Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of differentiation into any cell type of the organism (pluripotency) and indefinite self-renewal. Here, we show that the Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss- and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. We show that Rem2 mediates the fibroblastic growth factor 2 (FGF2) signaling pathway to maintain proliferation of hESCs. We demonstrate that Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D(1) to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, we also show that it is as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D(1) expression/localization and suppression of p53 transcription. We show that the effects of Rem2 on cyclin D(1) are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. Our studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use.

Linking the p53 tumour suppressor pathway to somatic cell reprogramming.

Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

Using targeted transgenic reporter mice to study promoter-specific p53 transcriptional activity.

The p53 transcription factor modulates gene expression programs that induce cell cycle arrest, senescence, or apoptosis, thereby preventing tumorigenesis. However, the mechanisms by which these fates are selected are unclear. Our objective is to understand p53 target gene selection and, thus, enable its optimal manipulation for cancer therapy. We have generated targeted transgenic reporter mice in which EGFP expression is driven by p53 transcriptional activity at a response element from either the p21 or Puma promoter, which induces cell cycle arrest/senescence and apoptosis, respectively. We demonstrate that we could monitor p53 activity in vitro and in vivo and detect variations in p53 activity depending on the response element, tissue type, and stimulus, thereby validating our reporter system and illustrating its utility for preclinical drug studies. Our results also show that the sequence of the p53 response element itself is sufficient to strongly influence p53 target gene selection. Finally, we use our reporter system to provide evidence for p53 transcriptional activity during early embryogenesis, showing that p53 is active as early as embryonic day 3.5 and that p53 activity becomes restricted to embryonic tissue by embryonic day 6.5. The data from this study demonstrate that these reporter mice could serve as powerful tools to answer questions related to basic biology of the p53 pathway, as well as cancer therapy and drug discovery.

Evidence-Based Implementation of the Family-Centered Model and the Use of Tele-Intervention in Early Childhood Services: A Systematic Review.

BACKGROUND: The purpose of this review is to explore the evidence and efficacy of two trends in early childhood intervention services: the family-centered model and the use of tele-intervention. METHODS: A systematic review was carried out following the PRISMA methodology and using three databases: Web of Science, PubMed and Scopus. The studies included were those aimed at children from 0 to 6 years of age, focused on early intervention, and which alluded to the family-centered model and/or tele-intervention. RESULTS: a total of 33 studies were included. Five main themes were identified: (1) The participation of children and family is facilitated and improved by the family-centered model of care; (2) the feeling of competence, self-efficacy, satisfaction and empowerment in professionals and families have a positive impact on quality of life; (3) the use of tele-intervention as a tool for prevention and intervention; (4) preparation for telepractice can improve the development of commitment; (5) tele-intervention as a possible solution to contextual barriers. CONCLUSIONS: Tele-intervention in pediatrics is presented as a tool inherent to the family-centered model since its implementation involves several common strategies. Future lines of research should explore the use of this tool as a possible solution to contextual barriers.

Assessing the clinical benefit, safety, and patient-reported outcomes with the use of the PAHcare™ digital platform in pulmonary arterial hypertension: a pilot study.

Introduction: Digital health interventions, particularly mobile health platforms, have shown promise in supporting patients with respiratory conditions, but their application in pulmonary arterial hypertension (PAH) remains limited. We aimed to assess the feasibility, acceptability, and potential clinical benefit of the novel PAHcare™ digital platform as a patient-centred intervention for PAH management through a prospective, single-arm, multicenter pilot study conducted on 53 patients diagnosed with PAH who used the platform for 6 months. Methods: The primary objective was to assess the impact on Health-Related Quality of Life (HRQoL) through questionnaires. Secondary objectives included evaluating clinical outcomes, including disease progression, PAH signs and symptoms, the 6-min walking test, and the patient's symptom perception. Additionally, we assessed patient satisfaction and engagement with the PAHcare™ platform, interaction with health coaches, retention, costs and healthcare resource utilisation (HCRU), and safety through monitoring device incidents. Results: Minimal changes in HRQoL and clinical outcomes were observed over 6 months. A noteworthy 92.4% of patients actively used the platform in the first month, maintaining high usage throughout the study. Patient satisfaction was substantial, with more than half of the patients expressing excellence in service quality, willingness to reuse the platform, and fulfilment of their needs. Health coach interaction was high, with 76% of patients initiating contact within the first week. User retention rates were 70%, with prevalent ongoing usage and interaction with healthcare professionals even after the study. In terms of HCRU and costs, the study showed no significant changes in PAH-related hospital admissions, clinical visits, or tests. Finally, the low number of device-related incidents indicated platform safety. Conclusion: This pilot study provides compelling evidence supporting the feasibility and acceptability of the PAHcare™ digital platform to empower patients to manage their disease and significantly enhance their overall experience with PAH.

Evaluation of a digital health system (PAHcare™) for routine care of patients with pulmonary arterial hypertension: The CBS-PAH study protocol.

Introduction: Pulmonary arterial hypertension (PAH) is a rare, multifactorial, chronic condition that requires ongoing monitoring and assessment. PAHcare™ is a novel, patient-centered digital platform that provides software intended for use on patients' mobile phones (downloadable application) and web-based dashboards for use by physicians and health coaches (HC). We describe herein the protocol of a clinical study aimed at evaluating the clinical benefit and safety of PAHcare™ for the routine management of patients with PAH. Methods and analysis: In this prospective, single cohort, multicenter study, 50 patients with PAH will be recruited at six specialized PAH units from reference hospitals of the public Spanish healthcare system. The PAHcare™ digital health platform allows patients to log health and lifestyle information while also providing structured content for patient education, medication reminders, and behavioral and lifestyle coaching from a remote HC. Evaluation will be primarily focused on the impact of the platform use on the patient's health-related quality of life (HRQoL) via questionnaires completion through electronic patient-reported outcomes. Moreover, the analysis of the impact on the patient's functional status, signs and symptoms of PAH, patient costs and healthcare resource utilization, satisfaction, knowledge of the disease and its management, and adherence to and safety of the platform will be secondary outcomes. The clinical investigation started in July 2021 and is expected to end by September 2022. Discussion: The PAHcare™ platform is anticipated to provide direct benefits to healthcare professionals, patients, and caregivers. These include the simplification of the multidisciplinary approach needed to tailor routine PAH management, enhancement of the patient/healthcare professional interaction, patient's empowerment to become more actively involved in the management and treatment of the disease, and increase of the patient's and caregiver's knowledge on PAH.

Psychosocial impact of mobility assistive technology on people with neurological conditions.

INTRODUCTION: A wide range of products are available to assist mobility, and it is, therefore, of great importance to obtain empirical information regarding the expected impact of the use of these products based on outcome measures. People affected by neurological disorders often use products to assist mobility such as wheelchairs (both manual self-propelled wheelchairs and externally propelled chairs such as electric wheelchairs), walkers, walking sticks, etc. It is important to conduct an assessment of the psychosocial impact of these products on the lives of affected people. METHODS: We performed this assessment using the Psychosocial Impact of Assistive Devices Scale (PIADS) and a socio-demographic questionnaire. RESULTS: The results showed greater psychosocial benefits relating to the use of electric wheelchairs in comparison with walking sticks or manual, non-self-propelled chairs. Moreover, significant differences are present in the three subscales of the PIADS in relation to variables such as age, training in the use of assistive technology (AT) and funding. CONCLUSION: Therefore, we conclude that the use of AT should be promoted among this group as a way to improve their adaptability, competency and self-esteem, and to reduce limits on participation deriving from the physical and contextual barriers faced by this collective.Implications for rehabilitationAmong the assessment of different mobility AT displaying a higher score in the three subscales of the PIADS amongst people using electric wheelchairs than among those using non-self-propelled manual wheelchairs. On the other hand, we found that the walker has a significant score only in the competence subscale. Canes, for example the stick 4-p is significant in the three subscales, or the walking cane in terms of competence. Despite the fact that crutches and walking sticks obtained the lowest score in this study.Not only the assessment is necessary, but also the adjustment of AT to the person who is going to use it, as well as some training on how to use it. Only 23.8% of the participants received training in the use of their main assistive device in this study.The results seem to indicate that for elder users, the score on adaptability with the AT is lower. That may result in future lines of research in usability and improving in terms of the needs of end-users and these AT since a high percentage of people with neurological conditions are elder people.The application of specific scales such as PIADS that helps to measure the use and capacity of the AT prescribed to patients with neurological disease provides more informed clinical reasoning.

Non invasive ventilation after extubation in paediatric patients: a preliminary study.

BACKGROUND: Non-invasive ventilation (NIV) may be useful after extubation in children. Our objective was to determine postextubation NIV characteristics and to identify risk factors of postextubation NIV failure. METHODS: A prospective observational study was conducted in an 8-bed pediatric intensive care unit (PICU). Following PICU protocol, NIV was applied to patients who had been mechanically ventilated for over 12 hours considered at high-risk of extubation failure -elective NIV (eNIV), immediately after extubation- or those who developed respiratory failure within 48 hours after extubation -rescue NIV (rNIV)-. Patients were categorized in subgroups according to their main underlying conditions. NIV was deemed successful when reintubation was avoided. Logistic regression analysis was performed in order to identify predictors of NIV failure. RESULTS: There were 41 episodes (rNIV in 20 episodes). Success rate was 50% in rNIV and 81% in eNIV (p = 0.037). We found significant differences in univariate analysis between success and failure groups in respiratory rate (RR) decrease at 6 hours, FiO2 at 1 hour and PO2/FiO2 ratio at 6 hours. Neurologic condition was found to be associated with NIV failure. Multiple logistic regression analysis identified no variable as independent NIV outcome predictor. CONCLUSIONS: Our data suggest that postextubation NIV seems to be useful in avoiding reintubation in high-risk children when applied immediately after extubation. NIV was more likely to fail when ARF has already developed (rNIV), when RR at 6 hours did not decrease and if oxygen requirements increased. Neurologic patients seem to be at higher risk of reintubation despite NIV use.

Relationship between tillage management and DMPSA nitrification inhibitor efficiency.

Agricultural sustainability is compromised by nitrogen (N) losses caused by soil microbial activity. Nitrous oxide (N2O) is a potent greenhouse gas (GHG) produced as consequence of nitrification and denitrification processes in soils. Nitrification inhibitors (NI) as 3,4-dimethylpyrazole-succinic acid (DMPSA) are useful tools to reduce these N losses from fertilization. The objective of this work was to test the efficiency of DMPSA in two different tillage management systems, conventional tillage (CT) and no-tillage (NT), in a winter wheat crop under Humid Mediterranean conditions. N fertilizer was applied as ammonium sulphate (AS) with or without DMPSA in a single or split application, including an unfertilized treatment. GHG fluxes (N2O, CO2 and CH4) were measured by the closed chamber method. amoA and nosZI genes were quantified by qPCR as indicators of nitrifying and denitrifying populations. Nitrification was inhibited by DMPSA in both CT and NT, while the higher water filled pore space (WFPS) in NT promoted a better efficiency of DMPSA in this system. This higher efficiency might be due to a greater N2O reduction to N2 as result of the nosZI gene induction. Consequently, DMPSA was able to reduce N2O emissions down to the unfertilized levels in NT. Provided that NT reduced CO2 emissions and maintained crop yield compared to CT, the application DMPSA under NT management is a promising strategy to increase agro-systems sustainability under Humid Mediterranean conditions.

Retropharyngeal abscess and mediastinitis as an uncommon complication of varicella infection.

Varicella infection is one of the most common and contagious infection in children and could course with severe complications. We report the case of a 4-year-old patient derived to our hospital for suspicion of suppurative complication in the context of a varicella infection. A computerized tomographic scanning was performed, showing a large retropharyngeal abscess with mediastinitis. Complications of varicella are up to 2% of patients, but this is the first report of a retropharyngeal and mediastinal abscess in this context. In the face of clinical suspicion, early intervention is important through imaging, intravenous antibiotics and surgical drainage in necessary cases.

Evolution of Patients with Pulmonary Arterial Hypertension Starting Macitentan After the Discontinuation of Other Endothelin-Receptor Antagonists: Results of a Retrospective Study.

BACKGROUND: Macitentan is the latest endothelin-receptor antagonist (ERA) approved for the treatment of pulmonary arterial hypertension (PAH), presenting enhanced properties over previous ERAs. OBJECTIVE: We describe the clinical and echocardiographic evolution of patients with PAH who started macitentan after discontinuing bosentan/ambrisentan. METHODS: This was a retrospective series of patients with different etiologies who started macitentan after the suspension of other ERAs under routine clinical practice at five Spanish hospitals. World Health Organization functional class (WHO-FC), 6-min walk distance (6MWD), levels of N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and cardiac imaging data were collected and described at baseline (before macitentan initiation) and after 3, 6, and 12 months, when available. RESULTS: In total, 12 patients (ten women; mean age 65.63 ± 13.27 years) were observed. At baseline, most patients were receiving concomitant PAH medications, and five patients were classed as WHO-FC III. After 3 months of macitentan treatment, WHO-FC had improved in four patients, 6MWD increased in eight patients, and NT-proBNP levels and right atrial area were lowered in seven and eight patients, respectively. Similar results were observed after 6 and 12 months. Macitentan was well-tolerated, with no PAH hospitalizations, septostomies, transplants, or deaths registered. CONCLUSIONS: Our results suggest that switching to macitentan in patients with PAH who discontinued bosentan/ambrisentan was well-tolerated and effective. Further studies are needed to confirm these observations.

Dimethyl pyrazol-based nitrification inhibitors effect on nitrifying and denitrifying bacteria to mitigate N2O emission.

Nitrous oxide (N2O) emissions have been increasing as a result of intensive nitrogen (N) fertilisation. Soil nitrification and denitrification are the main sources of N2O, and the use of ammonium-based fertilisers combined with nitrification inhibitors (NIs) could be useful in mitigating N2O emissions from agricultural systems. In this work we looked at the N2O mitigation capacity of two dimethylpyrazol-based NIs, 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA), on soil nitrifying and denitrifying microbial populations under two contrasting soil water contents (40% and 80% soil water filled pore space; WFPS). Our results show that DMPP and DMPSA are equally efficient at reducing N2O emissions under 40% WFPS conditions by inhibiting bacterial ammonia oxidation. In contrast, at 80% WFPS DMPSA was less efficient than DMPP at reducing N2O emissions. Interestingly, at 80% WFPS, where lowered oxygen availability limits nitrification, both DMPP and DMPSA not only inhibited nitrification but also stimulated N2O reduction to molecular nitrogen (N2) via nitrous oxide reductase activity (Nos activity). Therefore, in this work we observed that DMP-based NIs stimulated the reduction of N2O to N2 by nitrous oxide reductase during the denitrification process.

Reprogramming Roadblocks Are System Dependent.

Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog(-/-) fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.

Fatal lactic acidosis during antiretroviral therapy.

OBJECTIVE: To describe the first pediatric case of fatal lactic acidosis in an antiretroviral-treated child with human immunodeficiency virus (HIV) infection. DESIGN: Case report. SETTING: Pediatric intensive care unit. PATIENTS: A patient with fatal antiretroviral therapy-associated type B lactic acidosis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We report the case of a 5-yr-old girl with HIV infection, receiving ritonavir, stavudine, and didanosine, who presented with a 10-day history of nausea and vomiting. Severe lactic acidosis was found. Her clinical condition worsened, with progressive increase in serum lactate, despite aggressive supportive therapy, including intravenous alkali and continuous arteriovenous hemodiafiltration. CONCLUSIONS: Fatal lactic acidosis is a complication of antiretroviral therapy in pediatric HIV patients, which has not been previously reported in children. Early recognition of mitochondrial dysfunction in these patients could prevent the development of fatal lactic acidosis.

Correlation of oxygen saturation as measured by pulse oximetry/fraction of inspired oxygen ratio with Pao2/fraction of inspired oxygen ratio in a heterogeneous sample of critically ill children.

PURPOSE: Oxygen saturation as measured by pulse oximetry (Spo2)/fraction of inspired oxygen (Fio2) (SF) ratio has demonstrated to be an adequate marker for lung disease severity in children under mechanical ventilation. We sought to validate the utility of SF ratio in a population of critically ill children under mechanical ventilation, noninvasive ventilation support, and breathing spontaneously. MATERIALS AND METHODS: A retrospective database study was conducted in a pediatric intensive care unit of a university hospital. Children with Spo2 less than or equal to 97% and an indwelling arterial catheter were included. Simultaneous blood gas and pulse oximetry were collected in a database. Derivation and validation data sets were generated, and a linear mixed modeling was used to derive predictive equations. Model performance and fit were evaluated using the validation data set. RESULTS: Three thousand two hundred forty-eight blood gas and Spo2 values from 298 patients were included. 1/SF ratio had a strong linear association with 1/Pao2/Fio2 (PF) ratio in both derivation and validation data sets, given by the equation 1/SF = 0.00164 + 0.521/PF (derivation). Oxygen saturation as measured by pulse oximetry/Fio2 values for PF criteria of 100, 200, and 300 were 146 (95% confidence interval [CI], 142-150), 236 (95% CI, 228-244), and 296 (95% CI, 285-308). Areas under receiver operating characteristic curves for diagnosis of PF ratio less than 100, 200, and 300 with the SF ratio were 0.978, 0.952, and 0.951, respectively, in the validation data set. CONCLUSIONS: Oxygen saturation as measured by pulse oximetry/Fio2 ratio is an adequate noninvasive surrogate marker for PF ratio. Oxygen saturation as measured by pulse oximetry/Fio2 ratio may be an ideal noninvasive marker for patients with acute hypoxemic respiratory failure.

The p53 isoforms are differentially modified by Mdm2.

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53ß. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.

Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency.

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a promising therapeutic tool for many diseases, including aged tissues and organs at high risk of failure. However, the intrinsic self-renewal and pluripotency of ES and iPS cells make them tumorigenic, and hence, the risk of tumor development hinders their clinical application. Here, we present a novel approach to limit their tumorigenicity and increase their safety through increased copy number of tumor suppressors. iPS containing an extra copy of the p53 or Ink4a/ARF locus show normal pluripotency, as determined by in vitro and in vivo differentiation assays. Yet, while retaining full pluripotency, they also possess an improved engagement of the p53 pathway during teratocarcinoma formation, which leads to a reduced tumorigenic potential in various in vitro and in vivo assays. Furthermore, they show an improved response to anticancer drugs, which could aid in their elimination in case tumors arise with no adverse effects on cell function or aging. Our system provides a model for studying tumor suppressor pathways during reprogramming, differentiation, and cell therapy applications. This offers an improved understanding of the pathways involved in tumor growth from engrafted pluripotent stem cells, which could facilitate the use of ES and iPS cells in regenerative medicine.

Non-invasive ventilation in pediatric status asthmaticus: a prospective observational study.

OBJECTIVE: Non-invasive ventilation (NIV) has been shown to be effective in different causes of respiratory failure in both adult and pediatric patients. However, its role in status asthmaticus (SA) remains unclear. We designed a prospective study to assess the feasibility of NIV in children with SA. STUDY DESIGN: Prospective observational study, over a 4.5-year period. PATIENT SELECTION: Children with SA unresponsive to conventional therapy with a modified Wood's clinical asthma score (m-WCAS) ≥4 and marked increased work of breathing, were included. METHODOLOGY: Patients were placed on pressure support NIV. During NIV therapy, salbutamol was nebulized continuously and ipratropium bromide every 2 hr; methyl-prednisolone was given at a dose of 1-2 mg/kg/6 hr. Clinical variables were measured at baseline and at 1, 6, 12, 24, and 48 hr. RESULTS: During the study period, there were 122 PICU admissions due to SA; 72 episodes fulfilled inclusion criteria. Baseline mean values were as follows: m-WCAS of 5.7 points, heart rate (HR) of 166.7 beats/min, respiratory rate (RR) of 49.5 breaths/min and FiO(2) of 45.3%. In the first hour m-WCAS fell 2.3 ± 1.5 points, HR 13.5 ± 14 beats/min, and RR 9.8 ± 10 breaths/min (P < 0.01). After institution of NIV therapy, 5 children required intubation due to increasing respiratory distress. There was one case of massive subcutaneous emphysema, with no other serious adverse effects associated with NIV. CONCLUSIONS: These results show that NIV is a feasible therapy in children with SA unresponsive to conventional treatment. Pediatr. Pulmonol. 2011; 46:949-955. © 2011 Wiley-Liss, Inc.

MDM4 downregulates p53 transcriptional activity and response to stress during differentiation.

Embryonic stem (ES) cells are invaluable for their therapeutic potential as well as for the study of early development. Their clinical use demands an understanding of ES cell differentiation, particularly with respect to cell proliferation and the maintenance of genomic integrity, processes for which the transcription factor p53 is essential. However, although the function of p53 as a tumor suppressor has been extensively studied, its role in ES cell biology has not been clearly elucidated. To study p53 activity and regulation in differentiating ES cells, we used knock-in constructs to create a novel reporter system that provides a direct readout of p53 transcriptional activity. We thereby determine that the p53 pathway is active in ES cells, but that p53 activity and the p53-dependent stress response decrease upon differentiation. Although p53 protein levels and activity are usually primarily controlled by the ubiquitin ligase MDM2, we identify the MDM2 homolog MDM4 as the key modulator of p53 activity in differentiating ES cells. Our results provide a better understanding of ES cell regulation and could help to optimize ES cell differentiation protocols for their use in regenerative medicine.