Cancer-Related Mutations Are Not Enriched in Naive Human Pluripotent Stem Cells.

Previous analysis of RNA sequencing (RNA-seq) data from human naive pluripotent stem cells reported multiple point "mutations" in cancer-related genes and implicated selective culture conditions. We observed, however, that those mutations were only present in co-cultures with mouse feeder cells. Inspection of reads containing the polymorphisms revealed complete identity to the mouse reference genome. After we filtered reads to remove sequences of mouse origin, the actual incidence of oncogenic polymorphisms arising in naive pluripotent stem cells is close to zero.We are grateful to James Clarke for cell culture support and to Vicki Murry and Maike Paramor for generating sequencing libraries. This research was funded by the Medical Research Council (MRC) of the United Kingdom. The Wellcome-MRC Cambridge Stem Cell Institute receives core support from Wellcome and MRC. AS is a Medical Research Council Professor

2022: Are We Back to the pre-COVID-19 Pandemic Period in the Management of out-of-Hospital Cardiac Arrest?

Introduction: The COVID-19 pandemic caused a significant strain on the Emergency system, particularly for time-dependent diseases like Out of Hospital Cardiac Arrest (OHCA). Studies have shown an increase in the incidence of OHCA during different waves of the pandemic, but there is limited evidence on how survival rates and rescue efforts have been affected in the post-pandemic period. Methods: We performed a retrospective observational cohort study of all OHCA rescues by AREU (Agenzia Regionale Emergenza Urgenza), in the Lombardy region in March in three different years (2019, 2021 and 2022). We used rescue mission data collected in AREU’s database, where logistic information of patient rescue missions managed by the Lombardy Region’s 112 system is recorded.Results: This study was an epidemiology analysis of OHCA after the pandemic. The results showed no significant changes in the probability of receiving bystander cardiopulmonary resuscitation (22.5% vs 24.0%; p=0.41) and public access defibrillation (3.6 vs 3.2; p=0.50) compared to pre-pandemic period. However, there was a decrease in the probability of ROSC (11.5% vs 6.2%; p<0.01).Conclusion: According to our analysis, there appears to be a return to the pre-pandemic phase with regard to the OHCA network. However, it remains to be pointed out that a careful study of disease networks is essential to understand the resilience of our health system and to understand whether we have returned to a system similar to the pre-pandemic phase after the COVID-19 pandemic

The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1) and European Commission Framework 7 (HEALTH-F4-2013-602423, PluriMes). The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.

Pluripotent cells emerge as a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here, we investigate the intermediate formative stage. Using minimal exposure to specification cues, we derive stem cells from formative mouse epiblast. Unlike ESCs or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonize somatic tissues and germline in chimeras. Whole-transcriptome analyses show similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells show distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.The Cambridge Stem Cell Institute receives core funding from Wellcome and the Medical Research Council. This research was funded by the Biotechnology and Biological Sciences Research Council and the Medical Research Council of the United Kingdom. AS is a Medical Research Council Professor

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment.We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germlayer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stemcells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestockmammals, pluripotent stemcells related to the formative embryonic disc are reliably established using a common and defined signalling environment

Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition

Spring, it’s time to ROSC: Analysis of months in Out hospital cardiac arrest on ROSC achievement

Out-hospital cardiac arrest (OHCA) is a multifactor disease. Many studies have correlated OHCA with a patient's lifestyle; unfortunately, less evidence highlights the correlation with meteorological factors. Methods: We analysed 23959 OHCA rescue performed by the emergency medical system (EMS) of Lombardy Region, the most Italian populated region, in 2018 and 2019, the pre-pandemic era. The aim was to evaluate the probability of Return of spontaneous circulation (ROSC) in different months. In March and April, we highlight an increase of ROSC (OR: 1.20 IC 95% 1.04 - 1.31; p<0.001) compared to other months. During March and April, we highlight an increase of public access defibrillation (PAD) (3,5% vs 2,5%; p<0.001), and a reduction of overage time of first vehicle on scene (11,5 vs 11,8; p<0,001) and age of patient (73,5 vs 74,2; p<0,01). Finally, we highlight a slight reduction of cancer patient (1,6% vs 1,1%; p=0,01). the significant difference didn't register in the other nine variables analysed. We highlight a difference in ROSC probability during the first month of spring. We register few differences in patient characteristics and EMS rescue, though just PAD use and age clinically impact OHCA patients. In this study, we are unable to fully understand the modification of the probability of ROSC in these months. Even though four variables have a statistically significant difference, they can't fully explain this modification. Different variables like meteorological and seasonal factor must be considered. We propose more research on this item