic moduli, is degradable, can be easily surface modified and can be manufactured into porous three dimensional scaffolds or micropatterned substrates. We demonstrate that the PPGT substrates can modulate hMSC morphology, growth, and differentiation, and that they can produce similar outcomes as observed for a non-degradable polyacrylamide substrate, confirming their utility as a degradable elastomer for tissue engineering and other biomedical applications.
S4_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
genomic coordinates of the mouse CAGE peaks
Publisher: No publisher found
Date: 2017
ets-2 Promotes the Activation of a Mitochondrial Death Pathway in Down's Syndrome Neurons
Publisher: Society for Neuroscience
Date: 02-03-2005
DOI: 10.1523/JNEUROSCI.5107-04.2005
Abstract: Down's syndrome (DS) is characterized by mental retardation and development of Alzheimer's disease (AD). Oxidative stress and mitochondrial dysfunction are both related to neurodegeneration in DS. Several genes in chromosome 21 have been linked to neuronal death, including the transcription factor ets-2. Cortical cultures derived from normal and DS fetal brains were used to study the role of ets-2 in DS neuronal degeneration. ets-2 was expressed in normal human cortical neurons (HCNs) and was markedly upregulated by oxidative stress. When overexpressed in normal HCNs, ets-2 induced a stereotyped sequence of apoptotic changes leading to neuronal death. DS HCNs exhibit intracellular oxidative stress and increased apoptosis after the first week in culture (Busciglio and Yankner, 1995). ets-2 levels were increased in DS HCNs, and, between 7 and 14 d in vitro , DS HCNs showed increased bax, cytoplasmic translocation of cytochrome c and apoptosis inducing factor, and active caspases 3 and 7, consistent with activation of an apoptotic mitochondrial death pathway. Degeneration of DS neurons was reduced by dominant-negative ets-2, suggesting that increased ets-2 expression promotes DS neuronal apoptosis. In the human brain, ets-2 expression was found in neurons and astrocytes. Strong ets-2 immunoreactivity was observed in DS/AD and sporadic AD brains associated with degenerative markers such as bax, intracellular Aβ, and hyperphosphorylated tau. Thus, in DS/AD and sporadic AD brains, converging pathological mechanisms leading to chronic oxidative stress and ets-2 upregulation in susceptible neurons may result in increased vulnerability by promoting the activation of a mitochondrial-dependent proapoptotic pathway of cell death.
Induced pluripotent stem cells: A new technology to study human diseases
Publisher: Elsevier BV
Date: 06-2011
DOI: 10.1016/J.BIOCEL.2011.03.013
Abstract: Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed to a pluripotent state by the introduction of specific factors. They can be generated from cells of different origins such as fibroblasts, keratinocytes, hepatocytes and blood. iPS cells are similar to embryonic stem cells in several aspects such as morphology, expression of pluripotency markers and the capacity to develop teratomas tumors containing cells of the three germ layers. As pluripotent stem cells they can be differentiated into several lineages including neuronal, cardiac and blood cells. Recently, several groups have successfully generated patient-specific iPS cells from donors suffering different disorders and differentiated them into the cell type affected by the disease. These new human cell-based models cannot only be used to study the dynamics of diseases but also as systems to screen new drugs. Moreover, iPS cells promise to be good candidates for regenerative medicine.
Autophagy in Human Embryonic Stem Cells
Publisher: Public Library of Science (PLoS)
Date: 14-11-2011
Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis
Publisher: Springer Science and Business Media LLC
Date: 07-10-2015
DOI: 10.1038/NATURE15695
Abstract: The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, in idual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts
Publisher: Mary Ann Liebert Inc
Date: 07-2014
S10_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells
Publisher: Oxford University Press (OUP)
Date: 02-2012
Abstract: The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
The Impact of APP on Alzheimer-like Pathogenesis and Gene Expression in Down Syndrome iPSC-Derived Neurons
Publisher: Elsevier BV
Date: 07-2018
S13_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Concise review: New paradigms for down syndrome research using induced pluripotent stem cells: Tackling complex human genetic disease
Publisher: Oxford University Press (OUP)
Date: 14-02-2013
S12_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
S11_Table.docx
Publisher: Public Library of Science (PLoS)
Date: 2015
Didemnin B Induces Cell Death by Apoptosis: The Fastest Induction of Apoptosis Ever Described
Publisher: Elsevier BV
Date: 10-1995
Abstract: Didemnin B, a cyclic N-methylated peptolide induces apoptosis in human HL-60 cells. When incubated with 1 microM didemnin B, unsynchronized HL-60 cultures undergo apoptosis to 100% within 140 minutes. Apoptosis has been assessed by the typical apoptotic morphology, the presence of double-stranded DNA fragments within the cytosol and the generation of DNA ladders. None of these characteristics of apoptosis are seen when HL-60 cells are pretreated with 1mM Zn2+ immediately before treatment with didemnin B.
CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells
Publisher: Springer Science and Business Media LLC
Date: 26-02-2006
DOI: 10.1038/NBT1197
Abstract: The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small lifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.
The statistical geometry of transcriptome divergence in cell-type evolution and cancer
Publisher: Springer Science and Business Media LLC
Date: 14-01-2015
DOI: 10.1038/NCOMMS7066
Abstract: In evolution, body plan complexity increases due to an increase in the number of in idualized cell types. Yet, there is very little understanding of the mechanisms that produce this form of organismal complexity. One model for the origin of novel cell types is the sister cell-type model. According to this model, each cell type arises together with a sister cell type through specialization from an ancestral cell type. A key prediction of the sister cell-type model is that gene expression profiles of cell types exhibit tree structure. Here we present a statistical model for detecting tree structure in transcriptomic data and apply it to transcriptomes from ENCODE and FANTOM5. We show that transcriptomes of normal cells harbour substantial amounts of hierarchical structure. In contrast, cancer cell lines have less tree structure, suggesting that the emergence of cancer cells follows different principles from that of evolutionary cell-type origination.
Therapeutic targets and investigated treatments for Ataxia-Telangiectasia
Publisher: Informa UK Limited
Date: 11-11-2016
Defined high protein content surfaces for stem cell culture
Publisher: Elsevier BV
Date: 07-2010
DOI: 10.1016/J.BIOMATERIALS.2010.03.015
Abstract: Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.
Thyroid hormone and ALK5 inhibitor improve maturation of human pluripotent stem cell derived hepatocytes
Publisher: Cold Spring Harbor Laboratory
Date: 30-04-2022
DOI: 10.1101/2022.04.28.489845
Abstract: Hepatocytes derived from human pluripotent stem cells (PSCs) hold great promise for modeling human liver disease, in vitro hepatotoxicity testing, and future cellular therapy. However, current protocols generate hepatocyte-like cells (HLCs) that resemble fetal hepatocytes, and thus do not accurately recapitulate the molecular identity and functions of the adult liver. To address this, we compared the transcriptomes of human fetal and adult liver to PSC-derived HLCs during progressive stages of in vitro differentiation. This revealed that during the final stages of in vitro differentiation the hepatic transcription factors HNF4A and CEBPA were sub-optimally expressed. Computational analyses predicted that ALK5i II (TGF-β receptor inhibitor) and thyroid hormone (T3) would be able to rectify this and improve HLC maturation. We next show that application of these molecules during hepatocyte differentiation indeed increases CEBPA and HNF4A mRNA and protein expression, and that these HLCs show enhanced albumin secretion, a 25-fold increase in CYP3A4 activity, and 10 to 100-fold increased expression of mature hepatic markers. We demonstrate that this improved maturation is effective across different cell lines and HLC differentiation protocols, and exemplifies that our approach provides a tractable template for identifying and targeting additional factors that that will fully mature human liver cells from human pluripotent stem cells.
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