Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes.

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration.

Molecular Criteria for Defining the Naive Human Pluripotent State.

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development.

Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues.

Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency.

Drawing a fine line on endogenous retroelement activity.

Endogenous retroelements (EREs) are essential motors of evolution yet require careful control to prevent genomic catastrophes, notably during the vulnerable phases of epigenetic reprogramming that occur immediately after fertilization and in germ cells. Accordingly, a variety of mechanisms restrict these mobile genetic units. Previous studies have revealed the importance of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor, KAP1, in the early embryonic silencing of endogenous retroviruses and so-called SVAs, but the implication of this transcriptional repression system in the control of LINE-1, the only known active autonomous retrotransposon in the human genome, was thought to be marginal.

The developmental control of transposable elements and the evolution of higher species.

Transposable elements (TEs) account for at least 50% of the human genome. They constitute essential motors of evolution through their ability to modify genomic architecture, mutate genes and regulate gene expression. Accordingly, TEs are subject to tight epigenetic control during the earliest phases of embryonic development via histone and DNA methylation.

Evolutionally dynamic L1 regulation in embryonic stem cells.

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with, at any given time, a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage, likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis.

Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency.

Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes.

De novo DNA methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells.

Chromosome conformation capture uncovers potential genome-wide interactions between human conserved non-coding sequences.

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions.

A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus.

Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model.

Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening.

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs.

Human nocturnal frontal lobe epilepsy: pharmocogenomic profiles of pathogenic nicotinic acetylcholine receptor beta-subunit mutations outside the ion channel pore.

Certain mutations in specific parts of the neuronal nicotinic acetylcholine receptor (nAChR) subunit genes CHRNA4, CHRNB2, and probably CHRNA2, can cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). All but one of the known causative mutations are located in the second transmembrane region (TM2), which serves as the major ion poreforming domain of the receptor. Functional characterization of these ADNFLE mutations has shown that although each mutant exhibits specific properties, they all confer a gain of function with increased sensitivity to acetylcholine.

Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3.

SHFM3 is a limb malformation characterized by the absence of central digits. It has been shown that this condition is associated with tandem duplications of about 500 kb at 10q24. The Dactylaplasia mice display equivalent limb defects and the two corresponding alleles (Dac1j and Dac2j) map in the region syntenic with the duplications in SHFM3.

Identification of a novel member of the CLIC family, CLIC6, mapping to 21q22.12.

Five members of the newly identified chloride intracellular channel (CLIC) gene family of intracellular chloride channels (CLIC1-CLIC5) have previously been described in humans. Here we report the molecular cloning and initial characterisation of two splice forms of a novel member of this family, CLIC6, mapping to human chromosome 21. Two essential features distinguish CLIC6 from other members of the family.

Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts.

Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious.