Developing a model system to investigate the epigenetic mechanisms underlying pluripotency in human cells

Pluripotent human embryonic stem cells (hESCs) are a valuable tool for clinical therapies, drug testing and investigation of developmental pathways. Recently, over-expression of four pluripotency-associated genes (OCT4, NANOG, SOX2, and LIN28) has proven sufficient to reprogram differentiated cells into pluripotent stem cells, potentially alleviating the need for human embryos to isolate hESCs and opening new avenues for the investigation of pluripotency. This project aimed to generate an in vitro model system to study the epigenetic mechanisms regulating pluripotency transcription factors. hESCs were differentiated into fibroblasts (hESC-Fib) and subsequently reprogrammed to induced pluripotency stem cells (iPSCs) by lentiviral over-expression of human OCT4, NANOG, SOX2 and LIN28. iPSC colonies were positively identified by live staining with the surface marker TRA-1-81 and expanded in culture. They were then further differentiated into a fibroblast line to allow comparison with hESC-Fib. All cells in the model system shared the same genotype and were cultured under similar conditions, enabling unbiased analysis of epigenetic characteristics. DNA methylation analysis of key pluripotency-genes such as OCT4, SOX2, NANOG, and REX1 by bisulfite sequencing, revealed that these were hypomethylated in hESCs and iPSCs, and hypermethylated in their fibroblast derivatives. A gradual increase in the number of CpGs gaining DNA methylation was observed when hESCs and iPSCs were differentiated into fibroblasts, while TaqMan real-time PCR and fluorescence staining revealed that expression of these genes was inversely related to the levels of DNA methylation in their promoters. The master pluripotency regulators OCT4, SOX2 and NANOG all showed differential methylation in their OCT/SOX binding regions, suggesting a common regulatory mechanism between them. This is, to our knowledge, the first report for SOX2 differential methylation in human non-cancerous cells. Reactivation of REX1 was not found to be necessary for the reprogramming of hESC-Fib to iPSCs, calling for re-evaluation of its role in human pluripotency. Based on the observation that the DNA methylation levels of pluripotency genes were higher in fibroblast cell lines compared to hESCs and iPSCs, we hypothesised that reduction in DNA methylation could render differentiated cells more permissive to reprogramming. Stable knock-downs of the DNA methyltransferases (DNMTs) DNMT1 and DNMT3A were, thus, performed in hESC-Fib. Knock-down of DNMT1, the most abundant DNMT in hESC-Fib, resulted in global reduction of DNA methylation levels as determined by restriction digests with methylation specific enzymes. Reprogramming of hESC-Fib carrying a DNMT1 knock-down showed a 40% reduction in generation of iPSC colonies compared to untreated controls, perhaps owing to the delay in progression of S phase in the cell cycle caused by DNMT1 knock-down. In contrast, knock-down of DNMT3A resulted in a >80% increase in iPSC colony formation, potentially indicating differences in mechanism of action and specificity between the two DNMT enzymes. Through this study, we have gained new insights into the epigenetic mechanisms underlying cell phenotype and provided the foundation for further improving reprogramming efficiency

Developing a model system to investigate the epigenetic mechanisms underlying pluripotency in human cells

Pluripotent human embryonic stem cells (hESCs) are a valuable tool for clinical therapies, drug testing and investigation of developmental pathways. Recently, over-expression of four pluripotency-associated genes (OCT4, NANOG, SOX2, and LIN28) has proven sufficient to reprogram differentiated cells into pluripotent stem cells, potentially alleviating the need for human embryos to isolate hESCs and opening new avenues for the investigation of pluripotency. This project aimed to generate an in vitro model system to study the epigenetic mechanisms regulating pluripotency transcription factors. hESCs were differentiated into fibroblasts (hESC-Fib) and subsequently reprogrammed to induced pluripotency stem cells (iPSCs) by lentiviral over-expression of human OCT4, NANOG, SOX2 and LIN28. iPSC colonies were positively identified by live staining with the surface marker TRA-1-81 and expanded in culture. They were then further differentiated into a fibroblast line to allow comparison with hESC-Fib. All cells in the model system shared the same genotype and were cultured under similar conditions, enabling unbiased analysis of epigenetic characteristics. DNA methylation analysis of key pluripotency-genes such as OCT4, SOX2, NANOG, and REX1 by bisulfite sequencing, revealed that these were hypomethylated in hESCs and iPSCs, and hypermethylated in their fibroblast derivatives. A gradual increase in the number of CpGs gaining DNA methylation was observed when hESCs and iPSCs were differentiated into fibroblasts, while TaqMan real-time PCR and fluorescence staining revealed that expression of these genes was inversely related to the levels of DNA methylation in their promoters. The master pluripotency regulators OCT4, SOX2 and NANOG all showed differential methylation in their OCT/SOX binding regions, suggesting a common regulatory mechanism between them. This is, to our knowledge, the first report for SOX2 differential methylation in human non-cancerous cells. Reactivation of REX1 was not found to be necessary for the reprogramming of hESC-Fib to iPSCs, calling for re-evaluation of its role in human pluripotency. Based on the observation that the DNA methylation levels of pluripotency genes were higher in fibroblast cell lines compared to hESCs and iPSCs, we hypothesised that reduction in DNA methylation could render differentiated cells more permissive to reprogramming. Stable knock-downs of the DNA methyltransferases (DNMTs) DNMT1 and DNMT3A were, thus, performed in hESC-Fib. Knock-down of DNMT1, the most abundant DNMT in hESC-Fib, resulted in global reduction of DNA methylation levels as determined by restriction digests with methylation specific enzymes. Reprogramming of hESC-Fib carrying a DNMT1 knock-down showed a 40% reduction in generation of iPSC colonies compared to untreated controls, perhaps owing to the delay in progression of S phase in the cell cycle caused by DNMT1 knock-down. In contrast, knock-down of DNMT3A resulted in a >80% increase in iPSC colony formation, potentially indicating differences in mechanism of action and specificity between the two DNMT enzymes. Through this study, we have gained new insights into the epigenetic mechanisms underlying cell phenotype and provided the foundation for further improving reprogramming efficiency

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development

5-hydroxymethyl-cytosine (5-hmc) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (Esc) derived from mammalian blastocysts. Recent observations imply that both 5-hmc and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmc, may play an important role in self renewal and differentiation of Escs. here we assessed the distribution of 5-hmc in zebrafish and chick embryos and found that, unlike in mammals, 5-hmc is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. however, 5-hmc is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmc enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmc in its regulation

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Developing a model system to investigate the epigenetic mechanisms underlying pluripotency in human cells

Pluripotent human embryonic stem cells (hESCs) are a valuable tool for clinical therapies, drug testing and investigation of developmental pathways. Recently, over-expression of four pluripotency-associated genes (OCT4, NANOG, SOX2, and LIN28) has proven sufficient to reprogram differentiated cells into pluripotent stem cells, potentially alleviating the need for human embryos to isolate hESCs and opening new avenues for the investigation of pluripotency. This project aimed to generate an in vitro model system to study the epigenetic mechanisms regulating pluripotency transcription factors. hESCs were differentiated into fibroblasts (hESC-Fib) and subsequently reprogrammed to induced pluripotency stem cells (iPSCs) by lentiviral over-expression of human OCT4, NANOG, SOX2 and LIN28. iPSC colonies were positively identified by live staining with the surface marker TRA-1-81 and expanded in culture. They were then further differentiated into a fibroblast line to allow comparison with hESC-Fib. All cells in the model system shared the same genotype and were cultured under similar conditions, enabling unbiased analysis of epigenetic characteristics. DNA methylation analysis of key pluripotency-genes such as OCT4, SOX2, NANOG, and REX1 by bisulfite sequencing, revealed that these were hypomethylated in hESCs and iPSCs, and hypermethylated in their fibroblast derivatives. A gradual increase in the number of CpGs gaining DNA methylation was observed when hESCs and iPSCs were differentiated into fibroblasts, while TaqMan real-time PCR and fluorescence staining revealed that expression of these genes was inversely related to the levels of DNA methylation in their promoters. The master pluripotency regulators OCT4, SOX2 and NANOG all showed differential methylation in their OCT/SOX binding regions, suggesting a common regulatory mechanism between them. This is, to our knowledge, the first report for SOX2 differential methylation in human non-cancerous cells. Reactivation of REX1 was not found to be necessary for the reprogramming of hESC-Fib to iPSCs, calling for re-evaluation of its role in human pluripotency. Based on the observation that the DNA methylation levels of pluripotency genes were higher in fibroblast cell lines compared to hESCs and iPSCs, we hypothesised that reduction in DNA methylation could render differentiated cells more permissive to reprogramming. Stable knock-downs of the DNA methyltransferases (DNMTs) DNMT1 and DNMT3A were, thus, performed in hESC-Fib. Knock-down of DNMT1, the most abundant DNMT in hESC-Fib, resulted in global reduction of DNA methylation levels as determined by restriction digests with methylation specific enzymes. Reprogramming of hESC-Fib carrying a DNMT1 knock-down showed a 40% reduction in generation of iPSC colonies compared to untreated controls, perhaps owing to the delay in progression of S phase in the cell cycle caused by DNMT1 knock-down. In contrast, knock-down of DNMT3A resulted in a >80% increase in iPSC colony formation, potentially indicating differences in mechanism of action and specificity between the two DNMT enzymes. Through this study, we have gained new insights into the epigenetic mechanisms underlying cell phenotype and provided the foundation for further improving reprogramming efficiency.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Current status of drug screening and disease modelling in human pluripotent stem cells

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes

Aims Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. Methods and results We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K+ currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). Conclusions These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart