Manipulating the Mediator complex to induce naïve pluripotency

Human naïve pluripotent stem cells (PSCs) represent an optimal homogenous starting point for molecular interventions and differentiation strategies. This is in contrast to the standard primed PSCs which fluctuate in identity and are transcriptionally heterogeneous. However, despite many efforts, the maintenance and expansion of human naïve PSCs remains a challenge. Here, we discuss our recent strategy for the stabilization of human PSC in the naïve state based on the use of a single chemical inhibitor of the related kinases CDK8 and CDK19. These kinases phosphorylate and negatively regulate the multiprotein Mediator complex, which is critical for enhancer- driven recruitment of RNA Pol II. The net effect of CDK8/19 inhibition is a global stimulation of enhancers, which in turn reinforces transcriptional programs including those related to cellular identity. In the case of pluripotent cells, the presence of CDK8/19i ef ciently stabilizes the naïve state. Importantly, in contrast to previous chemical methods to induced the naïve state based on the inhibition of the FGF-MEK-ERK pathway, CDK8/19i-naïve human PSCs are chromosomally stable and retain developmental potential after long-term expansion. We suggest this could be related to the fact that CDK8/19 inhibition does not induce DNA demethylation. These principles may apply to other fate decisions

In Vivo Reprogramming Ameliorates Aging Features in Dentate Gyrus Cells and Improves Memory in Mice

Post-translational epigenetic modifications take place in mouse neurons of the dentate gyrus (DG) with age. Here, we report that age-dependent reduction in H3K9 trimethylation (H3K9me3) is prevented by cyclic induction of the Yamanaka factors used for cell reprogramming. Interestingly, Yamanaka factors elevated the levels of migrating cells containing the neurogenic markers doublecortin and calretinin, and the levels of the NMDA receptor subunit GluN2B. These changes could result in an increase in the survival of newborn DG neurons during their maturation and higher synaptic plasticity in mature neurons. Importantly, these cellular changes were accompanied by an improvement in mouse performance in the object recognition test over long time. We conclude that transient cyclic reprogramming in vivo in the central nervous system could be an effective strategy to ameliorate aging of the central nervous system and neurodegenerative diseases

Improved thermochemical energy storage behavior of manganese oxide by molybdenum doping

To improve the thermochemical energy storage (TCS) behavior of Mn2O3, several Mn–Mo oxides with varying amounts of MoO3 (0–30 wt%) were prepared by a precipitation method. The physico-chemical properties of the solids were studied byN2 adsorption–desorption, X-ray diffraction (XRD), scanning electron microscopy (SEM), and H2-temperature-programmed reduction (TPR), while their TCS behavior was determined by thermogravimetric analysis coupled with differential scanning calorimetry (TGA-DSC). Apart from Mn2O3 and MoO3 phases, XRD revealed a mixed MnMoO4 phase for MoO3 loadings equal or higher than 1.5 wt%. All samples showed a wellformed coral-like surface morphology, particularly those solids with low MoO3 contents. This coral morphology was progressively decorated with compact and Mo-enriched MnMoO4 particles as the MoO3 content increased. TPR revealed that the redox behavior of Mn2O3 was significantly altered upon addition of Mo. The TCS behavior of Mn2O3 (mostly oxidation kinetics and redox cyclability) was enhanced by addition of low amounts of Mo (0.6 and 1.5% MoO3) without significantly increasing the reduction temperature of the solids. The coral morphology (which facilitated oxygen diffusion) and a smoother transition from the reduced to oxidized phase were suggested to be responsible for this improved TCS behavior. The samples containing 0.6 and 1.5 wt% of MoO3 showed outstanding cyclability after 45 consecutive reduction–oxidation cycles at high temperatures (600–1000 C). These materials could potentially reach absorption efficiencies higher than 90% at concentration capacity values typical of concentrated solar power plants

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture

Dissection of two routes to naïve pluripotency using different kinase inhibitors

Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors

A Two-Photon Probe Based on Naphthalimide-Styrene Fluorophore for the In Vivo Tracking of Cellular Senescence

Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal β-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues

Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir/ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an anti-apoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence

Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC

Conservation of aging and cancer epigenetic signatures across human and mouse

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. While many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and non-tumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point towards the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits.© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution