Epigenomic Landscapes of hESC-Derived Neural Rosettes: Modeling Neural Tube Formation and Diseases

We currently lack a comprehensive understanding of the mechanisms underlying neural tube formation and their contributions to neural tube defects (NTDs). Developing a model to study such a complex morphogenetic process, especially one that models human-specific aspects, is critical. Three-dimensional, human embryonic stem cell (hESC)-derived neural rosettes (NRs) provide a powerful resource for in vitro modeling of human neural tube formation. Epigenomic maps reveal enhancer elements unique to NRs relative to 2D systems. A master regulatory network illustrates that key NR properties are related to their epigenomic landscapes. We found that folate-associated DNA methylation changes were enriched within NR regulatory elements near genes involved in neural tube formation and metabolism. Our comprehensive regulatory maps offer insights into the mechanisms by which folate may prevent NTDs. Lastly, our distal regulatory maps provide a better understanding of the potential role of neurological-disorder-associated SNPs.</p

An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3(K392R,) on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3(K392R) and healthy-control cells, but in later stages, NEUROG3 expressionwas upregulated prematurely in STAT3(K392R) cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3(K392R) -activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3(K392R). Collectively, our results demonstrate that the STAT3(K392R) mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.Peer reviewe

Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential

Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other. However, genetic variation imparts a donor-specific expression and methylation profile in reprogrammed cells that leads to variable functional capacities of iPSC lines. Our results suggest that integration-free, bona fide iPSC lines from fibroblasts and blood can be combined in repositories to form biobanks. Due to the impact of genetic variation on iPSC differentiation, biobanks should contain cells from large numbers of donors.Peer reviewe

Methylome Analysis of Human Bone Marrow MSCs Reveals Extensive Age- and Culture-Induced Changes at Distal Regulatory Elements

Human bone marrow stromal cells, or mesenchymal stem cells (BM-MSCs), need expansion prior to use as cell-based therapies in immunological and tissue repair applications. Aging and expansion of BM-MSCs induce epigenetic changes that can impact therapeutic outcomes. By applying sequencing-based methods, we reveal that the breadth of DNA methylation dynamics associated with aging and expansion is greater than previously reported. Methylation changes are enriched at known distal transcription factor binding sites such as enhancer elements, instead of CpG-rich regions, and are associated with changes in gene expression. From this, we constructed hypo- and hypermethylation-specific regulatory networks, including a sub-network of BM-MSC master regulators and their predicted target genes, and identified putatively disrupted signaling pathways. Our genome-wide analyses provide a broader overview of age- and expansion-induced DNA methylation changes and a better understanding of the extent to which these changes alter gene expression and functionality of human BM-MSCs.</p

Additional file 1: of cChIP-seq: a robust small-scale method for investigation of histone modifications

The following additional data are available with the online version of this paper. Additional data file 1 contains the following figures. Figure S1. illustrates the optimization on sonication down to 30,000 crosslinked cells using the Covaris LE220 ultrasonicator. Figure S2. provides p-values for the Pearson’s correlation heatmaps shown in Figs. 2, 3 and 4. Figure S3. relates to Fig. 2 and provides additional results for H3K4me3 in K562 cell line. Figure S4. relates to Fig. 3 and provides additional results for H3K4me1 in K562 cell line. Figure S5. shows results obtained for H3K4me1 in H1 hESC line. (PDF 3493 kb

TRIVAC decision-support model for evaluating the cost-effectiveness of Haemophilus influenzae type b, pneumococcal and rotavirus vaccination.

The TRIVAC decision support model has been used widely in Latin America and other regions to help national teams evaluate the cost-effectiveness of Haemophilus influenzae type b (Hib) vaccine, pneumococcal conjugate vaccine (PCV) and rotavirus vaccine (RV). We describe the structure and functioning of this model, and identify the parameters with the greatest influence on the results. The TRIVAC model is a spreadsheet software program that calculates incremental cost-effectiveness ratios (ICERs) and other indicators for three childhood vaccines (Hib, PCV and RV) utilising parameters such as demography, disease burden, vaccine costs, vaccine coverage, vaccine efficacy, health service utilisation and costs. There is a good deal of uncertainty about the local values of many of the parameters that have most influence on the cost-effectiveness of these new vaccines. Cost-effectiveness models can be used to explore the implications of different values of these parameters. However, for such models to be seen as relevant and helpful by decision-makers, they need to be transparent, flexible, easy to use, and embedded in a process which is owned and led by national teams. In this paper the key drivers of cost-effectiveness in the model are identified by one-way sensitivity analyses, run for each vaccine in 147 countries. The data used are mainly from standard international sources and the published literature. The primary indicator was the discounted cost per Disability Adjusted Life-Year (DALY) averted, from a government perspective, over a 20-year period (2013-2032). For all three vaccines, the ICER was most sensitive to changes in relative coverage (the coverage of the children who would have become diseased or, more importantly, died if the population had not been vaccinated, as a % of overall national coverage) and the herd effect multiplier. Other influential parameters for all three vaccines were: the incidence and case fatality of disease, the baseline trend in disease mortality in the absence of vaccination, vaccine efficacy, vaccine price and the % decline in vaccine price per year. Important vaccine-specific parameters included the cost of Hib meningitis sequelae, PCV serotype coverage and the rotavirus gastro-enteritis (RVGE) admission rate. While vaccine efficacy, herd effects, disease mortality and vaccine price are commonly cited as important drivers of cost-effectiveness, this analysis highlights the potentially important influence of relative coverage, a parameter rarely considered in models of vaccine impact and cost-effectiveness

ProVac Global Initiative: a vision shaped by ten years of supporting evidence-based policy decisions.

INTRODUCTION: The Pan American Health Organization (PAHO) created the ProVac Initiative in 2004 with the goal of strengthening national technical capacity to make evidence-based decisions on new vaccine introduction, focusing on economic evaluations. In view of the 10th anniversary of the ProVac Initiative, this article describes its progress and reflects on lessons learned to guide the next phase. METHODS: We quantified the output of the Initiative's capacity-building efforts and critically assess its progress toward achieving the milestones originally proposed in 2004. Additionally, we reviewed how country studies supported by ProVac have directly informed and strengthened the deliberations around new vaccine introduction. RESULTS: Since 2004, ProVac has conducted four regional workshops and supported 24 health economic analyses in 15 Latin American and Caribbean countries. Five Regional Centers of Excellence were funded, resulting in six operational research projects and nine publications. Twenty four decisions on new vaccine introductions were supported with ProVac studies. Enduring products include the TRIVAC and CERVIVAC cost-effectiveness models, the COSTVAC program costing model, methodological guides, workshop training materials and the OLIVES on-line data repository. Ten NITAGs were strengthened through ProVac activities. DISCUSSION: The evidence accumulated suggests that initiatives with emphasis on sustainable training and direct support for countries to generate evidence themselves, can help accelerate the introduction of the most valuable new vaccines. International and Regional Networks of Collaborators are necessary to provide technical support and tools to national teams conducting analyses. Timeliness, integration, quality and country ownership of the process are four necessary guiding principles for national economic evaluations to have an impact on policymaking. It would be an asset to have a model that offers different levels of complexity to choose from depending on the vaccine being evaluated, the availability of data, and the time frame of the decision. CONCLUSION: Decision support for new vaccine introduction in low- and middle-income countries is critical to maximizing the efficiency and impact of vaccination programs. Global technical cooperation will be required. In the future, PAHO and WHO have an opportunity to expand the reach of the ProVac philosophy, models, and methods to additional regions and countries requiring real-time support. The ProVac Global Initiative is proposed as an effective mechanism to do so

Potential intussusception risk versus health benefits from rotavirus vaccination in Latin America.

BACKGROUND: With the recent postlicensure identification of an increased risk of intussusception with rotavirus vaccine, the 14 Latin American countries currently using rotavirus vaccine must now weigh the health benefits versus risks to assess whether to continue vaccination. To inform policy considerations, we estimated excess intussusception cases and mortality potentially caused by rotavirus vaccine for each of the 14 countries and compared these estimates to hospitalizations and deaths expected to be averted through vaccination. METHODS: We used regional rotavirus disease burden and rotavirus vaccine efficacy data, global natural intussusception and regional rotavirus vaccine-related risk estimates, and country-specific diphtheria, tetanus, and pertussus vaccination coverage rates to estimate rotavirus vaccine coverage rates. We performed a probabilistic sensitivity analysis to account for uncertainty in these parameters. RESULTS: For an aggregate hypothetical birth cohort of 9.5 million infants in these 14 countries, rotavirus vaccine would annually prevent 144 746 (90% confidence interval [CI], 128 821-156 707) hospitalizations and 4124 deaths (90% CI, 3740-4239) due to rotavirus in their first 5 years of life but could cause an additional 172 hospitalizations (90% CI, 126-293) and 10 deaths (90% CI, 6-17) due to intussusception, yielding benefit-risk ratios for hospitalization and death of 841:1 (90% CI, 479:1 to 1142:1) and 395:1 (90% CI, 207:1 to 526:1), respectively. In an uncertainty analysis using 10 000 simulations of our probabilistic parameters, in comparing rotavirus disease averted to intussusception events caused, the hospitalization ratio was never below 100:1, and our death ratio fell below 100:1 only once. CONCLUSIONS: The health benefits of vaccination far outweigh the short-term risks and support continued rotavirus vaccination in Latin America