ABSTRACT
Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.
Subject(s)
Colorectal Neoplasms , Induced Pluripotent Stem Cells , Brain Neoplasms , Clustered Regularly Interspaced Short Palindromic Repeats , Colorectal Neoplasms/genetics , DNA Damage/genetics , Humans , Mutation , Neoplastic Syndromes, HereditaryABSTRACT
Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.
Subject(s)
Dinoprostone/metabolism , Inflammatory Bowel Diseases/metabolism , Interleukin-10 Receptor beta Subunit/metabolism , Interleukin-10/metabolism , Macrophages/metabolism , Salmonella typhimurium/metabolism , Signal Transduction/genetics , Cell Differentiation/genetics , Cells, Cultured , Dinoprostone/antagonists & inhibitors , Female , Gene Knockout Techniques , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/chemically induced , Inflammation/metabolism , Inflammatory Bowel Diseases/pathology , Interleukin-10 Receptor alpha Subunit/genetics , Interleukin-10 Receptor beta Subunit/genetics , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophage Activation/genetics , Macrophages/drug effects , Mutation , Phosphorylation/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effectsABSTRACT
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Subject(s)
Basement Membrane/pathology , Blood Vessels/pathology , Diabetic Angiopathies/pathology , Models, Biological , Organoids/pathology , Organoids/transplantation , Adaptor Proteins, Signal Transducing , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Animals , Arteries/cytology , Arteries/drug effects , Arterioles/cytology , Arterioles/drug effects , Basement Membrane/cytology , Basement Membrane/drug effects , Blood Vessels/cytology , Blood Vessels/drug effects , Blood Vessels/growth & development , Calcium-Binding Proteins , Diabetic Angiopathies/enzymology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Hyperglycemia/complications , In Vitro Techniques , Inflammation Mediators/pharmacology , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Organoids/cytology , Organoids/drug effects , Pericytes/cytology , Pericytes/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Receptor, Notch3/metabolism , Signal Transduction , Venules/cytology , Venules/drug effectsABSTRACT
Progressive microcephaly and neurodegeneration are genetically heterogenous conditions, largely associated with genes that are essential for the survival of neurons. In this study, we interrogate the genetic etiology of two siblings from a non-consanguineous family with severe early onset of neurological manifestations. Whole exome sequencing identified novel compound heterozygous mutations in VARS that segregated with the proband: a missense (c.3192G>A; p.Met1064Ile) and a splice site mutation (c.1577-2A>G). The VARS gene encodes cytoplasmic valyl-tRNA synthetase (ValRS), an enzyme that is essential during eukaryotic translation. cDNA analysis on patient derived fibroblasts revealed that the splice site acceptor variant allele led to nonsense mediated decay, thus resulting in a null allele. Three-dimensional modeling of ValRS predicts that the missense mutation lies in a highly conserved region and could alter side chain packing, thus affecting tRNA binding or destabilizing the interface between the catalytic and tRNA binding domains. Further quantitation of the expression of VARS showed remarkably reduced levels of mRNA and protein in skin derived fibroblasts. Aminoacylation experiments on patient derived cells showed markedly reduced enzyme activity of ValRS suggesting the mutations to be loss of function. Bi-allelic mutations in cytoplasmic amino acyl tRNA synthetases are well-known for their role in neurodegenerative disorders, yet human disorders associated with VARS mutations have not yet been clinically well characterized. Our study describes the phenotype associated with recessive VARS mutations and further functional delineation of the pathogenicity of novel variants identified, which widens the clinical and genetic spectrum of patients with progressive microcephaly.
Subject(s)
Atrophy/genetics , Microcephaly/genetics , Seizures/genetics , Valine-tRNA Ligase/genetics , Alleles , Amino Acyl-tRNA Synthetases/genetics , Atrophy/physiopathology , Child, Preschool , Gene Expression Regulation, Enzymologic , Humans , Infant , Loss of Function Mutation/genetics , Male , Microcephaly/physiopathology , Pedigree , RNA, Transfer/genetics , RNA-Binding Proteins/genetics , Seizures/physiopathology , Transfer RNA Aminoacylation/genetics , Exome SequencingABSTRACT
The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.
Subject(s)
Biological Specimen Banks , Genomics/methods , Haploidy , Mouse Embryonic Stem Cells/metabolism , Mutation , Animals , Blood Vessels/cytology , Cell Lineage/genetics , Common Cold/genetics , Common Cold/virology , Genes, Essential/genetics , Genetic Testing , HEK293 Cells , Homozygote , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Neovascularization, Physiologic/genetics , Phospholipases A2, Calcium-Independent/genetics , Phospholipases A2, Calcium-Independent/metabolism , Rhinovirus/pathogenicityABSTRACT
This corrects the article DOI: 10.1038/nature22403.
ABSTRACT
Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.
Subject(s)
Genetic Variation/genetics , Induced Pluripotent Stem Cells/metabolism , Cells, Cultured , Cellular Reprogramming/genetics , DNA Copy Number Variations/genetics , Gene Expression Regulation/genetics , Genotype , Humans , Organ Specificity , Phenotype , Quality Control , Quantitative Trait Loci/genetics , Transcriptome/geneticsABSTRACT
The collection sites of human primary tissue samples and the receiving laboratories, where the human induced pluripotent stem cells (hIPSCs) are derived, are often not on the same site. Thus, the stability of samples prior to derivation constrains the distance between the collection site and the receiving laboratory. To investigate sample stability, we collected blood and held it at room temperature for 5, 24, or 48 hr before isolating peripheral blood mononuclear cells (PBMCs) and reprogramming into IPSCs. Additionally, PBMC samples at 5- and 48-hr time points were frozen in liquid nitrogen for 4 months and reprogrammed into IPSCs. hIPSC lines derived from all time points were pluripotent, displayed no marked difference in chromosomal aberration rates, and differentiated into three germ layers. Reprogramming efficiency at 24- and 48-hr time points was 3- and 10-fold lower, respectively, than at 5 hr; the freeze-thaw process of PBMCs resulted in no obvious change in reprogramming efficiency.
Subject(s)
Cellular Reprogramming Techniques/methods , Induced Pluripotent Stem Cells/cytology , Leukocytes, Mononuclear/cytology , Blood Preservation , Cell Differentiation , Cell Separation/methods , Cells, Cultured , Cellular Reprogramming , Cryopreservation , Genomic Instability , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , TemperatureABSTRACT
Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum ß-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.
Subject(s)
Cell Division/physiology , DNA Replication/physiology , Drug Resistance, Bacterial/physiology , Escherichia coli Proteins/metabolism , Escherichia coli/physiology , R Factors/physiology , Base Sequence , Blotting, Western , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Microscopy, Electron , Molecular Sequence Data , Oligonucleotides/genetics , R Factors/metabolism , Sequence Analysis, DNAABSTRACT
The bacteriophage-encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage lambda-holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of lambda-holin-induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle-specific fluorescence immunocytochemistry indicated the presence of the lambda-holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria-specific fluorochrome JC-1 demonstrated a loss of mitochondrial transmembrane potential in response to lambda-holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP-ribose) polymerase (PARP) in an apoptosis-specific manner. These findings suggest that the lambda-holin protein mediates a caspase-independent non-apoptotic mode of cell death.
Subject(s)
Bacteriophage lambda/pathogenicity , Caspases/metabolism , Eukaryotic Cells/pathology , Necrosis , Viral Proteins/toxicity , Apoptosis , Bacteriophage lambda/metabolism , Cell Line, Tumor/pathology , Endoplasmic Reticulum/metabolism , HeLa Cells/pathology , Humans , Membrane Potentials/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Viral Proteins/metabolism , Viral Proteins/pharmacologyABSTRACT
BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.