Showing papers by "Ludovic Vallier published in 2007"
TL;DR: It is shown that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells.
Abstract: Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.
1,945 citations
TL;DR: Findings reveal the underlying importance of PTN in hESC survival and its usefulness in the clonal manipulation and large‐scale propagation of hESCs.
Abstract: To identify additional growth factors for optimizing propagation of human embryonic stem cells (hESCs), we mined publicly available data sets for the transcriptomes of murine and human ESCs and feeder cells, thereby generating a list of growth factors and complementary receptors. We identified the major pathways previously reported to be important, as well as several new ones. One pathway is the Pleiotrophin (PTN)-Pleiotrophin receptor (PTPRZ1) axis. Murine fibroblasts secrete Ptn, whereas hESCs expressed PTPRZ1, which is downregulated upon differentiation. Depletion of PTPRZ1 resulted in decreased colony formation and lower recovery of hESCs. Supplementation of chemically defined medium for feeder-free propagation of hESCs with PTN allowed higher recovery of hESCs without loss of pluripotency. PTN-PTPRZ1 functions here predominantly via an antiapoptotic effect mediated in part by the activation of Akt. These findings reveal the underlying importance of PTN in hESC survival and its usefulness in the clonal manipulation and large-scale propagation of hESCs. Disclosure of potential conflicts of interest is found at the end of this article.
56 citations
TL;DR: A high yield of definitive endoderm has been achieved, and although beta-like cells can be generated in a step-wise manner, the efficiency is still low and the final product is not fully differentiated.
45 citations
TL;DR: It is demonstrated that Cre‐ERT2 can be used to control gene expression in undifferentiated and differentiated cells, thereby providing the first conditional transgene expression system that works effectively in hESCs.
Abstract: Human embryonic stem cells (hESCs) possess unique properties for studying mechanisms controlling cell fate commitment during early mammalian development. Gain of function is a common strategy to study the function of specific genes involved in these mechanisms. However, transgene toxicity can be a major limitation, especially with factors influencing proliferation or differentiation. Here, we describe an efficient method based on the inducible recombinase Cre-ERT2 for conditional gene expression in hESCs and their differentiated derivatives. Using this approach, we have established several hESC sublines inducible for the expression of the enhanced green fluorescent protein and the transforming growth factor beta family member Nodal. Together, these results demonstrate that Cre-ERT2 can be used to control gene expression in undifferentiated and differentiated cells, thereby providing the first conditional transgene expression system that works effectively in hESCs. Disclosure of potential conflicts of interest is found at the end of this article.
35 citations
Patent•
09 Nov 2007
TL;DR: In this paper, the authors describe the culture of pluripotent cells in a fully humanised chemically defined medium, where cells may be cultured over a prolonged period of time and can be controllably induced to differentiate into progenitor cells of the three primary germ layers by the addition of differentiation factors.
Abstract: This invention relates to the culture of pluripotent cells in a fully humanised chemically defined medium. Cells may be cultured over a prolonged period of time without losing their pluripotent status or may be controllably induced to differentiate into progenitor cells of the three primary germ layers by the addition of differentiation factors, for example differentiation factors which modulate one or more of the Activin/Nodal, FGF, Wnt or BMP signalling pathways.
22 citations
TL;DR: A novel protocol to use real-time PCR to map DNaseI-hypersensitive sites within the genome and presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells.
Abstract: Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS. Advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low-cost experiments with minimal need for specialist equipment. Presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells.
18 citations
Patent•
09 Nov 2007
TL;DR: The Epiblast Stem Cells' (EpiSCs) as discussed by the authors are derived from the mammalian late epiblast layer and are useful in a range of applications, including the generation of transgenic animal species.
Abstract: This invention relates to the isolation and propagation of pluripotent cells isolated from the mammalian late epiblast layer, termed Epiblast Stem Cells' (EpiSCs). These cells are useful in a range of applications, including the generation of transgenic animal species.
13 citations
Patent•
09 Nov 2007
TL;DR: In this paper, the authors concerne la culture de cellules pluripotentes dans un milieu entierement humanise chimiquement defini les cellules peuvent etre cultivees pendant une periode prolongee sans perdre leur pluripotence.
Abstract: L'invention concerne la culture de cellules pluripotentes dans un milieu entierement humanise chimiquement defini Les cellules peuvent etre cultivees pendant une periode prolongee sans perdre leur pluripotence, ou peuvent etre amenees, par induction regulee, a se differencier en cellules progenitrices des trois couches germinales primaires par addition de facteurs de differenciation, tels que des facteurs de differenciation modulant l'une ou plusieurs des voies de signalisation Activine/Nodal, FGF, Wnt ou BMP