Hyaluronic acid-based hydrogels with tunable mechanics improved structural and contractile properties of cells.

Extracellular matrix (ECM) regulates cellular responses through mechanotransduction. The standard approach of in vitro culturing on plastic surfaces overlooks this phenomenon, so there is a need for biocompatible materials that exhibit adjustable mechanical and structural properties, promote cell adhesion and proliferation at low cost and for use in 2D or 3D cell cultures. This study presents a new tunable hydrogel system prepared from high-molecular hyaluronic acid (HA), Bovine serum albumin (BSA), and gelatin cross-linked using EDC/NHS. Hydrogels with Young's moduli (E) ranging from subunit to units of kilopascals were prepared by gradually increasing HA and BSA concentrations. Concentrated high-molecular HA network led to stiffer hydrogel with lower cluster size and swelling capacity. Medium and oxygen diffusion capability of all hydrogels showed they are suitable for 3D cell cultures. Mechanical and structural changes of mouse embryonic fibroblasts (MEFs) on hydrogels were compared with cells on standard cultivation surfaces. Experiments showed that hydrogels have suitable mechanical and cell adhesion capabilities, resulting in structural changes of actin filaments. Lastly, applying hydrogel for a more complex HL-1 cell line revealed improved mechanical and electrophysiological contractile properties.

Computer-aided engineering of stabilized fibroblast growth factor 21.

FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

α1-Adrenoceptor agonist methoxamine inhibits base excision repair via inhibition of apurinic/apyrimidinic endonuclease 1 (APE1).

Methoxamine (Mox) is a well-known α1-adrenoceptor agonist, clinically used as a longer-acting analogue of epinephrine. 1R,2S-Mox (NRL001) has been also undergoing clinical testing to increase the canal resting pressure in patients with bowel incontinence. Here we show, that Mox hydrochloride acts as an inhibitor of base excision repair (BER). The effect is mediated by the inhibition of apurinic/apyrimidinic endonuclease APE1. We link this observation to our previous report showing the biologically relevant effect of Mox on BER - prevention of converting oxidative DNA base damage to double-stranded breaks. We demonstrate that its effect is weaker, but still significant when compared to a known BER inhibitor methoxyamine (MX). We further determined Mox's relative IC 50 at 19 mmol L-1, demonstrating a significant effect of Mox on APE1 activity in clinically relevant concentrations.

hESC derived cardiomyocyte biosensor to detect the different types of arrhythmogenic properties of drugs.

In the present work, we introduce a new cell-based biosensor for detecting arrhythmias based on a novel utilization of the combination of the Atomic Force Microscope (AFM) lateral force measurement as a nanosensor with a dual 3D cardiomyocyte syncytium. Two spontaneously coupled clusters of cardiomyocytes form this. The syncytium's functional contraction behavior was assessed using video sequences analyzed with Musclemotion ImageJ/Fiji software, and immunocytochemistry evaluated phenotype composition. The application of caffeine solution induced arrhythmia as a model drug, and its spontaneous resolution was monitored by AFM lateral force recording and interpretation and calcium fluorescence imaging as a reference method describing non-synchronized contractions of cardiomyocytes. The phenotypic analysis revealed the syncytium as a functional contractile and conduction cardiac behavior model. Calcium fluorescence imaging was used to validate that AFM fully enabled to discriminate cardiac arrhythmias in this in vitro cellular model. The described novel 3D hESCs-based cellular biosensor is suitable to detect arrhythmic events on the level of cardiac contractile and conduction tissue cellular model. The resulting biosensor allows for screening of arrhythmogenic properties of tailored drugs enabling its use in precision medicine.

Atomic force spectroscopy is a promising tool to study contractile properties of cardiac cells.

Atomic Force Microscopy (AFM) is a rather new method with increasing potential in analyzing various biosamples. Moreover, it can serve as a multi-functional device in the studies of biological specimens under physiological conditions. However, it is becoming increasingly popular among biochemists and biologists, it is not often used in cardiology. Heart disease causes millions of deaths every year. A common point in all heart diseases is the inferior function of cardiomyocytes, which are the contracting unit of the heart. Therefore, these cells are a frequent target of scientific studies. However, few of them use innovative techniques such as AFM and related methods or parallel combinations with complementary techniques such as cell potential measurements. The aim of this review is to illustrate the potential of AFM microscopy in the study of cardiac cells, comparing it with related methods and other techniques used to study the biomechanics and electrophysiology of this cell type. A better understanding of these methods may lead to a better description of the pathophysiology of the heart disease and an improved understanding of the effect of selected drugs.

Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Cellular pathology of the human heart in Duchenne muscular dystrophy (DMD): lessons learned from in vitro modeling.

Duchenne muscular dystrophy is a genetic disorder where an X-linked mutation in the DMD gene initiates pathogenic development caused by the absence of dystrophin protein. This impacts primarily the evolution of a functional muscle tissue resulting in muscle weakness and later severe disability in young male patients leading to an early death. Patients in the final stage develop dilated cardiomyopathy leading ultimately to cardiac or respiratory failure as the cause of death. This review discusses recent advances in modeling the DMD pathology in vitro. It describes in detail the molecular abnormalities found on the cellular and organoid levels. The in vitro pathology is compared to that found in patients. Likewise, the drawbacks and limitations of current models are discussed.

Evidence for discrete modes of YAP1 signaling via mRNA splice isoforms in development and diseases.

Yes-associated protein 1 (YAP1) is a transcriptional co-activator downstream of Hippo pathway. The pathway exerts crucial roles in organogenesis and its dysregulation is associated with the spreading of different cancer types. YAP1 gene encodes for multiple protein isoforms, whose specific functions are not well defined. We demonstrate the splicing of isoform-specific mRNAs is controlled in a stage- and tissue-specific fashion. We designed expression vectors encoding for the most-represented isoforms of YAP1 with either one or two WW domains and studied their specific signaling activities in YAP1 knock-out cell lines. YAP1 isoforms display both common and unique functions and activate distinct transcriptional programs, as the result of their unique protein interactomes. By generating TEAD-based transcriptional reporter cell lines, we demonstrate individual YAP1 isoforms display unique effects on cell proliferation and differentiation. Finally, we illustrate the complexity of the regulation of Hippo-YAP1 effector in physiological and in pathological conditions of the heart.

Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids.

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiac side effects of some pulmonary drugs are observed in clinical practice. Aminophylline, a methylxanthine bronchodilator with documented proarrhythmic action, may serve as an example. Data on the action of aminophylline on cardiac cell electrophysiology and contractility are not available. Hence, this study was focused on the analysis of changes in the beat rate and contraction force of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and HL-1 cardiomyocytes in the presence of increasing concentrations of aminophylline (10 µM-10 mM in hPSC-CM and 8-512 µM in HL-1 cardiomyocytes). Basic biomedical parameters, namely, the beat rate (BR) and contraction force, were assessed in hPSC-CMs using an atomic force microscope (AFM). The beat rate changes under aminophylline were also examined on the HL-1 cardiac muscle cell line via a multielectrode array (MEA). Additionally, calcium imaging was used to evaluate the effect of aminophylline on intracellular Ca2+ dynamics in HL-1 cardiomyocytes. The BR was significantly increased after the application of aminophylline both in hPSC-CMs (with 10 mM aminophylline) and in HL-1 cardiomyocytes (with 256 and 512 µM aminophylline) in comparison with controls. A significant increase in the contraction force was also observed in hPSC-CMs with 10 µM aminophylline (a similar trend was visible at higher concentrations as well). We demonstrated that all aminophylline concentrations significantly increased the frequency of rhythm irregularities (extreme interbeat intervals) both in hPSC-CMs and HL-1 cells. The occurrence of the calcium sparks in HL-1 cardiomyocytes was significantly increased with the presence of 512 µM aminophylline. We conclude that the observed aberrant cardiomyocyte response to aminophylline suggests an arrhythmogenic potential of the drug. The acquired data represent a missing link between the arrhythmic events related to the aminophylline/theophylline treatment in clinical practice and describe cellular mechanisms of methylxanthine arrhythmogenesis. An AFM combined with hPSC-CMs may serve as a robust platform for direct drug effect screening.

AC Pulsed Field Ablation Is Feasible and Safe in Atrial and Ventricular Settings: A Proof-of-Concept Chronic Animal Study.

INTRODUCTION: Pulsed field ablation (PFA) exploits the delivery of short high-voltage shocks to induce cells death via irreversible electroporation. The therapy offers a potential paradigm shift for catheter ablation of cardiac arrhythmia. We designed an AC-burst generator and therapeutic strategy, based on the existing knowledge between efficacy and safety among different pulses. We performed a proof-of-concept chronic animal trial to test the feasibility and safety of our method and technology. METHODS: We employed 6 female swine - weight 53.75 ± 4.77 kg - in this study. With fluoroscopic and electroanatomical mapping assistance, we performed ECG-gated AC-PFA in the following settings: in the left atrium with a decapolar loop catheter with electrodes connected in bipolar fashion; across the interventricular septum applying energy between the distal electrodes of two tip catheters. After procedure and 4-week follow-up, the animals were euthanized, and the hearts were inspected for tissue changes and characterized. We perform finite element method simulation of our AC-PFA scenarios to corroborate our method and better interpret our findings. RESULTS: We applied square, 50% duty cycle, AC bursts of 100 µs duration, 100 kHz internal frequency, 900 V for 60 pulses in the atrium and 1500 V for 120 pulses in the septum. The inter-burst interval was determined by the native heart rhythm - 69 ± 9 bpm. Acute changes in the atrial and ventricular electrograms were immediately visible at the sites of AC-PFA - signals were elongated and reduced in amplitude (p < 0.0001) and tissue impedance dropped (p = 0.011). No adverse event (e.g., esophageal temperature rises or gas bubble streams) was observed - while twitching was avoided by addition of electrosurgical return electrodes. The implemented numerical simulations confirmed the non-thermal nature of our AC-PFA and provided specific information on the estimated treated area and need of pulse trains. The postmortem chest inspection showed no peripheral damage, but epicardial and endocardial discolorations at sites of ablation. T1-weighted scans revealed specific tissue changes in atria and ventricles, confirmed to be fibrotic scars via trichrome staining. We found isolated, transmural and continuous scars. A surviving cardiomyocyte core was visible in basal ventricular lesions. CONCLUSION: We proved that our method and technology of AC-PFA is feasible and safe for atrial and ventricular myocardial ablation, supporting their systematic investigation into effectiveness evaluation for the treatment of cardiac arrhythmia. Further optimization, with energy titration or longer follow-up, is required for a robust atrial and ventricular AC-PFA.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells.

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

DMD Pluripotent Stem Cell Derived Cardiac Cells Recapitulate in vitro Human Cardiac Pathophysiology.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted ß-adrenergic response connected with remodeling of ß-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies in vitro.

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

Cardiovascular progenitor cells and tissue plasticity are reduced in a myocardium affected by Becker muscular dystrophy.

We describe the association of Becker muscular dystrophy (BMD) derived heart failure with the impairment of tissue homeostasis and remodeling capabilities of the affected heart tissue. We report that BMD heart failure is associated with a significantly decreased number of cardiovascular progenitor cells, reduced cardiac fibroblast migration, and ex vivo survival. BACKGROUND: Becker muscular dystrophy belongs to a class of genetically inherited dystrophin deficiencies. It affects male patients and results in progressive skeletal muscle degeneration and dilated cardiomyopathy leading to heart failure. It is a relatively mild form of dystrophin deficiency, which allows patients to be on a heart transplant list. In this unique situation, the explanted heart is a rare opportunity to study the degenerative process of dystrophin-deficient cardiac tissue. Heart tissue was excised, dissociated, and analyzed. The fractional content of c-kit+/CD45- cardiovascular progenitor cells (CVPCs) and cardiac fibroblast migration were compared to control samples of atrial tissue. Control tissue was obtained from the hearts of healthy organ donor's during heart transplantation procedures. RESULTS: We report significantly decreased CVPCs (c-kit+/CD45-) throughout the heart tissue of a BMD patient, and reduced numbers of phase-bright cells presenting c-kit positivity in the dystrophin-deficient cultured explants. In addition, ex vivo CVPCs survival and cardiac fibroblasts migration were significantly reduced, suggesting reduced homeostatic support and irreversible tissue remodeling. CONCLUSIONS: Our findings associate genetically derived heart failure in a dystrophin-deficient patient with decreased c-kit+/CD45- CVPCs and their resilience, possibly hinting at a lack of cardioprotective capability and/or reduced homeostatic support. This also correlates with reduced plasticity of the explanted cardiac tissue, related to the process of irreversible remodeling in the BMD patient's heart.

Both Hypoxia-Inducible Factor 1 and MAPK Signaling Pathway Attenuate PI3K/AKT via Suppression of Reactive Oxygen Species in Human Pluripotent Stem Cells.

Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.

Generation of two Duchenne muscular dystrophy patient-specific induced pluripotent stem cell lines DMD02 and DMD03 (MUNIi001-A and MUNIi003-A).

Duchenne muscular dystrophy (DMD) affects 1:3500-5000 newborn boys and manifests with progressive skeletal muscle wasting, respiratory failure and eventual heart failure. Symptoms show different onset from patients' childhood to the second decade of age. We reprogrammed fibroblasts from two independent DMD patients with a complete loss of dystrophin expression, carrying deletions of exons 45-50 and 48-50. The resulting hiPSCs show expression of pluripotency markers (NANOG, OCT4, SSEA4), differentiation capacity into all three germ layers, normal karyotype, genetic identity to the originating parental fibroblasts and the patient-specific dystrophin mutation.

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.