A powerful way to discover key genes playing causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here, we report high-resolution analyses of somatic copy-number alterations (SCNAs) from 3131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across multiple cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κB pathway. We show that cancer cells harboring amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend upon expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in multiple cancer types.
Summary Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question whether an earlier ‘naive’ state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.
Human embryonic stem cells and induced pluripotent stem cells (hESCs and hiPSCs) are powerful tools for biomedical research. Realizing the full potential of these cells requires efficient genetic modification. However, techniques to generate cell type specific lineage reporters as well as reliable tools to disrupt, repair or overexpress genes by gene targeting are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc finger nuclease (ZFN) mediated genome editing. First, using ZFNs specific for the OCT4 locus we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Secondly, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here, we engineered Transcription Activation-Like Effector Nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained hESC and iPSC single-cell-derived clones carrying transgenic cassettes solely at the TALEN-specified location. Thus, TALENs mediate site-specific genome modifications in human pluripotent cells with comparable efficiency and precision as zinc finger nucleases (ZFNs).
SUMMARY Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson’s disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
Summary Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable raising the question whether these enzymes have overlapping roles in development. Here, we have generated Tet1 and Tet2 double knockout (DKO) ESCs and mice. DKO ESCs remained pluripotent, but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double mutant embryos exhibited mid-gestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2 deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. It also suggests a significant contribution of Tet3 to hydroxylation of 5mC during development.
Summary The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development we have generated Tet1 mutant mESCs and mice. Tet1−/− ESCs have reduced levels of 5hmC, subtle changes in global gene expression, are pluripotent and support development of live-born mice in tetraploid complementation assay but display skewed differentiation towards trophectoderm in vitro. Tet1 mutant mice are viable, fertile and grossly normal though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.
SUMMARY Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell-replacement therapies. However one crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age-onset disorders where in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this problem by generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson’s disease by modifying the underlying point mutations (A53T/E46K) in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell-replacement therapies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.