Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

• Keywords
• DMD, NO synthases, ROS, dystrophin, genome stability, pluripotent stem cells,
• MeSH
• Cell Line MeSH
• Muscular Dystrophy, Duchenne genetics   MeSH
• Dystrophin deficiency   genetics   MeSH
• Induced Pluripotent Stem Cells metabolism   pathology   MeSH
• Humans MeSH
• Genomic Instability * MeSH
• Oxidative Stress MeSH
• Reactive Oxygen Species metabolism   MeSH
• Nitric Oxide Synthase Type I metabolism   MeSH
• Nitric Oxide Synthase Type II metabolism   MeSH
• Nitric Oxide Synthase Type III metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dystrophin MeSH
• NOS1 protein, human MeSH Browser
• NOS2 protein, human MeSH Browser
• NOS3 protein, human MeSH Browser
• Reactive Oxygen Species MeSH
• Nitric Oxide Synthase Type I MeSH
• Nitric Oxide Synthase Type II MeSH
• Nitric Oxide Synthase Type III MeSH

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.

• Keywords
• CRISPR/Cas9, gene engineering, human embryonic stem cells, knockout,
• MeSH
• Cell Line MeSH
• CRISPR-Cas Systems * MeSH
• Down-Regulation MeSH
• Embryonic Stem Cells cytology   metabolism   MeSH
• Gene Knockout Techniques methods   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• MicroRNAs genetics   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MicroRNAs MeSH
• MIRN34 microRNA, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.

• Keywords
• Cell cycle, Chromatin, DNA replication, H3K56ac, Mammalian cells, Nucleosome,
• MeSH
• Cell Cycle genetics   physiology   MeSH
• Chromatin metabolism   MeSH
• G2 Phase genetics   MeSH
• Histones metabolism   MeSH
• HL-60 Cells MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Nucleosomes metabolism   MeSH
• DNA Replication genetics   physiology   MeSH
• S Phase genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH
• Nucleosomes MeSH
• Saccharomyces cerevisiae Proteins MeSH

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

• MeSH
• Cell Differentiation radiation effects   MeSH
• Cell Line MeSH
• Genetic Loci * MeSH
• Hypoxanthine Phosphoribosyltransferase genetics   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Humans MeSH
• Mutation Rate * MeSH
• Gamma Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hypoxanthine Phosphoribosyltransferase MeSH

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD(+), NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD(+)/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD(+)/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD(+)/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.

• MeSH
• Cell Extracts MeSH
• Electrophoresis, Capillary MeSH
• Embryonic Stem Cells metabolism   MeSH
• Calibration MeSH
• Humans MeSH
• NAD metabolism   MeSH
• Nonlinear Dynamics * MeSH
• Oxazines metabolism   MeSH
• Regression Analysis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cell Extracts MeSH
• NAD MeSH
• Oxazines MeSH
• resorufin MeSH Browser