A Cut above the Rest: Targeted Genome Editing Technologies in Human Pluripotent Stem Cells

Li, Mo; Suzuki, Keiichiro; Kim, Na Young; Liu, Guanghui; Belmonte, Juan Carlos Izpisúa

doi:10.1074/jbc.r113.488247

Cited by 115 publications

(100 citation statements)

References 62 publications

(69 reference statements)

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“…Various genome editing tools such as zinc fingers, TALENs, and CRISPR nucleases [42] have been developed to genetically engineer and correct genetic defects in patient-derived iPS cells. The gene transfer may correct disease-causing point mutations [43] or rescue the phenotype by providing a deficient gene into the safe harbor AAVS1 locus [44].…”

Section: Live Screening Of Successfully Reprogrammed Murine Ttfs To Imentioning

confidence: 99%

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Lahm

Doppler²,

Dreßen³

et al. 2015

Stem Cells

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The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells. STEM CELLS 2015;33:392-402

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Section: Live Screening Of Successfully Reprogrammed Murine Ttfs To Imentioning

confidence: 99%

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Lahm

Doppler²,

Dreßen³

et al. 2015

Stem Cells

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“…Another point on the list of advantages for pluripotent stem cells is the ability to genetically engineer their genome in an increasingly efficient manner [81]. This is of course based on the great expandability of iPS and ES cells as discussed above.…”

Section: Direct Conversion Of Somatic Cells Versus Induction Of Plurimentioning

confidence: 99%

Direct somatic lineage conversion

Tanabe¹,

Haag²,

Wernig³

2015

Phil. Trans. R. Soc. B

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The predominant view of embryonic development and cell differentiation has been that rigid and even irreversible epigenetic marks are laid down along the path of cell specialization ensuring the proper silencing of unrelated lineage programmes. This model made the prediction that specialized cell types are stable and cannot be redirected into other lineages. Accordingly, early attempts to change the identity of somatic cells had little success and was limited to conversions between closely related cell types. Nuclear transplantation experiments demonstrated, however, that specialized cells even from adult mammals can be reprogrammed into a totipotent state. The discovery that a small combination of transcription factors can reprogramme cells to pluripotency without the need of oocytes further supported the view that these epigenetic barriers can be overcome much easier than assumed, but the extent of this flexibility was still unclear. When we showed that a differentiated mesodermal cell can be directly converted to a differentiated ectodermal cell without a pluripotent intermediate, it was suggested that in principle any cell type could be converted into any other cell type. Indeed, the work of several groups in recent years has provided many more examples of direct somatic lineage conversions. Today, the question is not anymore whether a specific cell type can be generated by direct reprogramming but how it can be induced.

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“…There are numerous methods for genomic engineering, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). Interested readers are referred to this review on genome-editing technologies [66]. Figure 3 illustrates genome engineering of iPSCs using some of these methods where these engineered lines can either be differentiated into various cell types for research purposes or propagated and banked for future experiments.…”

Section: Figmentioning

confidence: 99%

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

Hunsberger

Efthymiou

Malik

et al. 2015

Stem Cells and Development

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There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.Disease Modeling D iseases of the central nervous system (CNS) affect a large number of people, but therapeutic intervention is hampered by the lack of useful models for many of these diseases. Current research on human subjects, particularly for drug discovery for CNS diseases, is severely (and appropriately) limited by ethical guidelines. Therefore, surrogate models are needed that share important anatomical, physiological, and genetic features to advance new treatments and therapies for CNS diseases [1].Developing rapid and effective therapies for CNS diseases requires the availability of in vitro models that accurately recapitulate disease phenotypes and predict patient treatment response. A proper model must be both sensitive and predictive while reflecting both normal and disease processes. Equally important, these models should enable the investigation of genetic and environmental risk factors contributing to diseases in a rapid and economical way. Currently used models often do not reflect a typical human response [2-4], despite efforts underway to better characterize these models and increase their preclinical value in predicting safety and efficacy in the clinic [5,6]. Therefore, there is a great need to develop disease-and patient-specific models from cells directly affected in CNS disorders. These cellbased models, we envision, could either replace or supplement current animal models and enable the efficient translation of basic research into the clinical setting. Limitations with current CNS modelsCurrently, drug discovery relies on the use of animalbased or cell-based models, ...

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A Cut above the Rest: Targeted Genome Editing Technologies in Human Pluripotent Stem Cells

Cited by 115 publications

References 62 publications

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Direct somatic lineage conversion

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

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