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

This invention relates to the use of late out-growth endothelial progenitor cells (L-EPCs) as a cellular substrate for the generation of Induced pluripotent stem cells (iPSCs). This may be useful in the production of patient-specific tissues for disease modelling, drug and toxicology screening, tissue replacement and delivery of gene therapy.

Cellular substrate for nuclear reprogramming

A novel organoid model of human liver bud development

Characterization of bioengineered liver constructs using hepatoblast organoids (HBOs) and human liver extracellular matrix (ECM) 3D-platforms

Cilia density, distribution and beating frequency are important parameters lung tissues, for example in diagnostics of Primary Ciliary Dyskinesia, and in the study of in vitro models, e.g. derived from induced Pluripotent Stem Cells. Video microscopy can be used to characterise these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the ciliary beat frequency of very small areas, while requiring human intervention and training for their use. We propose a novel and open source method to fully characterise cilia beating frequency and motile cilia coverage in an automated fashion without user intervention. We demonstrate the ability to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterise the cilia beating frequency of all moving areas. We also show that the method can be used to combine multiple fields of view to better describe a sample without relying on small pre-selected regions of interest. This is released with a simple graphical user interface for file handling, enabling a full analysis of individual fields of view in a few minutes on a typical personal computer.

https://www.biorxiv.org/content/biorxiv/early/2023/03/24/2023.03.22.533861.full.pdf

Ludovic Vallier

Papers

Cellular substrate for nuclear reprogramming

A novel organoid model of human liver bud development

Characterization of bioengineered liver constructs using hepatoblast organoids (HBOs) and human liver extracellular matrix (ECM) 3D-platforms

Assessing motile cilia coverage and beat frequency in mammalian in-vitro cell culture tissues

Forward programming to produce hepatocytes organoids for cell based therapy