Background: Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings. Objectives: To investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell dependent macrophage activation. Methods: We used human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users. Results: The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β (IL-1β) and IL-6, leading to increased ROS. After exposure of iPSC-ECs to serum of e-cigarette users, we observed increased ROS linked to endothelial dysfunction, as indicated by impaired pro-angiogenic properties. We also noted an increase in inflammatory cytokine expression in serum of e-cigarette users. Conclusions: Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.Abbreviations CRP = c-reactive protein CVD = cardiovascular disease E-cigarette = electronic cigarette E-liquids = electronic cigarette liquids iPSC-ECs = induced pluripotent stem cell-derived endothelial cells PG = propylene glycol PLT = platelet ROS = reactive oxygen species VG = vegetable glycerin Condensed Abstract E-cigarettes have seen a rapid increase in use although their effects on vascular health remain understudied. Here, we investigated the effects of flavored e-cigarette liquids (e-liquids) on endothelial health by exposing human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) to different e-liquids with varying nicotine concentrations. While cytotoxicity varied among the tested flavors, the cinnamon-flavored product was most potent leading to decreased cell viability, increased oxidative stress and caspase activity, and impaired tube formation confirming endothelial dysfunction; results which were further corroborated using e-cigarette

Highlights d TBX5 Clover2 and NKX2-5 TagRFP reporter enables purification of 4 cardiac subpopulations d Different cardiac lineages differentiate into specific cardiac cell types d CORIN is a cell-surface marker for the TBX5+NKX2-5+ subpopulation d Lineage-specific cardiomyocyte subtypes can be used for precise drug testing

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC selfrenewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.Erk | Mek | embryonic stem cells | self-renewal | genomic stability E mbryonic stem cells (ESCs) are pluripotent and, hence, promising donor cell sources for regenerative medicine. Transcriptional regulation plays an essential role in pluripotency maintenance of ESCs, and a transcriptional regulation network for pluripotency has been characterized (1, 2). The core component of the pluripotency transcriptional regulation network is a feed-forward selfregulating circuitry formed by transcription factors Oct4, Sox2, and Nanog (3, 4). ESCs are cultured in media supplemented with growth factors. Through signaling pathways, growth factors affect the pluripotency transcriptional regulation network and regulate the self-renewal and differentiation of ESCs. For example, LIF and BMP signaling controls the transcriptional activities of the downstream effectors Stat3 and Smad1 to promote the self-renewal of mouse ESCs (5-7).The extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) signal-transduction cascade mediates the effect of growth factors by sequential activation of Ras-like GTPase, Raf kinase, serine/threonine protein kinase Mek, and Erk to regulate cell-cycle progression, proliferation, differentiation, and carcinogenesis (8, 9). Erk signaling also plays a pivotal role in pluripotency maintenance. Inhibition of Mek/Erk signaling constrains the differentiation of mouse ESCs (10). Mouse ESCs can be derived and maintained in medium supplemented with inhibitors of Mek and Gsk3 signaling (2i) (11). Moreover, inhibition of Mek facilitates the conversion of mouse epiblast stem cells (epiSCs) to ESC-like cells (12). Similarly, the Mek inhibitor PD0325901 is used in establishing and maintaining human ground-state pluripotent stem cells (13-16). Conversely, activation of Mek/Erk signaling promotes the differentiation of ESCs. Ectopic expression of an activated H-RAS mutant leads to mouse ESC differentiation toward the trophectodermal lineage. Mek/Erk signaling is the downstream effector of Ras medi...

Summary Nicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine-induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. In addition, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca 2+ signaling was found in muscle cells upon nicotine exposure, as verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggest direct adverse effects of nicotine on hESC differentiation at the single-cell level and offer a new method for evaluating drug and environmental toxicity on human embryonic development in utero .

Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.

Background:Transcriptional regulation plays an important role in pluripotency maintenance. Results: Knockdown of the nuclear receptor coactivator 3 (Ncoa3) compromises the pluripotency of mouse embryonic stem cells. Conclusion: Ncoa3 binds to and activates the Nanog promoter, thus promoting self-renewal of mouse embryonic stem cells. Significance: Uncovering the novel role and mechanism of Ncoa3 in pluripotency maintenance.

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Aims The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. Methods and results Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. Conclusion This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.