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George Q Daley

Publications and source records attributed to George Q Daley.

At least 37 records · Page 2Linked to original sources
PubMed2005

High-efficiency RNA interference in human embryonic stem cells.

RNA interference methodology suppresses gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. In this study, we used retroviral and lentiviral vectors to deliver small interfering RNAs and report high-efficiency silencing of a green fluorescent protein (GFP) trans gene and the stem cell-specific transcription factors Oct4/POU5F1 and Nanog in human embryonic stem cells. Gene knockdown of Oct4 and Nanog promotes differentiation, thereby demonstrating a role for these factors in human embryonic stem cell self-renewal.

Antigens, Surface↗
PubMed2004

Chronic myeloid leukemia: proving ground for cancer stem cells.

A central question in cancer biology is whether malignancy arises in self-renewing tissue stem cells that suffer oncogene activation or in differentiated cells that acquire properties of unremitting self-renewal? In two papers, Weissman and colleagues document both mechanisms: chronic leukemia arising by mutation affecting the hematopoietic stem cell (HSC) and acute leukemia evolving from committed granulocyte-macrophage progenitors that have acquired the self-renewal machinery of HSCs.

Hematopoietic Stem Cells↗
PubMed2004

Bayesian analysis of signaling networks governing embryonic stem cell fate decisions.

MOTIVATION: Signaling events that direct mouse embryonic stem (ES) cell self-renewal and differentiation are complex and accordingly difficult to understand in an integrated manner. We address this problem by adapting a Bayesian network learning algorithm to model proteomic signaling data for ES cell fate responses to external cues. Using this model we were able to characterize the signaling pathway influences as quantitative, logic-circuit type interactions. Our experimental dataset includes measurements for 28 signaling protein phosphorylation states across 16 different factorial combinations of cytokine and matrix stimuli as reported previously. RESULTS: The Bayesian network modeling approach allows us to uncover previously reported signaling activities related to mouse ES cell self-renewal, such as the roles of LIF and STAT3 in maintaining undifferentiated ES cell populations. Furthermore, the network predicts novel influences such as between ERK phosphorylation and differentiation, or RAF phosphorylation and differentiated cell proliferation. Visualization of the influences detected by the Bayesian network provides intuition about the underlying physiology of the signaling pathways. We demonstrate that the Bayesian networks can capture the linear, nonlinear and multistate logic interactions that connect extracellular cues, intracellular signals and consequent cell functional responses.

Algorithms↗
PubMed2004

A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth.

Many mouse models of breast cancer form large primary tumors that rarely metastasize. Models with aggressive metastasis express oncoproteins that simultaneously affect growth and apoptosis pathways. To define the role of apoptotic resistance and to model a challenge faced by tumor cells during metastatic dissemination, we focused on apoptosis induced by cell shape change. Inhibiting actin polymerization with Latrunculin-A causes cell rounding and death within hours in nontumorigenic human 10A-Ras mammary epithelial cells. In contrast, MDA-MB-231 metastatic breast tumor cells resist LA-induced death, and survive for days despite cell rounding. Infecting 10A-Ras cells with a MDA-MB-231 retroviral expression library, and selecting with Latrunculin-A repeatedly identified Bcl-xL as a suppressor of cytoskeleton-dependent death. Although Bcl-xL enhances the spread of metastatic breast tumor cell lines, the distinct effects of apoptotic resistance on tumor growth in the mammary gland and during metastasis have not been compared directly. We find that Bcl-xL overexpression in mouse mammary epithelial cells does not induce primary tumor formation or enhance MEK-induced tumorigenesis within the mammary gland environment. However, it strongly enhances metastatic potential. These results with Bcl-xL provide novel evidence that isolated apoptotic resistance can increase metastatic potential, but remain overlooked by assays based on breast tumor growth.

Animals↗
PubMed2004

Taking stock and planning for the next decade: realistic prospects for stem cell therapies for the nervous system.

In thinking about the practical application of stem cell biology to clinical situations--particularly for the central nervous system (CNS)-it is instructive to remember that the neural stem cell (NSC) field--as a prototype for somatic stem cells in general-emerged as the unanticipated byproduct of investigations by developmental neurobiologists into fundamental aspects of neural determination, commitment, and plasticity. Stem cell behavior is ultimately an expression of developmental principles, an alluring vestige from the more plastic and generative stages of organogenesis. In attempting to apply stem cell biology therapeutically, it is instructive always to bear in mind what role the stem cell plays in development and to what cues it was "designed" to respond in trying to understand the "logic" behind its behavior (both what investigators want to see and what investigators do not want to see). Furthermore, in transplantation paradigms, the interaction between engrafted NSCs and recipient host is a dynamic, complex, ongoing reciprocal interaction where both entities are constantly in flux. In this review, we propose a "roadmap" to the clinic, with a particular emphasis on flagging the "potholes" and "speed bumps" through which we must navigate. Despite the admonitions to be circumspect, we also suggest disease processes that may be within the grasp of proven stem cell properties and might be approachable in the relatively near future.

Animals↗
PubMed2004

Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling.

A number of extracellular stimuli, including soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self-renewal and differentiation behavioral responses via intracellular signaling pathways, but their net effects in combination are difficult to understand. To gain insight concerning key intracellular signals governing these behavioral responses, we employ a multivariate systems analysis of proteomic data generated from combinatorial stimulation of mouse embryonic stem cells by fibronectin, laminin, leukemia-inhibitory factor, and fibroblast growth factor 4. Phosphorylation states of 31 intracellular signaling network components were obtained across 16 different stimulus conditions at three time points by quantitative Western blotting, and partial-least-squares modeling was used to determine which components were most strongly correlated with cell proliferation and differentiation rate constants obtained from flow cytometry measurements of Oct-4 expression levels. This data-driven, multivariate (16 conditions x 31 components x 3 time points = approximately 1,500 values) proteomic approach identified a set of signaling network components most critically associated (positively or negatively) with differentiation (Stat3, Raf1, MEK, and ERK), proliferation of undifferentiated cells (MEK and ERK), and proliferation of differentiated cells (PKB alpha, Stat3, Src, and PKC epsilon). These predictions were found to be consistent with previous in vivo literature, along with direct in vitro test here by a peptide inhibitor of PKC epsilon. Our results demonstrate how a computational systems biology approach can elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic responses to extracellular cues.

Animals↗
PubMed2004

Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation.

Molecular evidence suggests a multistep process in the development of acute leukemia. Since inappropriate activation of cytokine signaling cascades is a recurring theme in human leukemia, we performed expression screens to identify genes that transform cytokine-dependent cells. Using retroviral cDNA libraries derived from peripheral blood mononuclear cells of patients with myeloproliferative disorders, we isolated numerous genes that genetically complement cytokine requirements for proliferation of BaF/3 and TF-1 cells. The majority of recovered genes represent members of the kinase family, including several previously linked to leukemogenesis. Our unbiased screen highlights the central role of kinase activation in hematopoietic cell proliferation and identifies a number of potential leukemic oncoproteins.

Animals↗
PubMed2004

In vitro gametogenesis from embryonic stem cells.

Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments.

Animals↗
PubMed2004

Origins of mammalian hematopoiesis: in vivo paradigms and in vitro models.

Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or "blood-forming" tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and differentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their differentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suffering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain.

Animals↗
PubMed2004

Nuclear transplantation, embryonic stem cells and the potential for cell therapy.

Nuclear transfer experiments in mammals have shown that the nucleus of an adult cell has the ability to direct the development of an entire organism, id est its genome is totipotent. However, these experiments did not conclusively demonstrate that the nuclei of terminally differentiated adult cells remain totipotent. It is possible that rare adult stem cells served as donors for the few surviving clones. To address this question, we have generated monoclonal mice from terminally differentiated lymphocytes that carry a single antigen receptor rearrangement in all tissues. Nuclear transfer technology may provide a powerful method for obtaining autologous cells for replacement therapy. We have demonstrated the feasibility of this concept by combining nuclear transfer with gene and cell therapy to treat the immune deficiency of Rag2 mutant mice, thus establishing a paradigm for 'therapeutic cloning'. Moreover, we will discuss the potential use of nuclear transfer to study the role of reversible genomic (epigenetic) modifications during tumorigenesis.

Animals↗
PubMed2004

Mechanisms and implications of imatinib resistance mutations in BCR-ABL.

PURPOSE OF REVIEW: Aside from bone marrow transplantation, a definitive cure for Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) has yet to be developed. Although Imatinib, the first molecularly targeted drug developed for CML has achieved a remarkable success, the emergence of resistance to this agent mitigates the prospect of a cure for this leukemia. Though a variety of resistance mechanisms can arise, in the majority of patients resistance coincides with reactivation of the tyrosine kinase activity of the BCR-ABL fusion oncoprotein. This can result from gene amplification and, more importantly, point mutations that disrupt the bind of imatinib to BCR-ABL itself. In this review, we aim to define and illuminate mechanisms of resistance and describe how drug resistance is shedding new light on kinase domain regulation. RECENT FINDINGS: In light of recent studies and publications, it is now clear that Imatinib exerts its inhibitory action by stabilizing the inactive non ATP-binding conformation of BCR-ABL and that mutations even outside the kinase domain can lead to enhanced autophosphorylation of the kinase, thereby stabilizing the active conformation that resists imatinib binding. So far, 25 different substitutions of 21 amino acid residues of BCR-ABL have been detected in CML patients. In addition, it has been recently illustrated that mutations preexist the onset of treatment and that some confer a more aggressive disease phenotype. Finally it has been shown that molecular remission is almost never reached through Imatinib therapy. SUMMARY: The most common mechanism of relapse for CML patients treated with Imatinib is the appearance of point mutations in the BCR-ABL oncogene that confer resistance to this drug. Insights into the emerging problem of resistance should promote the rational development of alternative, synergistic, and potentially curative treatment strategies.

Antineoplastic Agents↗
PubMed2004

LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.

Murine embryonic stem (mES) cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF), and activation of signal transducer and activator of transcription 3 (STAT3) via LIF receptor (LIFR) signaling appears sufficient for maintenance of mES cell pluripotency. Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored. In this study, we show that the LIFRbeta and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs. Nevertheless, despite the functional activation of the LIF-STAT3 signaling pathway, human LIF is unable to maintain the pluripotent state of hESCs. Feeder-free culture conditions that maintain hESCs in an undifferentiated state do not show activation of STAT3, suggesting that distinct signaling mechanisms govern the self-renewal of hESCs.

Active Transport, Cell Nucleus↗
PubMed2003

Derivation of embryonic germ cells and male gametes from embryonic stem cells.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Animals↗
PubMed2003

cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes.

Organogenesis is dependent on the formation of distinct cell types within the embryo. Important to this process are the hox genes, which are believed to confer positional identities to cells along the anteroposterior axis. Here, we have identified the caudal-related gene cdx4 as the locus mutated in kugelig (kgg), a zebrafish mutant with an early defect in haematopoiesis that is associated with abnormal anteroposterior patterning and aberrant hox gene expression. The blood deficiency in kgg embryos can be rescued by overexpressing hoxb7a or hoxa9a but not hoxb8a, indicating that the haematopoietic defect results from perturbations in specific hox genes. Furthermore, the haematopoietic defect in kgg mutants is not rescued by scl overexpression, suggesting that cdx4 and hox genes act to make the posterior mesoderm competent for blood development. Overexpression of cdx4 during zebrafish development or in mouse embryonic stem cells induces blood formation and alters hox gene expression. Taken together, these findings demonstrate that cdx4 regulates hox genes and is necessary for the specification of haematopoietic cell fate during vertebrate embryogenesis.

Animals↗