Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different ...mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.
Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor ...cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.
Diverse types of blood cell (lineages) are produced from rare haematopoietic stem cells that reside in the bone marrow. This process, known as haematopoiesis, provides a valuable model for examining ...how genetic programs are established and executed in vertebrates, and also how homeostasis of blood formation is altered in leukaemias. So, how does an apparently small group of critical lineage-restricted nuclear regulatory factors specify the diversity of haematopoietic cells? Recent findings not only indicate how this may be achieved but also show the extraordinary plasticity of tissue stem cells in vivo.
Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their ...selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.
Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review ...describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes.
Much attention has focused on a small set of transcription factors that maintain human or mouse embryonic stem (ES) cells in a pluripotent state. To gain a more complete understanding of the ...regulatory network that maintains this state, we identified target promoters of nine transcription factors, including somatic cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) and others (Nanog, Dax1, Rex1, Zpf281, and Nac1), on a global scale in mouse ES cells. We found that target genes fall into two classes: promoters bound by few factors tend to be inactive or repressed, whereas promoters bound by more than four factors are largely active in the pluripotent state and become repressed upon differentiation. Furthermore, we propose a transcriptional hierarchy for reprogramming factors and broadly distinguish targets of c-Myc versus other factors. Our data provide a resource for exploration of the complex network maintaining pluripotency.
We disrupted the FOG-2 gene in mice to define its requirement in vivo. FOG-2−/− embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common ...atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2−/− hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2−/− vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. Our findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart.
The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies ...revealed that gene-disrupted GATA-1$^{-}$ embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1$^{-}$ embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1$^{-}$ embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1$^{+/-}$) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1$^{-}$ embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1$^{+/-}$ mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.
GATA-family transcription factors are critical to the development of diverse tissues. In particular, GATA-4 has been implicated in formation of the vertebrate heart. As the mouse Gata-4 knock-out is ...early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, we created mice harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2. Gata4(ki/ki) mice die just after embryonic day (E) 12.5 exhibiting features in common with Fog2(-/-) embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. We also infer that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation.
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