Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells.
Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics, and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems, including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence, and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, nonintegrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, RNA virion, RNA replicon, nonintegrating replicating episomal plasmids, minicircles, polycistron, and preintegration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
Childhood trauma and post-childhood chronic/repeated stress could increase the risk of a substance use disorder by affecting five stages of addiction illness-course: (a) initial experimentation with substances; (b) shifting from experimental to regular use; (c) escalation from regular use to abuse or dependence; (d) motivation to quit; and (e) risk of (re-)lapse. We reviewed the human literature on relationships between stress and addiction illness-course. We explored per illness-course stage: (i) whether childhood trauma and post-childhood chronic/repeated stress have comparable effects and (ii) whether effects cut across classes of substances of abuse. We further discuss potential underlying mechanisms by which stressors may affect illness-course stages for which we relied on evidence from studies in animals and humans. Stress and substances of abuse both activate stress and dopaminergic motivation systems, and childhood trauma and post-childhood stressful events are more chronic and occur more frequently in people who use substances. Stressors increase risk to initiate early use potentially by affecting trait-like factors of risk-taking, decision making, and behavioral control. Stressors also accelerate transition to regular use potentially due to prior effects of stress on sensitization of dopaminergic motivation systems, cross-sensitizing with substances of abuse, especially in people with high trait impulsivity who are more prone to sensitization. Finally, stressors increase risk for abuse and dependence, attenuate motivation to quit, and increase relapse risk potentially by intensified sensitization of motivational systems, by a shift from positive to negative reinforcement due to sensitization of the amygdala by corticotropin releasing factor, and by increased sensitization of noradrenergic systems. Stress generally affects addiction illness-course across stressor types and across classes of substances of abuse.
Several theoretical frameworks have suggested that anxiety/stress impairs cognitive performance. A competing prediction is made by attentional narrowing models that predict that stress decreases the processing of task-irrelevant items, thus benefiting performance when task-irrelevant information interferes with behavior. Critically, previous studies have not evaluated these competing frameworks when potent emotional manipulations are involved. Here, we used threat of bodily harm preceding a color-word Stroop task to test these claims. We found a basic effect of threat consisting of a slowing down of performance during neutral Stroop trials. Furthermore, both facilitation and interference scores were affected by threat of shock in a way that was consistent with a reduced-distractor effect. Taken together, we interpret our findings in terms of two opposing effects of stress on cognitive performance. Although partly consistent with the attentional narrowing hypothesis, both resource models and cognitive breadth models require revision in order to account for the results.
Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10-30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols.
Striated muscle development in vertebrates requires the redundant functions of multiple members of the MyoD family. Invertebrates such as Drosophila and Caenorhabditis elegans contain only one MyoD homolog in each organism. Earlier observations suggest that factors outside of the MyoD family might function redundantly with MyoD in striated muscle fate specification in these organisms. However, the identity of these factors has remained elusive. Here, we describe the identification and characterization of FOZI-1, a putative transcription factor that functions redundantly with CeMyoD (HLH-1) in striated body wall muscle (BWM) fate specification in the C. elegans postembryonic mesoderm. fozi-1 encodes a novel nuclear-localized protein with motifs characteristic of both transcription factors and actin-binding proteins. We show that FOZI-1 shares the same expression pattern as CeMyoD in the postembryonic mesodermal lineage, the M lineage, and that fozi-1-null mutants exhibit similar M lineage-null defects to those found in animals lacking CeMyoD in the M lineage (e.g. loss of a fraction of M lineage-derived BWMs). Interestingly, fozi-1-null mutants with a reduced level of CeMyoD lack most, if not all, M lineage-derived BWMs. Our results indicate that FOZI-1 and the Hox factor MAB-5 function redundantly with CeMyoD in the specification of the striated BWM fate in the C. elegans postembryonic mesoderm, implicating a remarkable level of complexity for the production of a simple striated musculature in C. elegans.
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.