scholarly journals Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells

Cell Reports ◽  
2017 ◽  
Vol 21 (2) ◽  
pp. 308-315 ◽  
Author(s):  
Masahito Yoshihara ◽  
Ryoko Araki ◽  
Yasuji Kasama ◽  
Misato Sunayama ◽  
Masumi Abe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Point Mutations ◽  
Induced Pluripotent

scholarly journals Insights into the mutational burden of human induced pluripotent stem cells using an integrative omics approach

2018 ◽  
Author(s):  
Matteo D’Antonio ◽  
Paola Benaglio ◽  
David Jakubosky ◽  
William W. Greenwald ◽  
Hiroko Matsui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Point Mutations ◽  
Mutational Burden ◽  
Altered Gene ◽  
Induced Pluripotent ◽  
Transplantation Therapy ◽  
Integrative Omics

SummaryTo understand the mutational burden of human induced pluripotent stem cells (iPSCs), we whole genome sequenced 18 fibroblast-derived iPSC lines and identified different classes of somatic mutations based on structure, origin and frequency. Copy number alterations affected 295 kb in each sample and strongly impacted gene expression. UV-damage mutations were present in ~45% of the iPSCs and accounted for most of the observed heterogeneity in mutation rates across lines. Subclonal mutations (not present in all iPSCs within a line) composed 10% of point mutations, and compared with clonal variants, showed an enrichment in active promoters and increased association with altered gene expression. Our study shows that, by combining WGS, transcriptome and epigenome data, we can understand the mutational burden of each iPSC line on an individual basis and suggests that this information could be used to prioritize iPSC lines for models of specific human diseases and/or transplantation therapy.

scholarly journals Highly Efficient Knockin in Human iPS Cells and Rat Embryos by CRISPR/Cas9 Molecular Optimization

2021 ◽  
Author(s):  
Vanessa Chenouard ◽  
Isabelle Leray ◽  
Laurent Tesson ◽  
Severine Remy ◽  
Agnes Fortun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Point Mutations ◽  
Molar Ratio ◽  
Rational Approach ◽  
Rat Embryos ◽  
Guide Rna ◽  
Induced Pluripotent ◽  
Transgenic Embryos

The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2µM and 0.4µM of Cas9, coupled with 2µM of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

scholarly journals Effects of Ceramide Accumulation in Cardiomyocytes Derived from human-induced Pluripotent Stem Cells on Mitochondrial Function and Mitophagy

2020 ◽  
Author(s):  
Mohamed Bekhite ◽  
Andres González-Delgado ◽  
Tom Kretzschma ◽  
Tina Müller ◽  
Jasmine M.F. Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mitochondrial Function ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Mitochondrial Fission ◽  
Serine Palmitoyltransferase ◽  
Induced Pluripotent ◽  
Ceramide Accumulation ◽  
Lipotoxic Cardiomyopathy

Abstract BackgroundOversupply of fatty acids (FAs) to cardiomyocytes (CMs) is associated with increased ceramide content and elevated the risk of lipotoxic cardiomyopathy. Here we investigate the role of ceramide accumulation on mitochondrial function and mitophagy in cardiac lipotoxicity using CMs derived from human-induced pluripotent stem cells (h-iPSCs). Methods and resultsMature CMs derived from h-iPSCs exposed to the diabetic-like environment or transfected with plasmids overexpressing serine-palmitoyltransferase long chain base subunit 1 (SPTLC1), a subunit of the serine-palmitoyltransferase (SPT) complex, resulted in increased intracellular ceramide levels. Accumulation of ceramides impaired insulin-dependent phosphorylation of Akt through activating protein phosphatase 2A (PP2A) and disturbed gene and protein levels of key metabolic enzymes including GLUT4, AMPK, PGC-1α, PPARα, CD36, PDK4, and PPARγ compared to controls. Analysis of CMs oxidative metabolism using a Seahorse analyzer showed a significant reduction in ATP synthesis-related O2 consumption, mitochondrial basal respiration and respiratory capacity, indicating an impaired mitochondrial function under diabetic-like conditions or SPTLC1-overexpression. Further, ceramide accumulation increased mitochondrial fission regulators such as dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) as well as auto/mitophagic proteins LC3B and PINK-1 compared to control. Incubation of CMs with the specific SPT inhibitor (myriocin) showed a significant increase in mitochondrial fusion regulators the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) as well as p-Akt, PGC-1 α, GLUT-4, and ATP production. In addition, a significant decrease in auto/mitophagy and apoptosis was found in CMs treated with myriocin.ConclusionsOur results suggest that ceramide accumulation has important implications in driving insulin resistance, oxidative stress, increased auto/mitophagy, and mitochondrial dysfunction in the setting of lipotoxic cardiomyopathy. Therefore, modulation of the de novo ceramide synthesis pathway may serve as a novel therapeutic target to treat metabolic cardiomyopathy.

scholarly journals Precision genome-editing with CRISPR/Cas9 in human induced pluripotent stem cells

2017 ◽  
Author(s):  
John P. Budde ◽  
Rita Martinez ◽  
Simon Hsu ◽  
Natalie Wen ◽  
Jason A. Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Genetic Variants ◽  
De Novo ◽  
Mutational Burden ◽  
Target Sites ◽  
Allele Specific ◽  
Induced Pluripotent

AbstractGenome engineering in human induced pluripotent stem cells (iPSCs) represent an opportunity to examine the contribution of pathogenic and disease modifying alleles to molecular and cellular phenotypes. However, the practical application of genome-editing approaches in human iPSCs has been challenging. We have developed a precise and efficient genome-editing platform that relies on allele-specific guideRNAs (gRNAs) paired with a robust method for culturing and screening the modified iPSC clones. By applying an allele-specific gRNA design strategy, we have demonstrated greatly improved editing efficiency without the introduction of additional modifications of unknown consequence in the genome. Using this approach, we have modified nine independent iPSC lines at five loci associated with neurodegeneration. This genome-editing platform allows for efficient and precise production of isogenic cell lines for disease modeling. Because the impact of CRISPR/Cas9 on off-target sites remains poorly understood, we went on to perform thorough off-target profiling by comparing the mutational burden in edited iPSC lines using whole genome sequencing. The bioinformatically predicted off-target sites were unmodified in all edited iPSC lines. We also found that the numbers of de novo genetic variants detected in the edited and unedited iPSC lines were similar. Thus, our CRISPR/Cas9 strategy does not specifically increase the mutational burden. Furthermore, our analyses of the de novo genetic variants that occur during iPSC culture and genome-editing indicate an enrichment of de novo variants at sites identified in dbSNP. Taken together, we propose that this enrichment represents regions of the genome more susceptible to mutation. Herein, we present an efficient and precise method for allele-specific genome-editing in iPSC and an analyses pipeline to distinguish off-target events from de novo mutations occurring with culture.

scholarly journals Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Sabine Klawitter ◽  
Nina V. Fuchs ◽  
Kyle R. Upton ◽  
Martin Muñoz-Lopez ◽  
Ruchi Shukla ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Embryonic Stem ◽  
Protein Coding ◽  
Induced Pluripotent ◽  
Hesc Lines

Abstract Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

scholarly journals De novo generation of a functional human thymus from induced pluripotent stem cells

2019 ◽  
Vol 144 (5) ◽  
pp. 1416-1419.e7 ◽  
Author(s):  
Amiet R. Chhatta ◽  
Martijn Cordes ◽  
Maaike A.J. Hanegraaf ◽  
Sandra Vloemans ◽  
Tom Cupedo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Human Thymus ◽  
Induced Pluripotent

scholarly journals Mesenchymal and Induced Pluripotent Stem Cells-Derived Extracellular Vesicles: The New Frontier for Regenerative Medicine?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1163 ◽  
Author(s):  
Maria Magdalena Barreca ◽  
Patrizia Cancemi ◽  
Fabiana Geraci
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Cell Communication ◽  
Self Healing ◽  
Induced Pluripotent

Regenerative medicine aims to repair damaged, tissues or organs for the treatment of various diseases, which have been poorly managed with conventional drugs and medical procedures. To date, multimodal regenerative methods include transplant of healthy organs, tissues, or cells, body stimulation to activate a self-healing response in damaged tissues, as well as the combined use of cells and bio-degradable scaffold to obtain functional tissues. Certainly, stem cells are promising tools in regenerative medicine due to their ability to induce de novo tissue formation and/or promote organ repair and regeneration. Currently, several studies have shown that the beneficial stem cell effects, especially for mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) in damaged tissue restore are not dependent on their engraftment and differentiation on the injury site, but rather to their paracrine activity. It is now well known that paracrine action of stem cells is due to their ability to release extracellular vesicles (EVs). EVs play a fundamental role in cell-to-cell communication and are directly involved in tissue regeneration. In the present review, we tried to summarize the molecular mechanisms through which MSCs and iPSCs-derived EVs carry out their therapeutic action and their possible application for the treatment of several diseases.

Directed differentiation of mouse-induced pluripotent stem cells generates dopaminergic nerve

2010 ◽  
Vol 34 (8) ◽  
pp. S36-S36
Author(s):  
Ping Duan ◽  
Xuelin Ren ◽  
Wenhai Yan ◽  
Xuefei Han ◽  
Xu Yan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Induced Pluripotent
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