Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations ...poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
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► Transcriptional analysis of iPSC formation reveals biphasic process ► c-Myc/Klf4 drive first phase whereas Oct4/Sox2/Klf4 drive second phase ► Bivalent genes form gradually whereas DNA methylation changes occur late ► Refractory cells can be rescued by elevated reprogramming factor expression
By sorting fibroblasts induced to reprogram to iPSCs, the molecular characteristics of cells in the early and late stages of reprogramming are revealed, as well as the attributes of the cells refractory to reprogramming. New genes that enhance reprogramming efficiency are identified.
Aims
Data on the impact of COVID‐19 in chronic heart failure (CHF) patients and its potential to trigger acute heart failure (AHF) are lacking. The aim of this work was to study characteristics, ...cardiovascular outcomes and mortality in patients with confirmed COVID‐19 infection and a prior diagnosis of heart failure (HF). Further aims included the identification of predictors and prognostic implications for AHF decompensation during hospital admission and the determination of a potential correlation between the withdrawal of HF guideline‐directed medical therapy (GDMT) and worse outcomes during hospitalization.
Methods and results
Data for a total of 3080 consecutive patients with confirmed COVID‐19 infection and follow‐up of at least 30 days were analysed. Patients with a previous history of CHF (n = 152, 4.9%) were more prone to the development of AHF (11.2% vs. 2.1%; P < 0.001) and had higher levels of N‐terminal pro brain natriuretic peptide. In addition, patients with previous CHF had higher mortality rates (48.7% vs. 19.0%; P < 0.001). In contrast, 77 patients (2.5%) were diagnosed with AHF, which in the vast majority of cases (77.9%) developed in patients without a history of HF. Arrhythmias during hospital admission and CHF were the main predictors of AHF. Patients developing AHF had significantly higher mortality (46.8% vs. 19.7%; P < 0.001). Finally, the withdrawal of beta‐blockers, mineralocorticoid receptor antagonists and angiotensin‐converting enzyme inhibitors or angiotensin receptor blockers was associated with a significant increase in in‐hospital mortality.
Conclusions
Patients with COVID‐19 have a significant incidence of AHF, which is associated with very high mortality rates. Moreover, patients with a history of CHF are prone to developing acute decompensation after a COVID‐19 diagnosis. The withdrawal of GDMT was associated with higher mortality.
Heart failure in COVID‐19 patients: prevalence, incidence and prognostic implications.
Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR ...can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.
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•Germline transcripts peak prior to GC formation and rapidly decline in GCs•IgM-dominated clones are found in late GCs, arguing against ongoing Ig switching•CSR largely ceases upon the onset of somatic hypermutation•CSR decline due to low GLT and APE1 expression is possibly orchestrated by BCL6
Germinal centers (GCs) have long been considered sites in which Ig class-switch recombination (CSR) is favored. Roco et al. show that CSR occurs during the initial T cell:B cell interaction prior to GC formation and rapidly declines as B cells differentiate into GC cells and somatic hypermutation commences.
The transcription factor Sox2 maintains the pluripotency of early embryonic cells and regulates the formation of several epithelia during fetal development. Whether Sox2 continues to play a role in ...adult tissues remains largely unknown. We show here that Sox2 marks adult cells in several epithelial tissues where its expression has not previously been characterized, including the stomach, cervix, anus, testes, lens, and multiple glands. Genetic lineage tracing and transplantation experiments demonstrate that Sox2-expressing cells continuously give rise to mature cell types within these tissues, documenting their self-renewal and differentiation potentials. Consistent with these findings, ablation of Sox2
+ cells in mice results in a disruption of epithelial tissue homeostasis and lethality. Developmental fate mapping reveals that Sox2
+ adult stem cells originate from fetal Sox2
+ tissue progenitors. Thus, our results identify Sox2 expression in numerous adult endodermal and ectodermal stem cell compartments, which are critical for normal tissue regeneration and survival.
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► Stem cell factor Sox2 is expressed in multiple adult self-renewing epithelia ► Adult Sox2
+ stem cells are derived from fetal Sox2
+ progenitors ► Transplantation of Sox2
+ testis cells restores spermatogenesis in infertile mice ► Ablation of adult Sox2
+ cells disrupts tissue integrity and results in lethality
Human development is a highly coordinated process, with any abnormalities during the early embryonic stages that can often have detrimental consequences. The complexity and nuances of human ...development underpin its significance in embryo research. However, this research is often hindered by limited availability and ethical considerations associated with the use of donated blastocysts from in vitro fertilization (IVF) surplus. Human blastoids offer promising alternatives as they can be easily generated and manipulated in the laboratory while preserving key characteristics of human blastocysts. In this way, they hold the potential to serve as a scalable and ethically permissible resource in embryology research. By utilizing such human embryo models, we can establish a transformative platform that complements the study with IVF embryos, ultimately enhancing our understanding of human embryogenesis.
Aims
Extensive research regarding the association of troponin and prognosis in coronavirus disease 2019 (COVID‐19) has been performed. However, data regarding natriuretic peptides are scarce. ...N‐terminal pro B‐type natriuretic peptide (NT‐proBNP) reflects haemodynamic stress and has proven useful for risk stratification in heart failure (HF) and other conditions such as pulmonary embolism and pneumonia. We aimed to adequately characterize NT‐proBNP concentrations using a large cohort of patients with COVID‐19, and to investigate its association with prognosis.
Methods and results
Consecutive patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and available NT‐proBNP determinations, from March 1st to April 20th, 2020 who completed at least 1‐month follow‐up or died, were studied. Of 3080 screened patients, a total of 396 (mean age 71.8 ± 14.6 years, 61.1% male) fulfilled all the selection criteria and were finally included, with a median follow‐up of 53 (18–62) days. Of those, 192 (48.5%) presented NT‐proBNP levels above the recommended cut‐off for the identification of HF. However, only 47 fulfilled the clinical criteria for the diagnosis of HF. Patients with higher NT‐proBNP during admission experienced more frequent bleeding, arrhythmias and HF decompensations. NT‐proBNP was associated with mortality both in the whole study population and after excluding patients with HF. A multivariable Cox model confirmed that NT‐proBNP was independently associated with mortality after adjusting for all relevant confounders (hazard ratio 1.28, 95% confidence interval 1.13–1.44, per logarithmic unit).
Conclusion
NT‐proBNP is frequently elevated in COVID‐19. It is strongly and independently associated with mortality after adjusting for relevant confounders, including chronic HF and acute HF. Therefore, its use may improve early prognostic stratification in this condition.
NT‐proBNP is frequently elevated in patients with COVID‐19 and is strongly associated with myocardial injury and all‐cause mortality.
The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain ...largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin “interactome” for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.
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•The Nanog promoter forms a pluripotency-specific, genome-wide chromatin interactome•Nanog interactions in ESCs are enriched for active marks and pluripotency factors•Mediator and cohesin are essential for the maintenance of the Nanog interactome•Many Nanog interactions form before increased gene expression during reprogramming
The Nanog promoter interacts with the rest of the genome in an unexpectedly complex pluripotency-specific chromatin interaction network that depends on Mediator and cohesin.
Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been ...extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.
Generation of induced pluripotent stem cells (iPSCs) is a process whose mechanistic underpinnings are only beginning to emerge. Here, we applied in-depth quantitative proteomics to monitor proteome ...changes during the course of reprogramming of fibroblasts to iPSCs. We uncover a two-step resetting of the proteome during the first and last 3 days of reprogramming, with multiple functionally related proteins changing in expression in a highly coordinated fashion. This comprised several biological processes, including changes in the stoichiometry of electron transport-chain complexes, repressed vesicle-mediated transport during the intermediate stage, and an EMT-like process in the late phase. In addition, we demonstrate that the nucleoporin Nup210 is essential for reprogramming by its permitting of rapid cellular proliferation and subsequent progression through MET. Along with the identification of proteins expressed in a stage-specific manner, this study provides a rich resource toward an enhanced mechanistic understanding of cellular reprogramming.
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► Major proteome changes occur in two steps, early and late during reprogramming ► Expression changes of functionally related proteins are tightly coordinated ► Intermediate cells are characterized by stage-specific protein expression ► Enhanced expression of Nup210 is required for reprogramming
Little is known about proteomic events that underlie reprogramming of fibroblasts to induce pluripotent stem cells. Hansson and colleagues performed a quantitative proteomic time-course analysis, showing that multiple functionally related proteins change in expression in a highly coordinated fashion, comprising several biological processes with a previously unknown role in reprogramming. Along with the identification of proteins expressed in a stage-specific manner, they show that Nup210 is essential for reprogramming. This resource contributes to an enhanced understanding of cellular reprogramming.
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