Our capacity to sequence human genomes has exceeded our ability to interpret genetic variation. Current genomic annotations tend to exploit a single information type (e.g. conservation) and/or are restricted in scope (e.g. to missense changes). Here, we describe Combined Annotation Dependent Depletion (CADD), a framework that objectively integrates many diverse annotations into a single, quantitative score. We implement CADD as a support vector machine trained to differentiate 14.7 million high-frequency human derived alleles from 14.7 million simulated variants. We pre-compute “C-scores” for all 8.6 billion possible human single nucleotide variants and enable scoring of short insertions/deletions. C-scores correlate with allelic diversity, annotations of functionality, pathogenicity, disease severity, experimentally measured regulatory effects, and complex trait associations, and highly rank known pathogenic variants within individual genomes. The ability of CADD to prioritize functional, deleterious, and pathogenic variants across many functional categories, effect sizes and genetic architectures is unmatched by any current annotation.

/pdf/a-general-framework-for-estimating-the-relative-zgrfr3u3u8.pdf

A general framework for estimating the relative pathogenicity of human genetic variants

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Integrative analysis of 111 reference human epigenomes

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(13)00087-5.pdf

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

/pdf/highly-efficient-neural-conversion-of-human-es-and-ips-cells-5ewq762oqw.pdf

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Tumors are cellularly and molecularly heterogeneous, with subsets of undifferentiated cancer cells exhibiting stem cell-like features (CSCs). Epithelial to mesenchymal transitions (EMT) are transdifferentiation programs that are required for tissue morphogenesis during embryonic development. The EMT process can be regulated by a diverse array of cytokines and growth factors, such as transforming growth factor (TGF)-β, whose activities are dysregulated during malignant tumor progression. Thus, EMT induction in cancer cells results in the acquisition of invasive and metastatic properties. Recent reports indicate that the emergence of CSCs occurs in part as a result of EMT, for example, through cues from tumor stromal components. Recent evidence now indicates that EMT of tumor cells not only causes increased metastasis, but also contributes to drug resistance. In this review, we will provide potential mechanistic explanations for the association between EMT induction and the emergence of CSCs. We will also highlight recent studies implicating the function of TGF-β-regulated noncoding RNAs in driving EMT and promoting CSC self-renewal. Finally we will discuss how EMT and CSCs may contribute to drug resistance, as well as therapeutic strategies to overcome this clinically.

/pdf/emt-cancer-stem-cells-and-drug-resistance-an-emerging-axis-3ztp4dopjd.pdf

EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer

Haematopoietic stem cells (HSC) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. In this study, we took advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrated that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. Therefore, we propose a direct link between the molecular machineries that control cell cycle progression and EHT.

/pdf/analysis-of-endothelial-to-haematopoietic-transition-at-the-2xrwl8h9uy.pdf

Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

Increasing brown adipose tissue (BAT) mass and activation has been proposed as a potential therapeutic strategy to treat obesity and associated cardiometabolic complications. Given that obese and diabetic patients possess low amounts of BAT, an efficient way to expand their BAT mass would be necessary if BAT is to be useful. Currently, there is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Moreover, to have access to human BAT is challenging, given its low volume and being anatomically dispersed. These constrain makes detailed mechanistic studies related to BAT development and function in humans virtually impossible. To overcome these limitations, we have developed a human-relevant new protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs). Unique to this protocol is that it is chemically-defined to recapitulate a physiological step-by-step developmental path of BAT that captures transient paraxial mesoderm and BAT progenitor states, on its way to reaching the adipocyte stage finally. These hPSC-derived BAs express brown adipocyte and thermogenic markers, are insulin sensitive, and respond to β-adrenergic stimuli. This new protocol is a scalable tool to study human BAs during development.

/pdf/unravelling-the-developmental-roadmap-towards-human-brown-3pv6ecfe8a.pdf

Unravelling the developmental roadmap towards human brown adipose tissue

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting multiple cell types of the human liver. The high prevalence of NAFLD and the lack of approved therapies increase the demand for reliable models for the preclinical discovery of drug targets. In the last decade, multiple proof-of-principle studies have demonstrated human-specific NAFLD modeling in the dish. These systems have included technologies based on human induced pluripotent stem cell derivatives, liver tissue section cultures, intrahepatic cholangiocyte organoids, and liver-on-a-chip. These platforms differ in functional maturity, multicellularity, scalability, and spatial organization. Identifying an appropriate model for a specific NAFLD-related research question is challenging. Therefore, we review different platforms for their strengths and limitations in modeling NAFLD. To define the fidelity of the current human in vitro NAFLD models in depth, we define disease hallmarks within the NAFLD spectrum that range from steatosis to severe fibroinflammatory tissue injury. We discuss how the most common methods are efficacious in modeling genetic contributions and aspects of the early NAFLD-related tissue response. We also highlight the shortcoming of current models to recapitulate the complexity of inter-organ crosstalk and the chronic process of liver fibrosis-to-cirrhosis that usually takes decades in patients. Importantly, we provide methodological overviews and discuss implementation hurdles (eg, reproducibility or costs) to help choose the most appropriate NAFLD model for the individual research focus: hepatocyte injury, ductular reaction, cellular crosstalk, or other applications. In sum, we highlight current strategies and deficiencies to model NAFLD in the dish and propose a framework for the next generation of human-specific investigations. 

Modeling Nonalcoholic Fatty Liver Disease in the Dish Using Human-Specific Platforms: Strategies and Limitations

La presente invention concerne un procede d'obtention d'une population de cellules progenitrices hepatiques, ledit procede comprenant une etape consistant a cultiver des cellules endodermiques definitives avec un milieu de culture stimulant la specification hepatique. Dans un mode de realisation particulier, un tel milieu de culture stimulant la specification hepatique comprend un agoniste du recepteur de l'acide retinoique (RAR), un facteur de croissance de la famille du FGF et un inhibiteur de la voie de signalisation de l'activine.

Ludovic Vallier

Papers

Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

Unravelling the developmental roadmap towards human brown adipose tissue

Modeling Nonalcoholic Fatty Liver Disease in the Dish Using Human-Specific Platforms: Strategies and Limitations

Procédé de différenciation hépatique de cellules endodermiques définitives