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Induced pluripotent stem (iPS) cells have a great impact on biology and medicine, and they are expected to improve regenerative medicine. 
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In this study, Jalil and colleagues develop and test a method for simultaneous hiPSC
BigField GEG Tech's insight:
Researchers at the University of Helsinki and Helsinki University Hospital have developed a method to accurately and rapidly correct genetic alterations in cultured patient cells. The method produces genetically corrected autologous pluripotent stem cells from a 2-3 mm skin biopsy of patients with different genetic diseases. The scientists based the new method on two Nobel Prize-winning techniques. The first technique is the invention of induced pluripotent stem cells from differentiated cells, which won the Nobel Prize in 2012. The other technique is the CRISPR-Cas9 innovation that won the prize in 2020. The new method combines these techniques to correct genetic alterations that cause inherited diseases and at the same time create new, fully functional stem cells. Their new system is much faster and more precise than older methods for correcting DNA errors, and the speed makes it easier and also decreases the risk of unwanted changes.
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Murine induced pluripotent stem cells (iPSCs) lacking IL-1 receptor type 1 (IL-1R1), engineered using the CRISPR/Cas9 system, are able to form cartilaginous tissue that is resistant to IL-1α-mediated inflammation, a new study shows.
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BigField GEG Tech's insight:
Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogrammed iPSCs lines may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. Here, the scientists present optimized RNA based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic and translational research.
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Here the authors show that CRISPR/Cas9 system can be utilized to completely remove the large repeat expansion mutation within C9orf72 in patient-derived induced pluripotent stem (iPS) cells.
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BigField GEG Tech's insight:
This review addresses this need directly by providing both the up-to-date biochemical rationale of CRISPR-mediated genome engineering and detailed practical guidelines for the design and execution of CRISPR experiments in cell models. Ultimately, this review will serve as a timely and comprehensive guide for this fast developing technology.
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BigField GEG Tech's insight:
In this study, the scientists used the CRISPR system to do gene correction in reprogrammed fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Therefore, their findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy.
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BigField GEG Tech's insight:
To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screening of cells to confirm that viral material has been completely eliminated during cell passaging.
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In this study, the authors report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. This cellular model allows to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches.
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In this review the authors provide a detailed overview of versatile primary iPSC generation from mouse somatic cells using PB transposons, and the subsequent establishment of robust secondary reprogramming systems. These protocols are highlighted with examples from recent studies as to how PB has been, and continues to be, conducive to the dissection of reprogramming processes at the cellular and molecular levels.
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In this chapter, the authors outline the features of a panel of “All-in-One” PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.
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The authors describe a method for the derivation of bovine iPSCs employing Sleeping Beauty and piggyBac transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter.
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The scientists used cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q, a somatic cytogenetic abnormality present in myelodysplastic syndromes. They used a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Their approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.
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Indiana University School of Medicine researchers have identified a new therapeutic target that could lead to more effective treatment of glaucoma.
BigField GEG Tech's insight:
Glaucoma is a neurodegenerative disease that causes vision loss and blindness due to a damaged optic nerve. Unfortunately, there is currently no cure. In a paper recently published in Communications Biology, researchers found that restoring mitochondrial homeostasis in diseased neurons can protect optic nerve cells from damage. The research team used induced pluripotent stem cells from glaucoma and non-glaucoma patients as well as clustered regularly spaced short palindromic repeats (CRISPRs) from human embryonic stem cells with glaucoma mutation. Using optic nerve stem cell differentiated retinal ganglion cells, electron microscopy and metabolic analysis, the researchers identified glaucomatous retinal ganglion cells with mitochondrial deficiency with a higher metabolic load on each mitochondrion, which leads to mitochondrial damage and degeneration. However, the process could be reversed by enhancing mitochondrial biogenesis with a pharmacological agent. The team showed that retinal ganglion cells are very efficient at degrading bad mitochondria, but at the same time produce more to maintain homeostasis.
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Cellular reprogramming by transient expression of Yamanaka factors ameliorates age-associated
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Here, the scientists report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging.
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This protocol extends the use of genome editing technology to the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions. The authors use the CRISPR/Cas system to edit human induced pluripotent stem cells.
BigField GEG Tech's insight:
In this study, the scientists describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.
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BigField GEG Tech's insight:
For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs is a very important issue. Customized engineered nucleases could provide excellent tools for targeted genome editing and opening new perspectives for biomedical research and cellular therapies.
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The authors discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of the genome editing technology in human PSCs using designer nucleases.
Joye Shuist's curator insight,
March 14, 2016 9:52 PM
The authors discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of the genome editing technology in human PSCs using designer nucleases.
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In this study, the authors used the CRISPR system to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.
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In this study, the authors used the CRISPR/Cas9 system to transform Leu33-positive megakaryocyte-like DAMI cells and induced pluripotent stem (iPS) cells to the Pro33 allotype.
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BigField GEG Tech's insight:
In this review, the authors speak about the status of transposon-based methods to induce pluripotency. The advantages of transposon-based gene transfer are their increased safety, their large cargo capacity, their relatively simple design, and the availability of hyper-active and mutated transposase enzymes. For example, integration-deficient, excision-competent transposase variants allow the complete removal of the reprogramming transposon after successful reprogramming to obtain transposon-free reprogrammed cells. Transposon-based reprogramming will broaden the toolbox for iPS cell production and will advance the establishment of safe, non-viral methods.
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In this study, the scientists demonstrate that hPSC-derived cholangiocytes possess epithelial functions, including rhodamine efflux and CFTR-mediated fluid secretion. Furthermore, they show that functionally impaired hPSC-derived cholangiocytes from cystic fibrosis patients are rescued by CFTR correctors.
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BigField GEG Tech's insight:
The scientists describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), they succeed to select and expand corrected human iPS cell line with a great efficiency, more than 2 % of the sequenced colonies (and about 15% to obtain KO by NHEJ).
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In this study, the authors introduce CRISPR and TALEN genome editing techniques, compare and contrast each technical approach and discuss their potential to study the underlying mechanisms of human disease using patient-derived induced pluripotent stem cells.
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In this study, the authors design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 to generate reporter systems while retaining pluripotent characteristics. They demonstrate the possibility to use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. The results provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.
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Fabry disease is caused by a genetic deficiency of α-galactosidase A (GLA), leading to the accumulation of its substrates such as globotriaosylceramide and globotriaosylsphingosine. Researchers have therefore developed a modified enzyme, modified α-N-acetylgalactosaminidase (mNAGA), to cure Fabry disease by changing the substrate specificity of NAGA to that of GLA. In this study, researchers tested whether genome-editing transplantation of mNAGA-secreting induced pluripotent stem cells (iPS) cells could deliver GLA activity in vivo. They therefore generated mNAGA-secreting iPS cells by TALEN-mediated knock-in at the AAVS1 site, a refuge locus. Furthermore, to exclude possible immunogenic reactions caused by endogenous GLA from iPS cells in patients, they disrupted the GLA gene by CRISPR-Cas9. When cardiomyocytes and fibroblasts from the Fabry model without GLA activity were co-cultured with mNAGA-secreting iPS cells, GLA activity was restored by mNAGA-expressing cells in vitro. Next, they transplanted the mNAGA-secreting iPS cells into the testes of mouse models of Fabry disease. After 7 or 8 weeks, GLA activity in the liver was significantly improved, although no recovery of activity was observed in the heart, kidneys or blood plasma.