Showing papers by "Ludovic Vallier published in 2011"

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

Abstract: Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Pluripotency factors regulate definitive endoderm specification through eomesodermin

Abstract: Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective toward the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells and mouse epiblast stem cells to study specification of definitive endoderm in vitro. Using a combination of whole-genome expression and chromatin immunoprecipitation (ChIP) deep sequencing (ChIP-seq) analyses, we established an hierarchy of transcription factors regulating endoderm specification. Importantly, the pluripotency factors NANOG, OCT4, and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMESODERMIN (EOMES), which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development.

Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors.

Abstract: Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions. STEM CELLS 2011;29:1176–1185

The serpinopathies studying serpin polymerization in vivo.

Abstract: The serpinopathies result from point mutations in members of the serine protease inhibitor or serpin superfamily. They are characterized by the formation of ordered polymers that are retained within the cell of synthesis. This causes disease by a “toxic gain of function” from the accumulated protein and a “loss of function” as a result of the deficiency of inhibitors that control important proteolytic cascades. The serpinopathies are exemplified by the Z (Glu342Lys) mutant of α1-antitrypsin that results in the retention of ordered polymers within the endoplasmic reticulum of hepatocytes. These polymers form the intracellular inclusions that are associated with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. A second example results from mutations in the neurone-specific serpin–neuroserpin to form ordered polymers that are retained as inclusions within subcortical neurones as Collins’ bodies. These inclusions underlie the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. There are different pathways to polymer formation in vitro but not all form polymers that are relevant in vivo. It is therefore essential that protein-based structural studies are interpreted in the context of human samples and cell and animal models of disease. We describe here the biochemical techniques, monoclonal antibodies, cell biology, animal models, and stem cell technology that are useful to characterize the serpin polymers that form in vivo.

Mouse pluripotent stem cells at a glance.

Abstract: Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of an embryo at the blastocyst stage. They are characterised by the ability to proliferate indefinitely (or self-renew) in vitro while maintaining pluripotency, i. e. the capacity to differentiate into germ cells and a broad

Activin/Nodal Signaling and Pluripotency

Abstract: Maintenance of a pluripotent cell population during mammalian embryogenesis is crucial for the proper generation of extraembryonic and embryonic tissues to ensure intrauterine survival and fetal development. Pluripotent stem cells derived from early stage mammalian embryos are known as "embryonic stem cells." Such embryo-derived stem cells can proliferate indefinitely in vitro and give rise to derivatives of all three primary germ layers. Their potential for clinical and commercial applications has sparked great excitement within scientific and lay communities. Identification of the signaling pathways controlling stem cell pluripotency and differentiation provides knowledge-based approaches to manipulate stem cells for regenerative medicine. One of the signaling cascades that has been identified in the control of stem cell pluripotency and differentiation is the Activin/Nodal pathway. Here, we describe the differences among pluripotent cell types and discuss the latest findings on the molecular mechanisms involving Activin/Nodal signaling in controlling their pluripotency and differentiation.

Serum-free and feeder-free culture conditions for human embryonic stem cells.

Abstract: Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. Based on these properties of self-renewal and pluripotency, hESCs represent a unique source to generate a large quantity of certain specialized cell types with clinical interest for transplantation-based therapy. However, hESCs are usually grown in culture conditions using fetal bovine serum and mouse embryonic fibroblasts, two components that are not compatible with clinical applications. Consequently, the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here, we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders.

In vitro hepatic differentiation

Abstract: This invention relates to the induction of hepatic differentiation by culturing induced pluripotent stem (iPS) cells in an endoderm induction medium to produce a population of anterior definitive endoderm (ADE) cells and then culturing the population of ADE cells in a hepatic induction medium to produce a population of hepatic progenitor cells, which may be optionally differentiated into hepatocytes. The endoderm induction medium is a chemically defined medium which has fibroblast growth factor activity, stimulates SMAD2 and SMAD3 mediated signalling pathways and SMAD1, SMAD5 and SMAD9 mediated signalling pathways, and inhibits phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β); and the hepatic induction medium is a chemically defined medium which stimulates SMAD2 and SMAD3 mediated signalling pathways. These methods may be useful, for example, in producing hepatocytes and hepatic progenitor cells for cell-based therapies or disease modelling.

A practical guide to human stem cell biology

Abstract: Human Stem Cell Technology and Biology: A Research Guide and Laboratory Manual Edited by Gary S. Stein, Maria Borowski, Mai X. Luong, Meng-Jiao Shi, Kelly P. Smith, Priscilla Vasquez Wiley-Blackwell (2011) 419 pages ISBN 978-0-470-59545-9 £93.50/€112.20 (hardback) ![Figure][1] Do we