Project description:Induction of the Arf tumor suppressor in response to hyperproliferative stress following oncogene activation activates a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although Arf is not expressed in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm. We demonstrate that expression of the mouse p19Arf protein marks late stages of extraembryonic endoderm differentiation in cultured embryoid bodies derived from either embryonic stem cells or induced pluripotent stem cells, and that Arf inactivation specifically delays the differentiation of the extraembryonic endoderm lineage, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of extraembryonic endodermal cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure extraembryonic endoderm lineage development, but not maintenance. Remarkably, a significant temporal delay in extraembryonic endoderm differentiation detected during the maturation of Arf-null embryoid bodies is rescued by enforced expression of miR-205, a micro-RNA up-regulated by p19Arf and p53. Introduction of miR-205 into Arf-null embryonic stem cells rescues defective ExEn formation and elicits a program of gene expression that controls the migration and adhesion of embryonic endodermal cells. This occurs, at least in part, through atypical regulation of genes that control the epithelial-to-mesenchymal transition in cancer cells. Our findings suggest that noncanonical and canonical roles of Arf in extraembryonic endoderm development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell-to-cell attachment and migration. 4 samples total two each at two time points in ESC development At each time point one sample was treted with miR-205 and the other with a GFP control vector
Project description:Induction of the Arf tumor suppressor in response to hyperproliferative stress following oncogene activation activates a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although Arf is not expressed in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm. We demonstrate that expression of the mouse p19Arf protein marks late stages of extraembryonic endoderm differentiation in cultured embryoid bodies derived from either embryonic stem cells or induced pluripotent stem cells, and that Arf inactivation specifically delays the differentiation of the extraembryonic endoderm lineage, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of extraembryonic endodermal cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure extraembryonic endoderm lineage development, but not maintenance. Remarkably, a significant temporal delay in extraembryonic endoderm differentiation detected during the maturation of Arf-null embryoid bodies is rescued by enforced expression of miR-205, a micro-RNA up-regulated by p19Arf and p53. Introduction of miR-205 into Arf-null embryonic stem cells rescues defective ExEn formation and elicits a program of gene expression that controls the migration and adhesion of embryonic endodermal cells. This occurs, at least in part, through atypical regulation of genes that control the epithelial-to-mesenchymal transition in cancer cells. Our findings suggest that noncanonical and canonical roles of Arf in extraembryonic endoderm development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell-to-cell attachment and migration.
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm (DE) is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm. hESCs (H9) were differentiated in the presence of Activin A and Wnt3A under low serum conditions to induce DE formation. Samples were collected at day 0 (hESCs), and day 4 (DE).
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung) We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm hESCs (Cyt49) were differentiated in the presence of Activin A and Wnt3A under low serum conditions to induce DE. formation. Samples were collected at day 0 (hESCs), and day 4 (DE).
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm. hESCs (Cyt49) were differentiated in the presence of Activin A and Wnt3A under low serum conditions to induce DE formation. Samples were collected at day 0 (2 samples), day 2 (3 samples) and day 4 (3 samples).
Project description:hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm (DE) is the first step into the pathway to endoderm derived tissues: pancreas, liver, gut, lung. We used microarrays to detail the changes in mRNA expression during the transition from pluripotent hESCs into definitive endoderm. hESCs (Cyt49) were differentiated in the presence of Activin A and Wnt3A under low serum conditions to induce DE formation. Samples were collected at day 0, day 2 and day 4.
Project description:While the reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, they also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established alongside the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. Several studies have shown that endodermal genes are upregulated in fibroblasts undergoing reprogramming, although whether endodermal genes promote or inhibit acquisition of pluripotency is unclear. We show that, in fibroblasts undergoing conventional reprogramming, OSKM-induced expression of endodermal genes leads to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to iPS cells, indicating that OSKM are sufficient to drive cells to two distinct fates during reprogramming. Sequence-based mRNA transcriptional profiling of three different cell lines (MEF, XEN, iXEN) with multiple biological replicates, under two different growth medium conditions (ESC medium, XEN medium) for XEN and iXEN cells.
Project description:While the reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, they also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established alongside the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. Several studies have shown that endodermal genes are upregulated in fibroblasts undergoing reprogramming, although whether endodermal genes promote or inhibit acquisition of pluripotency is unclear. We show that, in fibroblasts undergoing conventional reprogramming, OSKM-induced expression of endodermal genes leads to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to iPS cells, indicating that OSKM are sufficient to drive cells to two distinct fates during reprogramming.
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung) We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm
Project description:Pluripotent hESCs can differentiate into the three primary embryonic lineages (endoderm, mesoderm, ectoderm) as well as extraembryonic tissues. Definitive endoderm is the first step into the pathway to endoderm dreived tissues (pancreas, liver, gut, lung). We used microarrays to detail the changes in microRNA expression during the transition from pluripotent hESCs into definitive endoderm.