Wellcome Trust Sanger Institute

About: Wellcome Trust Sanger Institute is a nonprofit organization based out in Cambridge, United Kingdom. It is known for research contribution in the topics: Population & Genome. The organization has 4009 authors who have published 9671 publications receiving 1224479 citations.

Production of hepatocyte-like cells from human pluripotent stem cells

Abstract: Large-scale production of hepatocytes from a variety of genetic backgrounds would be beneficial for drug screening and to provide a source of cells to be used as a substitute for liver transplantation. However, fully functional primary hepatocytes remain difficult to expand in vitro, and circumventing this problem by using an alternative source of cells is desirable. Here we describe a 25-d protocol to direct the differentiation of human pluripotent stem cells into a near-homogenous population of hepatocyte-like cells. As cells progress through this protocol, they express genes in a chronological manner similar to that described during in vivo hepatic development. The protocol relies on culture systems devoid of serum, feeders or complex extracellular matrices, which enable molecular analyses without interference from unknown factors. This approach works efficiently with human embryonic stem cells and human induced pluripotent stem cells and was recently used to model liver diseases in vitro.

Domain architectures of σ54-dependent transcriptional activators

Abstract: Transcription is the key control point for regulation of numerous cellular activities. Bacteria regulate levels of gene expression by using transcription factors that modulate the recruitment of RNA polymerase (RNAP) to promoter elements in the DNA. Many bacteria also control gene expression by using a second class of transcription factor that uses energy from nucleotide hydrolysis to actively promote transcription initiation. The sigma (σ) subunit is required for promoter recognition and initiation of transcription by the bacterial RNAP. Typically a bacterial cell may contain several alternative σ subunits with differing sequence specificities that direct the RNAP holoenzyme to different sets of promoters. The σ54 subunit (also known as RpoN, NtrA, and σN) is unique (42) in that it shares no detectable homology with any of the other known sigma factors (e.g., σ70 and σ28). σ54-RNAP binds to specific promoter sites, with the consensus DNA sequence YTGGCACGrNNNTTGCW (6), to form a transcriptionally inactive closed complex consisting of holoenzyme bound to double-stranded DNA. In contrast to σ70-RNAP bound at its cognate promoter sites, σ54-RNAP is unable to spontaneously isomerize from a closed complex to a transcriptionally competent open complex (11, 72). To proceed with initiation of transcription, the closed complex must participate in an interaction with a transcriptional activator, involving nucleotide hydrolysis. This transcriptional activator is usually bound at least 100 bp upstream of the promoter site, and DNA looping is required for the activator to contact the closed complex and catalyze formation of the open promoter complex. In this respect the activator resembles the enhancer-binding proteins (EBPs) found in eukaryotic systems, and for this reason such activators are known as bacterial EBPs. From a protein structural point of view, EBPs share in common a σ54 interaction module (Pfam accession number PF00158) but typically have at least one additional domain (Fig. ​(Fig.1).1). In nearly all of those investigated so far, there is a DNA-binding domain containing a helix-turn-helix sequence motif, enabling the protein to bind to specific DNA enhancer elements upstream of σ54-dependent promoters (44, 47, 52, 56, 72). One exception to this scenario was recently reported (30), where Pseudomonas aeruginosa FleQ can activate transcription while bound in the downstream vicinity of the promoter. FIG. 1. Major domain architectures of bacterial EBPs. Examples of each of the known domain organizations found in bacterial EBPs are given. Sequences are identified by SwissProt/trEMBL accession numbers, except for XAC3643, TTE0180, and {"type":"entrez-protein","attrs":{"text":"CPE23358","term_id":"896862659","term_text":"CPE23358"}} ... Although the physiological advantages conferred by the σ54-EBP mode of transcription are not yet clear, activation of σ54-dependent transcription is highly regulated by environmental cues through regulatory modules in the EBPs and in some cases by interactions with other regulatory proteins. Sensory modules in EBPs include CheY-like response regulator domains, PAS domains, GAF domains, PRD modules, and V4R domains, often represented within an N-terminal region (Fig. ​(Fig.1).1). These sequence features are described in more detail later in this article. Intriguingly, recent complete genome sequences have revealed some unusual EBPs containing regions of homology to other signal transduction domains and enzymes. With the large number of complete bacterial genomes now available, and with the importance of accurate annotation of future sequence data, we feel that it is timely to survey the variety of domain architectures found in these important proteins.

Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution

Abstract: Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation

Abstract: The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.