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Sample records for broad cellular reprogramming

  1. Cellular Reprogramming

    PubMed Central

    Takahashi, Kazutoshi

    2014-01-01

    Nuclear reprogramming technology was first established more than 50 years ago. It can rejuvenate somatic cells by erasing the epigenetic memories and reconstructing a new pluripotent order. The recent discovery reviewed here that induced pluripotency can be achieved by a small set of transcription factors has opened up unprecedented opportunities in the pharmaceutical industry, the clinic, and laboratories. This technology allows us to access pathological studies by using patient-specific induced pluripotent stem (iPS) cells. In addition, iPS cells are also expected to be a rising star for regenerative medicine, as sources of transplantation therapy. PMID:24492711

  2. Therapeutic cloning and cellular reprogramming.

    PubMed

    Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

    2012-01-01

    Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use. PMID:22457116

  3. Cellular reprogramming and hepatocellular carcinoma development.

    PubMed

    Zheng, Yun-Wen; Nie, Yun-Zhong; Taniguchi, Hideki

    2013-12-21

    Hepatocellular carcinoma (HCC) is one of the most common cancers, and is also the leading cause of death worldwide. Studies have shown that cellular reprogramming contributes to chemotherapy and/or radiotherapy resistance and the recurrence of cancers. In this article, we summarize and discuss the latest findings in the area of cellular reprogramming in HCC. The aberrant expression of transcription factors OCT4, KLF4, SOX2, c-MYC, NANOG, and LIN28 have been also observed, and the expression of these transcription factors is associated with unfavorable clinical outcomes in HCC. Studies indicate that cellular reprogramming may play a critical role in the occurrence and recurrence of HCC. Recent reports have shown that DNA methylation, miRNAs, tumor microenvironment, and signaling pathways can induce the expression of stemness transcription factors, which leads to cellular reprogramming in HCC. Furthermore, studies indicate that therapies based on cellular reprogramming could revolutionize HCC treatment. Finally, a novel therapeutic concept is discussed: reprogramming control therapy. A potential reprogramming control therapy method could be developed based on the reprogramming demonstrated in HCC studies and applied at two opposing levels: differentiation and reprogramming. Our increasing understanding and control of cellular programming should facilitate the exploitation of this novel therapeutic concept and its application in clinical HCC treatment, which may represent a promising strategy in the future that is not restricted to liver cancer. PMID:24379607

  4. Molecular features of cellular reprogramming and development.

    PubMed

    Smith, Zachary D; Sindhu, Camille; Meissner, Alexander

    2016-03-01

    Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation. PMID:26883001

  5. Genomic stability during cellular reprogramming: Mission impossible?

    PubMed

    von Joest, Mathieu; Búa Aguín, Sabela; Li, Han

    2016-06-01

    The generation of induced pluripotent stem cells (iPSCs) from adult somatic cells is one of the most exciting discoveries in recent biomedical research. It holds tremendous potential in drug discovery and regenerative medicine. However, a series of reports highlighting genomic instability in iPSCs raises concerns about their clinical application. Although the mechanisms cause genomic instability during cellular reprogramming are largely unknown, several potential sources have been suggested. This review summarizes current knowledge on this active research field and discusses the latest efforts to alleviate the genomic insults during cellular reprogramming to generate iPSCs with enhanced quality and safety. PMID:26851988

  6. Reprogramming cellular identity for regenerative medicine

    PubMed Central

    Cherry, Anne B.C.; Daley, George Q.

    2012-01-01

    The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

  7. In Vivo Cellular Reprogramming: The Next Generation.

    PubMed

    Srivastava, Deepak; DeWitt, Natalie

    2016-09-01

    Cellular reprogramming technology has created new opportunities in understanding human disease, drug discovery, and regenerative medicine. While a combinatorial code was initially found to reprogram somatic cells to pluripotency, a "second generation" of cellular reprogramming involves lineage-restricted transcription factors and microRNAs that directly reprogram one somatic cell to another. This technology was enabled by gene networks active during development, which induce global shifts in the epigenetic landscape driving cell fate decisions. A major utility of direct reprogramming is the potential of harnessing resident support cells within damaged organs to regenerate lost tissue by converting them into the desired cell type in situ. Here, we review the progress in direct cellular reprogramming, with a focus on the paradigm of in vivo reprogramming for regenerative medicine, while pointing to hurdles that must be overcome to translate this technology into future therapeutics. PMID:27610565

  8. Cellular reprogramming: a small molecule perspective

    PubMed Central

    Nie, Baoming; Wang, Haixia; Laurent, Timothy; Ding, Sheng

    2013-01-01

    The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the expression of a few transcription factors has attracted enormous interest in biomedical research and the field of regenerative medicine. iPSCs nearly identically resemble embryonic stem cells (ESCs) and can give rise to all cell types in the body, and thus have opened new opportunities for personalized regenerative medicine and new ways of modeling human diseases. Although some studies have raised concerns about genomic stability and epigenetic memory in the resulting cells, better understanding and control of the reprogramming process should enable enhanced efficiency and higher fidelity in reprogramming. Therefore, small molecules regulating reprogramming mechanisms are valuable tools to probe the process of reprogremming and harness cell fate transitions for various applications. PMID:22959962

  9. Anti-Aging Strategies Based on Cellular Reprogramming.

    PubMed

    Ocampo, Alejandro; Reddy, Pradeep; Izpisua Belmonte, Juan Carlos

    2016-08-01

    Aging can be defined as the progressive decline in the ability of a cell or organism to resist stress and disease. Recent advances in cellular reprogramming technologies have enabled detailed analyses of the aging process, often involving cell types derived from aged individuals, or patients with premature aging syndromes. In this review we discuss how cellular reprogramming allows the recapitulation of aging in a dish, describing novel experimental approaches to investigate the aging process. Finally, we explore the role of epigenetic dysregulation as a driver of aging, discussing how epigenetic reprogramming may be harnessed to ameliorate aging hallmarks, both in vitro and in vivo. A better understanding of the reprogramming process may indeed assist the development of novel therapeutic strategies to extend a healthy lifespan. PMID:27426043

  10. Cellular reprogramming for understanding and treating human disease.

    PubMed

    Kanherkar, Riya R; Bhatia-Dey, Naina; Makarev, Evgeny; Csoka, Antonei B

    2014-01-01

    In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called "cellular Rosetta stones" because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging, and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any disease. PMID

  11. Cellular reprogramming for understanding and treating human disease

    PubMed Central

    Kanherkar, Riya R.; Bhatia-Dey, Naina; Makarev, Evgeny; Csoka, Antonei B.

    2014-01-01

    In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called “cellular Rosetta stones” because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging, and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any disease. PMID

  12. Cellular Reprogramming Using Defined Factors and MicroRNAs

    PubMed Central

    Eguchi, Takanori; Kuboki, Takuo

    2016-01-01

    Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming. PMID:27382371

  13. Implications and limitations of cellular reprogramming for psychiatric drug development.

    PubMed

    Tobe, Brian T D; Brandel, Michael G; Nye, Jeffrey S; Snyder, Evan Y

    2013-01-01

    Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming confound interpretation, (4) developing efficient differentiation strategies to relevant cell types, (5) identifying discernible cellular phenotypes meaningful for cyclic, stress induced or relapsing-remitting diseases, (6) converting phenotypes to screening assays suitable for genome-wide mechanistic studies or large collection compound testing and (7) controlling for variability in relation to disease specificity amidst low sample numbers. Coordination of material for reprogramming from patients well-characterized clinically, genetically and with neuroimaging are beginning, and initial studies have begun to identify cellular phenotypes. Finally, several psychiatric drugs have been found to alter reprogramming efficiency in vitro, suggesting further complexity in applying hiPSCs to psychiatric diseases or that some drugs influence neural differentiation moreso than generally recognized. Despite these challenges, studies utilizing hiPSCs may eventually serve to fill essential niches in the translational pipeline for the discovery of new therapeutics. PMID:24232258

  14. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  15. The Importance of Ubiquitination and Deubiquitination in Cellular Reprogramming

    PubMed Central

    Suresh, Bharathi; Lee, Junwon; Kim, Kye-Seong; Ramakrishna, Suresh

    2016-01-01

    Ubiquitination of core stem cell transcription factors can directly affect stem cell maintenance and differentiation. Ubiquitination and deubiquitination must occur in a timely and well-coordinated manner to regulate the protein turnover of several stemness related proteins, resulting in optimal embryonic stem cell maintenance and differentiation. There are two switches: an E3 ubiquitin ligase enzyme that tags ubiquitin molecules to the target proteins for proteolysis and a second enzyme, the deubiquitinating enzyme (DUBs), that performs the opposite action, thereby preventing proteolysis. In order to maintain stemness and to allow for efficient differentiation, both ubiquitination and deubiquitination molecular switches must operate properly in a balanced manner. In this review, we have summarized the importance of the ubiquitination of core stem cell transcription factors, such as Oct3/4, c-Myc, Sox2, Klf4, Nanog, and LIN28, during cellular reprogramming. Furthermore, we emphasize the role of DUBs in regulating core stem cell transcriptional factors and their function in stem cell maintenance and differentiation. We also discuss the possibility of using DUBs, along with core transcription factors, to efficiently generate induced pluripotent stem cells. Our review provides a relatively new understanding regarding the importance of ubiquitination/deubiquitination of stem cell transcription factors for efficient cellular reprogramming. PMID:26880980

  16. Cell reprogramming. Histone chaperone ASF1A is required for maintenance of pluripotency and cellular reprogramming.

    PubMed

    Gonzalez-Muñoz, Elena; Arboleda-Estudillo, Yohanna; Otu, Hasan H; Cibelli, Jose B

    2014-08-15

    Unfertilized oocytes have the intrinsic capacity to remodel sperm and the nuclei of somatic cells. The discoveries that cells can change their phenotype from differentiated to embryonic state using oocytes or specific transcription factors have been recognized as two major breakthroughs in the biomedical field. Here, we show that ASF1A, a histone-remodeling chaperone specifically enriched in the metaphase II human oocyte, is necessary for reprogramming of human adult dermal fibroblasts (hADFs) into undifferentiated induced pluripotent stem cell. We also show that overexpression of just ASF1A and OCT4 in hADFs exposed to the oocyte-specific paracrine growth factor GDF9 can reprogram hADFs into pluripotent cells. Our Report underscores the importance of studying the unfertilized MII oocyte as a means to understand the molecular pathways governing somatic cell reprogramming. PMID:25035411

  17. Transcriptional and epigenetic mechanisms of cellular reprogramming to induced pluripotency.

    PubMed

    van den Hurk, Mark; Kenis, Gunter; Bardy, Cedric; van den Hove, Daniel L; Gage, Fred H; Steinbusch, Harry W; Rutten, Bart P

    2016-08-01

    Enforced ectopic expression of a cocktail of pluripotency-associated genes such as Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). The remarkable proliferation ability of iPSCs and their aptitude to redifferentiate into any cell lineage makes these cells a promising tool for generating a variety of human tissue in vitro. Yet, pluripotency induction is an inefficient process, as cells undergoing reprogramming need to overcome developmentally imposed epigenetic barriers. Recent work has shed new light on the molecular mechanisms that drive the reprogramming of somatic cells to iPSCs. Here, we present current knowledge on the transcriptional and epigenetic regulation of pluripotency induction and discuss how variability in epigenetic states impacts iPSCs' inherent biological properties. PMID:27419933

  18. From Stealing Fire to Cellular Reprogramming: A Scientific History Leading to the 2012 Nobel Prize

    PubMed Central

    Lensch, M. William; Mummery, Christine L.

    2013-01-01

    Cellular reprogramming was recently “crowned” with the award of the Nobel Prize to two of its groundbreaking researchers, Sir John Gurdon and Shinya Yamanaka. The recent link between reprogramming and stem cells makes this appear almost a new field of research, but its historical roots have actually spanned more than a century. Here, the Nobel Prize in Physiology or Medicine 2012 is placed in its historical context. PMID:24052937

  19. PROGRAMMING AND REPROGRAMMING CELLULAR AGE IN THE ERA OF INDUCED PLURIPOTENCY

    PubMed Central

    Studer, Lorenz; Vera, Elsa; Cornacchia, Daniela

    2015-01-01

    The ability to reprogram adult somatic cells back to pluripotency presents a powerful tool to study cell fate identity and model human disease. However the reversal of cellular age during reprogramming results in an embryonic-like state of induced pluripotent stem cells (iPSCs) and their derivatives, which presents specific challenges for modeling late onset disease. This age reset requires novel methods to mimic age-related changes, but also offers opportunities to study cellular rejuvenation in real time. Here, we discuss how iPSC research may transform studies of aging and enable the precise programming of cellular age in parallel to cell fate specification. PMID:26046759

  20. A case of cellular alchemy: lineage reprogramming and its potential in regenerative medicine.

    PubMed

    Asuelime, Grace E; Shi, Yanhong

    2012-08-01

    The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting. PMID:22371436

  1. Cellular reprogramming for pancreatic β-cell regeneration: clinical potential of small molecule control

    PubMed Central

    2014-01-01

    Recent scientific breakthroughs in stem cell biology suggest that a sustainable treatment approach to cure diabetes mellitus (DM) can be achieved in the near future. However, the transplantation complexities and the difficulty in obtaining the stem cells from adult cells of pancreas, liver, bone morrow and other cells is a major concern. The epoch-making strategy of transcription-factor based cellular reprogramming suggest that these barriers could be overcome, and it is possible to reprogram any cells into functional β cells. Contemporary biological and analytical techniques help us to predict the key transcription factors needed for β-cell regeneration. These β cell-specific transcription factors could be modulated with diverse reprogramming protocols. Among cellular reprogramming strategies, small molecule approach gets proclaimed to have better clinical prospects because it does not involve genetic manipulation. Several small molecules targeting certain epigenetic enzymes and/or signaling pathways have been successful in helping to induce pancreatic β-cell specification. Recently, a synthetic DNA-based small molecule triggered targeted transcriptional activation of pancreas-related genes to suggest the possibility of achieving desired cellular phenotype in a precise mode. Here, we give a brief overview of treating DM by regenerating pancreatic β-cells from various cell sources. Through a comprehensive overview of the available transcription factors, small molecules and reprogramming strategies available for pancreatic β-cell regeneration, this review compiles the current progress made towards the generation of clinically relevant insulin-producing β-cells. PMID:24679123

  2. SCL/TAL1 in Hematopoiesis and Cellular Reprogramming.

    PubMed

    Hoang, T; Lambert, J A; Martin, R

    2016-01-01

    SCL, a transcription factor of the basic helix-loop-helix family, is a master regulator of hematopoiesis. Scl specifies lateral plate mesoderm to a hematopoietic fate and establishes boundaries by inhibiting the cardiac lineage. A combinatorial interaction between Scl and Vegfa/Flk1 sets in motion the first wave of primitive hematopoiesis. Subsequently, definitive hematopoietic stem cells (HSCs) emerge from the embryo proper via an endothelial-to-hematopoietic transition controlled by Runx1, acting with Scl and Gata2. Past this stage, Scl in steady state HSCs is redundant with Lyl1, a highly homologous factor. However, Scl is haploinsufficient in stress response, when a rare subpopulation of HSCs with very long term repopulating capacity is called into action. SCL activates transcription by recruiting a core complex on DNA that necessarily includes E2A/HEB, GATA1-3, LIM-only proteins LMO1/2, LDB1, and an extended complex comprising ETO2, RUNX1, ERG, or FLI1. These interactions confer multifunctionality to a complex that can control cell proliferation in erythroid progenitors or commitment to terminal differentiation through variations in single component. Ectopic SCL and LMO1/2 expression in immature thymocytes activates of a stem cell gene network and reprogram cells with a finite lifespan into self-renewing preleukemic stem cells (pre-LSCs), an initiating event in T-cell acute lymphoblastic leukemias. Interestingly, fate conversion of fibroblasts to hematoendothelial cells requires not only Scl and Lmo2 but also Gata2, Runx1, and Erg, indicating a necessary collaboration between these transcription factors for hematopoietic reprogramming. Nonetheless, full reprogramming into self-renewing multipotent HSCs may require additional factors and most likely, a permissive microenvironment. PMID:27137657

  3. Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming

    PubMed Central

    Shchuka, Virlana M.; Malek-Gilani, Nakisa; Singh, Gurdeep; Langroudi, Lida; Dhaliwal, Navroop K.; Moorthy, Sakthi D.; Davidson, Scott; Macpherson, Neil N.; Mitchell, Jennifer A.

    2015-01-01

    Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells. PMID:26193323

  4. Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming.

    PubMed

    Shchuka, Virlana M; Malek-Gilani, Nakisa; Singh, Gurdeep; Langroudi, Lida; Dhaliwal, Navroop K; Moorthy, Sakthi D; Davidson, Scott; Macpherson, Neil N; Mitchell, Jennifer A

    2015-01-01

    Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells. PMID:26193323

  5. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  6. Cellular alchemy and the golden age of reprogramming.

    PubMed

    Daley, George Q

    2012-12-01

    The 2012 Nobel Prize in Medicine or Physiology recognizes the architects of two of the great paradigm-shifting discoveries of the last half-century of biology. In experiments performed nearly 50 years apart, Gurdon and Yamanaka made feasible the reawakening of pluripotency inherent in all cells and challenged forever our notions of cellular identity. PMID:23217698

  7. A molecular roadmap of cellular reprogramming into iPS cells

    PubMed Central

    Polo, Jose M.; Anderssen, Endre; Walsh, Ryan M.; Schwarz, Benjamin A.; Nefzger, Christian M.; Lim, Sue Mei; Borkent, Marti; Apostolou, Effie; Alaei, Sara; Cloutier, Jennifer; Bar-Nur, Ori; Cheloufi, Sihem; Stadtfeld, Matthias; Figueroa, Maria Eugenia; Robinton, Daisy; Natesan, Sridaran; Melnick, Ari; Zhu, Jinfang; Ramaswamy, Sridhar; Hochedlinger, Konrad

    2013-01-01

    Summary Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we studied defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, while changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming. PMID:23260147

  8. Investigation of the cellular reprogramming phenomenon referred to as stimulus-triggered acquisition of pluripotency (STAP)

    PubMed Central

    Niwa, Hitoshi

    2016-01-01

    In January 2014, it was reported that strong external stimuli, such as a transient low-pH stressor, was capable of inducing the reprogramming of mammalian somatic cells, resulting in the generation of pluripotent cells. This cellular reprograming event was designated ‘stimulus-triggered acquisition of pluripotency’ (STAP) by the authors of these reports. However, after multiple instances of scientific misconduct in the handling and presentation of the data were brought to light, both reports were retracted. To investigate the actual scientific significance of the purported STAP phenomenon, we sought to repeat the original experiments based on the methods presented in the retracted manuscripts and other relevant information. As a result, we have concluded that the STAP phenomenon as described in the original studies is not reproducible. PMID:27292224

  9. Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming

    PubMed Central

    Konno, Masamitsu; Koseki, Jun; Kawamoto, Koichi; Nishida, Naohiro; Matsui, Hidetoshi; Dewi, Dyah Laksmi; Ozaki, Miyuki; Noguchi, Yuko; Mimori, Koshi; Gotoh, Noriko; Tanuma, Nobuhiro; Shima, Hiroshi; Doki, Yuichiro

    2015-01-01

    Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming. PMID:26176628

  10. Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming

    PubMed Central

    Marthaler, Adele G.; Adachi, Kenjiro; Tiemann, Ulf; Wu, Guangming; Sabour, Davood; Velychko, Sergiy; Kleiter, Ingo; Schöler, Hans R.; Tapia, Natalia

    2016-01-01

    Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming. PMID:26762895

  11. Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis

    PubMed Central

    Calderón-Gómez, Elisabeth; Lampropoulou, Vicky; Shen, Ping; Neves, Patricia; Roch, Toralf; Stervbo, Ulrik; Rutz, Sascha; Kühl, Anja A.; Heppner, Frank L.; Loddenkemper, Christoph; Anderton, Stephen M.; Kanellopoulos, Jean M.; Charneau, Pierre; Fillatreau, Simon

    2011-01-01

    Activated B cells can regulate immunity, and have been envisaged as potential cell-based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered to express antigens in a remarkably simple, rapid, and effective way with lentiviral vectors. Notably, this neither required nor induced activation of the B cells. With that approach we were able to produce reprogrammed resting B cells that inhibited antigen-specific CD4+ T cells, CD8+ T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in experimental autoimmune encephalomyelitis, and even induced complete remission from disease in mice lacking functional natural regulatory T cells, which otherwise developed a chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity. PMID:21469107

  12. Maintenance of mouse hematopoietic stem cells ex vivo by reprogramming cellular metabolism.

    PubMed

    Liu, Xia; Zheng, Hong; Yu, Wen-Mei; Cooper, Todd M; Bunting, Kevin D; Qu, Cheng-Kui

    2015-03-01

    The difficulty in maintaining the reconstituting capabilities of hematopoietic stem cells (HSCs) in culture outside of the bone marrow microenvironment has severely limited their utilization for clinical therapy. This hurdle is largely due to the differentiation of long-term stem cells. Emerging evidence suggests that energy metabolism plays an important role in coordinating HSC self-renewal and differentiation. Here, we show that treatment with alexidine dihydrochloride, an antibiotic and a selective inhibitor of the mitochondrial phosphatase Ptpmt1, which is crucial for the differentiation of HSCs, reprogrammed cellular metabolism from mitochondrial aerobic metabolism to glycolysis, resulting in a remarkable preservation of long-term HSCs ex vivo in part through hyperactivation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In addition, inhibition of mitochondrial metabolism and activation of AMPK by metformin, a diabetes drug, also decreased differentiation and helped maintain stem cells in culture. Thus, manipulating metabolic pathways represents an effective new strategy for ex vivo maintenance of HSCs. PMID:25593337

  13. Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion

    PubMed Central

    Nguyen, Van T. M.; Barozzi, Iros; Faronato, Monica; Lombardo, Ylenia; Steel, Jennifer H.; Patel, Naina; Darbre, Philippa; Castellano, Leandro; Győrffy, Balázs; Woodley, Laura; Meira, Alba; Patten, Darren K.; Vircillo, Valentina; Periyasamy, Manikandan; Ali, Simak; Frige, Gianmaria; Minucci, Saverio; Coombes, R. Charles; Magnani, Luca

    2015-01-01

    Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients. PMID:26610607

  14. Chromatin remodeling system, cancer stem-like attractors, and cellular reprogramming.

    PubMed

    Zhang, Yue; Moriguchi, Hisashi

    2011-11-01

    The cancer cell attractors theory provides a next-generation understanding of carcinogenesis and natural explanation of punctuated clonal expansions of tumor progression. The impressive notion of atavism of cancer is now updated but more evidence is awaited. Besides, the mechanisms that the ectopic expression of some germline genes result in somatic tumors such as melanoma and brain tumors are emerging but are not well understood. Cancer could be triggered by cells undergoing abnormal cell attractor transitions, and may be reversible with "cyto-education". From mammals to model organisms like Caenorhabditis elegans and Drosophila melanogaster, the versatile Mi-2β/nucleosome remodeling and histone deacetylation complexes along with their functionally related chromatin remodeling complexes (CRCs), i.e., the dREAM/Myb-MuvB complex and Polycomb group complex are likely master regulators of cell attractors. The trajectory that benign cells switch to cancerous could be the reverse of navigation of embryonic cells converging from a series of intermediate transcriptional states to a final adult state, which is supported by gene expression dynamics inspector assays and some cross-species genetic evidence. The involvement of CRCs in locking cancer attractors may help find the recipes of perturbing genes to achieve successful reprogramming such that the reprogrammed cancer cell function in the same way as the normal cells. PMID:21909785

  15. Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

    PubMed Central

    Kuo, Tse-Chun; Chen, Chun-Ting; Baron, Desiree; Onder, Tamer T.; Loewer, Sabine; Almeida, Sandra; Weismann, Cara; Xu, Ping; Houghton, Jean-Marie; Gao, Fen-Biao; Daley, George Q.; Doxsey, Stephen

    2014-01-01

    The midbody (MB) is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. MBs persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells (iPSCs) and potential cancer ‘stem cells’ (CSCs) in vivo and in vitro. MBd loss accompanies stem cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor, NBR1, to the MB protein Cep55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to iPSCs and increases in vitro tumorigenicity of cancer cells. These results suggest unexpected roles for MBds in stem cells and CSCs. PMID:21909099

  16. Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

    PubMed Central

    McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

    2013-01-01

    Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of

  17. Massive Broad Ligament Cellular Leiomyoma with Cystic Change: A Diagnostic Dilemma

    PubMed Central

    Zaheer, Sufian; Yadav, Amit Kumar; Mandal, Ashish Kumar

    2016-01-01

    Leiomyomas are known to arise from uterus, but rarely from broad ligament. Further, cellular leiomyoma of broad ligament is the least common variant reported in literature. The diagnostic dilemma arises when leiomyomas undergo degenerative changes. This poses both clinical and radiological difficulty in differentiating with an ovarian tumour. We present an unusual case of a huge broad ligament mass measuring 29x19x09cm, mimicking an ovarian tumour both clinically and radiologically. Histopathology revealed cellular leiomyoma of broad ligament with cystic and myxoid degeneration hereby being the second case reported in literature. This case is being presented not only because of the rare incidence but also due to its diagnostic confusion with ovarian malignancy on clinical evaluation and radiological findings. PMID:27190813

  18. Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

    PubMed

    Jeon, Hyojung; Waku, Tsuyoshi; Azami, Takuya; Khoa, Le Tran Phuc; Yanagisawa, Jun; Takahashi, Satoru; Ema, Masatsugu

    2016-01-01

    Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming. PMID:26943822

  19. Cellular mechanisms of deep brain stimulation: activity-dependent focal circuit reprogramming?

    PubMed Central

    Veerakumar, Avin; Berton, Olivier

    2015-01-01

    Deep brain stimulation (DBS) is a well-established treatment modality for movement disorders. As more behavioral disorders are becoming understood as specific disruptions in neural circuitry, the therapeutic realm of DBS is broadening to encompass a wider range of domains, including disorders of compulsion, affect, and memory, but current understanding of the cellular mechanisms of DBS remains limited. We review progress made during the last decade focusing in particular on how recent methods for targeted circuit manipulations, imaging and reconstruction are fostering preclinical and translational advances that improve our neurobiological understanding of DBS’s action in psychiatric disorders. PMID:26719852

  20. Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells.

    PubMed

    Muñoz-López, Álvaro; van Roon, Eddy H J; Romero-Moya, Damià; López-Millan, Belén; Stam, Ronald W; Colomer, Dolors; Nakanishi, Mahito; Bueno, Clara; Menendez, Pablo

    2016-03-01

    Although B cells have been shown to be refractory to reprogramming into pluripotency, induced pluripotent stem cells (iPSCs) have been very recently generated, at very low efficiency, from human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20 + B cells using nonintegrative tetracistronic OSKM-expressing Sendai Virus (SeV). Here, we addressed whether cell ontogeny and hierarchy influence the reprogramming efficiency of the B-cell compartment. We demonstrate that human fetal liver (FL)-derived CD19 + B cells are 110-fold easier to reprogram into iPSCs than those from CB/PB. Similarly, FL-derived CD34+CD19 + B progenitors are reprogrammed much easier than mature B cells (0.46% vs. 0.11%). All FL B-cell iPSCs carry complete VDJH rearrangements while 55% and 45% of the FL B-progenitor iPSCs carry incomplete and complete VDJH rearrangements, respectively, reflecting the reprogramming of developmentally different B progenitors (pro-B vs. pre-B) within a continuous differentiation process. Finally, our data suggest that successful B-cell reprogramming relies on active cell proliferation, and it is MYC-dependent as identical nonintegrative polycistronic SeV lacking MYC (OSKL or OSKLN) fail to reprogram B cells. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances. Stem Cells 2016;34:581-587. PMID:26850912

  1. [Reprogramming equals gambling?].

    PubMed

    David, Laurent; De Vos, John

    2013-04-01

    While somatic cell reprogramming is now part of our text books, we ignore most of the mechanisms governing this cellular transformation. The most enigmatic question is why only rare cells undergo reprogramming, and whether this is governed by stochastc or deterministic events. In the late 2012, several major studies have addressed this question through a clonal analysis of the reprogramming process in murine MEF. In this mini-review, we describe these results and discuss future perspectives based on these date to optimize and secure the derivation of iPSC. PMID:23621936

  2. Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

    PubMed Central

    Grabundzija, Ivana; Wang, Jichang; Sebe, Attila; Erdei, Zsuzsanna; Kajdi, Robert; Devaraj, Anantharam; Steinemann, Doris; Szuhai, Károly; Stein, Ulrike; Cantz, Tobias; Schambach, Axel; Baum, Christopher; Izsvák, Zsuzsanna; Sarkadi, Balázs; Ivics, Zoltán

    2013-01-01

    The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells. PMID:23275558

  3. Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo

    PubMed Central

    Kagias, Konstantinos; Ahier, Arnaud; Fischer, Nadine; Jarriault, Sophie

    2012-01-01

    Differentiated cells can be forced to change identity, either to directly adopt another differentiated identity or to revert to a pluripotent state. Direct reprogramming events can also occur naturally. We recently characterized such an event in Caenorhabditis elegans, in which a rectal cell switches to a neuronal cell. Here we have used this single-cell paradigm to investigate the molecular requirements of direct cell-type conversion, with a focus on the early steps. Our genetic analyses revealed the requirement of sem-4/Sall, egl-27/Mta, and ceh-6/Oct, members of the NODE complex recently identified in embryonic stem (ES) cells, and of the OCT4 partner sox-2, for the initiation of this natural direct reprogramming event. These four factors have been shown to individually impact on ES cell pluripotency; however, whether they act together to control cellular potential during development remained an open question. We further found that, in addition to acting at the same time, these factors physically associate, suggesting that they could act together as a NODE-like complex during this in vivo process. Finally, we have elucidated the functional domains in EGL-27/MTA that mediate its reprogramming activity in this system and have found that modulation of the posterior HOX protein EGL-5 is a downstream event to allow the initiation of Y identity change. Our data reveal unique in vivo functions in a natural direct reprogramming event for these genes that impact on ES cells pluripotency and suggest that conserved nuclear events could be shared between different cell plasticity phenomena across phyla. PMID:22493276

  4. Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo.

    PubMed

    Kagias, Konstantinos; Ahier, Arnaud; Fischer, Nadine; Jarriault, Sophie

    2012-04-24

    Differentiated cells can be forced to change identity, either to directly adopt another differentiated identity or to revert to a pluripotent state. Direct reprogramming events can also occur naturally. We recently characterized such an event in Caenorhabditis elegans, in which a rectal cell switches to a neuronal cell. Here we have used this single-cell paradigm to investigate the molecular requirements of direct cell-type conversion, with a focus on the early steps. Our genetic analyses revealed the requirement of sem-4/Sall, egl-27/Mta, and ceh-6/Oct, members of the NODE complex recently identified in embryonic stem (ES) cells, and of the OCT4 partner sox-2, for the initiation of this natural direct reprogramming event. These four factors have been shown to individually impact on ES cell pluripotency; however, whether they act together to control cellular potential during development remained an open question. We further found that, in addition to acting at the same time, these factors physically associate, suggesting that they could act together as a NODE-like complex during this in vivo process. Finally, we have elucidated the functional domains in EGL-27/MTA that mediate its reprogramming activity in this system and have found that modulation of the posterior HOX protein EGL-5 is a downstream event to allow the initiation of Y identity change. Our data reveal unique in vivo functions in a natural direct reprogramming event for these genes that impact on ES cells pluripotency and suggest that conserved nuclear events could be shared between different cell plasticity phenomena across phyla. PMID:22493276

  5. Reprogramming mammalian somatic cells.

    PubMed

    Rodriguez-Osorio, N; Urrego, R; Cibelli, J B; Eilertsen, K; Memili, E

    2012-12-01

    Somatic cell nuclear transfer (SCNT), the technique commonly known as cloning, permits transformation of a somatic cell into an undifferentiated zygote with the potential to develop into a newborn animal (i.e., a clone). In somatic cells, chromatin is programmed to repress most genes and express some, depending on the tissue. It is evident that the enucleated oocyte provides the environment in which embryonic genes in a somatic cell can be expressed. This process is controlled by a series of epigenetic modifications, generally referred to as "nuclear reprogramming," which are thought to involve the removal of reversible epigenetic changes acquired during cell differentiation. A similar process is thought to occur by overexpression of key transcription factors to generate induced pluripotent stem cells (iPSCs), bypassing the need for SCNT. Despite its obvious scientific and medical importance, and the great number of studies addressing the subject, the molecular basis of reprogramming in both reprogramming strategies is largely unknown. The present review focuses on the cellular and molecular events that occur during nuclear reprogramming in the context of SCNT and the various approaches currently being used to improve nuclear reprogramming. A better understanding of the reprogramming mechanism will have a direct impact on the efficiency of current SCNT procedures, as well as iPSC derivation. PMID:22979962

  6. Brief report: human pluripotent stem cell models of fanconi anemia deficiency reveal an important role for fanconi anemia proteins in cellular reprogramming and survival of hematopoietic progenitors.

    PubMed

    Yung, Sun K; Tilgner, Katarzyna; Ledran, Maria H; Habibollah, Saba; Neganova, Irina; Singhapol, Chatchawan; Saretzki, Gabriele; Stojkovic, Miodrag; Armstrong, Lyle; Przyborski, Stefan; Lako, Majlinda

    2013-05-01

    Fanconi anemia (FA) is a genomic instability disorder caused by mutations in genes involved in replication-dependant-repair and removal of DNA cross-links. Mouse models with targeted deletions of FA genes have been developed; however, none of these exhibit the human bone marrow aplasia. Human embryonic stem cell (hESC) differentiation recapitulates many steps of embryonic hematopoietic development and is a useful model system to investigate the early events of hematopoietic progenitor specification. It is now possible to derive patient-specific human-induced pluripotent stem cells (hiPSC); however, this approach has been rather difficult to achieve in FA cells due to a requirement for activation of FA pathway during reprogramming process which can be bypassed either by genetic complementation or reprogramming under hypoxic conditions. In this study, we report that FA-C patient-specific hiPSC lines can be derived under normoxic conditions, albeit at much reduced efficiency. These disease-specific hiPSC lines and hESC with stable knockdown of FANCC display all the in vitro hallmarks of pluripotency. Nevertheless, the disease-specific hiPSCs show a much higher frequency of chromosomal abnormalities compared to parent fibroblasts and are unable to generate teratoma composed of all three germ layers in vivo, likely due to increased genomic instability. Both FANCC-deficient hESC and hiPSC lines are capable of undergoing hematopoietic differentiation, but the hematopoietic progenitors display an increased apoptosis in culture and reduced clonogenic potential. Together these data highlight the critical requirement for FA proteins in survival of hematopoietic progenitors, cellular reprogramming, and maintenance of genomic stability. PMID:23280624

  7. Cellular reprogramming in farm animals: an overview of iPSC generation in the mammalian farm animal species.

    PubMed

    Ogorevc, J; Orehek, S; Dovč, P

    2016-01-01

    Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism's body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven't received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format. PMID:26900466

  8. Reprogramming anti-tumor immunity

    PubMed Central

    Crompton, Joseph G.; Clever, David; Vizcardo, Raul; Rao, Mahendra; Restifo, Nicholas P.

    2014-01-01

    Regenerative medicine holds great promise in replacing tissues and organs lost to degenerative disease and injury. Applying principles of cellular reprogramming for the treatment of cancer, however, are not well established. Here we present an overview of cell-based reprogramming techniques (i.e. lineage reprogramming and stimulus-triggered acquisition of pluripotency) used in regenerative medicine, and within this context, envision how the scope of regenerative medicine may be expanded to treat metastatic cancer by revitalizing an exhausted and senescent immune system. PMID:24661777

  9. Advances in reprogramming to pluripotency.

    PubMed

    Alateeq, Suad; Fortuna, Patrick R J; Wolvetang, Ernst

    2015-01-01

    Pluripotent stem cells (PSCs) derived from somatic cells represent a powerful experimental tool for investigating the molecular mechanisms underlying the disease phenotype; with prospects to advance medical therapies. They also have significant potential as a renewable source of autologous cells for cellular therapy. Various approaches for PSC derivation from somatic cells have been reported in the literature. The method used for reprogramming is particularly relevant as it may affect the characteristics and quality of PSCs. This review will present an overview of the basic strategies and methods for reprogramming to pluripotency. These strategies will be briefly discussed in the context of how the mechanism of reprogramming could influence PSC characteristics with respect to safety and quality. Aspects of the reprogramming approach that can influence PSC properties, such as culture conditions and donor cell source, are also discussed. PMID:25697500

  10. Reprogramming cells with synthetic proteins.

    PubMed

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

  11. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells

    PubMed Central

    Viswanath, Pavithra; Chaumeil, Myriam M.; Ronen, Sabrina M.

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70–90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an “oncometabolite,” inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo. PMID:27014635

  12. Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway.

    PubMed

    Corominas-Faja, Bruna; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; López-Bonet, Eugeni; Lupu, Ruth; Alarcón, Tomás; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Ángel G; Vazquez-Martin, Alejandro; Menendez, Javier A

    2013-09-15

    Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg's theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka's stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR--the master switch of cellular catabolism and anabolism--in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1-60, and TRA-1-81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44(+) and ALDEFLUOR-stained ALDH(bright) cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells

  13. Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses.

    PubMed

    Mohr, Emma L; McMullan, Laura K; Lo, Michael K; Spengler, Jessica R; Bergeron, Éric; Albariño, César G; Shrivastava-Ranjan, Punya; Chiang, Cheng-Feng; Nichol, Stuart T; Spiropoulou, Christina F; Flint, Mike

    2015-08-01

    Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention. PMID:25986249

  14. Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

    PubMed Central

    Waheed, Syed Mohsin; Ghosh, Arnab; Chakravarti, Ritu; Biswas, Ashis; Haque, Mohammad Mahfuzul; Panda, Koustubh; Stuehr, Dennis J.

    2010-01-01

    Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time period, and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structure, heme ligation, and function (three NO synthases, two cytochrome P450’s, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme level, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process. PMID:20211245

  15. Dynamic culture improves cell reprogramming efficiency.

    PubMed

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling. PMID:27031931

  16. Controlled Measurement and Comparative Analysis of Cellular Components in E. coli Reveals Broad Regulatory Changes in Response to Glucose Starvation

    PubMed Central

    Houser, John R.; Barnhart, Craig; Boutz, Daniel R.; Carroll, Sean M.; Dasgupta, Aurko; Michener, Joshua K.; Needham, Brittany D.; Papoulas, Ophelia; Sridhara, Viswanadham; Sydykova, Dariya K.; Marx, Christopher J.; Trent, M. Stephen; Barrick, Jeffrey E.; Marcotte, Edward M.; Wilke, Claus O.

    2015-01-01

    How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated. PMID:26275208

  17. Looping around Reprogramming: The Topological Memory of Induced Pluripotency.

    PubMed

    Gonzales, Kevin Andrew Uy; Ng, Huck-Hui

    2016-05-01

    Genome architecture is associated with cellular identity, but how this organization changes during reprogramming is not well understood. Now in Cell Stem Cell, Krijger et al. (2016) and Beagan et al. (2016) report 3D chromatin interaction maps before and after reprogramming, providing evidence for topological memory in induced pluripotent stem cells. PMID:27152435

  18. Transient Expression of WNT2 Promotes Somatic Cell Reprogramming by Inducing β-Catenin Nuclear Accumulation.

    PubMed

    Kimura, Mizuki; Nakajima-Koyama, May; Lee, Joonseong; Nishida, Eisuke

    2016-06-14

    Treatment with several Wnt/β-catenin signaling pathway regulators can change the cellular reprogramming efficiency; however, the dynamics and role of endogenous Wnt/β-catenin signaling in reprogramming remain largely unanswered. Here we identify the upregulation of WNT2 and subsequent β-catenin nuclear accumulation as key events in reprogramming. Transient nuclear accumulation of β-catenin occurs early in MEF reprogramming. Wnt2 is strongly expressed in the early stage of reprogramming. Wnt2 knockdown suppresses the nuclear accumulation of β-catenin and reduces the reprogramming efficiency. WNT2 overexpression promotes β-catenin nuclear accumulation and enhances the reprogramming efficiency. WNT2 contributes to the promotion of cell proliferation. Experiments with several drugs that control the Wnt pathway also indicate the importance of β-catenin nuclear accumulation in reprogramming. Our findings reveal the role of WNT2/β-catenin signaling in reprogramming. PMID:27211212

  19. Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis.

    PubMed

    Kareta, Michael S; Gorges, Laura L; Hafeez, Sana; Benayoun, Bérénice A; Marro, Samuele; Zmoos, Anne-Flore; Cecchini, Matthew J; Spacek, Damek; Batista, Luis F Z; O'Brien, Megan; Ng, Yi-Han; Ang, Cheen Euong; Vaka, Dedeepya; Artandi, Steven E; Dick, Frederick A; Brunet, Anne; Sage, Julien; Wernig, Marius

    2015-01-01

    Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function. PMID:25467916

  20. Inhibition of Pluripotency Networks by the Rb Tumor Suppressor Restricts Reprogramming and Tumorigenesis

    PubMed Central

    Kareta, Michael S.; Gorges, Laura L.; Hafeez, Sana; Benayoun, Bérénice A.; Marro, Samuele; Zmoos, Anne-Flore; Cecchini, Matthew J.; Spacek, Damek; Batista, Luis F.Z.; O’Brien, Megan; Ng, Yi-Han; Ang, Cheen Euong; Vaka, Dedeepya; Artandi, Steven E.; Dick, Frederick A.; Brunet, Anne; Sage, Julien; Wernig, Marius

    2015-01-01

    SUMMARY Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function. PMID:25467916

  1. Epigenetic Landscapes Explain Partially Reprogrammed Cells and Identify Key Reprogramming Genes

    PubMed Central

    Lang, Alex H.; Li, Hu; Collins, James J.; Mehta, Pankaj

    2014-01-01

    A common metaphor for describing development is a rugged “epigenetic landscape” where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from spin-glass physics. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells are a natural consequence of high-dimensional landscapes, and predicts that partially reprogrammed cells should be hybrids that co-express genes from multiple cell fates. We verify this prediction by reanalyzing existing datasets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity. PMID:25122086

  2. Effect of biophysical cues on reprogramming to cardiomyocytes.

    PubMed

    Sia, Junren; Yu, Pengzhi; Srivastava, Deepak; Li, Song

    2016-10-01

    Reprogramming of fibroblasts to cardiomyocytes offers exciting potential in cell therapy and regenerative medicine, but has low efficiency. We hypothesize that physical cues may positively affect the reprogramming process, and studied the effects of periodic mechanical stretch, substrate stiffness and microgrooved substrate on reprogramming yield. Subjecting reprogramming fibroblasts to periodic mechanical stretch and different substrate stiffness did not improve reprogramming yield. On the other hand, culturing the cells on microgrooved substrate enhanced the expression of cardiomyocyte genes by day 2 and improved the yield of partially reprogrammed cells at day 10. By combining microgrooved substrate with an existing optimized culture protocol, yield of reprogrammed cardiomyocytes with striated cardiac troponin T staining and spontaneous contractile activity was increased. We identified the regulation of Mkl1 activity as a new mechanism by which microgroove can affect reprogramming. Biochemical approach could only partially recapitulate the effect of microgroove. Microgroove demonstrated an additional effect of enhancing organization of sarcomeric structure, which could not be recapitulated by biochemical approach. This study provides insights into new mechanisms by which topographical cues can affect cellular reprogramming. PMID:27376554

  3. Defining the Diversity of Phenotypic Respecification Using Multiple Cell Lines and Reprogramming Regimens

    PubMed Central

    Alicea, Bradly; Murthy, Shashanka; Keaton, Sarah A.; Cobbett, Peter; Cibelli, Jose B.

    2013-01-01

    To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens–induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally “weak grip” on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines—even lines with similar origins—is likely the most direct means of improving reprogramming efficiency. PMID:23672680

  4. Defining the diversity of phenotypic respecification using multiple cell lines and reprogramming regimens.

    PubMed

    Alicea, Bradly; Murthy, Shashanka; Keaton, Sarah A; Cobbett, Peter; Cibelli, Jose B; Suhr, Steven T

    2013-10-01

    To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally "weak grip" on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines--even lines with similar origins--is likely the most direct means of improving reprogramming efficiency. PMID:23672680

  5. Reprogramming for cardiac regeneration

    PubMed Central

    Raynaud, Christophe Michel; Ahmad, Faizzan Syed; Allouba, Mona; Abou-Saleh, Haissam; Lui, Kathy O.; Yacoub, Magdi

    2014-01-01

    Treatment of cardiovascular diseases remains challenging considering the limited regeneration capacity of the heart muscle. Developments of reprogramming strategies to create in vitro and in vivo cardiomyocytes have been the focus point of a considerable amount of research in the past decades. The choice of cells to employ, the state-of-the-art methods for different reprogramming strategies, and their promises and future challenges before clinical entry, are all discussed here. PMID:25763379

  6. A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments.

    PubMed

    Zhang, M; Søndergaard, R V; Kumar, E K P; Henriksen, J R; Cui, D; Hammershøj, P; Clausen, M H; Andresen, T L

    2015-11-01

    Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements of acidic intracellular pH (pH < 4) in living cells are still challenging due to the lack of suitable nanosensors. In this paper we successfully developed a multiple sensor, a fluorophore based nanosensor, with an unprecedented broad measurement range from pH 1.4 to 7.0. In this nanosensor, three pH-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured without being too close to the limits of its pH-range. The nanosensor exhibits no susceptibility to interference by other intracellular ions at physiological concentrations. Due to its positive surface charge it is spontaneously internalized by HeLa cells and localizes to the lysosomes where the mean pH was measured at 4.6. This quadruple-labelled nanosensor performs accurate measurements of fluctuations of lysosomal pH in both directions, which was shown by treatment with the V-ATPase inhibitor bafilomycin A1 or its substrate ATP in HeLa cells. These measurements indicate that this novel quadruple-labelled nanosensor is a promising new tool for measuring the pH of acidic compartments in living cells. PMID:26393332

  7. Reprogramming Roadblocks Are System Dependent

    PubMed Central

    Chantzoura, Eleni; Skylaki, Stavroula; Menendez, Sergio; Kim, Shin-Il; Johnsson, Anna; Linnarsson, Sten; Woltjen, Knut; Chambers, Ian; Kaji, Keisuke

    2015-01-01

    Summary Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog−/− fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming. PMID:26278041

  8. Broad MICA/B Expression in the Small Bowel Mucosa: A Link between Cellular Stress and Celiac Disease

    PubMed Central

    Allegretti, Yessica L.; Bondar, Constanza; Guzman, Luciana; Cueto Rua, Eduardo; Chopita, Nestor; Fuertes, Mercedes; Zwirner, Norberto W.; Chirdo, Fernando G.

    2013-01-01

    The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B+ T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+ B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role. PMID:24058482

  9. Epimedium koreanum Nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state.

    PubMed

    Cho, Won-Kyung; Weeratunga, Prasanna; Lee, Byeong-Hoon; Park, Jun-Seol; Kim, Chul-Joong; Ma, Jin Yeul; Lee, Jong-Soo

    2015-01-01

    Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant's known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakai markedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans. PMID:25609307

  10. Epimedium koreanum Nakai Displays Broad Spectrum of Antiviral Activity in Vitro and in Vivo by Inducing Cellular Antiviral State

    PubMed Central

    Cho, Won-Kyung; Weeratunga, Prasanna; Lee, Byeong-Hoon; Park, Jun-Seol; Kim, Chul-Joong; Ma, Jin Yeul; Lee, Jong-Soo

    2015-01-01

    Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant’s known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakaimarkedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans. PMID:25609307

  11. Advances in Reprogramming-Based Study of Neurologic Disorders

    PubMed Central

    Baldwin, Kristin K.

    2015-01-01

    The technology to convert adult human non-neural cells into neural lineages, through induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer, and direct lineage reprogramming or transdifferentiation has progressed tremendously in recent years. Reprogramming-based approaches aimed at manipulating cellular identity have enormous potential for disease modeling, high-throughput drug screening, cell therapy, and personalized medicine. Human iPSC (hiPSC)-based cellular disease models have provided proof of principle evidence of the validity of this system. However, several challenges remain before patient-specific neurons produced by reprogramming can provide reliable insights into disease mechanisms or be efficiently applied to drug discovery and transplantation therapy. This review will first discuss limitations of currently available reprogramming-based methods in faithfully and reproducibly recapitulating disease pathology. Specifically, we will address issues such as culture heterogeneity, interline and inter-individual variability, and limitations of two-dimensional differentiation paradigms. Second, we will assess recent progress and the future prospects of reprogramming-based neurologic disease modeling. This includes three-dimensional disease modeling, advances in reprogramming technology, prescreening of hiPSCs and creating isogenic disease models using gene editing. PMID:25749371

  12. Spin glass model for dynamics of cell reprogramming

    NASA Astrophysics Data System (ADS)

    Pusuluri, Sai Teja; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

    2015-03-01

    Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor to another attractor. We perform Monte Carlo simulations in a simple model of the landscape. This model is based on spin glass theory and it can be used to construct a simulated epigenetic landscape starting from the experimental genomic data. We re-analyse data from several cell reprogramming experiments and compare with our simulation results. We find that the model can reproduce some of the main features of the dynamics of cell reprogramming.

  13. Susceptibility of pancreatic cancer stem cells to reprogramming

    PubMed Central

    Noguchi, Kozo; Eguchi, Hidetoshi; Konno, Masamitsu; Kawamoto, Koichi; Nishida, Naohiro; Koseki, Jun; Wada, Hiroshi; Marubashi, Shigeru; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2015-01-01

    Previous reports have indicated that reprogramming technologies may be useful for altering the malignant phenotype of cancer cells. Although somatic stem cells in normal tissues are more sensitive to reprogramming induction than differentiated cells, it remains to be elucidated whether any specific subpopulations are sensitive to reprogramming in heterogeneous tumor tissues. Here we examined the susceptibility of pancreatic cancer stem cells (CSC) and non-CSC to reprogramming. To characterize CSC populations, we focused on c-Met signaling, which has been identified as a marker of CSC in mouse experiments in vivo. Cells that expressed high levels of c-Met showed higher CSC properties, such as tumor-initiating capacity, and resistance to gemcitabine. Real-time reverse transcription-polymerase chain reaction in cells expressing high levels of c-Met revealed endogenous expression of reprogramming factors, such as OCT3/4, SOX2, KLF4 and cMYC. Introduction of these four factors resulted in higher alkaline phosphatase staining in cells with high c-Met expression than in controls. Therefore, the study results demonstrate that cellular reprogramming may be useful for extensive epigenetic modification of malignant features of pancreatic CSC. PMID:26298849

  14. Reprogramming of human somatic cells by bacteria.

    PubMed

    Ito, Naofumi; Ohta, Kunimasa

    2015-05-01

    In general, it had been believed that the cell fate restriction of terminally differentiated somatic cells was irreversible. In 1952, somatic cell nuclear transfer (SCNT) was introduced to study early embryonic development in frogs. So far, various mammalian species have been successfully cloned using the SCNT technique, though its efficiency is very low. Embryonic stem (ES) cells were the first pluripotent cells to be isolated from an embryo and have a powerful potential to differentiate into more than 260 types of cells. The generation of induced pluripotent stem (iPS) cells was a breakthrough in stem cell research, and the use of these iPS cells has solved problems such as low efficiency and cell fate restriction. These cells have since been used for clinical application, disease investigation, and drug selection. As it is widely accepted that the endosymbiosis of Archaea into eukaryotic ancestors resulted in the generation of eukaryotic cells, we examined whether bacterial infection could alter host cell fate. We previously showed that when human dermal fibroblast (HDF) cells were incorporated with lactic acid bacteria (LAB), the LAB-incorporated HDF cells formed clusters and expressed a subset of common pluripotent markers. Moreover, LAB-incorporated cell clusters could differentiate into cells derived from each of the three germinal layers both in vivo and in vitro, indicating successful reprogramming of host HDF cells by LAB. In the current review, we introduce the existing examples of cellular reprogramming by bacteria and discuss their nuclear reprogramming mechanisms. PMID:25866152

  15. Small-Molecule-Based Lineage Reprogramming Creates Functional Astrocytes.

    PubMed

    Tian, E; Sun, Guoqiang; Sun, Guihua; Chao, Jianfei; Ye, Peng; Warden, Charles; Riggs, Arthur D; Shi, Yanhong

    2016-07-19

    Growing evidence indicates important roles for astrocytes in neurodevelopment and diseases. However, astrocytes and their roles in these processes remain poorly understood. Despite recent progress in reprogramming somatic cells into different types of neural cells, reprogramming to astrocytes has lagged. Here, we show that functional astrocytes can be generated from mammalian fibroblasts using only small molecules. Induced mouse astrocytes resemble primary astrocytes in astrocytic gene expression and epigenomic status and exhibit functional properties in promoting neuronal maturation, glutamate uptake, and calcium signaling. Moreover, these cells can recapitulate the Alexander disease phenotype of protein aggregation when expressing Gfap with a disease-causing mutation. The same compounds can also reprogram human fibroblasts into astroglial progenitor cells that can further mature into functional astrocytes. These chemically induced astrocytes may provide cellular models to uncover roles of astrocytes in normal neurodevelopment and pathogenesis of neurological diseases. PMID:27396343

  16. Toward reprogramming bacteria with small molecules and RNA.

    PubMed

    Gallivan, Justin P

    2007-12-01

    A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior. A remaining challenge is to control how and when the modified genes are expressed. Several recent studies have highlighted how synthetic riboswitches, which are RNA sequences that undergo a ligand-induced conformational change to alter gene expression, can be used to reprogram how bacteria respond to small molecules. PMID:17967431

  17. Pleurotus eryngii Polysaccharide Promotes Pluripotent Reprogramming via Facilitating Epigenetic Modification.

    PubMed

    Deng, Wenwen; Cao, Xia; Wang, Yan; Yu, Qingtong; Zhang, Zhijian; Qu, Rui; Chen, Jingjing; Shao, Genbao; Gao, Xiangdong; Xu, Ximing; Yu, Jiangnan

    2016-02-17

    Pleurotus eryngii is a medicinal/edible mushroom with great nutritional value and bioactivity. Its polysaccharide has recently been developed into an effective gene vector via cationic modification. In the present study, cationized P. eryngii polysaccharide (CPS), hybridized with calcium phosphate (CP), was used to codeliver plasmids (Oct4, Sox2, Klf4, c-Myc) for generating induced pluripotent stem cells (iPSCs). The results revealed that the hybrid nanoparticles could significantly enhance the process and efficiency of reprogramming (1.6-fold increase) compared with the CP nanoparticles. The hybrid CPS also facilitated epigenetic modification during the reprogramming. Moreover, these hybrid nanoparticles exhibited multiple pathways (both caveolae- and clathrin-mediated endocytosis) in their cellular internalization, which accounted for the improved iPSCs generation. These findings therefore present a novel application of P. eryngii polysaccharide in pluripotent reprogramming via active epigenetic modification. PMID:26809505

  18. The Role of microRNAs in Animal Cell Reprogramming.

    PubMed

    Cruz-Santos, María Concepción; Aragón-Raygoza, Alejandro; Espinal-Centeno, Annie; Arteaga-Vázquez, Mario; Cruz-Hernández, Andrés; Bako, Laszlo; Cruz-Ramírez, Alfredo

    2016-07-15

    Our concept of cell reprogramming and cell plasticity has evolved since John Gurdon transferred the nucleus of a completely differentiated cell into an enucleated Xenopus laevis egg, thereby generating embryos that developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4, and c-myc has evidenced the plasticity of the genome to change the expression program and cell phenotype by driving differentiated cells to the pluripotent state. Beyond these milestone achievements, research in artificial cell reprogramming has been focused on other molecules that are different than transcription factors. Among the candidate molecules, microRNAs (miRNAs) stand out due to their potential to control the levels of proteins that are involved in cellular processes such as self-renewal, proliferation, and differentiation. Here, we review the role of miRNAs in the maintenance and differentiation of mesenchymal stem cells, epimorphic regeneration, and somatic cell reprogramming to induced pluripotent stem cells. PMID:27224014

  19. Toward Reprogramming Bacteria with Small Molecules and RNA

    PubMed Central

    Gallivan, Justin P.

    2007-01-01

    Summary A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior. A remaining challenge is to control how and when the modified genes are expressed. Several recent studies have highlighted how synthetic riboswitches, which are RNA sequences that undergo a ligand-induced conformational change to alter gene expression, can be used to reprogram how bacteria respond to small molecules. PMID:17967431

  20. Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion.

    PubMed

    Poleganov, Marco Alexander; Eminli, Sarah; Beissert, Tim; Herz, Stephanie; Moon, Jung-Il; Goldmann, Johanna; Beyer, Arianne; Heck, Rosario; Burkhart, Isabell; Barea Roldan, Diana; Türeci, Özlem; Yi, Kevin; Hamilton, Brad; Sahin, Ugur

    2015-11-01

    mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections. PMID:26381596

  1. Reprogramming of somatic cells.

    PubMed

    Rajasingh, Johnson

    2012-01-01

    Reprogramming of adult somatic cells into pluripotent stem cells may provide an attractive source of stem cells for regenerative medicine. It has emerged as an invaluable method for generating patient-specific stem cells of any cell lineage without the use of embryonic stem cells. A revolutionary study in 2006 showed that it is possible to convert adult somatic cells directly into pluripotent stem cells by using a limited number of pluripotent transcription factors and is called as iPS cells. Currently, both genomic integrating viral and nonintegrating nonviral methods are used to generate iPS cells. However, the viral-based technology poses increased risk of safety, and more studies are now focused on nonviral-based technology to obtain autologous stem cells for clinical therapy. In this review, the pros and cons of the present iPS cell technology and the future direction for the successful translation of this technology into the clinic are discussed. PMID:22917226

  2. Wound signaling of regenerative cell reprogramming.

    PubMed

    Lup, Samuel Daniel; Tian, Xin; Xu, Jian; Pérez-Pérez, José Manuel

    2016-09-01

    Plants are sessile organisms that must deal with various threats resulting in tissue damage, such as herbivore feeding, and physical wounding by wind, snow or crushing by animals. During wound healing, phytohormone crosstalk orchestrates cellular regeneration through the establishment of tissue-specific asymmetries. In turn, hormone-regulated transcription factors and their downstream targets coordinate cellular responses, including dedifferentiation, cell cycle reactivation and vascular regeneration. By comparing different examples of wound-induced tissue regeneration in the model plant Arabidopsis thaliana, a number of key regulators of developmental plasticity of plant cells have been identified. We present the relevance of these findings and of the dynamic establishment of differential auxin gradients for cell reprogramming after wounding. PMID:27457994

  3. Directed Dedifferentiation Using Partial Reprogramming Induces Invasive Phenotype in Melanoma Cells.

    PubMed

    Knappe, Nathalie; Novak, Daniel; Weina, Kasia; Bernhardt, Mathias; Reith, Maike; Larribere, Lionel; Hölzel, Michael; Tüting, Thomas; Gebhardt, Christoffer; Umansky, Viktor; Utikal, Jochen

    2016-04-01

    The combination of cancer-focused studies and research related to nuclear reprogramming has gained increasing importance since both processes-reprogramming towards pluripotency and malignant transformation-share essential features. Studies have revealed that incomplete reprogramming of somatic cells leads to malignant transformation indicating that epigenetic regulation associated with iPSC generation can drive cancer development [J Mol Cell Biol 2011;341-350; Cell 2012;151:1617-1632; Cell 2014;156:663-677]. However, so far it is unclear whether incomplete reprogramming also affects cancer cells and their function. In the context of melanoma, dedifferentiation correlates to therapy resistance in mouse studies and has been documented in melanoma patients [Nature 2012;490:412-416; Clin Cancer Res 2014;20:2498-2499]. Therefore, we sought to investigate directed dedifferentiation using incomplete reprogramming of melanoma cells. Using a murine model we investigated the effects of partial reprogramming on the cellular plasticity of melanoma cells. We demonstrate for the first time that induced partial reprogramming results in a reversible phenotype switch in melanoma cells. Partially reprogrammed cells at day 12 after transgene induction display elevated invasive potential in vitro and increased lung colonization in vivo. Additionally, using global gene expression analysis of partially reprogrammed cells, we identified SNAI3 as a novel invasion-related marker in human melanoma. SNAI3 expression correlates with tumor thickness in primary melanomas and thus, may be of prognostic value. In summary, we show that investigating intermediate states during the process of reprogramming melanoma cells can reveal novel insights into the pathogenesis of melanoma progression. We propose that deeper analysis of partially reprogrammed melanoma cells may contribute to identification of yet unknown signaling pathways that can drive melanoma progression. Stem Cells 2016;34:832-846. PMID

  4. Metabolic reprogramming: a hallmark of viral oncogenesis.

    PubMed

    Lévy, P; Bartosch, B

    2016-08-11

    More than 1 in 10 cases of cancer in the world are due to chronic viral infections. Viruses induce oncogenesis by targeting the same pathways known to be responsible for neoplasia in tumor cells, such as control of cell cycle progression, cell migration, proliferation and evasion from cell death and the host's immune defense. In addition, metabolic reprogramming has been identified over a century ago as a requirement for growth of transformed cells. Renewed interest in this topic has emerged recently with the discovery that basically all metabolic changes in tumor cells are finely orchestrated by oncogenes and tumor suppressors. Indeed, cancer cells activate biosynthetic pathways in order to provide them with sufficient levels of energy and building blocks to proliferate. Interestingly, viruses introduce into their host cells similar metabolic adaptations, and importantly, it seems that they depend on these changes for their persistence and amplification. The central carbon metabolism, for example, is not only frequently altered in tumor cells but also modulated by human papillomavirus, hepatitis B and C viruses, Epstein-Barr virus and Kaposi's Sarcoma-associated virus. Moreover, adenoviruses (Ad) and human cytomegalovirus, which are not directly oncogenic but present oncomodulatory properties, also divert cellular metabolism in a tumor cell-like mnner. Thus, metabolic reprogramming appears to be a hallmark of viral infection and provides an interesting therapeutic target, in particular, for oncogenic viruses. Therapeutic targeting of metabolic pathways may not only allow to eliminate or control the viral infection but also to prevent virus-induced carcinogenesis. PMID:26686092

  5. Totipotency, Pluripotency and Nuclear Reprogramming

    NASA Astrophysics Data System (ADS)

    Mitalipov, Shoukhrat; Wolf, Don

    Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

  6. Totipotency, Pluripotency and Nuclear Reprogramming

    PubMed Central

    Mitalipov, Shoukhrat; Wolf, Don

    2009-01-01

    Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient’s own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations. PMID:19343304

  7. Vitamin C modulates TET1 function during somatic cell reprogramming.

    PubMed

    Chen, Jiekai; Guo, Lin; Zhang, Lei; Wu, Haoyu; Yang, Jiaqi; Liu, He; Wang, Xiaoshan; Hu, Xiao; Gu, Tianpeng; Zhou, Zhiwei; Liu, Jing; Liu, Jiadong; Wu, Hongling; Mao, Shi-Qing; Mo, Kunlun; Li, Yingying; Lai, Keyu; Qi, Jing; Yao, Hongjie; Pan, Guangjin; Xu, Guo-Liang; Pei, Duanqing

    2013-12-01

    Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation. PMID:24162740

  8. Transcriptional analysis of pluripotency reveals the Hippo pathway as a barrier to reprogramming

    PubMed Central

    Qin, Han; Blaschke, Kathryn; Wei, Grace; Ohi, Yuki; Blouin, Laure; Qi, Zhongxia; Yu, Jingwei; Yeh, Ru-Fang; Hebrok, Matthias; Ramalho-Santos, Miguel

    2012-01-01

    Pluripotent stem cells are derived from culture of early embryos or the germline and can be induced by reprogramming of somatic cells. Barriers to reprogramming that stabilize the differentiated state and have tumor suppression functions are expected to exist. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline with pluripotent and somatic cells, in vivo and in vitro. There is a remarkable global expression of the transcriptional program for pluripotency in primordial germ cells (PGCs). We identify parallels between PGC reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming. PMID:22286172

  9. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.

    PubMed

    Treutlein, Barbara; Lee, Qian Yi; Camp, J Gray; Mall, Moritz; Koh, Winston; Shariati, Seyed Ali Mohammad; Sim, Sopheak; Neff, Norma F; Skotheim, Jan M; Wernig, Marius; Quake, Stephen R

    2016-06-16

    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation. PMID:27281220

  10. Energy metabolism in nuclear reprogramming.

    PubMed

    Folmes, Clifford D L; Nelson, Timothy J; Terzic, Andre

    2011-12-01

    Nuclear reprogramming with stemness factors enables resetting of somatic differentiated tissue back to the pluripotent ground state. Recent evidence implicates mitochondrial restructuring and bioenergetic plasticity as key components underlying execution of orchestrated dedifferentiation and derivation of induced pluripotent stem cells. Aerobic to anaerobic transition of somatic oxidative energy metabolism into a glycolytic metabotype promotes proficient reprogramming, establishing a novel regulator of acquired stemness. Metabolomic profiling has further identified specific metabolic remodeling traits defining lineage redifferentiation of pluripotent cells. Therefore, mitochondrial biogenesis and energy metabolism comprise a vital axis for biomarker discovery, intimately reflecting the molecular dynamics fundamental for the resetting and redirection of cell fate. PMID:22103608

  11. Energy metabolism in nuclear reprogramming

    PubMed Central

    Folmes, Clifford DL; Nelson, Timothy J; Terzic, Andre

    2012-01-01

    Nuclear reprogramming with stemness factors enables resetting of somatic differentiated tissue back to the pluripotent ground state. Recent evidence implicates mitochondrial restructuring and bioenergetic plasticity as key components underlying execution of orchestrated dedifferentiation and derivation of induced pluripotent stem cells. Aerobic to anaerobic transition of somatic oxidative energy metabolism into a glycolytic metabotype promotes proficient reprogramming, establishing a novel regulator of acquired stemness. Metabolomic profiling has further identified specific metabolic remodeling traits defining lineage redifferentiation of pluripotent cells. Therefore, mitochondrial biogenesis and energy metabolism comprise a vital axis for biomarker discovery, intimately reflecting the molecular dynamics fundamental for the resetting and redirection of cell fate. PMID:22103608

  12. Epigenetic reprogramming in plant sexual reproduction.

    PubMed

    Kawashima, Tomokazu; Berger, Frédéric

    2014-09-01

    Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation. PMID:25048170

  13. A predictive computational framework for direct reprogramming between human cell types.

    PubMed

    Rackham, Owen J L; Firas, Jaber; Fang, Hai; Oates, Matt E; Holmes, Melissa L; Knaupp, Anja S; Suzuki, Harukazu; Nefzger, Christian M; Daub, Carsten O; Shin, Jay W; Petretto, Enrico; Forrest, Alistair R R; Hayashizaki, Yoshihide; Polo, Jose M; Gough, Julian

    2016-03-01

    Transdifferentiation, the process of converting from one cell type to another without going through a pluripotent state, has great promise for regenerative medicine. The identification of key transcription factors for reprogramming is currently limited by the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable. Here we present a predictive system (Mogrify) that combines gene expression data with regulatory network information to predict the reprogramming factors necessary to induce cell conversion. We have applied Mogrify to 173 human cell types and 134 tissues, defining an atlas of cellular reprogramming. Mogrify correctly predicts the transcription factors used in known transdifferentiations. Furthermore, we validated two new transdifferentiations predicted by Mogrify. We provide a practical and efficient mechanism for systematically implementing novel cell conversions, facilitating the generalization of reprogramming of human cells. Predictions are made available to help rapidly further the field of cell conversion. PMID:26780608

  14. Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

    PubMed Central

    Lina, Taslima T.; Farris, Tierra; Luo, Tian; Mitra, Shubhajit; Zhu, Bing; McBride, Jere W.

    2016-01-01

    Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival. PMID:27303657

  15. Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy.

    PubMed

    Lina, Taslima T; Farris, Tierra; Luo, Tian; Mitra, Shubhajit; Zhu, Bing; McBride, Jere W

    2016-01-01

    Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival. PMID:27303657

  16. Cell reprogramming: Into the groove

    NASA Astrophysics Data System (ADS)

    Xu, Yan; Liu, Longqi; Laslett, Andrew L.; Esteban, Miguel A.

    2013-12-01

    Adult cells can be routinely reprogrammed into pluripotent stem cells by chemical and genetic means, such as the expression of a cocktail of exogenous transcription factors. It is now shown that growing cells on substrates with aligned features such as microgrooves can enhance this process.

  17. A twist in zygotic reprogramming.

    PubMed

    Messerschmidt, Daniel M

    2016-02-01

    The first hours of mammalian embryogenesis are devoted to extensive epigenetic reprogramming. One hallmark is active demethylation of the paternal genome by Tet (ten-eleven translocation) enzymes. However, the process is now shown to be Tet-independent at first, with Tet enzymes only counteracting hitherto underappreciated de novo DNA methylation activity in later zygotic stages. PMID:26820436

  18. Terminal differentiation and loss of tumorigenicity of human cancers via pluripotency based reprogramming

    PubMed Central

    Zhang, Xi; Cruz, Filemon Dela; Terry, Melissa; Remotti, Fabrizio; Matushansky, Igor

    2012-01-01

    Pluripotent cells can be derived from various types of somatic cells by nuclear reprogramming using defined transcription factors. It is however unclear whether human cancer cells can be similarly reprogrammed and subsequently terminally differentiated with abrogation of tumorigenicity. Here, using sarcomas we show that human derived complex karyotype solid tumors: (1) can be reprogrammed into a pluripotent-like state as defined by all in vitro criteria used to define pluripotent stem cells generated from somatic cells; (2) can be terminally differentiated into mature connective tissue and red blood cells; and (3) terminal differentiation is accompanied with loss of both proliferation and tumorigenicity. We go on to perform the first global DNA promoter methylation and gene expression analyses comparing human cancers to their reprogrammed counterparts and report that reprogramming/differentiation results in significant epigenetic remodeling of oncogenes and tumor suppressors; while not significantly altering the differentiation status of the reprogrammed cancer cells, in essence de-differentiating them to a state slightly before the mesenchymal stem cell differentiation stage. Our data demonstrates that direct nuclear reprogramming can restore terminal differentiation potential to human derived cancer cells, with simultaneous loss of tumorigenicity, without the need to revert to an embryonic state. We anticipate that our models would serve as a starting point to more fully assess how nuclear reprogramming overcomes the multitude of genetic and epigenetic aberrancies inherent in human cancers to restore normal terminal differentiation pathways. Finally, these findings suggest that nuclear reprogramming may be a broadly applicable therapeutic strategy for the treatment of cancer. PMID:22777357

  19. Reprogramming of adult rod photoreceptors prevents retinal degeneration

    PubMed Central

    Montana, Cynthia L.; Kolesnikov, Alexander V.; Shen, Susan Q.; Myers, Connie A.; Kefalov, Vladimir J.; Corbo, Joseph C.

    2013-01-01

    A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration. PMID:23319618

  20. A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

    PubMed Central

    Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

    2015-01-01

    SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

  1. Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

    PubMed Central

    Mills, Jason C.; Sansom, Owen J.

    2016-01-01

    It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

  2. Knockdown of Brm and Baf170, Components of Chromatin Remodeling Complex, Facilitates Reprogramming of Somatic Cells.

    PubMed

    Jiang, Zongliang; Tang, Yong; Zhao, Xueming; Zhang, Mingyuan; Donovan, David M; Tian, Xiuchun Cindy

    2015-10-01

    The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells, esBAF contains Brg1 and Baf155, while their homologs, Brm and Baf170, are present in BAF of somatic cells. In this study, we sought to determine whether Brm and Baf170 play any roles in induced pluripotent stem cell (iPSC) reprogramming by using shRNA-mediated knockdown studies in the mouse model. We found that knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improve the numbers of iPSC colonies formed. We further showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed somatic cells (pre-iPSCs). Finally, we found that the expression of Brm and Baf170 during reprogramming was regulated by Jak/Stat3 activity. Taken together, these data suggest that inhibiting somatic BAF improves complete reprogramming by facilitating the activation of the pluripotency circuitry. PMID:26121422

  3. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors

    PubMed Central

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  4. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors.

    PubMed

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  5. The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

    PubMed Central

    Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

    2014-01-01

    Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

  6. JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells

    PubMed Central

    Neganova, Irina; Shmeleva, Evgenija; Munkley, Jennifer; Chichagova, Valeria; Anyfantis, George; Anderson, Rhys; Passos, Joao; Elliott, David J.; Armstrong, Lyle

    2016-01-01

    Abstract Reprogramming of somatic cells to the phenotypic state termed “induced pluripotency” is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c‐Jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well‐established molecular barriers in human somatic cell

  7. Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

    PubMed Central

    Suhr, Steven T.; Chang, Eun Ah; Rodriguez, Ramon M.; Wang, Kai; Ross, Pablo J.; Beyhan, Zeki; Murthy, Shashanka; Cibelli, Jose B.

    2009-01-01

    Background Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell “rejuvenation.” Methodology/Principal Findings We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. Conclusions/Significance While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age. PMID:19956585

  8. p53, Stem Cells, and Reprogramming

    PubMed Central

    Spike, Benjamin T.; Wahl, Geoffrey M.

    2011-01-01

    p53 is well recognized as a potent tumor suppressor. In its classic role, p53 responds to genotoxic insults by inducing cell cycle exit or programmed cell death to limit the propagation of cells with corrupted genomes. p53 is also implicated in a variety of other cellular processes in which its involvement is less well understood including self-renewal, differentiation, and reprogramming. These activities represent an emerging area of intense interest for cancer biologists, as they provide potential mechanistic links between p53 loss and the stem cell–like cellular plasticity that has been suggested to contribute to tumor cell heterogeneity and to drive tumor progression. Despite accumulating evidence linking p53 loss to stem-like phenotypes in cancer, it is not yet understood how p53 contributes to acquisition of “stemness” at the molecular level. Whether and how stem-like cells confer survival advantages to propagate the tumor also remain to be resolved. Furthermore, although it seems reasonable that the combination of p53 deficiency and the stem-like state could contribute to the genesis of cancers that are refractory to treatment, direct linkages and mechanistic underpinnings remain under investigation. Here, we discuss recent findings supporting the connection between p53 loss and the emergence of tumor cells bearing functional and molecular similarities to stem cells. We address several potential molecular and cellular mechanisms that may contribute to this link, and we discuss implications of these findings for the way we think about cancer progression. PMID:21779509

  9. FLCN: A new regulator of AMPK-dependent Warburg metabolic reprogramming

    PubMed Central

    Gingras, Marie-Claude; Pause, Arnim

    2014-01-01

    Tumor cells manage their energy to support aberrant proliferation by reprogramming their cellular metabolism, for example through the Warburg effect. Although AMPK is a major regulator of energy homeostasis, its role in cancer metabolic adaptation is unclear. We recently identified the tumor suppressor folliculin as a new regulator of AMPK-dependent metabolic transformation. PMID:27308336

  10. Phosphatidic Acid Improves Reprogramming to Pluripotency by Reducing Apoptosis.

    PubMed

    Jiang, Yuan; Du, Mingxia; Wu, Menghua; Zhu, Yanbing; Zhao, Xing; Cao, Xu; Li, Xin; Long, Peipei; Li, Wei; Hu, Baoyang

    2016-01-01

    Generation of induced pluripotent stem cells (iPSCs) requires a considerable amount of lipids, such as phosphatidic acid (PA), to meet the needs of subsequent rapid cell division and proliferation. However, it is unclear whether PA, a biosynthetic precursor of lipids, affects reprogramming. By using lentiviral expression of the Yamanaka factors in mouse embryonic fibroblasts for reprogramming, we identified that PA is beneficial for the generation of iPS colonies. Inhibiting the generation of cellular PA dramatically decreased the number of iPSCs. Consistently, 400 μM PA improved iPSC generation by more than 4- to 5-fold. iPSCs generated in the presence of PA (PA-iPS) expressed pluripotent markers such as Oct4 and Nanog, differentiated into cells of the three germ layers in vitro, and contributed to chimeric mice when injected into blastocysts. The improved efficiency was primarily due to reduction of apoptosis as sufficient PA increased the accumulation of cardiolipin in the inner membrane of the mitochondria, which reduced the release of cytochrome c and, in turn, suppressed apoptosis by inhibiting caspase-7. The relatively higher amount of Bcl-2 in PA treatment also inhibited apoptosis. In addition, an accompanied sequential change from epithelial-to-mesenchymal transition (EMT) at the initial phase of reprogramming to mesenchymal-to-epithelial transition (MET) was also detected. Our microarray data, which also supported our results, indicated the presence of significant membrane enrichment genes, thus suggesting that PA may function through membrane-anchored proteins. We thus identified a novel type of culture supplement that improves the efficiency of reprogramming and could be valuable for the generation of high-quality iPS cells. PMID:26451619

  11. Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate

    PubMed Central

    Ward, Patrick S.; Thompson, Craig B.

    2012-01-01

    Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria, but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer. PMID:22439925

  12. Chinese Herbs Interfering with Cancer Reprogramming Metabolism

    PubMed Central

    Zhong, Zhangfeng; Qiang, William W.; Tan, Wen; Zhang, Haotian; Wang, Shengpeng; Wang, Chunming; Qiang, Wenan; Wang, Yitao

    2016-01-01

    Emerging evidence promotes a reassessment of metabolic reprogramming regulation in cancer research. Although there exists a long history of Chinese herbs applied in cancer treatment, few reports have addressed the effects of Chinese herbal components on metabolic reprogramming, which is a central cancer hallmark involved in the slowing or prevention of chemoresistance in cancer cells. In this review, we have focused on four core elements altered by metabolic reprogramming in cancer cells. These include glucose transport, glycolysis, mitochondrial oxidative phosphorylation, and fatty acid synthesis. With this focus, we have summarized recent advances in metabolic reprogramming of cancer cells in response to specific Chinese herbal components. We propose that exploring Chinese herbal interference in cancer metabolic reprogramming might identify new therapeutic targets for cancer and more ways in which to approach metabolism-related diseases. PMID:27242914

  13. The human endogenous retrovirus K(HML-2) has a broad envelope-mediated cellular tropism and is prone to inhibition at a post-entry, pre-integration step.

    PubMed

    Kramer, Philipp; Lausch, Veronika; Volkwein, Alexander; Hanke, Kirsten; Hohn, Oliver; Bannert, Norbert

    2016-01-01

    The HERV-K(HML-2) family is the most recent addition to the collection of human endogenous retroviruses. It comprises proviruses that encode functional proteins that can assemble into replication defective particles carrying the envelope protein. Using a reconstituted HERV-K113 envelope sequence, we have analyzed its ability to mediate entry into a set of 33 cell lines from 10 species. Of these, 30 were permissive, demonstrating an amphotropism consistent with a broad expression of receptor protein(s). In an initial effort to identify a receptor for HERV-K(HML-2) we investigated whether transferrin receptor 1 and hyaluronidase 2, known cellular receptors of the closely related betaretroviruses mouse mammary tumor virus (MMTV) and Jaagsiekte sheep retrovirus (JSRV), could facilitate HERV-K(HML-2) entry. However, neither of these proteins could serve as a receptor for HERV-K(HML-2). Moreover, during attempts to further characterize the tropism of HERV-K(HML-2), we identified a cellular activity that inhibits infection at a post-entry, pre-integration step. PMID:26517399

  14. A Continuous Molecular Roadmap to iPSC Reprogramming through Progression Analysis of Single-Cell Mass Cytometry

    PubMed Central

    Zunder, Eli R.; Lujan, Ernesto; Goltsev, Yury; Wernig, Marius; Nolan, Garry P.

    2015-01-01

    SUMMARY To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4highKlf4high cells transitioned to a CD73highCD104highCD54low partially reprogrammed state. Ki67low cells from this intermediate population reverted to a MEF-like phenotype, but Ki67high cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like NanoghighSox2highCD54high population and a mesendoderm-like NanoglowSox2lowLin28high CD24highPDGFR-αhigh population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development. PMID:25748935

  15. Isonitrosoacetophenone drives transcriptional reprogramming in Nicotiana tabacum cells in support of innate immunity and defense.

    PubMed

    Djami-Tchatchou, Arnaud T; Maake, Mmapula P; Piater, Lizelle A; Dubery, Ian A

    2015-01-01

    Plants respond to various stress stimuli by activating broad-spectrum defense responses both locally as well as systemically. As such, identification of expressed genes represents an important step towards understanding inducible defense responses and assists in designing appropriate intervention strategies for disease management. Genes differentially expressed in tobacco cell suspensions following elicitation with isonitrosoacetophenone (INAP) were identified using mRNA differential display and pyro-sequencing. Sequencing data produced 14579 reads, which resulted in 198 contigs and 1758 singletons. Following BLAST analyses, several inducible plant defense genes of interest were identified and classified into functional categories including signal transduction, transcription activation, transcription and protein synthesis, protein degradation and ubiquitination, stress-responsive, defense-related, metabolism and energy, regulation, transportation, cytoskeleton and cell wall-related. Quantitative PCR was used to investigate the expression of 17 selected target genes within these categories. Results indicate that INAP has a sensitising or priming effect through activation of salicylic acid-, jasmonic acid- and ethylene pathways that result in an altered transcriptome, with the expression of genes involved in perception of pathogens and associated cellular re-programming in support of defense. Furthermore, infection assays with the pathogen Pseudomonas syringae pv. tabaci confirmed the establishment of a functional anti-microbial environment in planta. PMID:25658943

  16. Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

    PubMed Central

    Hu, Gangqing; Yu, Zu-Xi; Liu, Chengyu

    2013-01-01

    Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. PMID:24100015

  17. Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation.

    PubMed

    Ariyachet, Chaiyaboot; Tovaglieri, Alessio; Xiang, Guanjue; Lu, Jiaqi; Shah, Manasvi S; Richmond, Camilla A; Verbeke, Catia; Melton, Douglas A; Stanger, Ben Z; Mooney, David; Shivdasani, Ramesh A; Mahony, Shaun; Xia, Qing; Breault, David T; Zhou, Qiao

    2016-03-01

    The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to β cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control. PMID:26908146

  18. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

    PubMed Central

    Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

    2015-01-01

    Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

  19. Reprogramming cell fate: a changing story

    PubMed Central

    Chin, Michael T.

    2014-01-01

    Direct reprogramming of adult, lineage-determined cells from one cell fate to another has long been an elusive goal in developmental biology. Recent studies have demonstrated that forced expression of lineage-specific transcription factors in various differentiated cell types can promote the adoption of different lineages. These seminal findings have the potential to revolutionize the field of regenerative medicine by providing replacement cells for various degenerative disorders. Current reprogramming protocols, however, are inefficient in that relatively few cells in a given population can be made to undergo reprogramming and the completeness and extent of reprogramming that occurs has been questioned. At present, the fundamental molecular mechanisms involved are still being elucidated. Although the potential clinical applications are extensive, these issues will need to be addressed before direct reprogramming may be used clinically. This review will give an overview of pioneering studies in the field, will describe what is known about direct reprogramming to specific lineage types, will summarize what is known about the molecular mechanisms involved in reprogramming and will discuss challenges for the future. PMID:25364753

  20. Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

    PubMed Central

    Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

    2015-01-01

    Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

  1. Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints.

    PubMed

    Monk, David

    2015-10-01

    DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that "ubiquitous" imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans. This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease. PMID:25966912

  2. MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

    PubMed

    Ogawa, Hisataka; Wu, Xin; Kawamoto, Koichi; Nishida, Naohiro; Konno, Masamitsu; Koseki, Jun; Matsui, Hidetoshi; Noguchi, Kozou; Gotoh, Noriko; Yamamoto, Tsuyoshi; Miyata, Kanjiro; Nishiyama, Nobuhiro; Nagano, Hiroaki; Yamamoto, Hirofumi; Obika, Satoshi; Kataoka, Kazunori; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2015-01-01

    Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors. PMID:25970424

  3. Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

    PubMed

    Easley, Charles A; Miki, Toshio; Castro, Carlos A; Ozolek, John A; Minervini, Crescenzio F; Ben-Yehudah, Ahmi; Schatten, Gerald P

    2012-06-01

    Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. PMID:22686477

  4. Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells.

    PubMed

    Zhang, Yiqiang; Zhong, Jiang F; Qiu, Hongyu; MacLellan, W Robb; Marbán, Eduardo; Wang, Charles

    2015-01-01

    It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. PMID:26657817

  5. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

    PubMed Central

    Chiribao, María Laura; Libisch, Gabriela; Parodi-Talice, Adriana; Robello, Carlos

    2014-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response), a great number of transcription factors (including the majority of NFκB family members), and host metabolism (cholesterol, fatty acids, and phospholipids). These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination. PMID:24812617

  6. Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells

    PubMed Central

    Zhang, Yiqiang; Zhong, Jiang F; Qiu, Hongyu; Robb MacLellan, W.; Marbán, Eduardo; Wang, Charles

    2015-01-01

    It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. PMID:26657817

  7. Mechanisms and models of somatic cell reprogramming

    PubMed Central

    Buganim, Yosef; Faddah, Dina A.; Jaenisch, Rudolf

    2014-01-01

    Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic stem cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodeling the genome, including new insights into the function of c-Myc, and describe the different phases, markers and emerging models of reprogramming. PMID:23681063

  8. Restoring totipotency through epigenetic reprogramming

    PubMed Central

    Wasson, Jadiel A.; Ruppersburg, Chelsey C.

    2013-01-01

    Epigenetic modifications are implicated in the maintenance and regulation of transcriptional memory by marking genes that were previously transcribed to facilitate transmission of these expression patterns through cell division. During germline specification and maintenance, extensive epigenetic modifications are acquired. Yet somehow at fertilization, the fusion of the highly differentiated sperm and egg results in formation of the totipotent zygote. This massive change in cell fate implies that the selective erasure and maintenance of epigenetic modifications at fertilization may be critical for the re-establishment of totipotency. In this review, we discuss recent studies that provide insight into the extensive epigenetic reprogramming that occurs around fertilization and the mechanisms that may be involved in the re-establishment of totipotency in the embryo. PMID:23117862

  9. Reprogramming of energy metabolism as a driver of aging

    PubMed Central

    Feng, Zhaoyang; Berger, Nathan A.; Trubitsyn, Alexander

    2016-01-01

    Aging is characterized by progressive loss of cellular function and integrity. It has been thought to be driven by stochastic molecular damage. However, genetic and environmental maneuvers enhancing mitochondrial function or inhibiting glycolysis extend lifespan and promote healthy aging in many species. In post-fertile Caenorhabditis elegans, a progressive decline in phosphoenolpyruvate carboxykinase with age, and a reciprocal increase in pyruvate kinase shunt energy metabolism from oxidative metabolism to anaerobic glycolysis. This reduces the efficiency and total of energy generation. As a result, energy-dependent physical activity and other cellular functions decrease due to unmatched energy demand and supply. In return, decrease in physical activity accelerates this metabolic shift, forming a vicious cycle. This metabolic event is a determinant of aging, and is retarded by caloric restriction to counteract aging. In this review, we summarize these and other evidence supporting the idea that metabolic reprogramming is a driver of aging. We also suggest strategies to test this hypothesis PMID:26919253

  10. Reprogramming of energy metabolism as a driver of aging.

    PubMed

    Feng, Zhaoyang; Hanson, Richard W; Berger, Nathan A; Trubitsyn, Alexander

    2016-03-29

    Aging is characterized by progressive loss of cellular function and integrity. It has been thought to be driven by stochastic molecular damage. However, genetic and environmental maneuvers enhancing mitochondrial function or inhibiting glycolysis extend lifespan and promote healthy aging in many species. In post-fertile Caenorhabditis elegans, a progressive decline in phosphoenolpyruvate carboxykinase with age, and a reciprocal increase in pyruvate kinase shunt energy metabolism from oxidative metabolism to anaerobic glycolysis. This reduces the efficiency and total of energy generation. As a result, energy-dependent physical activity and other cellular functions decrease due to unmatched energy demand and supply. In return, decrease in physical activity accelerates this metabolic shift, forming a vicious cycle. This metabolic event is a determinant of aging, and is retarded by caloric restriction to counteract aging. In this review, we summarize these and other evidence supporting the idea that metabolic reprogramming is a driver of aging. We also suggest strategies to test this hypothesis. PMID:26919253

  11. Non-genetic direct reprogramming and biomimetic platforms in a preliminary study for adipose-derived stem cells into corneal endothelia-like cells.

    PubMed

    Dai, Ying; Guo, Yonglong; Wang, Chan; Liu, Qing; Yang, Yan; Li, Shanyi; Guo, Xiaoling; Lian, Ruiling; Yu, Rongjie; Liu, Hongwei; Chen, Jiansu

    2014-01-01

    Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells. PMID:25333522

  12. Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Study for Adipose-Derived Stem Cells into Corneal Endothelia-Like Cells

    PubMed Central

    Wang, Chan; Liu, Qing; Yang, Yan; Li, Shanyi; Guo, Xiaoling; Lian, Ruiling; Yu, Rongjie; Liu, Hongwei; Chen, Jiansu

    2014-01-01

    Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells. PMID:25333522

  13. Stem cell reprogramming: A 3D boost

    NASA Astrophysics Data System (ADS)

    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

    Biophysical factors in an optimized three-dimensional microenvironment enhance the reprogramming efficiency of human somatic cells into pluripotent stem cells when compared to traditional cell-culture substrates.

  14. Systems biology of stem cell fate and cellular reprogramming

    PubMed Central

    MacArthur, Ben D.; Ma’ayan, Avi; Lemischka, Ihor R.

    2010-01-01

    Stem cell differentiation and the maintenance of self-renewal are intrinsically complex processes requiring the coordinated dynamic expression of hundreds of genes and proteins in precise response to external signalling cues. Numerous recent reports have used both experimental and computational techniques to dissect this complexity. These reports suggest that the control of cell fate has both deterministic and stochastic elements: complex underlying regulatory networks define stable molecular ‘attractor’ states towards which individual cells are drawn over time, whereas stochastic fluctuations in gene and protein expression levels drive transitions between coexisting attractors, ensuring robustness at the population level. PMID:19738627

  15. Single-cell transcriptome and epigenomic reprogramming of cardiomyocyte-derived cardiac progenitor cells.

    PubMed

    Chen, Xin; Chakravarty, Tushar; Zhang, Yiqiang; Li, Xiaojin; Zhong, Jiang F; Wang, Charles

    2016-01-01

    The molecular basis underlying the dedifferentiation of mammalian adult cardiomyocytes (ACMs) into myocyte-derived cardiac progenitor cells (mCPCs) during cardiac tissue regeneration is poorly understood. We present data integrating single-cell transcriptome and whole-genome DNA methylome analyses of mouse mCPCs to understand the epigenomic reprogramming governing their intrinsic cellular plasticity. Compared to parental cardiomyocytes, mCPCs display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlating well with the methylome, our single-cell transcriptomic data show that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implanting mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. This dataset suggests that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. Understanding cardiomyocyte epigenomic reprogramming may enable the design of future clinical therapies that induce cardiac regeneration, and prevent heart failure. PMID:27622691

  16. Reprogramming of cell fate: epigenetic memory and the erasure of memories past

    PubMed Central

    Nashun, Buhe; Hill, Peter WS; Hajkova, Petra

    2015-01-01

    Cell identity is a reflection of a cell type-specific gene expression profile, and consequently, cell type-specific transcription factor networks are considered to be at the heart of a given cellular phenotype. Although generally stable, cell identity can be reprogrammed in vitro by forced changes to the transcriptional network, the most dramatic example of which was shown by the induction of pluripotency in somatic cells by the ectopic expression of defined transcription factors alone. Although changes to cell fate can be achieved in this way, the efficiency of such conversion remains very low, in large part due to specific chromatin signatures constituting an epigenetic barrier to the transcription factor-mediated reprogramming processes. Here we discuss the two-way relationship between transcription factor binding and chromatin structure during cell fate reprogramming. We additionally explore the potential roles and mechanisms by which histone variants, chromatin remodelling enzymes, and histone and DNA modifications contribute to the stability of cell identity and/or provide a permissive environment for cell fate change during cellular reprogramming. PMID:25820261

  17. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  18. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    SciTech Connect

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-04-08

    Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  19. Therapeutic potential of targeting acinar cell reprogramming in pancreatic cancer

    PubMed Central

    Wong, Chi-Hin; Li, You-Jia; Chen, Yang-Chao

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a common pancreatic cancer and the fourth leading cause of cancer death in the United States. Treating this life-threatening disease remains challenging due to the lack of effective prognosis, diagnosis and therapy. Apart from pancreatic duct cells, acinar cells may also be the origin of PDAC. During pancreatitis or combined with activating KRasG12D mutation, acinar cells lose their cellular identity and undergo a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), forming duct cells which may then transform into pancreatic intraepithelial neoplasia (PanIN) and eventually PDAC. During ADM, the activation of mitogen-activated protein kinases, Wnt, Notch and phosphatidylinositide 3-kinases/Akt signaling inhibits the transcription of acinar-specific genes, including Mist and amylase, but promotes the expression of ductal genes, such as cytokeratin-19. Inhibition of this transdifferentiation process hinders the development of PanIN and PDAC. In addition, the transdifferentiated cells regain acinar identity, indicating ADM may be a reversible process. This provides a new therapeutic direction in treating PDAC through cancer reprogramming. Many studies have already demonstrated the success of switching PanIN/PDAC back to normal cells through the use of PD325901, the expression of E47, and the knockdown of Dickkopf-3. In this review, we discuss the signaling pathways involved in ADM and the therapeutic potential of targeting reprogramming in order to treat PDAC. PMID:27610015

  20. Fibromodulin reprogrammed cells: A novel cell source for bone regeneration.

    PubMed

    Li, Chen-Shuang; Yang, Pu; Ting, Kang; Aghaloo, Tara; Lee, Soonchul; Zhang, Yulong; Khalilinejad, Kambiz; Murphy, Maxwell C; Pan, Hsin Chuan; Zhang, Xinli; Wu, Benjamin; Zhou, Yan-Heng; Zhao, Zhihe; Zheng, Zhong; Soo, Chia

    2016-03-01

    Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However, currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. Previously, we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan, fibromodulin (FMOD), circumventing gene transduction. In this study, we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence, engraftment, and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration. PMID:26774565

  1. Therapeutic potential of targeting acinar cell reprogramming in pancreatic cancer.

    PubMed

    Wong, Chi-Hin; Li, You-Jia; Chen, Yang-Chao

    2016-08-21

    Pancreatic ductal adenocarcinoma (PDAC) is a common pancreatic cancer and the fourth leading cause of cancer death in the United States. Treating this life-threatening disease remains challenging due to the lack of effective prognosis, diagnosis and therapy. Apart from pancreatic duct cells, acinar cells may also be the origin of PDAC. During pancreatitis or combined with activating KRas(G12D) mutation, acinar cells lose their cellular identity and undergo a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), forming duct cells which may then transform into pancreatic intraepithelial neoplasia (PanIN) and eventually PDAC. During ADM, the activation of mitogen-activated protein kinases, Wnt, Notch and phosphatidylinositide 3-kinases/Akt signaling inhibits the transcription of acinar-specific genes, including Mist and amylase, but promotes the expression of ductal genes, such as cytokeratin-19. Inhibition of this transdifferentiation process hinders the development of PanIN and PDAC. In addition, the transdifferentiated cells regain acinar identity, indicating ADM may be a reversible process. This provides a new therapeutic direction in treating PDAC through cancer reprogramming. Many studies have already demonstrated the success of switching PanIN/PDAC back to normal cells through the use of PD325901, the expression of E47, and the knockdown of Dickkopf-3. In this review, we discuss the signaling pathways involved in ADM and the therapeutic potential of targeting reprogramming in order to treat PDAC. PMID:27610015

  2. Cell fate reprogramming by control of intracellular network dynamics

    NASA Astrophysics Data System (ADS)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  3. Broad-spectrum antiviral agents

    PubMed Central

    Zhu, Jun-Da; Meng, Wen; Wang, Xiao-Jia; Wang, Hwa-Chain R.

    2015-01-01

    Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents. PMID:26052325

  4. Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

    PubMed

    Perera, P I P; Ordoñez, C A; Dedicova, B; Ortega, P E M

    2014-01-01

    Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in

  5. Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development

    PubMed Central

    Perera, P. I. P.; Ordoñez, C. A.; Dedicova, B.; Ortega, P. E. M.

    2014-01-01

    Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu2+ and Fe2+) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4′,6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in

  6. The maternal-to-zygotic transition during vertebrate development: a model for reprogramming

    PubMed Central

    Yartseva, Valeria; Giraldez, Antonio J.

    2015-01-01

    Cellular transitions occur at all stages of organismal life from conception to adult regeneration. Changing cellular state involves three main features: activating gene expression necessary to install the new cellular state, modifying the chromatin status to stabilize the new gene expression program, and removing existing gene products to clear out the previous cellular program. The maternal-to-zygotic transition (MZT) is one of the most profound changes in the life of an organism. It involves gene expression remodeling at all levels, including the active clearance of the maternal oocyte program to adopt the embryonic totipotency. In this chapter we provide an overview of molecular mechanisms driving maternal mRNA clearance during the MZT, describe the developmental consequences of losing components of this gene regulation, and illustrate how remodeling of gene expression during the MZT is common to other cellular transitions with parallels to cellular reprogramming. PMID:26358874

  7. Oncometabolic Nuclear Reprogramming of Cancer Stemness

    PubMed Central

    Menendez, Javier A.; Corominas-Faja, Bruna; Cuyàs, Elisabet; García, María G.; Fernández-Arroyo, Salvador; Fernández, Agustín F.; Joven, Jorge; Fraga, Mario F.; Alarcón, Tomás

    2016-01-01

    Summary By impairing histone demethylation and locking cells into a reprogramming-prone state, oncometabolites can partially mimic the process of induced pluripotent stem cell generation. Using a systems biology approach, combining mathematical modeling, computation, and proof-of-concept studies with live cells, we found that an oncometabolite-driven pathological version of nuclear reprogramming increases the speed and efficiency of dedifferentiating committed epithelial cells into stem-like states with only a minimal core of stemness transcription factors. Our biomathematical model, which introduces nucleosome modification and epigenetic regulation of cell differentiation genes to account for the direct effects of oncometabolites on nuclear reprogramming, demonstrates that oncometabolites markedly lower the “energy barriers” separating non-stem and stem cell attractors, diminishes the average time of nuclear reprogramming, and increases the size of the basin of attraction of the macrostate occupied by stem cells. These findings establish the concept of oncometabolic nuclear reprogramming of stemness as a bona fide metabolo-epigenetic mechanism for generation of cancer stem-like cells. PMID:26876667

  8. Reprogrammed pluripotent stem cells from somatic cells.

    PubMed

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-06-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction. PMID:24298328

  9. Oncometabolic Nuclear Reprogramming of Cancer Stemness.

    PubMed

    Menendez, Javier A; Corominas-Faja, Bruna; Cuyàs, Elisabet; García, María G; Fernández-Arroyo, Salvador; Fernández, Agustín F; Joven, Jorge; Fraga, Mario F; Alarcón, Tomás

    2016-03-01

    By impairing histone demethylation and locking cells into a reprogramming-prone state, oncometabolites can partially mimic the process of induced pluripotent stem cell generation. Using a systems biology approach, combining mathematical modeling, computation, and proof-of-concept studies with live cells, we found that an oncometabolite-driven pathological version of nuclear reprogramming increases the speed and efficiency of dedifferentiating committed epithelial cells into stem-like states with only a minimal core of stemness transcription factors. Our biomathematical model, which introduces nucleosome modification and epigenetic regulation of cell differentiation genes to account for the direct effects of oncometabolites on nuclear reprogramming, demonstrates that oncometabolites markedly lower the "energy barriers" separating non-stem and stem cell attractors, diminishes the average time of nuclear reprogramming, and increases the size of the basin of attraction of the macrostate occupied by stem cells. These findings establish the concept of oncometabolic nuclear reprogramming of stemness as a bona fide metabolo-epigenetic mechanism for generation of cancer stem-like cells. PMID:26876667

  10. X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency

    PubMed Central

    Pasque, Vincent; Tchieu, Jason; Karnik, Rahul; Uyeda, Molly; Dimashkie, Anupama Sadhu; Case, Dana; Papp, Bernadett; Bonora, Giancarlo; Patel, Sanjeet; Ho, Ritchie; Schmidt, Ryan; McKee, Robin; Sado, Takashi; Tada, Takashi; Meissner, Alexander; Plath, Kathrin

    2014-01-01

    SUMMARY Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming. PMID:25525883

  11. Optical reprogramming with ultrashort femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Breunig, Hans G.; Batista, Ana; König, Karsten

    2015-03-01

    The use of sub-15 femtosecond laser pulses in stem cell research is explored with particular emphasis on the optical reprogramming of somatic cells. The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.

  12. Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

    PubMed Central

    Liao, Mei-Chih; Prigione, Alessandro; Jozefczuk, Justyna; Lichtner, Björn; Wolfrum, Katharina; Haltmeier, Manuela; Flöttmann, Max; Schaefer, Martin; Hahn, Alexander; Mrowka, Ralf; Klipp, Edda; Andrade-Navarro, Miguel A.; Adjaye, James

    2011-01-01

    Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal. PMID:21909390

  13. A molecular roadmap of reprogramming somatic cells into iPS cells.

    PubMed

    Polo, Jose M; Anderssen, Endre; Walsh, Ryan M; Schwarz, Benjamin A; Nefzger, Christian M; Lim, Sue Mei; Borkent, Marti; Apostolou, Effie; Alaei, Sara; Cloutier, Jennifer; Bar-Nur, Ori; Cheloufi, Sihem; Stadtfeld, Matthias; Figueroa, Maria Eugenia; Robinton, Daisy; Natesan, Sridaran; Melnick, Ari; Zhu, Jinfang; Ramaswamy, Sridhar; Hochedlinger, Konrad

    2012-12-21

    Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming. PMID:23260147

  14. Neurogenin1 effectively reprograms cultured chick RPE cells to differentiate towards photoreceptors

    PubMed Central

    Yan, Run-Tao; Liang, Lina; Ma, Wenxin; Li, Xiumei; Xie, Wenlian; Wang, Shu-Zhen

    2009-01-01

    Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor-like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0 ± 5.9%) began to differentiate towards photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRγ, and phototransduction pathway components, including transducin, cGMP-gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca2+) levels and responded to 9-cis-retinal by increasing their Ca2+ levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies. PMID:20029995

  15. Zebrafish small molecule screen in reprogramming/cell fate modulation

    PubMed Central

    Munson, Kathleen M.; Yeh, Jing-Ruey J.

    2010-01-01

    Embryonic zebrafish have long been used for lineage tracing studies. In zebrafish embryos, the cell fate identities can be determined by whole-mount in situ hybridization, or by visualization of live embryos if using fluorescent reporter lines. We use embryonic zebrafish to study the effects of a leukemic oncogene AML1-ETO on modulating hematopoietic cell fate. Induced expression of AML1-ETO is able to efficiently reprogram hematopoietic progenitor cells from erythroid to myeloid cell fate. Using the zebrafish model of AML1-ETO, we performed a chemical screen to identify small molecules that suppress the cell fate switch in the presence of AML1-ETO. The methods discussed herein may be broadly applicable for identifying small molecules that modulate other cell fate decisions. PMID:20336532

  16. Open chromatin in pluripotency and reprogramming

    PubMed Central

    Meshorer, Eran; Ramalho-Santos, Miguel

    2013-01-01

    Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique from any other stem cell in that they can give rise to all cell types of the body. Recent findings indicate that a particularly open chromatin state contributes to maintenance of pluripotency. Two emerging principles are that: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer. PMID:21179060

  17. Metabolic reprogramming of the tumour microenvironment.

    PubMed

    Xing, Yazhi; Zhao, Shimin; Zhou, Binhua P; Mi, Jun

    2015-10-01

    Tumour cells, stromal cells and the stroma comprise the tumour microenvironment. The metabolism of both tumour cells and several types of tumour stromal cells, such as cancer-associated fibroblasts and tumour-associated macrophages, is reprogrammed. Current studies have found that stromal cells promote tumour progression and metastasis, through not only the paracrine secretion of cytokines or chemokines, but also intermediate metabolites. Here, we summarize the latest insights into the mechanism of metabolic reprogramming in cancer cells, cancer-associated fibroblasts and tumour-associated macrophages, and their potential roles in tumour progression and metastasis. PMID:26255648

  18. Direct lineage reprogramming to neural cells

    PubMed Central

    Kim, Janghwan; Ambasudhan, Rajesh; Ding, Sheng

    2016-01-01

    Recently we have witnessed an array of studies on direct reprogramming that describe induced inter conversion of mature cell types from higher organisms including human. While these studies reveal an unexpected level of plasticity of differentiated somatic cells, they also provide unprecedented opportunities to develop regenerative therapies for many debilitating disorders and model these ‘diseases-in-a-dish’ for studying their pathophysiology. Here we review the current state of the art in direct lineage reprogramming to neural cells, and discuss the challenges that need to be addressed toward achieving the full potential of this exciting new technology. PMID:22652035

  19. Direct Reprogramming of RESTing Astrocytes.

    PubMed

    Wang, Chengzhong; Fong, Helen; Huang, Yadong

    2015-07-01

    Understanding the mechanisms underlying neuronal fate determination will provide important insights into brain development and regenerative approaches to neurological diseases. Now in Cell Stem Cell, Masserdotti et al. (2015) use neuronal conversion of astrocytes to dissect transcriptional mechanisms of fate determination and identify circuits that mediate cellular identity. PMID:26140600

  20. Pluripotent reprogramming and lineage reprogramming: promises and challenges in cardiovascular regeneration.

    PubMed

    He, Wen-Jun; Hou, Qian; Han, Qing-Wang; Han, Wei-Dong; Fu, Xiao-Bing

    2014-08-01

    Cardiovascular disease is a leading cause of death in industrialized countries. Scientists are trying to generate cardiomyocytes in vitro and in vivo to repair damaged heart tissue. Pluripotent reprogramming brings an alternative source of embryonic-like stem cells, and the possibility of regenerating mammalian tissues by first reverting somatic cells to induced pluripotent stem cells, followed by redifferentiating these cells into cardiomyocytes. More recently, lineage reprogramming of fibroblasts directly into functional cardiomyocytes has been reported. The procedure does not involve reverting cells back to a pluripotent stage, and, thus, would presumably reduce tumorigenic potential. Interestingly, lineage reprogramming could be used for in situ conversion of cell fate. Moreover, zebrafish-like regenerative mechanism in mammalian heart tissue, which was observed in mice within the first week of postpartum, should be further addressed. Here, we review the landmark progresses of the two major reprogramming strategies, compare their pros and cons in cardiovascular regeneration, and forecast the future directions of cardiac repair. PMID:24063625

  1. Direct Cardiomyocyte Reprogramming: A New Direction for Cardiovascular Regenerative Medicine

    PubMed Central

    Yi, B. Alexander; Mummery, Christine L.; Chien, Kenneth R.

    2013-01-01

    The past few years have seen unexpected new developments in direct cardiomyocyte reprogramming. Direct cardiomyocyte reprogramming potentially offers an entirely novel approach to cardiovascular regenerative medicine by converting cardiac fibroblasts into functional cardiomyocytes in situ. There is much to be learned, however, about the mechanisms of direct reprogramming in order that the process can be made more efficient. Early efforts have suggested that this new technology can be technically challenging. Moreover, new methods of inducing heart reprogramming will need to be developed before this approach can be translated to the bedside. Despite this, direct cardiomyocyte reprogramming may lead to new therapeutic options for sufferers of heart disease. PMID:24003244

  2. Blood pressure reprogramming adapter assists signal recording

    NASA Technical Reports Server (NTRS)

    Vick, H. A.

    1967-01-01

    Blood pressure reprogramming adapter separates the two components of a blood pressure signal, a dc pressure signal and an ac Korotkoff sounds signal, so that the Korotkoff sounds are recorded on one channel as received while the dc pressure signal is converted to FM and recorded on a second channel.

  3. The microenvironment reprograms circuits in tumor cells

    PubMed Central

    Cai, Qingchun; Xu, Yan

    2015-01-01

    In the course of multistep oncogenesis, initially normal cells acquire several new functions that render them malignant. We have recently demonstrated that the peritoneal microenvironment promotes resistance to anoikis in ovarian cancer cells by reprogramming SRC/AKT/ERK signaling and metabolism. These findings have prognostic and therapeutic implications. PMID:27308400

  4. Reprogramming chick RPE progeny cells to differentiate towards retinal neurons by ash1

    PubMed Central

    Mao, Weiming; Yan, Run-Tao

    2008-01-01

    Purpose Harnessing a cell culture of retinal pigment epithelium (RPE) to give rise to retinal neurons may offer a source of developing neurons for cell-replacement studies. This study explores the possibility of reprogramming RPE progeny cells to differentiate toward retinal neurons with achaete-scute homolog 1 (ash1), a proneural gene that is expressed in progenitor cells in the developing retina and promotes amacrine cell production when overexpressed in the chick retina. Methods Replication Competent Avian Splice (RCAS) retrovirus was used to drive the ectopic expression of ash1 in cell cultures of dissociated RPE isolated from day 6 chick embryos. RCAS expressing green fluorescent protein (RCAS-GFP) was used as control. The cultures were examined for de novo generation of neuron-like cells by molecular, cellular, and physiologic criteria. Results In control cultures infected with RCAS-GFP, RPE cells appeared cobblestone-like and often darkly pigmented. In cultures infected with RCAS-ash1, however, cells remained de-pigmented and frequently formed clusters. Further examination at the morphological and molecular levels showed the development of elaborate processes characteristic of neurons and the expression of genes/markers that identify different types of retinal neurons. The most prevalently expressed neural marker was calretinin, which in the chick retina identifies amacrine, ganglion, and horizontal cells. As an assay for functional maturation, the reprogrammed cells were analyzed for the presence of functional, ionotropic glutamate receptors that lead to a rise in the cytosolic free calcium (Ca2+) concentration. Calcium imaging showed that reprogrammed cells responded to glutamate and N-methyl-D-aspartate (NMDA) by increasing their Ca2+ concentrations, which, after reaching a peak level, returned to the basal level. The response curves of reprogrammed cells resembled those of cultured retinal neurons. Conclusions These results suggest that RPE progeny cells

  5. Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

    PubMed Central

    Vidal, Simon E.; Amlani, Bhishma; Chen, Taotao; Tsirigos, Aristotelis; Stadtfeld, Matthias

    2014-01-01

    Summary The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs). To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β) together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP) kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells. PMID:25358786

  6. Sox9 Reprogrammed Dermal Fibroblasts Undergo Hypertrophic Differentiation In Vitro and Trigger Endochondral Ossification In Vivo

    PubMed Central

    Tam, Wai Long; O, Dorien F.; Hiramatsu, Kunihiko; Tsumaki, Noriyuki; Luyten, Frank P.

    2014-01-01

    Abstract Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies. PMID:24459991

  7. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

    NASA Astrophysics Data System (ADS)

    Bared, Kalia

    The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

  8. Brain repair and reprogramming: the route to clinical translation.

    PubMed

    Grealish, S; Drouin-Ouellet, J; Parmar, M

    2016-09-01

    The adult brain has a very limited capacity for generation of new neurons, and neurogenesis only takes place in restricted regions. Some evidence for neurogenesis after injury has been reported, but few, if any, neurons are replaced after brain injury or degeneration, and the permanent loss of neurons leads to long-term disability and loss of brain function. For decades, researchers have been developing cell transplantation using exogenous cell sources for brain repair, and this method has now been shown to successfully restore lost function in experimental and clinical trials. Here, we review the development of cell-replacement strategies for brain repair in Parkinson's disease using the example of human foetal brain cells being successfully translated from preclinical findings to clinical trials. These trials demonstrate that cell-replacement therapy is a viable option for patients with Parkinson's disease, but more importantly also show how the limited availability of foetal cells calls for development of novel cell sources and methods for generating new neurons for brain repair. We focus on new stem cell sources that are on the threshold of clinical application for brain repair and discuss emerging cellular reprogramming technologies. Reviewing the current status of direct neural conversion, both in vitro and in vivo, where somatic cells are directly reprogrammed into functional neurons without passing through a stem cell intermediate, we conclude that both methods result in the successful replacement of new neurons that mature and integrate into the host brain. Thus, this new field shows great promise for future brain repair, although much work is still needed in preclinical animal models before it can be seriously considered for clinical applications. PMID:27539906

  9. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    SciTech Connect

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  10. The cytotoxic and immunogenic hurdles associated with non-viral mRNA-mediated reprogramming of human fibroblasts.

    PubMed

    Drews, Katharina; Tavernier, Geertrui; Demeester, Joseph; Lehrach, Hans; De Smedt, Stefaan C; Rejman, Joanna; Adjaye, James

    2012-06-01

    Delivery of reprogramming factor-encoding mRNAs by means of lipofection in somatic cells is a desirable method for deriving integration-free iPSCs. However, the lack of reproducibility implies there are major hurdles to overcome before this protocol becomes universally accepted. This study demonstrates the functionality of our in-house synthesized mRNAs expressing the reprogramming factors (OCT4, SOX2, KLF4, c-MYC) within the nucleus of human fibroblasts. However, upon repeated transfections, the mRNAs induced severe loss of cell viability as demonstrated by MTT cytotoxicity assays. Microarray-derived transcriptome data revealed that the poor cell survival was mainly due to the innate immune response triggered by the exogenous mRNAs. We validated the influence of mRNA transfection on key immune response-associated transcript levels, including IFNB1, RIG-I, PKR, IL12A, IRF7 and CCL5, by quantitative real-time PCR and directly compared these with the levels induced by other methods previously published to mediate reprogramming in somatic cells. Finally, we evaluated chemical compounds (B18R, chloroquine, TSA, Pepinh-TRIF, Pepinh-MYD), known for their ability to suppress cellular innate immune responses. However, none of these had the desired effect. The data presented here should provide the basis for further investigations into other immunosuppressing strategies that might facilitate efficient mRNA-mediated cellular reprogramming in human cells. PMID:22381475

  11. Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

    PubMed

    Ursu, Andrei; Illich, Damir J; Takemoto, Yasushi; Porfetye, Arthur T; Zhang, Miao; Brockmeyer, Andreas; Janning, Petra; Watanabe, Nobumoto; Osada, Hiroyuki; Vetter, Ingrid R; Ziegler, Slava; Schöler, Hans R; Waldmann, Herbert

    2016-04-21

    The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs. PMID:27049670

  12. Reprogramming of germ cells into pluripotency

    PubMed Central

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-01-01

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  13. Reprogramming of germ cells into pluripotency.

    PubMed

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-08-26

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  14. Transgenic expression of Telomerase reverse transcriptase (Tert) improves cell proliferation of primary cells and enhances reprogramming efficiency into the induced pluripotent stem cell.

    PubMed

    Hidema, Shizu; Fukuda, Tomokazu; Date, Shiori; Tokitake, Yuko; Matsui, Yasuhisa; Sasaki, Hiroki; Nishimori, Katsuhiko

    2016-10-01

    The enzymatic activity of telomerase is important for the extension of the telomere repeat sequence and overcoming cellular senescence. We generated a conditional transgenic mouse line, carrying the telomerase reverse transcriptase (Tert) expression cassette, controlled by the Cre-loxP-mediated recombination. In our study, Cre recombinase expression efficiently activated Tert expression, resulting in its increased enzymatic activity, which extended the period of cellular proliferation until the keratinocytes entered senescence. This suggests that transgenic Tert expression is effective in enhancing primary cell proliferation. Notably, Tert expression increased colony formation of induced pluripotent stem (iPS) cells after the introduction of four reprogramming factors, Oct-4, klf4, SOX-2, and c-Myc into the transgenic fibroblasts. To the best of our knowledge, this is the first study to show that the transgenic Tert expression enhances reprogramming efficiency of iPS cells, which indicates a critical role for Tert in the reprogramming process. PMID:27297181

  15. Matrix identity and tractional forces influence indirect cardiac reprogramming

    PubMed Central

    Kong, Yen P.; Carrion, Bita; Singh, Rahul K.; Putnam, Andrew J.

    2013-01-01

    Heart regeneration through in vivo cardiac reprogramming has been demonstrated as a possible regenerative strategy. While it has been reported that cardiac reprogramming in vivo is more efficient than in vitro, the influence of the extracellular microenvironment on cardiac reprogramming remains incompletely understood. This understanding is necessary to improve the efficiency of cardiac reprogramming in order to implement this strategy successfully. Here we have identified matrix identity and cell-generated tractional forces as key determinants of the dedifferentiation and differentiation stages during reprogramming. Cell proliferation, matrix mechanics, and matrix microstructure are also important, but play lesser roles. Our results suggest that the extracellular microenvironment can be optimized to enhance cardiac reprogramming. PMID:24326998

  16. Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos.

    PubMed

    Sepulveda-Rincon, Lessly P; Solanas, Edgar Del Llano; Serrano-Revuelta, Elisa; Ruddick, Lydia; Maalouf, Walid E; Beaujean, Nathalie

    2016-07-01

    Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased-thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization-and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation-the parthenote-and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research. PMID:27156679

  17. Constitutive heterochromatin reorganization during somatic cell reprogramming

    PubMed Central

    Fussner, Eden; Djuric, Ugljesa; Strauss, Mike; Hotta, Akitsu; Perez-Iratxeta, Carolina; Lanner, Fredrik; Dilworth, F Jeffrey; Ellis, James; Bazett-Jones, David P

    2011-01-01

    Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing repressive epigenetic marks. In contrast to loci-specific epigenetic changes, heterochromatin domains undergo epigenetic resetting during the reprogramming process, but the effect on the heterochromatin ultrastructure is not known. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative electron spectroscopic imaging. In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocentre structures of densely packed chromatin fibres. In contrast, chromocentre boundaries are poorly defined in pluripotent embryonic stem and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibres in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst before differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by MEK/GSK3 2i inhibitor treatment. Thus, constitutive heterochromatin is compacted in partial iPS cells but reorganizes into dispersed 10 nm chromatin fibres as the fully reprogrammed iPS cell state is acquired. PMID:21468033

  18. Understanding the molecular mechanisms of reprogramming.

    PubMed

    Krause, Marie N; Sancho-Martinez, Ignacio; Izpisua Belmonte, Juan Carlos

    2016-05-01

    Despite the profound and rapid advancements in reprogramming technologies since the generation of the first induced pluripotent stem cells (iPSCs) in 2006[1], the molecular basics of the process and its implications are still not fully understood. Recent work has suggested that a subset of TFs, so called "Pioneer TFs", play an important role during the stochastic phase of iPSC reprogramming [2-6]. Pioneer TFs activities differ from conventional transcription factors in their mechanism of action. They bind directly to condensed chromatin and elicit a series of chromatin remodeling events that lead to opening of the chromatin. Chromatin decondensation by pioneer factors progressively occurs during cell division and in turn exposes specific gene promoters in the DNA to which TFs can now directly bind to promoters that are readily accessible[2, 6]. Here, we will summarize recent advancements on our understanding of the molecular mechanisms underlying reprogramming to iPSC as well as the implications that pioneer Transcription Factor activities might play during different lineage conversion processes. PMID:26655812

  19. Metabolic reprogramming during neuronal differentiation.

    PubMed

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-09-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

  20. Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

    PubMed

    Quintanilla, Rene H; Asprer, Joanna; Sylakowski, Kyle; Lakshmipathy, Uma

    2016-01-01

    Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming. PMID:27500543

  1. Reprogramming the genome to totipotency in mouse embryos.

    PubMed

    Zhou, Li-quan; Dean, Jurrien

    2015-02-01

    Despite investigative interest, the artificial derivation of pluripotent stem cells remains inefficient and incomplete reprogramming hinders its potential as a reliable tool in regenerative medicine. By contrast, fusion of terminally differentiated gametes at fertilization activates efficient epigenetic reprogramming to ensure totipotency of early embryos. Understanding the epigenetic mechanisms required for the transition from the fertilized egg to the embryo can improve efforts to reprogram differentiated cells to pluripotent/totipotent cells for therapeutic use. We review recent discoveries that are providing insight into the molecular mechanisms required for epigenetic reprogramming to totipotency in vivo. PMID:25448353

  2. Reprogramming the genome to totipotency in mouse embryos

    PubMed Central

    Zhou, Li-quan; Dean, Jurrien

    2014-01-01

    Despite investigative interest, the artificial derivation of pluripotent stem cells remains inefficient and incomplete reprogramming hinders its potential as a reliable tool in regenerative medicine. By contrast, fusion of terminally differentiated gametes at fertilization activates efficient epigenetic reprogramming to ensure totipotency of early embryos. Understanding the epigenetic mechanisms required for the transition from the fertilized egg to the embryo can improve efforts to reprogram differentiated cells to pluripotent/totipotent cells for therapeutic use. We review recent discoveries that are providing insight into the molecular mechanisms required for epigenetic reprogramming to totipotency in vivo. PMID:25448353

  3. Reprogramming stem cells is a microenvironmental task

    SciTech Connect

    Bissell, Mina J; Inman, Jamie

    2008-10-14

    That tumor cells for all practical purposes are unstable and plastic could be expected. However, the astonishing ability of the nuclei from cells of normal adult tissues to be reprogrammed - given the right embryonic context - found its final truth even for mammals in the experiments that allowed engineering Dolly (1). The landmark experiments showed that nuclei originating from cells of frozen mammary tissues were capable of being reprogrammed by the embryonic cytoplasm and its microenvironment to produce a normal sheep. The rest is history. However, whether microenvironments other than those of the embryos can also reprogram adult cells of different tissue origins still containing their cytoplasm is of obvious interest. In this issue of PNAS, the laboratory of Gilbert Smith (2) reports on how the mammary gland microenvironment can reprogram both embryonic and adult stem neuronal cells. The work is a follow-up to their previous report on testis stem cells that were reprogrammed by the mammary microenvironment (3). They demonstrated that cells isolated from the seminiferous tubules of the mature testis, mixed with normal mammary epithelial cells, contributed a sizable number of epithelial progeny to normal mammary outgrowths in transplanted mammary fat pads. However, in those experiments they were unable to distinguish which subpopulation of the testis cells contributed progeny to the mammary epithelial tree. The current work adds new, compelling, and provocative information to our understanding of stem cell plasticity. Booth et al. (2) use neuronal stem cells (NSCs) isolated from WAP-cre/R26R mice combined with unlabeled mammary epithelial cells that subsequently are implanted in cleared mammary fat pads. In this new microenvironment, the NSCs that are incorporated into the branching mammary tree make chimeric glands (Fig. 1) that remarkably can also express the milk protein {beta}-casein, progesterone receptor, and estrogen receptor {alpha}. Remarkably, the

  4. Targeted alternative splicing of TAF4: a new strategy for cell reprogramming.

    PubMed

    Kazantseva, Jekaterina; Sadam, Helle; Neuman, Toomas; Palm, Kaia

    2016-01-01

    Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression. PMID:27499390

  5. Targeted alternative splicing of TAF4: a new strategy for cell reprogramming

    PubMed Central

    Kazantseva, Jekaterina; Sadam, Helle; Neuman, Toomas; Palm, Kaia

    2016-01-01

    Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression. PMID:27499390

  6. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

    PubMed Central

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future. PMID:26179207

  7. NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

    PubMed Central

    Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

    2016-01-01

    Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

  8. NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming.

    PubMed

    Hawkins, Kate E; Joy, Shona; Delhove, Juliette M K M; Kotiadis, Vassilios N; Fernandez, Emilio; Fitzpatrick, Lorna M; Whiteford, James R; King, Peter J; Bolanos, Juan P; Duchen, Michael R; Waddington, Simon N; McKay, Tristan R

    2016-03-01

    The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

  9. Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds

    PubMed Central

    Carlson, Aaron L.; Bennett, Neal K.; Francis, Nicola L.; Halikere, Apoorva; Clarke, Stephen; Moore, Jennifer C.; Hart, Ronald P.; Paradiso, Kenneth; Wernig, Marius; Kohn, Joachim; Pang, Zhiping P.; Moghe, Prabhas V.

    2016-01-01

    Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. PMID:26983594

  10. Early reprogramming regulators identified by prospective isolation and mass cytometry

    PubMed Central

    Lujan, Ernesto; Zunder, Eli R.; Ng, Yi Han; Goronzy, Isabel N.; Nolan, Garry P.; Wernig, Marius

    2015-01-01

    In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1–11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. PMID:25830878

  11. Chromatin-modifying enzymes as modulators of reprogramming.

    PubMed

    Onder, Tamer T; Kara, Nergis; Cherry, Anne; Sinha, Amit U; Zhu, Nan; Bernt, Kathrin M; Cahan, Patrick; Marcarci, B Ogan; Unternaehrer, Juli; Gupta, Piyush B; Lander, Eric S; Armstrong, Scott A; Daley, George Q

    2012-03-29

    Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. PMID:22388813

  12. MicroRNAs and oncogenic transcriptional regulatory networks controlling metabolic reprogramming in cancers.

    PubMed

    Pinweha, Pannapa; Rattanapornsompong, Khanti; Charoensawan, Varodom; Jitrapakdee, Sarawut

    2016-01-01

    Altered cellular metabolism is a fundamental adaptation of cancer during rapid proliferation as a result of growth factor overstimulation. We review different pathways involving metabolic alterations in cancers including aerobic glycolysis, pentose phosphate pathway, de novo fatty acid synthesis, and serine and glycine metabolism. Although oncoproteins, c-MYC, HIF1α and p53 are the major drivers of this metabolic reprogramming, post-transcriptional regulation by microRNAs (miR) also plays an important role in finely adjusting the requirement of the key metabolic enzymes underlying this metabolic reprogramming. We also combine the literature data on the miRNAs that potentially regulate 40 metabolic enzymes responsible for metabolic reprogramming in cancers, with additional miRs from computational prediction. Our analyses show that: (1) a metabolic enzyme is frequently regulated by multiple miRs, (2) confidence scores from prediction algorithms might be useful to help narrow down functional miR-mRNA interaction, which might be worth further experimental validation. By combining known and predicted interactions of oncogenic transcription factors (TFs) (c-MYC, HIF1α and p53), sterol regulatory element binding protein 1 (SREBP1), 40 metabolic enzymes, and regulatory miRs we have established one of the first reference maps for miRs and oncogenic TFs that regulate metabolic reprogramming in cancers. The combined network shows that glycolytic enzymes are linked to miRs via p53, c-MYC, HIF1α, whereas the genes in serine, glycine and one carbon metabolism are regulated via the c-MYC, as well as other regulatory organization that cannot be observed by investigating individual miRs, TFs, and target genes. PMID:27358718

  13. Label-free, live optical imaging of reprogrammed bipolar disorder patient-derived cells reveals a functional correlate of lithium responsiveness.

    PubMed

    Wang, J L; Shamah, S M; Sun, A X; Waldman, I D; Haggarty, S J; Perlis, R H

    2014-01-01

    Development of novel treatments and diagnostic tools for psychiatric illness has been hindered by the absence of cellular models of disease. With the advent of cellular reprogramming, it may be possible to recapitulate the disease biology of psychiatric disorders using patient skin cells transdifferentiated to neurons. However, efficiently identifying and characterizing relevant neuronal phenotypes in the absence of well-defined pathophysiology remains a challenge. In this study, we collected fibroblast samples from patients with bipolar 1 disorder, characterized by their lithium response (n=12), and healthy control subjects (n=6). We identified a cellular phenotype in reprogrammed neurons using a label-free imaging assay based on a nanostructured photonic crystal biosensor and found that an optical measure of cell adhesion was associated with clinical response to lithium treatment. This cellular phenotype may represent a useful biomarker to evaluate drug response and screen for novel therapeutics. PMID:25158003

  14. Identification of Oct4-activating compounds that enhance reprogramming efficiency.

    PubMed

    Li, Wendong; Tian, E; Chen, Zhao-Xia; Sun, Guoqiang; Ye, Peng; Yang, Su; Lu, Dave; Xie, Jun; Ho, Thach-Vu; Tsark, Walter M; Wang, Charles; Horne, David A; Riggs, Arthur D; Yip, M L Richard; Shi, Yanhong

    2012-12-18

    One of the hurdles for practical application of induced pluripotent stem cells (iPSC) is the low efficiency and slow process of reprogramming. Octamer-binding transcription factor 4 (Oct4) has been shown to be an essential regulator of embryonic stem cell (ESC) pluripotency and key to the reprogramming process. To identify small molecules that enhance reprogramming efficiency, we performed a cell-based high-throughput screening of chemical libraries. One of the compounds, termed Oct4-activating compound 1 (OAC1), was found to activate both Oct4 and Nanog promoter-driven luciferase reporter genes. Furthermore, when added to the reprogramming mixture along with the quartet reprogramming factors (Oct4, Sox2, c-Myc, and Klf4), OAC1 enhanced the iPSC reprogramming efficiency and accelerated the reprogramming process. Two structural analogs of OAC1 also activated Oct4 and Nanog promoters and enhanced iPSC formation. The iPSC colonies derived using the Oct4-activating compounds along with the quartet factors exhibited typical ESC morphology, gene-expression pattern, and developmental potential. OAC1 seems to enhance reprogramming efficiency in a unique manner, independent of either inhibition of the p53-p21 pathway or activation of the Wnt-β-catenin signaling. OAC1 increases transcription of the Oct4-Nanog-Sox2 triad and Tet1, a gene known to be involved in DNA demethylation. PMID:23213213

  15. Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos

    PubMed Central

    Zhang, Sheng; Chen, Xin; Wang, Fang; An, Xinglan; Tang, Bo; Zhang, Xueming; Sun, Liguang; Li, Ziyi

    2016-01-01

    DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner. PMID:27456302

  16. Combining small molecules for cell reprogramming through an interatomic analysis.

    PubMed

    Feltes, Bruno César; Bonatto, Diego

    2013-11-01

    The knowledge available about the application and generation of induced pluripotent stem cells (iPSC) has grown since their discovery, and new techniques to enhance the reprogramming process have been described. Among the new approaches to induce iPSC that have gained great attention is the use of small molecules for reprogramming. The application of small molecules, unlike genetic manipulation, provides for control of the reprogramming process through the shifting of concentrations and the combination of different molecules. However, different researchers have reported the use of "reprogramming cocktails" with variable results and drug combinations. Thus, the proper combination of small molecules for successful and enhanced reprogramming is a matter for discussion. However, testing all potential drug combinations in different cell lineages is very costly and time-consuming. Therefore, in this article, we discuss the use of already employed molecules for iPSC generation, followed by the application of systems chemo-biology tools to create different data sets of protein-protein (PPI) and chemical-protein (CPI) interaction networks based on the knowledge of already used and new reprogramming cocktail combinations. We further analyzed the biological processes associated with PPI-CPI networks and provided new potential protein targets to be inhibited or expressed for stem cell reprogramming. In addition, we applied a new interference analysis to prospective targets that could negatively affect the classical pluripotency-associated factors (SOX2, NANOG, KLF4 and OCT4) and thus potentially improve reprogramming protocols. PMID:24056910

  17. Sir John Gurdon: father of nuclear reprogramming.

    PubMed

    Blau, Helen M

    2014-07-01

    Sir John Gurdon founded the field of nuclear reprogramming. His work set the stage for the ever burgeoning area of stem cell biology and regenerative medicine. Here I provide personal reflections on times I shared with John Gurdon and professional reflections of the impact of his ground-breaking research on my own development as a scientist and on the field in general. His paradigm-shifting experiments will continue to provoke scientists to think outside the box for many years to come. PMID:24954777

  18. Sir John Gurdon: Father of nuclear reprogramming

    PubMed Central

    Blau, Helen M.

    2015-01-01

    Sir John Gurdon founded the field of nuclear reprogramming. His work set the stage for the ever burgeoning area of stem cell biology and regenerative medicine. Here I provide personal reflections on times I shared with John Gurdon and professional reflections of the impact of his ground-breaking research on my own development as a scientist and on the field in general. His paradigm-shifting experiments will continue to provoke scientists to think outside the box for many years to come. PMID:24954777

  19. Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

    PubMed Central

    Esteves, Telma C.; Psathaki, Olympia E.; Pfeiffer, Martin J.; Balbach, Sebastian T.; Zeuschner, Dagmar; Shitara, Hiroshi; Yonekawa, Hiromichi; Siatkowski, Marcin; Fuellen, Georg; Boiani, Michele

    2012-01-01

    While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism. PMID:22693623

  20. Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion

    PubMed Central

    Jang, Hyun Sik; Hong, Yean Ju; Choi, Hyun Woo; Song, Hyuk; Byun, Sung June; Uhm, Sang Jun; Seo, Han Geuk; Do, Jeong Tae

    2016-01-01

    Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner. PMID:27232503

  1. Molecular barriers to processes of genetic reprogramming and cell transformation.

    PubMed

    Chestkov, I V; Khomyakova, E A; Vasilieva, E A; Lagarkova, M A; Kiselev, S L

    2014-12-01

    Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar "molecular barriers" that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation. PMID:25716723

  2. mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: a roadmap from energy metabolism to stem cell renewal and aging.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Vazquez-Martin, Alejandro

    2011-11-01

    Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced Pluripotent Stem Cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of

  3. Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast.

    PubMed

    Isasa, Marta; Suñer, Clara; Díaz, Miguel; Puig-Sàrries, Pilar; Zuin, Alice; Bichman, Anne; Gygi, Steven P; Rebollo, Elena; Crosas, Bernat

    2016-01-22

    Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway. PMID:26601941

  4. Direct Cardiac Reprogramming: Advances in Cardiac Regeneration

    PubMed Central

    Chen, Olivia; Qian, Li

    2015-01-01

    Heart disease is one of the lead causes of death worldwide. Many forms of heart disease, including myocardial infarction and pressure-loading cardiomyopathies, result in irreversible cardiomyocyte death. Activated fibroblasts respond to cardiac injury by forming scar tissue, but ultimately this response fails to restore cardiac function. Unfortunately, the human heart has little regenerative ability and long-term outcomes following acute coronary events often include chronic and end-stage heart failure. Building upon years of research aimed at restoring functional cardiomyocytes, recent advances have been made in the direct reprogramming of fibroblasts toward a cardiomyocyte cell fate both in vitro and in vivo. Several experiments show functional improvements in mouse models of myocardial infarction following in situ generation of cardiomyocyte-like cells from endogenous fibroblasts. Though many of these studies are in an early stage, this nascent technology holds promise for future applications in regenerative medicine. In this review, we discuss the history, progress, methods, challenges, and future directions of direct cardiac reprogramming. PMID:26176012

  5. Forward engineering neuronal diversity using direct reprogramming.

    PubMed

    Tsunemoto, Rachel K; Eade, Kevin T; Blanchard, Joel W; Baldwin, Kristin K

    2015-06-01

    The nervous system is comprised of a vast diversity of distinct neural cell types. Differences between neuronal subtypes drive the assembly of neuronal circuits and underlie the subtype specificity of many neurological diseases. Yet, because neurons are irreversibly post-mitotic and not readily available from patients, it has not been feasible to study specific subtypes of human neurons in larger numbers. A powerful means to study neuronal diversity and neurological disease is to establish methods to produce desired neuronal subtypes in vitro. Traditionally this has been accomplished by treating pluripotent or neural stem cells with growth factors and morphogens that recapitulate exogenous developmental signals. These approaches often require extended periods of culture, which can limit their utility. However, more recently, it has become possible to produce neurons directly from fibroblasts using transcription factors and/or microRNAs. This technique referred to as direct reprogramming or transdifferentiation has proven to be a rapid, robust, and reproducible method to generate mature neurons of many different subtypes from multiple cell sources. Here, we highlight recent advances in generating neurons of specific subtypes using direct reprogramming and outline various scenarios in which induced neurons may be applied to studies of neuronal function and neurological disease. PMID:25908841

  6. Parkinson's Disease in a Dish: What Patient Specific-Reprogrammed Somatic Cells Can Tell Us about Parkinson's Disease, If Anything?

    PubMed

    Drouin-Ouellet, J; Barker, R A

    2012-01-01

    Technologies allowing for the derivation of patient-specific neurons from somatic cells are emerging as powerful in vitro tools to investigate the intrinsic cellular pathological behaviours of the diseases that affect these patients. While the use of patient-derived neurons to model Parkinson's disease (PD) has only just begun, these approaches have allowed us to begin investigating disease pathogenesis in a unique way. In this paper, we discuss the advances made in the field of cellular reprogramming to model PD and discuss the pros and cons associated with the use of such cells. PMID:23316244

  7. Tumorigenic potential is restored during differentiation in fusion-reprogrammed cancer cells

    PubMed Central

    Yao, J; Zhang, L; Hu, L; Guo, B; Hu, X; Borjigin, U; Wei, Z; Chen, Y; Lv, M; Lau, J T Y; Wang, X; Li, G; Hu, Y-P

    2016-01-01

    Detailed understanding of the mechanistic steps underlying tumor initiation and malignant progression is critical for insights of potentially novel therapeutic modalities. Cellular reprogramming is an approach of particular interest because it can provide a means to reset the differentiation state of the cancer cells and to revert these cells to a state of non-malignancy. Here, we investigated the relationship between cellular differentiation and malignant progression by the fusion of four independent mouse cancer cell lines from different tissues, each with differing developmental potentials, to pluripotent mouse embryonic stem (ES) cells. Fusion was accompanied by loss of differentiated properties of the four parental cancer cell lines and concomitant emergence of pluripotency, demonstrating the feasibility to reprogram the malignant and differentiative properties of cancer cells. However, the original malignant and differentiative phenotypes re-emerge upon withdrawal of the fused cells from the embryonic environment in which they were maintained. cDNA array analysis of the malignant hepatoma progression implicated a role for Foxa1, and silencing Foxa1 prevented the re-emergence of malignant and differentiation-associated gene expression. Our findings support the hypothesis that tumor progression results from deregulation of stem cells, and our approach provides a strategy to analyze possible mechanisms in the cancer initiation. PMID:27468690

  8. Direct neuronal reprogramming: learning from and for development.

    PubMed

    Masserdotti, Giacomo; Gascón, Sergio; Götz, Magdalena

    2016-07-15

    The key signalling pathways and transcriptional programmes that instruct neuronal diversity during development have largely been identified. In this Review, we discuss how this knowledge has been used to successfully reprogramme various cell types into an amazing array of distinct types of functional neurons. We further discuss the extent to which direct neuronal reprogramming recapitulates embryonic development, and examine the particular barriers to reprogramming that may exist given a cell's unique developmental history. We conclude with a recently proposed model for cell specification called the 'Cook Islands' model, and consider whether it is a fitting model for cell specification based on recent results from the direct reprogramming field. PMID:27436039

  9. Metabolic reprogramming, caloric restriction and aging

    PubMed Central

    Anderson, Rozalyn M.; Weindruch, Richard

    2009-01-01

    Caloric restriction (CR) without malnutrition slows the aging process and extends lifespan in diverse species by unknown mechanisms. The inverse linear relationship between calorie intake and lifespan suggests that regulators of energy metabolism are important in CR’s actions. Studies in several species reveal tissue-specific changes in energy metabolism with CR and suggest that metabolic reprogramming plays a critical role in its mechanism of aging retardation. We herein describe common signatures of CR and suggest how they may slow aging. We discuss recent advances in understanding the function of key metabolic regulators that likely coordinate the response to altered nutrient availability with CR, and how the pathways they regulate may retard the aging process. PMID:20004110

  10. Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

    PubMed Central

    Afzal, Muhammad Z.; Strande, Jennifer L.

    2015-01-01

    Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2–3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening. PMID:25650629

  11. Two new routes to make blood: Hematopoietic specification from pluripotent cell lines versus reprogramming of somatic cells.

    PubMed

    Singbrant, Sofie; van Galen, Peter; Lucas, Daniel; Challen, Grant; Rossi, Derrick J; Daley, George Q

    2015-09-01

    Transplantation of hematopoietic stem cells (HSCs) to treat hematologic disorders is routinely used in the clinic. However, HSC therapy is hindered by the requirements of finding human leukocyte antigen (HLA)-matched donors and attaining sufficient numbers of long-term HSCs in the graft. Therefore, ex vivo expansion of transplantable HSCs remains one of the "holy grails" of hematology. Without the ability to maintain and expand human HSCs in vitro, two complementary approaches involving cellular reprogramming to generate transplantable HSCs have emerged. Reprogrammed HSCs represent a potentially inexhaustible supply of autologous tissue. On March 18th, 2015, Dr. George Q. Daley and Dr. Derrick J. Rossi, two pioneers in the field, presented and discussed their most recent research on these topics in a webinar organized by the International Society for Experimental Hematology (ISEH). Here, we summarize these seminars and discuss the possibilities and challenges in the field of hematopoietic specification. PMID:26143581

  12. The Broad Way

    ERIC Educational Resources Information Center

    Butler, Kevin

    2008-01-01

    In the world of corporate philanthropy, there are those who give to educational causes, and this article describes one such philanthropist, Eli Broad, who shares his take on schools in America. Broad is in a category unto himself not only because of the amount of money he has given--more than $280 million since 1999--but also for his unique…

  13. The 2010 Broad Prize

    ERIC Educational Resources Information Center

    Education Digest: Essential Readings Condensed for Quick Review, 2011

    2011-01-01

    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  14. Genetic background affects susceptibility to tumoral stem cell reprogramming

    PubMed Central

    García-Ramírez, Idoia; Ruiz-Roca, Lucía; Martín-Lorenzo, Alberto; Blanco, Óscar; García-Cenador, María Begoña; García-Criado, Francisco Javier; Vicente-Dueñas, Carolina; Sánchez-García, Isidro

    2013-01-01

    The latest studies of the interactions between oncogenes and its target cell have shown that certain oncogenes may act as passengers to reprogram tissue-specific stem/progenitor cell into a malignant cancer stem cell state. In this study, we show that the genetic background influences this tumoral stem cell reprogramming capacity of the oncogenes using as a model the Sca1-BCRABLp210 mice, where the type of tumor they develop, chronic myeloid leukemia (CML), is a function of tumoral stem cell reprogramming. Sca1-BCRABLp210 mice containing FVB genetic components were significantly more resistant to CML. However, pure Sca1-BCRABLp210 FVB mice developed thymomas that were not seen in the Sca1-BCRABLp210 mice into the B6 background. Collectively, our results demonstrate for the first time that tumoral stem cell reprogramming fate is subject to polymorphic genetic control. PMID:23839033

  15. In Vivo Reprogramming for Brain and Spinal Cord Repair.

    PubMed

    Chen, Gong; Wernig, Marius; Berninger, Benedikt; Nakafuku, Masato; Parmar, Malin; Zhang, Chun-Li

    2015-01-01

    Cell reprogramming technologies have enabled the generation of various specific cell types including neurons from readily accessible patient cells, such as skin fibroblasts, providing an intriguing novel cell source for autologous cell transplantation. However, cell transplantation faces several difficult hurdles such as cell production and purification, long-term survival, and functional integration after transplantation. Recently, in vivo reprogramming, which makes use of endogenous cells for regeneration purpose, emerged as a new approach to circumvent cell transplantation. There has been evidence for in vivo reprogramming in the mouse pancreas, heart, and brain and spinal cord with various degrees of success. This mini review summarizes the latest developments presented in the first symposium on in vivo reprogramming glial cells into functional neurons in the brain and spinal cord, held at the 2014 annual meeting of the Society for Neuroscience in Washington, DC. PMID:26730402

  16. A Cell Electrofusion Chip for Somatic Cells Reprogramming

    PubMed Central

    Wu, Wei; Zeng, Yuxiao; Yang, Jun; Xu, Haiwei; Yin, Zheng Qin

    2015-01-01

    Cell fusion is a potent approach to explore the mechanisms of somatic cells reprogramming. However, previous fusion methods, such as polyethylene glycol (PEG) mediated cell fusion, are often limited by poor fusion yields. In this study, we developed a simplified cell electrofusion chip, which was based on a micro-cavity/ discrete microelectrode structure to improve the fusion efficiency and to reduce multi-cell electrofusion. Using this chip, we could efficiently fuse NIH3T3 cells and mouse embryonic stem cells (mESCs) to induce somatic cells reprogramming. We also found that fused cells demethylated gradually and 5-hydroxymethylcytosine (5hmC) was involved in the demethylation during the reprogramming. Thus, the cell electrofusion chip would facilitate reprogramming mechanisms research by improving efficiency of cell fusion and reducing workloads. PMID:26177036

  17. Conversion of genomic imprinting by reprogramming and redifferentiation.

    PubMed

    Kim, Min Jung; Choi, Hyun Woo; Jang, Hyo Jin; Chung, Hyung Min; Arauzo-Bravo, Marcos J; Schöler, Hans R; Do, Jeong Tae

    2013-06-01

    Induced pluripotent stem cells (iPSCs), generated from somatic cells by overexpression of transcription factors Oct4, Sox2, Klf4 and c-Myc have the same characteristics as pluripotent embryonic stem cells (ESCs). iPSCs reprogrammed from differentiated cells undergo epigenetic modification during reprogramming, and ultimately acquire a similar epigenetic state to that of ESCs. In this study, these epigenetic changes were observed in reprogramming of uniparental parthenogenetic somatic cells. The parthenogenetic pattern of imprinted genes changes during the generation of parthenogenetic maternal iPSCs (miPSCs), a process referred to as pluripotent reprogramming. We determined whether altered imprinted genes are maintained or revert to the parthenogenetic state when the reprogrammed cells are redifferentiated into specialized cell types. To address this question, we redifferentiated miPSCs into neural stem cells (miPS-NSCs) and compared them with biparental female NSCs (fNSCs) and parthenogenetic NSCs (pNSCs). We found that pluripotent reprogramming of parthenogenetic somatic cells could reset parthenogenetic DNA methylation patterns in imprinted genes, and that alterations in DNA methylation were maintained even after miPSCs were redifferentiated into miPS-NSCs. Notably, maternally methylated imprinted genes (Peg1, Peg3, Igf2r, Snrpn and Ndn), whose differentially methylated regions were fully methylated in pNSCs, were demethylated and their expression levels were found to be close to the levels in normal biparental fNSCs after reprogramming and redifferentiation. Our findings suggest that pluripotent reprogramming of parthenogenetic somatic cells followed by redifferentiation leads to changes in DNA methylation of imprinted genes and the reestablishment of gene expression levels to those of normal biparental cells. PMID:23525019

  18. Signaling involved in stem cell reprogramming and differentiation

    PubMed Central

    Tanabe, Shihori

    2015-01-01

    Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have revealed that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell programming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review, the molecular interactions and signaling pathways related to stem cell differentiation are discussed. PMID:26328015

  19. Advanced Technologies Lead iNto New Reprogramming Routes.

    PubMed

    Zhou, Yang; Qian, Li

    2016-09-01

    Two recent papers provide new insights on transcriptional dynamics and epigenetic remodeling during reprogramming of induced neurons (iNs). Treutlein et al. (2016) applied single-cell transcriptomics to identify routes and detours during early iN induction, while in this issue of Cell Stem Cell, Black et al. (2016) employed gene editing to activate endogenous loci of the reprogramming factors. PMID:27588744

  20. Reprogramming of Xist against the pluripotent state in fusion hybrids.

    PubMed

    Do, Jeong Tae; Han, Dong Wook; Gentile, Luca; Sobek-Klocke, Ingeborg; Wutz, Anton; Schöler, Hans R

    2009-11-15

    The fusion of somatic cells with pluripotent cells results in the generation of pluripotent hybrid cells. Because the ;memory' of somatic cells seems to be erased during fusion-induced reprogramming, genetic reprogramming is thought to be a largely unidirectional process. Here we show that fusion-induced reprogramming, which brings about the formation of pluripotent hybrids, does not always follow a unidirectional route. Xist is a unique gene in that it is reprogrammed to the state of somatic cells in fusion-induced pluripotent hybrids. In hybrids formed from the cell fusion of embryonal carcinoma cells (ECCs) with male neural stem cells (mNSCs), the Xist gene was found to be reprogrammed to the somatic cell state, whereas the pluripotency-related and tissue-specific marker genes were reprogrammed to the pluripotent cell state. Specifically, Xist is not expressed in hybrids, because the ;memory' of the somatic cell has been retained (i.e. mNSCs do not exhibit Xist expression) and that of the pluripotent cell erased (i.e. inactivation of the partially active Xist gene of ECCs, complete methylation of the Xist region). The latter phenomenon is induced by male, but not by female, NSCs. PMID:19843582

  1. Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

    PubMed

    Sun, Ming; Liao, Bing; Tao, Yu; Chen, Hao; Xiao, Feng; Gu, Junjie; Gao, Shaorong; Jin, Ying

    2016-05-01

    Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. PMID:26448199

  2. Developmental exposure to endocrine disrupting chemicals alters the epigenome: Identification of reprogrammed targets

    PubMed Central

    Prusinski, Lauren; Al-Hendy, Ayman; Yang, Qiwei

    2016-01-01

    Endocrine disruptions induced by environmental toxicants have placed an immense burden on society to properly diagnose, treat and attempt to alleviate symptoms and disease. Environmental exposures during critical periods of development can permanently reprogram normal physiological responses, thereby increasing susceptibility to disease later in life - a process known as developmental reprogramming. During development, organogenesis and tissue differentiation occur through a continuous series of tightly-regulated and precisely-timed molecular, biochemical and cellular events. Humans may encounter endocrine disrupting chemicals (EDCs) daily and during all stages of life, from conception and fetal development through adulthood and senescence. Though puberty and perimenopausal periods may be affected by endocrine disruption due to hormonal effects, prenatal and early postnatal windows are most critical for proper development due to rapid changes in system growth. Developmental reprogramming is shown to be caused by alterations in the epigenome. Development is the time when epigenetic programs are ‘installed’ on the genome by ‘writers’, such as histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs), which add methyl groups to lysine and arginine residues on histone tails and to CpG sites in DNA, respectively. A number of environmental compounds, referred to as estrogenic endocrine disruptors (EEDs), are able to bind to estrogen receptors (ERs) and interfere with the normal cellular development in target tissues including the prostate and uterus. These EEDs, including diethylstilbestrol (DES), bisphenol A (BPA), and genistein (a phytoestrogen derived from soybeans), have been implicated in the malformation of reproductive organs and later development of disease. Due to the lack of fully understanding the underlying mechanisms of how environmental toxicants and their level of exposure affect the human genome, it can be challenging to create clear

  3. Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

    PubMed Central

    Asensi, Karina D; Fortunato, Rodrigo S; dos Santos, Danúbia S; Pacheco, Thaísa S; de Rezende, Danielle F; Rodrigues, Deivid C; Mesquita, Fernanda C P; Kasai-Brunswick, Tais H; de Carvalho, Antonio C Campos; Carvalho, Denise P; Carvalho, Adriana B; Goldenberg, Regina C dos S

    2014-01-01

    Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS. PMID:24528612

  4. Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

    PubMed Central

    Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

    2012-01-01

    Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

  5. Reprogramming: A Preventive Strategy in Hypertension Focusing on the Kidney

    PubMed Central

    Tain, You-Lin; Joles, Jaap A.

    2015-01-01

    Adulthood hypertension can be programmed in response to a suboptimal environment in early life. However, developmental plasticity also implies that one can prevent hypertension in adult life by administrating appropriate compounds during early development. We have termed this reprogramming. While the risk of hypertension has been assessed in many mother-child cohorts of human developmental programming, interventions necessary to prove causation and provide a reprogramming strategy are lacking. Since the developing kidney is particularly vulnerable to environmental insults and blood pressure is determined by kidney function, renal programming is considered key in developmental programming of hypertension. Common pathways, whereby both genetic and acquired developmental programming converge into the same phenotype, have been recognized. For instance, the same reprogramming interventions aimed at shifting nitric oxide (NO)-reactive oxygen species (ROS) balance, such as perinatal citrulline or melatonin supplements, can be protective in both genetic and developmentally programmed hypertension. Furthermore, a significantly increased expression of gene Ephx2 (soluble epoxide hydrolase) was noted in both genetic and acquired animal models of hypertension. Since a suboptimal environment is often multifactorial, such common reprogramming pathways are a practical finding for translation to the clinic. This review provides an overview of potential clinical applications of reprogramming strategies to prevent programmed hypertension. We emphasize the kidney in the following areas: mechanistic insights from human studies and animal models to interpret programmed hypertension; identified risk factors of human programmed hypertension from mother-child cohorts; and the impact of reprogramming strategies on programmed hypertension from animal models. It is critical that the observed effects on developmental reprogramming in animal models are replicated in human studies. PMID

  6. Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming.

    PubMed

    Zimmer, Sebastian; Grebe, Alena; Bakke, Siril S; Bode, Niklas; Halvorsen, Bente; Ulas, Thomas; Skjelland, Mona; De Nardo, Dominic; Labzin, Larisa I; Kerksiek, Anja; Hempel, Chris; Heneka, Michael T; Hawxhurst, Victoria; Fitzgerald, Michael L; Trebicka, Jonel; Björkhem, Ingemar; Gustafsson, Jan-Åke; Westerterp, Marit; Tall, Alan R; Wright, Samuel D; Espevik, Terje; Schultze, Joachim L; Nickenig, Georg; Lütjohann, Dieter; Latz, Eicke

    2016-04-01

    Atherosclerosis is an inflammatory disease linked to elevated blood cholesterol concentrations. Despite ongoing advances in the prevention and treatment of atherosclerosis, cardiovascular disease remains the leading cause of death worldwide. Continuous retention of apolipoprotein B-containing lipoproteins in the subendothelial space causes a local overabundance of free cholesterol. Because cholesterol accumulation and deposition of cholesterol crystals (CCs) trigger a complex inflammatory response, we tested the efficacy of the cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), a compound that increases cholesterol solubility in preventing and reversing atherosclerosis. We showed that CD treatment of murine atherosclerosis reduced atherosclerotic plaque size and CC load and promoted plaque regression even with a continued cholesterol-rich diet. Mechanistically, CD increased oxysterol production in both macrophages and human atherosclerotic plaques and promoted liver X receptor (LXR)-mediated transcriptional reprogramming to improve cholesterol efflux and exert anti-inflammatory effects. In vivo, this CD-mediated LXR agonism was required for the antiatherosclerotic and anti-inflammatory effects of CD as well as for augmented reverse cholesterol transport. Because CD treatment in humans is safe and CD beneficially affects key mechanisms of atherogenesis, it may therefore be used clinically to prevent or treat human atherosclerosis. PMID:27053774

  7. Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming

    PubMed Central

    Zimmer, Sebastian; Grebe, Alena; Bakke, Siril S.; Bode, Niklas; Halvorsen, Bente; Ulas, Thomas; Skjelland, Mona; De Nardo, Dominic; Labzin, Larisa I.; Kerksiek, Anja; Hempel, Chris; Heneka, Michael T.; Hawxhurst, Victoria; Fitzgerald, Michael L; Trebicka, Jonel; Gustafsson, Jan-Åke; Westerterp, Marit; Tall, Alan R.; Wright, Samuel D.; Espevik, Terje; Schultze, Joachim L.; Nickenig, Georg; Lütjohann, Dieter; Latz, Eicke

    2016-01-01

    Atherosclerosis is an inflammatory disease linked to elevated blood cholesterol levels. Despite ongoing advances in the prevention and treatment of atherosclerosis, cardiovascular disease remains the leading cause of death worldwide. Continuous retention of apolipoprotein B-containing lipoproteins in the subendothelial space causes a local overabundance of free cholesterol. Since cholesterol accumulation and deposition of cholesterol crystals (CCs) triggers a complex inflammatory response, we tested the efficacy of the cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), a compound that increases cholesterol solubility, in preventing and reversing atherosclerosis. Here we show that CD treatment of murine atherosclerosis reduced atherosclerotic plaque size and CC load, and promoted plaque regression even with a continued cholesterol-rich diet. Mechanistically, CD increased oxysterol production in both macrophages and human atherosclerotic plaques, and promoted liver X receptor (LXR)-mediated transcriptional reprogramming to improve cholesterol efflux and exert anti-inflammatory effects. In vivo, this CD-mediated LXR agonism was required for the anti-atherosclerotic and anti-inflammatory effects of CD as well as for augmented reverse cholesterol transport. Since CD treatment in humans is safe and CD beneficially affects key mechanisms of atherogenesis, it may therefore be used clinically to prevent or treat human atherosclerosis. PMID:27053774

  8. A Proposed Quantitative Index for Assessing the Potential Contribution of Reprogramming to Cancer Stem Cell Kinetics

    PubMed Central

    Naidu, Mamta; Hahnfeldt, Philip; Hlatky, Lynn

    2014-01-01

    Enrichment of cancer stem cells (CSCs) is thought to be responsible for glioblastoma multiforme (GBM) recurrence after radiation therapy. Simulation results from our agent-based cellular automata model reveal that the enrichment of CSCs may result either from an increased symmetric self-renewal division rate of CSCs or a reprogramming of non-stem cancer cells (CCs) to a stem cell state. Based on plateau-to-peak ratio of the CSC fraction in the tumor following radiation, a downward trend from peak to subsequent plateau (i.e., a plateau-to-peak ratio exceeding 1.0) was found to be inconsistent with increased symmetric division alone and favors instead a strong reprogramming component. The two contributions together are seen to be the product of a dynamic equilibrium between CSCs and CCs that is highly regulated by the kinetics of single cells, including the potential for CCs to reacquire a stem cell state and confer phenotypic plasticity to the population as a whole. We conclude that tumor malignancy can be gauged by a degree of cancer cell plasticity. PMID:24955094

  9. Gaining myocytes or losing fibroblasts: Challenges in cardiac fibroblast reprogramming for infarct repair.

    PubMed

    Nagalingam, Raghu S; Safi, Hamza A; Czubryt, Michael P

    2016-04-01

    Unlike most somatic tissues, the heart possesses a very limited inherent ability to repair itself following damage. Attempts to therapeutically salvage the myocardium after infarction, either by sparing surviving myocytes or by injection of exogenous cells of varied provenance, have met with limited success. Cardiac fibroblasts are numerous, resistant to hypoxia, and amenable to phenotype reprogramming to cardiomyocytes - a potential panacea to an intractable problem. However, the long-term effects of mass conversion of fibroblasts are as-yet unknown. Since fibroblasts play key roles in normal cardiac function, treating these cells as a ready source of replacements for myocytes may have the effect of swapping one problem for another. This review briefly examines the roles of cardiac fibroblasts, recaps the strides made so far in their reprogramming to cardiomyocytes both in vitro and in vivo, and discusses the potential ramifications of large-scale cellular identity swapping. While such therapy offers great promise, the potential repercussions require consideration and careful study. PMID:26640115

  10. MicroRNA-Mediated Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells.

    PubMed

    Sandmaier, Shelley E S; Telugu, Bhanu Prakash V L

    2015-01-01

    MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttranscriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are then processed into a smaller 18-23-nucleotide mature miRNAs that bind to the target transcripts and induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expression in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4, Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced pluripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for establishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC. PMID:26621586

  11. A combination of small molecules directly reprograms mouse fibroblasts into neural stem cells.

    PubMed

    Zheng, Jie; Choi, Kyung-Ah; Kang, Phil Jun; Hyeon, Solji; Kwon, Suhyun; Moon, Jai-Hee; Hwang, Insik; Kim, Yang In; Kim, Yoon Sik; Yoon, Byung Sun; Park, Gyuman; Lee, JangBo; Hong, SungHoi; You, Seungkwon

    2016-07-15

    The generation of induced neural stem cells (iNSCs) from somatic cells using defined factors provides new avenues for basic research and cell therapies for various neurological diseases, such as Parkinson's disease, Huntington's disease, and spinal cord injuries. However, the transcription factors used for direct reprogramming have the potential to cause unexpected genetic modifications, which limits their potential application in cell therapies. Here, we show that a combination of four chemical compounds resulted in cells directly acquiring a NSC identity; we termed these cells chemically-induced NSCs (ciNSCs). ciNSCs expressed NSC markers (Pax6, PLZF, Nestin, Sox2, and Sox1) and resembled NSCs in terms of their morphology, self-renewal, gene expression profile, and electrophysiological function when differentiated into the neuronal lineage. Moreover, ciNSCs could differentiate into several types of mature neurons (dopaminergic, GABAergic, and cholinergic) as well as astrocytes and oligodendrocytes in vitro. Taken together, our results suggest that stably expandable and functional ciNSCs can be directly reprogrammed from mouse fibroblasts using a combination of small molecules without any genetic manipulation, and will provide a new source of cells for cellular replacement therapy of neurodegenerative diseases. PMID:27207831

  12. Re-programming tumour cell metabolism to treat cancer: no lone target for lonidamine.

    PubMed

    Bhutia, Yangzom D; Babu, Ellappan; Ganapathy, Vadivel

    2016-06-01

    Tumour cell metabolism is very different from normal cell metabolism; cancer cells re-programme the metabolic pathways that occur in normal cells in such a manner that it optimizes their proliferation, growth and survival. Although this metabolic re-programming obviously operates to the advantage of the tumour, it also offers unique opportunities for effective cancer therapy. Molecules that target the tumour cell-specific metabolic pathways have potential as novel anti-cancer drugs. Lonidamine belongs to this group of molecules and is already in use in some countries for cancer treatment. It has been known for a long time that lonidamine interferes with energy production in tumour cells by inhibiting hexokinase II (HKII), a glycolytic enzyme. However, subsequent studies have uncovered additional pharmacological targets for the drug, which include the electron transport chain and the mitochondrial permeability transition pore, thus expanding the pharmacological effects of the drug on tumour cell metabolism. A study by Nancolas et al. in a recent issue of the Biochemical Journal identifies two additional new targets for lonidamine: the pyruvate transporter in the mitochondria and the H(+)-coupled monocarboxylate transporters in the plasma membrane (PM). It is thus becoming increasingly apparent that the anti-cancer effects of lonidamine do not occur through a single target; the drug works at multiple sites. Irrespective of the molecular targets, what lonidamine does in the end is to undo what the tumour cells have done in terms of re-programming cellular metabolism and mitochondrial function. PMID:27234586

  13. Re-programming tumour cell metabolism to treat cancer: no lone target for lonidamine

    PubMed Central

    Bhutia, Yangzom D.; Babu, Ellappan; Ganapathy, Vadivel

    2016-01-01

    Tumour cell metabolism is very different from normal cell metabolism; cancer cells re-programme the metabolic pathways that occur in normal cells in such a manner that it optimizes their proliferation, growth and survival. Although this metabolic re-programming obviously operates to the advantage of the tumour, it also offers unique opportunities for effective cancer therapy. Molecules that target the tumour cell-specific metabolic pathways have potential as novel anti-cancer drugs. Lonidamine belongs to this group of molecules and is already in use in some countries for cancer treatment. It has been known for a long time that lonidamine interferes with energy production in tumour cells by inhibiting hexokinase II (HKII), a glycolytic enzyme. However, subsequent studies have uncovered additional pharmacological targets for the drug, which include the electron transport chain and the mitochondrial permeability transition pore, thus expanding the pharmacological effects of the drug on tumour cell metabolism. A study by Nancolas et al. in a recent issue of the Biochemical Journal identifies two additional new targets for lonidamine: the pyruvate transporter in the mitochondria and the H+-coupled monocarboxylate transporters in the plasma membrane (PM). It is thus becoming increasingly apparent that the anti-cancer effects of lonidamine do not occur through a single target; the drug works at multiple sites. Irrespective of the molecular targets, what lonidamine does in the end is to undo what the tumour cells have done in terms of re-programming cellular metabolism and mitochondrial function. PMID:27234586

  14. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth.

    PubMed

    Cox, Andrew G; Hwang, Katie L; Brown, Kristin K; Evason, Kimberley J; Beltz, Sebastian; Tsomides, Allison; O'Connor, Keelin; Galli, Giorgio G; Yimlamai, Dean; Chhangawala, Sagar; Yuan, Min; Lien, Evan C; Wucherpfennig, Julia; Nissim, Sahar; Minami, Akihiro; Cohen, David E; Camargo, Fernando D; Asara, John M; Houvras, Yariv; Stainier, Didier Y R; Goessling, Wolfram

    2016-08-01

    The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. PMID:27428308

  15. Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

    PubMed

    Tseng, Ting-Chen; Hsieh, Fu-Yu; Dai, Niann-Tzyy; Hsu, Shan-Hui

    2016-09-01

    Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future. PMID:27341268

  16. Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

    PubMed

    Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

    2015-01-01

    Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells. PMID:25965553

  17. Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

    PubMed

    Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

    2014-01-01

    In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors

  18. Radiation-Induced Reprogramming of Pre-Senescent Mammary Epithelial Cells Enriches Putative CD44+/CD24−/low Stem Cell Phenotype

    PubMed Central

    Gao, Xuefeng; Sishc, Brock J.; Nelson, Christopher B.; Hahnfeldt, Philip; Bailey, Susan M.; Hlatky, Lynn

    2016-01-01

    The enrichment of putative CD44+/CD24−/low breast stem cell populations following exposure to ionizing radiation (IR) has been ascribed to their inherent radioresistance and an elevated frequency of symmetric division during repopulation. However, recent studies demonstrating radiation-induced phenotypic reprogramming (the transition of non-CD44+/CD24−/low cells into the CD44+/CD24−/low phenotype) as a potential mechanism of CD44+/CD24−/low cell enrichment have raised the question of whether a higher survival and increased self-renewal of existing CD44+/CD24−/low cells or induced reprogramming is an additional mode of enrichment. To investigate this question, we combined a cellular automata model with in vitro experimental data using both MCF-10A non-tumorigenic human mammary epithelial cells and MCF-7 breast cancer cells, with the goal of identifying the mechanistic basis of CD44+/CD24−/low stem cell enrichment in the context of radiation-induced cellular senescence. Quantitative modeling revealed that incomplete phenotypic reprogramming of pre-senescent non-stem cells (reprogramming whereby the CD44+/CD24−/low phenotype is conveyed, along with the short-term proliferation capacity of the original cell) could be an additional mode of enriching the CD44+/CD24−/low subpopulation. Furthermore, stem cell enrichment in MCF-7 cells occurs both at lower doses and earlier time points, and has longer persistence, than that observed in MCF-10A cells, suggesting that phenotypic plasticity appears to be less regulated in breast cancer cells. Taken together, these results suggest that reprogramming of pre-senescent non-stem cells may play a significant role in both cancer and non-tumorigenic mammary epithelial populations following exposure to IR, a finding with important implications for both radiation therapy and radiation carcinogenesis. PMID:27379202

  19. Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

    PubMed Central

    Ozmadenci, Duygu; Féraud, Olivier; Markossian, Suzy; Kress, Elsa; Ducarouge, Benjamin; Gibert, Benjamin; Ge, Jian; Durand, Isabelle; Gadot, Nicolas; Plateroti, Michela; Bennaceur-Griscelli, Annelise; Scoazec, Jean-Yves; Gil, Jesus; Deng, Hongkui; Bernet, Agnes; Mehlen, Patrick; Lavial, Fabrice

    2015-01-01

    The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells. PMID:26154507

  20. Non-stochastic reprogramming from a privileged somatic cell state

    PubMed Central

    Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P.; Cheng, Jijun; Zhong, Mei; Koochaki, Sebastian H.J.; Megyola, Cynthia M.; Pan, Xinghua; Heydari, Kartoosh; Weissman, Sherman M.; Gallagher, Patrick G.; Krause, Diane S.; Fan, Rong; Lu, Jun

    2014-01-01

    SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PMID:24486105

  1. Epigenetic reprogramming in mammalian species after SCNT-based cloning.

    PubMed

    Niemann, Heiner

    2016-07-01

    The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. PMID:27160443

  2. Regulation of somatic cell reprogramming through inducible mir-302 expression.

    PubMed

    Lin, Shi-Lung; Chang, Donald C; Lin, Chun-Hung; Ying, Shao-Yao; Leu, Davey; Wu, David T S

    2011-02-01

    Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation. PMID:20870751

  3. Upping the Ante: Recent Advances in Direct Reprogramming

    PubMed Central

    Müller, Lars UW; Daley, George Q; Williams, David A

    2009-01-01

    The concept of reversing the characteristics of differentiated tissues to pluripotency through reprogramming was introduced over 50 years ago in the first somatic cell nuclear transfer (SCNT) experiments. More recently, direct reprogramming of differentiated somatic cells by gene transfer of a small number of defined transcription factors has been shown to yield cells that are indistinguishable from inner cell mass–derived embryonic stem (ES) cells. These cells, termed induced pluripotent stem (iPS) cells, offer exciting possibilities for studying mechanism of pluripotency, establishing models for disease-specific investigations, and enabling future applications in regenerative medicine. In this review, we discuss the basic foundation of reestablishing pluripotency and recent progress toward enhancing the efficiency and safety of the process through optimization of the reprogramming factor combination, identification of small molecules that augment efficiency, and assessment of distinct target cells in reprogramming efficiency. We also highlight recent advances that eliminate stable genetic modification from the reprogramming process, and summarize preclinical models that provide proof-of-concept for ES/iPS cell-based regenerative medicine. PMID:19337233

  4. Metabolic Reprogramming by the PI3K-Akt-mTOR Pathway in Cancer.

    PubMed

    Lien, Evan C; Lyssiotis, Costas A; Cantley, Lewis C

    2016-01-01

    In the past decade, there has been a resurgence of interest in elucidating how metabolism is altered in cancer cells and how such dependencies can be targeted for therapeutic gain. At the core of this research is the concept that metabolic pathways are reprogrammed in cancer cells to divert nutrients toward anabolic processes to facilitate enhanced growth and proliferation. Importantly, physiological cellular signaling mechanisms normally tightly regulate the ability of cells to gain access to and utilize nutrients, posing a fundamental barrier to transformation. This barrier is often overcome by aberrations in cellular signaling that drive tumor pathogenesis by enabling cancer cells to make critical cellular decisions in a cell-autonomous manner. One of the most frequently altered pathways in human cancer is the PI3K-Akt-mTOR signaling pathway. Here, we describe mechanisms by which this signaling network is responsible for controlling cellular metabolism. Through both the post-translational regulation and the induction of transcriptional programs, the PI3K-Akt-mTOR pathway coordinates the uptake and utilization of multiple nutrients, including glucose, glutamine, nucleotides, and lipids, in a manner best suited for supporting the enhanced growth and proliferation of cancer cells. These regulatory mechanisms illustrate how metabolic changes in cancer are closely intertwined with oncogenic signaling pathways that drive tumor initiation and progression. PMID:27557534

  5. The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis

    PubMed Central

    Shao, Zhicheng; Zhang, Ruowen; Khodadadi-Jamayran, Alireza; Chen, Bo; Crowley, Michael R.; Festok, Muhamad A.; Crossman, David K.; Townes, Tim M.; Hu, Kejin

    2016-01-01

    It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming. PMID:26947130

  6. The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.

    PubMed

    Shao, Zhicheng; Zhang, Ruowen; Khodadadi-Jamayran, Alireza; Chen, Bo; Crowley, Michael R; Festok, Muhamad A; Crossman, David K; Townes, Tim M; Hu, Kejin

    2016-01-01

    It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming. PMID:26947130

  7. The Broad Foundations, 2006

    ERIC Educational Resources Information Center

    Broad Foundation, 2006

    2006-01-01

    The mission of the Broad Foundations is to transform K-12 urban public education through better governance, management, labor relations and competition; make significant contributions to advance major scientific and medical research; foster public appreciation of contemporary art by increasing access for audiences worldwide; and lead and…

  8. Broad Bandwidth Telecommunications Systems.

    ERIC Educational Resources Information Center

    Sodolski, John

    Broad bandwidth transmission systems have been around for years. They include microwave, assorted cable systems, and recently, satellites. With the exception of some privately owned systems, broadband services have been furnished by the common carriers. Recently, a new element has been added--Cable Antenna Television (CATV) distribution systems.…

  9. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.

    PubMed

    Muraoka, Naoto; Ieda, Masaki

    2014-01-01

    Heart disease is a major cause of morbidity and mortality worldwide. The low regenerative capacity of adult human hearts has thus far limited the available therapeutic approaches for heart failure. Therefore, new therapies that can regenerate damaged myocardium and improve heart function are urgently needed. Although cell transplantation-based therapies may hold great potential, direct reprogramming of endogenous cardiac fibroblasts, which represent more than half of the cells in the heart, into functional cardiomyocytes in situ may be an alternative strategy by which to regenerate the heart. We and others demonstrated that functional cardiomyocytes can be directly generated from fibroblasts by using several combinations of cardiac-enriched factors in mouse and human. In vivo gene delivery of cardiac reprogramming factors generates new cardiac muscle and improved heart function after myocardial infarction in mouse. This article reviews recent progress in cardiac reprogramming research and discusses the perspectives and challenges of this new technology for future regenerative therapy. PMID:24079415

  10. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos.

    PubMed

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Jeon, Kyuheum; Kang, Yong-Kook

    2016-01-01

    Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT) embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts. PMID:26976441

  11. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    PubMed Central

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Jeon, Kyuheum; Kang, Yong-Kook

    2016-01-01

    Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT) embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts. PMID:26976441

  12. Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

    PubMed Central

    Ibarretxe, Gaskon; Alvarez, Antonia; Cañavate, Maria-Luz; Hilario, Enrique; Aurrekoetxea, Maitane; Unda, Fernando

    2012-01-01

    The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration. PMID:22690226

  13. Electromagnetic fields mediate efficient cell reprogramming into a pluripotent state.

    PubMed

    Baek, Soonbong; Quan, Xiaoyuan; Kim, Soochan; Lengner, Christopher; Park, Jung-Keug; Kim, Jongpil

    2014-10-28

    Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency. PMID:25248035

  14. DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice.

    PubMed

    Shi, Sally Yu; Lu, Shun-Yan; Sivasubramaniyam, Tharini; Revelo, Xavier S; Cai, Erica P; Luk, Cynthia T; Schroer, Stephanie A; Patel, Prital; Kim, Raymond H; Bombardier, Eric; Quadrilatero, Joe; Tupling, A Russell; Mak, Tak W; Winer, Daniel A; Woo, Minna

    2015-01-01

    Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinson's disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders. PMID:26077864

  15. DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice

    PubMed Central

    Shi, Sally Yu; Lu, Shun-Yan; Sivasubramaniyam, Tharini; Revelo, Xavier S.; Cai, Erica P.; Luk, Cynthia T.; Schroer, Stephanie A.; Patel, Prital; Kim, Raymond H.; Bombardier, Eric; Quadrilatero, Joe; Tupling, A. Russell; Mak, Tak W.; Winer, Daniel A.; Woo, Minna

    2015-01-01

    Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinson's disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders. PMID:26077864

  16. Nuclear Reprogramming and Mitosis – how does mitosis enhance changes in gene expression?

    PubMed Central

    Halley-Stott, Richard P

    2015-01-01

    Abstract Nuclear reprogramming changes the identity of cells by changing gene expression programmes. Two recent pieces of work have highlighted the role that mitosis plays in enhancing the success of nuclear reprogramming. This Point of View article examines this work in the context of nuclear reprogramming. PMID:25668203

  17. CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION

    PubMed Central

    Haspel, Jeffrey A.; Chettimada, Sukrutha; Shaik, Rahamthulla S.; Chu, Jen-Hwa; Raby, Benjamin A.; Cernadas, Manuela; Carey, Vincent; Process, Vanessa; Hunninghake, G. Matthew; Ifedigbo, Emeka; Lederer, James A.; Englert, Joshua; Pelton, Ashley; Coronata, Anna; Fredenburgh, Laura E.; Choi, Augustine M. K.

    2014-01-01

    Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here, we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian program exhibits unique features, including a divergent group of rhythmic genes and metabolites compared to the basal state and a distinct periodicity and phase distribution. At the cellular level endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex reorganization of cellular and molecular circadian rhythms that are relevant to early events in lung injury. PMID:25208554

  18. 25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages*

    PubMed Central

    Shibata, Norihito; Carlin, Aaron F.; Spann, Nathanael J.; Saijo, Kaoru; Morello, Christopher S.; McDonald, Jeffrey G.; Romanoski, Casey E.; Maurya, Mano R.; Kaikkonen, Minna U.; Lam, Michael T.; Crotti, Andrea; Reichart, Donna; Fox, Jesse N.; Quehenberger, Oswald; Raetz, Christian R. H.; Sullards, M. Cameron; Murphy, Robert C.; Merrill, Alfred H.; Brown, H. Alex; Dennis, Edward A.; Fahy, Eoin; Subramaniam, Shankar; Cavener, Douglas R.; Spector, Deborah H.; Russell, David W.; Glass, Christopher K.

    2013-01-01

    25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity. PMID:24189069

  19. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    PubMed Central

    Hawkins, Kate; Joy, Shona; McKay, Tristan

    2014-01-01

    Induced pluripotent stem (iPS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and cMyc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few “stabilisation competent” cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-iPS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, iPS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As iPS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon. PMID:25426259

  20. RNA-binding proteins in pluripotency, differentiation, and reprogramming

    PubMed Central

    GUALLAR, Diana; WANG, Jianlong

    2014-01-01

    Embryonic stem cell maintenance, differentiation, and somatic cell reprogramming require the interplay of multiple pluripotency factors, epigenetic remodelers, and extracellular signaling pathways. RNA-binding proteins (RBPs) are involved in a wide range of regulatory pathways, from RNA metabolism to epigenetic modifications. In recent years we have witnessed more and more studies on the discovery of new RBPs and the assessment of their functions in a variety of biological systems, including stem cells. We review the current studies on RBPs and focus on those that have functional implications in pluripotency, differentiation, and/or reprogramming in both the human and mouse systems. PMID:25554730

  1. HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

    PubMed Central

    McNamara, Ryan P; Forst, Christian V; D'Orso, Iván

    2015-01-01

    HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular ‘TAR-like’ motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but ‘multitasking’ protein to simultaneously control viral and cellular transcription. DOI: http://dx.doi.org/10.7554/eLife.08955.001 PMID:26488441

  2. Bench-to-bedside review: Endotoxin tolerance as a model of leukocyte reprogramming in sepsis

    PubMed Central

    Cavaillon, Jean-Marc; Adib-Conquy, Minou

    2006-01-01

    Endotoxin tolerance is defined as a reduced responsiveness to a lipopolysaccharide (LPS) challenge following a first encounter with endotoxin. Endotoxin tolerance protects against a lethal challenge of LPS and prevents infection and ischemia-reperfusion damage. Endotoxin tolerance is paralleled by a dramatic reduction of tumor necrosis factor (TNF) production and some other cytokines in response to LPS. Endotoxin tolerance involves the participation of macrophages and mediators, such as glucocorticoids, prostaglandins, IL-10, and transforming growth factor-β. Endotoxin tolerance is accompanied by the up-regulation of inhibitory molecules that down-regulate the Toll-like receptor (TLR)4-dependent signaling pathway. Cross-tolerance between LPS and other TLR specific ligands, as well as IL-1 and TNF, has been regularly reported. A similar loss of LPS reactivity has been repeatedly reported in circulating leukocytes of septic patients and in patients with non-infectious systemic inflammation response syndrome (SIRS). Studies on cellular signaling within leukocytes from septic and SIRS patients reveal numerous alterations reminiscent of those observed in endotoxin tolerant cells. However, altered responsiveness to LPS of leukocytes from sepsis and SIRS patients is not synonymous with a global down-regulation of cellular reactivity. The term 'cellular reprogramming', which has been proposed to qualify the process of endotoxin tolerance, defines well the immune status of circulating leukocytes in septic and SIRS patients. PMID:17044947

  3. Broad band waveguide spectrometer

    DOEpatents

    Goldman, Don S.

    1995-01-01

    A spectrometer for analyzing a sample of material utilizing a broad band source of electromagnetic radiation and a detector. The spectrometer employs a waveguide possessing an entry and an exit for the electromagnetic radiation emanating from the source. The waveguide further includes a surface between the entry and exit portions which permits interaction between the electromagnetic radiation passing through the wave guide and a sample material. A tapered portion forms a part of the entry of the wave guide and couples the electromagnetic radiation emanating from the source to the waveguide. The electromagnetic radiation passing from the exit of the waveguide is captured and directed to a detector for analysis.

  4. Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

    PubMed

    Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

    2014-01-01

    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture. PMID:25170299

  5. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

    PubMed Central

    Qian, Li; Huang, Yu; Spencer, C. Ian; Foley, Amy; Vedantham, Vasanth; Liu, Lei; Conway, Simon J.; Fu, Ji-dong; Srivastava, Deepak

    2012-01-01

    SUMMARY The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here, we use genetic lineage-tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became bi-nucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast activating peptide, Thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. PMID:22522929

  6. Reprogram to pluripotency: a new logic and a chemical cocktail

    PubMed Central

    Song, Hongjun; Ming, Guo-li

    2015-01-01

    Somatic cells from animals and humans can be reprogrammed into pluripotent stem cells by pluripotency factors. Hongkui Deng and colleagues discovered that pluripotency can also be induced with exogenous lineage specifiers via balancing competing differentiation forces. In a related study they achieved, for the first time, restoration of pluripotency in adult somatic cells using a chemical cocktail alone. PMID:26998393

  7. How microRNAs facilitate reprogramming to pluripotency

    PubMed Central

    Anokye-Danso, Frederick; Snitow, Melinda; Morrisey, Edward E.

    2012-01-01

    Summary The ability to generate pluripotent stem cells from a variety of cell and tissue sources through the ectopic expression of a specific set of transcription factors has revolutionized regenerative biology. The development of this reprogramming technology not only makes it possible to perform basic research on human stem cells that do not have to be derived from embryos, but also allows patient-specific cells and tissues to be generated for therapeutic use. Optimizing this process will probably lead to a better and more efficient means of generating pluripotent stem cells. Here, we discuss recent findings that show that, in addition to transcription factors, microRNAs can promote pluripotent reprogramming and can even substitute for these pluripotency transcription factors in some cases. Taking into consideration that microRNAs have the potential to be used as small-molecule therapeutics, such findings open new possibilities for both pluripotent stem cell reprogramming and the reprogramming of cells into other cell lineages. PMID:23077173

  8. Somatic Cell Dedifferentiation/Reprogramming for Regenerative Medicine

    PubMed Central

    Ramesh, Thiyagarajan; Lee, Sun-Hee; Lee, Choon-Soo; Kwon, Yoo-Wook; Cho, Hyun-Jai

    2009-01-01

    The concept of dedifferentiation or reprogramming of a somatic cell into a pluripotent embryonic stem cell-like cell (ES-like cell), which give rise to three germ layers and differentiate various cell types, opens a new era in stem cell biology and provides potential therapeutic modality in regenerative medicine. Here, we outline current dedifferentiation/reprogramming methods and their technical hurdles, and the safety and therapeutic applications of reprogrammed pluripotent stem cells in regenerative medicine. This review summarizes the concept and data of somatic cell nuclear transfer, fusion of somatic cells with ES cells, viral or non-viral transduction of pluripotency-related genes into somatic cells, introduction of extract (or proteins) of pluripotent cells into somatic cells. Dedifferentiated/reprogrammed ES-like cells could be a perfect genetic match (autologous or tailored pluripotent stem cells) for future applications. Further studies regarding technical refinements as well as mechanistic analysis of dedifferentiation induction and re-differentiation into specific cell types will provide us with the substantial application of pluripotent stem cells to therapeutic purposes. PMID:24855516

  9. Broad spectrum bioactive sunscreens.

    PubMed

    Velasco, Maria Valéria Robles; Sarruf, Fernanda Daud; Salgado-Santos, Idalina Maria Nunes; Haroutiounian-Filho, Carlos Alberto; Kaneko, Telma Mary; Baby, André Rolim

    2008-11-01

    The development of sunscreens containing reduced concentration of chemical UV filters, even though, possessing broad spectrum effectiveness with the use of natural raw materials that improve and infer UV absorption is of great interest. Due to the structural similarities between polyphenolic compounds and organic UV filters, they might exert photoprotection activity. The objective of the present research work was to develop bioactive sunscreen delivery systems containing rutin, Passiflora incarnata L. and Plantago lanceolata extracts associated or not with organic and inorganic UV filters. UV transmission of the sunscreen delivery system films was performed by using diffuse transmittance measurements coupling to an integrating sphere. In vitro photoprotection efficacy was evaluated according to the following parameters: estimated sun protection factor (SPF); Boot's Star Rating category; UVA/UVB ratio; and critical wavelength (lambda(c)). Sunscreen delivery systems obtained SPF values ranging from 0.972+/-0.004 to 28.064+/-2.429 and bioactive compounds interacted with the UV filters positive and negatively. This behavior may be attributed to: the composition of the delivery system; the presence of inorganic UV filter and quantitative composition of the organic UV filters; and the phytochemical composition of the P. incarnata L. and P. lanceolata extracts. Among all associations of bioactive compounds and UV filters, we found that the broad spectrum sunscreen was accomplished when 1.68% (w/w) P. incarnata L. dry extract was in the presence of 7.0% (w/w) ethylhexyl methoxycinnamate, 2.0% (w/w) benzophenone-3 and 2.0% (w/w) TiO(2). It was demonstrated that this association generated estimated SPF of 20.072+/-0.906 and it has improved the protective defense against UVA radiation accompanying augmentation of the UVA/UVB ratio from 0.49 to 0.52 and lambda(c) from 364 to 368.6nm. PMID:18662760

  10. Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors.

    PubMed

    Planello, Aline C; Ji, Junfeng; Sharma, Vivek; Singhania, Rajat; Mbabaali, Faridah; Müller, Fabian; Alfaro, Javier A; Bock, Christoph; De Carvalho, Daniel D; Batada, Nizar N

    2014-01-01

    The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities. PMID:25408883

  11. Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells

    PubMed Central

    2014-01-01

    Integrating viruses represent robust tools for cellular reprogramming; however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. PMID:24713299

  12. Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes

    PubMed Central

    Powley, Ian R.; Kondrashov, Alexander; Young, Lucy A.; Dobbyn, Helen C.; Hill, Kirsti; Cannell, Ian G.; Stoneley, Mark; Kong, Yi-Wen; Cotes, Julia A.; Smith, Graeme C.M.; Wek, Ron; Hayes, Christopher; Gant, Timothy W.; Spriggs, Keith A.; Bushell, Martin; Willis, Anne E.

    2009-01-01

    UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5′ untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis. PMID:19451221

  13. Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection

    PubMed Central

    Cubas, Rafael; van Grevenynghe, Julien; Wills, Saintedym; Kardava, Lela; Santich, Brian H.; Buckner, Clarisa M.; Muir, Roshell; Tardif, Virginie; Nichols, Carmen; Procopio, Francesco; He, Zhong; Metcalf, Talibah; Ghneim, Khader; Locci, Michela; Ancuta, Petronella; Routy, Jean-Pierre; Trautmann, Lydie; Li, Yuxing; McDermott, Adrian B.; Koup, Rick A.; Petrovas, Constantinos; Migueles, Steven A.; Connors, Mark; Tomaras, Georgia D.; Moir, Susan; Crotty, Shane

    2015-01-01

    Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2–responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART. PMID:26546609

  14. MUC16-mediated activation of mTOR and c-MYC reprograms pancreatic cancer metabolism

    PubMed Central

    Shukla, Surendra K.; Gunda, Venugopal; Abrego, Jaime; Haridas, Dhanya; Mishra, Anusha; Souchek, Joshua; Chaika, Nina V.; Yu, Fang; Sasson, Aaron R.; Lazenby, Audrey J.; Batra, Surinder K.; Singh, Pankaj K.

    2015-01-01

    MUC16, a transmembrane mucin, facilitates pancreatic adenocarcinoma progression and metastasis. In the current studies, we observed that MUC16 knockdown pancreatic cancer cells exhibit reduced glucose uptake and lactate secretion along with reduced migration and invasion potential, which can be restored by supplementing the culture media with lactate, an end product of aerobic glycolysis. MUC16 knockdown leads to inhibition of mTOR activity and reduced expression of its downstream target c-MYC, a key player in cellular growth, proliferation and metabolism. Ectopic expression of c-MYC in MUC16 knockdown pancreatic cancer cells restores the altered cellular physiology. Our LC-MS/MS based metabolomics studies indicate global metabolic alterations in MUC16 knockdown pancreatic cancer cells, as compared to the controls. Specifically, glycolytic and nucleotide metabolite pools were significantly decreased. We observed similar metabolic alterations that correlated with MUC16 expression in primary tumor tissue specimens from human pancreatic adenocarcinoma cancer patients. Overall, our results demonstrate that MUC16 plays an important role in metabolic reprogramming of pancreatic cancer cells by increasing glycolysis and enhancing motility and invasiveness. PMID:26046375

  15. Deacetylation by SIRT1 Reprograms Inflammation and Cancer

    PubMed Central

    McCall, Charles E.

    2013-01-01

    NAD+-dependent deacetylase SIRT1 is a master regulator of nucleosome positioning and chromatin structure, thereby reprogramming gene expression. In acute inflammation, chromatin departs from, and returns to, homeostasis in an orderly sequence. This sequence depends on shifts in NAD+ availability for SIRT1 activation and deacetylation of signaling proteins, which support orderly gene reprogramming during acute inflammation by switching between euchromatin and heterochromatin. In contrast, in chronic inflammation and cancer, limited availability of NAD+ and reduced expression of SIRT1 may sustain aberrant chromatin structure and functions. SIRT1 also influences inflammation and cancer by directly deacetylating targets like NFκB p65 and p53. Here, we review SIRT1 in the context of inflammation and cancer. PMID:24020005

  16. Development-Inspired Reprogramming of the Mammalian Central Nervous System

    PubMed Central

    Amamoto, Ryoji; Arlotta, Paola

    2014-01-01

    In 2012, John Gurdon and Shinya Yamanaka shared the Nobel Prize for the exciting demonstration that the identity of differentiated cells is not irreversibly determined but can be changed back to a pluripotent state under appropriate instructive signals. The principle that differentiated cells can revert to an embryonic state and even be converted directly from one cell-type into another not only turns fundamental principles of development on their head but also has profound implications for regenerative medicine. Replacement of diseased tissue with newly reprogrammed cells and modeling of human disease are concrete opportunities. Here, we focus on the central nervous system to consider whether and how reprogramming of cell identity may impact regeneration and modeling of a system historically considered immutable and hardwired. PMID:24482482

  17. Metabolic reprogramming in macrophages and dendritic cells in innate immunity

    PubMed Central

    Kelly, Beth; O'Neill, Luke AJ

    2015-01-01

    Activation of macrophages and dendritic cells (DCs) by pro-inflammatory stimuli causes them to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS), similar to the Warburg effect in tumors. However, it is only recently that the mechanisms responsible for this metabolic reprogramming have been elucidated in more detail. The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role under conditions of both hypoxia and normoxia. The withdrawal of citrate from the tricarboxylic acid (TCA) cycle has been shown to be critical for lipid biosynthesis in both macrophages and DCs. Interference with this process actually abolishes the ability of DCs to activate T cells. Another TCA cycle intermediate, succinate, activates HIF-1α and promotes inflammatory gene expression. These new insights are providing us with a deeper understanding of the role of metabolic reprogramming in innate immunity. PMID:26045163

  18. Regeneration through reprogramming adult cell identity in vivo.

    PubMed

    Smith, Derek K; Zhang, Chun-Li

    2015-10-01

    The discovery and in vivo application of cell fate reprogramming concepts have jumpstarted new technologies aimed at the functional regeneration of damaged tissues. As most adult organ systems retain only a limited potential for self-regeneration after trauma, the production of fate-specific cells by in vivo transdifferentiation offers a targeted method for tissue bioengineering. Proof-of-principle studies have demonstrated the induction of neural precursor cells, neurons, cardiomyocytes, and insulin-producing β islet cells. Each of these induced cell types survive, mature, and integrate into the local environment in a functionally meaningful manner. Here, we briefly highlight recent advances in the in vivo reprogramming of cell identity and the current challenges that face the clinical relevance of these methods. PMID:26056931

  19. Exploring the Mechanisms of Differentiation, Dedifferentiation, Reprogramming and Transdifferentiation

    PubMed Central

    Xu, Li; Zhang, Kun; Wang, Jin

    2014-01-01

    We explored the underlying mechanisms of differentiation, dedifferentiation, reprogramming and transdifferentiation (cell type switchings) from landscape and flux perspectives. Lineage reprogramming is a new regenerative method to convert a matured cell into another cell including direct transdifferentiation without undergoing a pluripotent cell state and indirect transdifferentiation with an initial dedifferentiation-reversion (reprogramming) to a pluripotent cell state. Each cell type is quantified by a distinct valley on the potential landscape with higher probability. We investigated three driving forces for cell fate decision making: stochastic fluctuations, gene regulation and induction, which can lead to cell type switchings. We showed that under the driving forces the direct transdifferentiation process proceeds from a differentiated cell valley to another differentiated cell valley through either a distinct stable intermediate state or a certain series of unstable indeterminate states. The dedifferentiation process proceeds through a pluripotent cell state. Barrier height and the corresponding escape time from the valley on the landscape can be used to quantify the stability and efficiency of cell type switchings. We also uncovered the mechanisms of the underlying processes by quantifying the dominant biological paths of cell type switchings on the potential landscape. The dynamics of cell type switchings are determined by both landscape gradient and flux. The flux can lead to the deviations of the dominant biological paths for cell type switchings from the naively expected landscape gradient path. As a result, the corresponding dominant paths of cell type switchings are irreversible. We also classified the mechanisms of cell fate development from our landscape theory: super-critical pitchfork bifurcation, sub-critical pitchfork bifurcation, sub-critical pitchfork with two saddle-node bifurcation, and saddle-node bifurcation. Our model showed good

  20. Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer.

    PubMed

    Hutton, Josiah E; Wang, Xiaojing; Zimmerman, Lisa J; Slebos, Robbert J C; Trenary, Irina A; Young, Jamey D; Li, Ming; Liebler, Daniel C

    2016-09-01

    Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

  1. Oxamflatin Treatment Enhances Cloned Porcine Embryo Development and Nuclear Reprogramming*

    PubMed Central

    Mao, Jiude; Zhao, Ming-Tao; Whitworth, Kristin M.; Spate, Lee D.; Walters, Eric M.; O'Gorman, Chad; Lee, Kiho; Samuel, Melissa S.; Murphy, Clifton N.; Wells, Kevin; Rivera, Rocio M.

    2015-01-01

    Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency. PMID:25548976

  2. Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

    PubMed

    Hirsch, Calley L; Coban Akdemir, Zeynep; Wang, Li; Jayakumaran, Gowtham; Trcka, Dan; Weiss, Alexander; Hernandez, J Javier; Pan, Qun; Han, Hong; Xu, Xueping; Xia, Zheng; Salinger, Andrew P; Wilson, Marenda; Vizeacoumar, Frederick; Datti, Alessandro; Li, Wei; Cooney, Austin J; Barton, Michelle C; Blencowe, Benjamin J; Wrana, Jeffrey L; Dent, Sharon Y R

    2015-04-15

    Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming. PMID:25877919

  3. The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis

    PubMed Central

    Pomerantz, Mark M.; Li, Fugen; Takeda, David; Lenci, Romina; Chonkar, Apurva; Chabot, Matthew; Cejas, Paloma; Vazquez, Francisca; Cook, Jennifer; Shivdasani, Ramesh A.; Bowden, Michaela; Lis, Rosina; Hahn, William C.; Kantoff, Philip W.; Brown, Myles; Loda, Massimo; Long, Henry W.; Freedman, Matthew L.

    2015-01-01

    Master transcription factors interact with DNA to establish cell-type identity and to regulate gene expression in mammalian cells1,2. The genome-wide map of these transcription factor binding sites has been termed the cistrome3. Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13, co-localized with the reprogrammed AR sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium towards transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis. PMID:26457646

  4. Prostate cancer metastasis: roles of recruitment and reprogramming, cell signal network and three-dimensional growth characteristics

    PubMed Central

    Ziaee, Shabnam; Chu, Gina Chia-Yi; Huang, Jen-Ming; Sieh, Shirly

    2015-01-01

    Prostate cancer (PCa) metastasizes to bone and soft tissues, greatly decreasing quality of life, causing bone pain, skeletal complications, and mortality in PCa patients. While new treatment strategies are being developed, the molecular and cellular basis of PCa metastasis and the “cross-talk” between cancer cells and their microenvironment and crucial cell signaling pathways need to be successfully dissected for intervention. In this review, we introduce a new concept of the mechanism of PCa metastasis, the recruitment and reprogramming of bystander and dormant cells (DCs) by a population of metastasis-initiating cells (MICs). We provide evidence that recruited and reprogrammed DCs gain MICs phenotypes and can subsequently metastasize to bone and soft tissues. We show that MICs can also recruit and reprogram circulating tumor cells (CTCs) and this could contribute to cancer cell evolution and the acquisition of therapeutic resistance. We summarize relevant molecular signaling pathways, including androgen receptors (ARs) and their variants and growth factors (GFs) and cytokines that could contribute to the predilection of PCa for homing to bone and soft tissues. To understand the etiology and the biology of PCa and the effectiveness of therapeutic targeting, we briefly summarize the animal and cell models that have been employed. We also report our experience in the use of three-dimensional (3-D) culture and co-culture models to understand cell signaling networks and the use of these attractive tools to conduct drug screening exercises against already-identified molecular targets. Further research into PCa growth and metastasis will improve our ability to target cancer metastasis more effectively and provide better rationales for personalized oncology. PMID:26816842

  5. Stress Response and Perinatal Reprogramming: Unraveling (Mal)adaptive Strategies.

    PubMed

    Musazzi, Laura; Marrocco, Jordan

    2016-01-01

    Environmental stressors induce coping strategies in the majority of individuals. The stress response, involving the activation of the hypothalamic-pituitary-adrenocortical axis and the consequent release of corticosteroid hormones, is indeed aimed at promoting metabolic, functional, and behavioral adaptations. However, behavioral stress is also associated with fast and long-lasting neurochemical, structural, and behavioral changes, leading to long-term remodeling of glutamate transmission, and increased susceptibility to neuropsychiatric disorders. Of note, early-life events, both in utero and during the early postnatal life, trigger reprogramming of the stress response, which is often associated with loss of stress resilience and ensuing neurobehavioral (mal)adaptations. Indeed, adverse experiences in early life are known to induce long-term stress-related neuropsychiatric disorders in vulnerable individuals. Here, we discuss recent findings about stress remodeling of excitatory neurotransmission and brain morphology in animal models of behavioral stress. These changes are likely driven by epigenetic factors that lie at the core of the stress-response reprogramming in individuals with a history of perinatal stress. We propose that reprogramming mechanisms may underlie the reorganization of excitatory neurotransmission in the short- and long-term response to stressful stimuli. PMID:27057367

  6. Metabolic reprogramming orchestrates cancer stem cell properties in nasopharyngeal carcinoma

    PubMed Central

    Shen, Yao-An; Wang, Chia-Yu; Hsieh, Yi-Tao; Chen, Yann-Jang; Wei, Yau-Huei

    2015-01-01

    Cancer stem cells (CSCs) represent a subpopulation of tumor cells endowed with self-renewal capacity and are considered as an underlying cause of tumor recurrence and metastasis. The metabolic signatures of CSCs and the mechanisms involved in the regulation of their stem cell-like properties still remain elusive. We utilized nasopharyngeal carcinoma (NPC) CSCs as a model to dissect their metabolic signatures and found that CSCs underwent metabolic shift and mitochondrial resetting distinguished from their differentiated counterparts. In metabolic shift, CSCs showed a greater reliance on glycolysis for energy supply compared with the parental cells. In mitochondrial resetting, the quantity and function of mitochondria of CSCs were modulated by the biogenesis of the organelles, and the round-shaped mitochondria were distributed in a peri-nuclear manner similar to those seen in the stem cells. In addition, we blocked the glycolytic pathway, increased the ROS levels, and depolarized mitochondrial membranes of CSCs, respectively, and examined the effects of these metabolic factors on CSC properties. Intriguingly, the properties of CSCs were curbed when we redirected the quintessential metabolic reprogramming, which indicates that the plasticity of energy metabolism regulated the balance between acquisition and loss of the stemness status. Taken together, we suggest that metabolic reprogramming is critical for CSCs to sustain self-renewal, deter from differentiation and enhance the antioxidant defense mechanism. Characterization of metabolic reprogramming governing CSC properties is paramount to the design of novel therapeutic strategies through metabolic intervention of CSCs. PMID:25483072

  7. Biophysical regulation of epigenetic state and cell reprogramming

    NASA Astrophysics Data System (ADS)

    Downing, Timothy L.; Soto, Jennifer; Morez, Constant; Houssin, Timothee; Fritz, Ashley; Yuan, Falei; Chu, Julia; Patel, Shyam; Schaffer, David V.; Li, Song

    2013-12-01

    Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells’ epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.

  8. Identification of Spectral Modifications Occurring during Reprogramming of Somatic Cells

    PubMed Central

    Sandt, Christophe; Féraud, Olivier; Oudrhiri, Noufissa; Bonnet, Marie Laure; Meunier, Marie Claude; Valogne, Yannick; Bertrand, Angelina; Raphaël, Martine; Griscelli, Frank; Turhan, Ali G.; Dumas, Paul; Bennaceur-Griscelli, Annelise

    2012-01-01

    Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose. PMID:22514597

  9. Direct reprogramming of human neural stem cells by OCT4.

    PubMed

    Kim, Jeong Beom; Greber, Boris; Araúzo-Bravo, Marcos J; Meyer, Johann; Park, Kook In; Zaehres, Holm; Schöler, Hans R

    2009-10-01

    Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells. PMID:19718018

  10. Stress Response and Perinatal Reprogramming: Unraveling (Mal)adaptive Strategies

    PubMed Central

    Musazzi, Laura; Marrocco, Jordan

    2016-01-01

    Environmental stressors induce coping strategies in the majority of individuals. The stress response, involving the activation of the hypothalamic-pituitary-adrenocortical axis and the consequent release of corticosteroid hormones, is indeed aimed at promoting metabolic, functional, and behavioral adaptations. However, behavioral stress is also associated with fast and long-lasting neurochemical, structural, and behavioral changes, leading to long-term remodeling of glutamate transmission, and increased susceptibility to neuropsychiatric disorders. Of note, early-life events, both in utero and during the early postnatal life, trigger reprogramming of the stress response, which is often associated with loss of stress resilience and ensuing neurobehavioral (mal)adaptations. Indeed, adverse experiences in early life are known to induce long-term stress-related neuropsychiatric disorders in vulnerable individuals. Here, we discuss recent findings about stress remodeling of excitatory neurotransmission and brain morphology in animal models of behavioral stress. These changes are likely driven by epigenetic factors that lie at the core of the stress-response reprogramming in individuals with a history of perinatal stress. We propose that reprogramming mechanisms may underlie the reorganization of excitatory neurotransmission in the short- and long-term response to stressful stimuli. PMID:27057367

  11. Metabolic reprogramming orchestrates cancer stem cell properties in nasopharyngeal carcinoma.

    PubMed

    Shen, Yao-An; Wang, Chia-Yu; Hsieh, Yi-Tao; Chen, Yann-Jang; Wei, Yau-Huei

    2015-01-01

    Cancer stem cells (CSCs) represent a subpopulation of tumor cells endowed with self-renewal capacity and are considered as an underlying cause of tumor recurrence and metastasis. The metabolic signatures of CSCs and the mechanisms involved in the regulation of their stem cell-like properties still remain elusive. We utilized nasopharyngeal carcinoma (NPC) CSCs as a model to dissect their metabolic signatures and found that CSCs underwent metabolic shift and mitochondrial resetting distinguished from their differentiated counterparts. In metabolic shift, CSCs showed a greater reliance on glycolysis for energy supply compared with the parental cells. In mitochondrial resetting, the quantity and function of mitochondria of CSCs were modulated by the biogenesis of the organelles, and the round-shaped mitochondria were distributed in a peri-nuclear manner similar to those seen in the stem cells. In addition, we blocked the glycolytic pathway, increased the ROS levels, and depolarized mitochondrial membranes of CSCs, respectively, and examined the effects of these metabolic factors on CSC properties. Intriguingly, the properties of CSCs were curbed when we redirected the quintessential metabolic reprogramming, which indicates that the plasticity of energy metabolism regulated the balance between acquisition and loss of the stemness status. Taken together, we suggest that metabolic reprogramming is critical for CSCs to sustain self-renewal, deter from differentiation and enhance the antioxidant defense mechanism. Characterization of metabolic reprogramming governing CSC properties is paramount to the design of novel therapeutic strategies through metabolic intervention of CSCs. PMID:25483072

  12. Reprogramming microbes to be pathogen-seeking killers.

    PubMed

    Hwang, In Young; Tan, Mui Hua; Koh, Elvin; Ho, Chun Loong; Poh, Chueh Loo; Chang, Matthew Wook

    2014-04-18

    Recent examples of new genetic circuits that enable cells to acquire biosynthetic capabilities, such as specific pathogen killing, present an attractive therapeutic application of synthetic biology. Herein, we demonstrate a novel genetic circuit that reprograms Escherichia coli to specifically recognize, migrate toward, and eradicate both dispersed and biofilm-encased pathogenic Pseudomonas aeruginosa cells. The reprogrammed E. coli degraded the mature biofilm matrix and killed the latent cells encapsulated within by expressing and secreting the antimicrobial peptide microcin S and the nuclease DNaseI upon the detection of quorum sensing molecules naturally secreted by P. aeruginosa. Furthermore, the reprogrammed E. coli exhibited directed motility toward the pathogen through regulated expression of CheZ in response to the quorum sensing molecules. By integrating the pathogen-directed motility with the dual antimicrobial activity in E. coli, we achieved signifincantly improved killing activity against planktonic and mature biofilm cells due to target localization, thus creating an active pathogen seeking killer E. coli. PMID:24020906

  13. Polycomb Repressed Genes have Permissive Enhancers that Initiate Reprogramming

    PubMed Central

    Taberlay, Phillippa C.; Kelly, Theresa K.; Liu, Chun-Chi; You, Jueng Soo; de Carvalho, Daniel D.; Miranda, Tina B.; Zhou, Xianghong J.; Liang, Gangning; Jones, Peter A.

    2011-01-01

    SUMMARY Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer, but has a different effect on the cognate promoter, where the monovalent H3K27me3-marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting they may provide a widespread avenue for the initiation of cell-fate reprogramming. PMID:22153073

  14. New tools for experimental diabetes research: Cellular reprogramming and genome editing

    PubMed Central

    2016-01-01

    Isolated human islets are a rare and precious material for diabetes research. However, their availability is limited, and it is impossible to obtain them from patients with specific genotypes. Human pluripotent stem cells provide an alternative. Induced pluripotent stem cells can be generated from any individual’s somatic cells and differentiated into pancreatic cells. Currently, this approach is limited by the immaturity of the islet-like cells derived from stem cells. However, this approach can already be used to model developmental defects, and the possibilities for studying insulin secretion are continuously improving. In addition, genome editing using the CRISPR/Cas9 technology provides powerful possibilities to study the impact of specific genotypes. The same technology can also be used for transcriptional regulation in order to improve the functional maturation of stem cell-derived islets. These tools are today becoming available for tomorrow’s translational diabetes research. PMID:27007444

  15. STAT3-Mediated Metabolic Reprograming in Cellular Transformation and Implications for Drug Resistance

    PubMed Central

    Poli, Valeria; Camporeale, Annalisa

    2015-01-01

    Signal transducer and activator of transcription (STAT)3 mediates the signaling downstream of cytokine and growth factor receptors, regulating the expression of target genes. It is constitutively phosphorylated on tyrosine (Y-P) in many tumors, where its transcriptional activity can induce a metabolic switch toward aerobic glycolysis and down-regulate mitochondrial activity, a prominent metabolic feature of most cancer cells, correlating with reduced production of ROS, delayed senescence, and protection from apoptosis. STAT3 can, however, also localize to mitochondria, where its serine-phosphorylated (S-P) form preserves mitochondrial oxidative phosphorylation and controls the opening of the mitochondrial permeability transition pore, also promoting survival and resistance to apoptosis in response to specific signals/oncogenes such as RAS. Thus, downstream of different signals, both nuclear, Y-P STAT3, and mitochondrial, S-P STAT3, can act by promoting cell survival and reducing ROS production. Here, we discuss these properties in the light of potential connections between STAT3-driven alterations of mitochondrial metabolism and the development of drug resistance in cancer patients. PMID:26106584

  16. MiR-31/SDHA Axis Regulates Reprogramming Efficiency through Mitochondrial Metabolism.

    PubMed

    Lee, Man Ryul; Mantel, Charlie; Lee, Sang A; Moon, Sung-Hwan; Broxmeyer, Hal E

    2016-07-12

    Metabolism is remodeled when somatic cells are reprogrammed into induced pluripotent stem cells (iPSCs), but the majority of iPSCs are not fully reprogrammed. In a shift essential for reprogramming, iPSCs use less mitochondrial respiration but increased anaerobic glycolysis for bioenergetics. We found that microRNA 31 (miR-31) suppressed succinate dehydrogenase complex subunit A (SDHA) expression, vital for mitochondrial electron transport chain (ETC) complex II. MiR-31 overexpression in partially reprogrammed iPSCs lowered SDHA expression levels and oxygen consumption rates to that of fully reprogrammed iPSCs, but did not increase the proportion of fully reprogrammed TRA1-60(+) cells in colonies unless miR-31 was co-transduced with Yamanaka factors, which resulted in a 2.7-fold increase in full reprogramming. Thus switching from mitochondrial respiration to glycolytic metabolism through regulation of the miR-31/SDHA axis is critical for lowering the reprogramming threshold. This is supportive of multi-stage reprogramming whereby metabolic remodeling is fundamental. PMID:27346679

  17. Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions

    PubMed Central

    Yamakawa, Hiroyuki; Muraoka, Naoto; Miyamoto, Kazutaka; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Umei, Tomohiko; Akiyama, Mizuha; Kuishi, Yuki; Kurokawa, Junko; Furukawa, Tetsushi; Fukuda, Keiichi; Ieda, Masaki

    2015-01-01

    Summary Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming. PMID:26626177

  18. The Mitochondrial Uncoupler DNP Triggers Brain Cell mTOR Signaling Network Reprogramming and CREB Pathway Upregulation

    PubMed Central

    Liu, Dong; Zhang, Yongqing; Gharavi, Robert; Park, Hee Ra; Lee, Jaewon; Siddiqui, Sana; Telljohann, Richard; Nassar, Matthew R.; Cutler, Roy G.; Becker, Kevin G.; Mattson, Mark P.

    2015-01-01

    Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2, 4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca2+ levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mTOR and insulin – PI3K – MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and BDNF, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal and synaptic plasticity. PMID:26010875

  19. Cellular resilience.

    PubMed

    Smirnova, Lena; Harris, Georgina; Leist, Marcel; Hartung, Thomas

    2015-01-01

    Cellular resilience describes the ability of a cell to cope with environmental changes such as toxicant exposure. If cellular metabolism does not collapse directly after the hit or end in programmed cell death, the ensuing stress responses promote a new homeostasis under stress. The processes of reverting "back to normal" and reversal of apoptosis ("anastasis") have been studied little at the cellular level. Cell types show astonishingly similar vulnerability to most toxicants, except for those that require a very specific target, metabolism or mechanism present only in specific cell types. The majority of chemicals triggers "general cytotoxicity" in any cell at similar concentrations. We hypothesize that cells differ less in their vulnerability to a given toxicant than in their resilience (coping with the "hit"). In many cases, cells do not return to the naive state after a toxic insult. The phenomena of "pre-conditioning", "tolerance" and "hormesis" describe this for low-dose exposures to toxicants that render the cell more resistant to subsequent hits. The defense and resilience programs include epigenetic changes that leave a "memory/scar" - an alteration as a consequence of the stress the cell has experienced. These memories might have long-term consequences, both positive (resistance) and negative, that contribute to chronic and delayed manifestations of hazard and, ultimately, disease. This article calls for more systematic analyses of how cells cope with toxic perturbations in the long-term after stressor withdrawal. A technical prerequisite for these are stable (organotypic) cultures and a characterization of stress response molecular networks. PMID:26536287

  20. Master regulators in primary skin fibroblast fate reprogramming in a human ex vivo model of chronic wounds.

    PubMed

    Noizet, Maïté; Lagoutte, Emilie; Gratigny, Marlène; Bouschbacher, Marielle; Lazareth, Isabelle; Roest Crollius, Hugues; Darzacq, Xavier; Dugast-Darzacq, Claire

    2016-03-01

    Fibroblasts are important players in regulating tissue homeostasis. In the dermis, they are involved in wound healing where they differentiate into contractile myofibroblasts leading to wound closure. In nonhealing chronic wounds, fibroblasts fail to undertake differentiation. We established and used a human ex vivo model of chronic wounds where fibroblasts can undergo normal myofibroblast differentiation, or take on a nondifferentiable pathological state. At the whole genome scale, we identified the genes that are differentially regulated in these two cell fates. By coupling the search of evolutionary conserved regulatory elements with global gene network expression changes, we identified transcription factors (TF) potentially involved in myofibroblast differentiation, and constructed a network of relationship between these key factors. Among these, we found that TCF4, SOX9, EGR2, and FOXS1 are major regulators of fibroblast to myofibroblast differentiation. Conversely, down-regulation of MEOX2, SIX2, and MAF causes reprogramming of fibroblasts to myofibroblasts even in absence of TGF-β, the natural inducer of myofibroblast differentiation. These results provide insight into the fibroblast differentiation program and reveal a TF network essential for cellular reprogramming. They could lead to the development of new therapeutics to treat fibroblast-related human pathologies. PMID:26663515

  1. Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots.

    PubMed

    Hartmann, Laura; Pedrotti, Lorenzo; Weiste, Christoph; Fekete, Agnes; Schierstaedt, Jasper; Göttler, Jasmin; Kempa, Stefan; Krischke, Markus; Dietrich, Katrin; Mueller, Martin J; Vicente-Carbajosa, Jesus; Hanson, Johannes; Dröge-Laser, Wolfgang

    2015-08-01

    Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity. PMID:26276836

  2. Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots

    PubMed Central

    Hartmann, Laura; Pedrotti, Lorenzo; Weiste, Christoph; Fekete, Agnes; Schierstaedt, Jasper; Göttler, Jasmin; Kempa, Stefan; Krischke, Markus; Dietrich, Katrin; Mueller, Martin J.; Vicente-Carbajosa, Jesus; Hanson, Johannes; Dröge-Laser, Wolfgang

    2015-01-01

    Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity. PMID:26276836

  3. The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

    PubMed

    Coppotelli, Giuseppe; Mughal, Nouman; Callegari, Simone; Sompallae, Ramakrishna; Caja, Laia; Luijsterburg, Martijn S; Dantuma, Nico P; Moustakas, Aristidis; Masucci, Maria G

    2013-03-01

    Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation. PMID:23358825

  4. Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells.

    PubMed

    Hung, Sandy S C; Van Bergen, Nicole J; Jackson, Stacey; Liang, Helena; Mackey, David A; Hernández, Damián; Lim, Shiang Y; Hewitt, Alex W; Trounce, Ian; Pébay, Alice; Wong, Raymond C B

    2016-05-01

    Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology. PMID:27127184

  5. Single transcription factor reprogramming of hair follicle dermal papilla cells to induced pluripotent stem cells.

    PubMed

    Tsai, Su-Yi; Bouwman, Britta Am; Ang, Yen-Sin; Kim, Soo Jeong; Lee, Dung-Fang; Lemischka, Ihor R; Rendl, Michael

    2011-06-01

    Reprogramming patient-specific somatic cells into induced pluripotent stem (iPS) cells has great potential to develop feasible regenerative therapies. However, several issues need to be resolved such as ease, efficiency, and safety of generation of iPS cells. Many different cell types have been reprogrammed, most conveniently even peripheral blood mononuclear cells. However, they typically require the enforced expression of several transcription factors, posing mutagenesis risks as exogenous genetic material. To reduce this risk, iPS cells were previously generated with Oct4 alone from rather inaccessible neural stem cells that endogenously express the remaining reprogramming factors and very recently from fibroblasts with Oct4 alone in combination with additional small molecules. Here, we exploit that dermal papilla (DP) cells from hair follicles in the skin express all but one reprogramming factors to show that these accessible cells can be reprogrammed into iPS cells with the single transcription factor Oct4 and without further manipulation. Reprogramming was already achieved after 3 weeks and with efficiencies similar to other cell types reprogrammed with four factors. Dermal papilla-derived iPS cells are comparable to embryonic stem cells with respect to morphology, gene expression, and pluripotency. We conclude that DP cells may represent a preferred cell type for reprogramming accessible cells with less manipulation and for ultimately establishing safe conditions in the future by replacing Oct4 with small molecules. PMID:21563278

  6. p18 inhibits reprogramming through inactivation of Cdk4/6

    PubMed Central

    Zhu, Shaohua; Cao, Jiani; Sun, Hongyan; Liu, Kun; Li, Yaqiong; Zhao, Tongbiao

    2016-01-01

    Pluripotent stem cells (PSCs), including embryonic and induced pluripotent stem cells (iPSCs), show atypical cell cycle regulation characterized by a high proliferation rate and a shorter G1 phase compared with somatic cells. The mechanisms by which somatic cells remodel their cell cycle to achieve the high proliferation rate of PSCs during reprogramming are unclear. Here we identify that the Ink4 protein p18, which is expressed at high levels in somatic cells but at low levels in PSCs, is a roadblock to successful reprogramming. Mild inhibition of p18 expression enhances reprogramming efficiency, while ectopic expression of p18 completely blocks reprogramming. Mechanistic studies show that expression of wild-type p18, but not a p18D68N mutant which cannot inhibit Cdk4/6, down-regulates expression of Cdk4/6 target genes involved in DNA synthesis (TK, TS, DHFR, PCNA) and cell cycle regulation (CDK1 and CCNA2) and thus inhibits reprogramming. These results indicate that p18 blocks reprogramming by targeting Cdk4/6-mediated cell cycle regulation. Taken together, our results define a novel pathway that inhibits somatic cell reprogramming, and provide a new target to enhance reprogramming efficiency. PMID:27484146

  7. Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells

    PubMed Central

    Hung, Sandy S.C.; Van Bergen, Nicole J.; Jackson, Stacey; Liang, Helena; Mackey, David A.; Hernández, Damián; Lim, Shiang Y.; Hewitt, Alex W.; Trounce, Ian; Pébay, Alice; Wong, Raymond C.B.

    2016-01-01

    Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology. PMID:27127184

  8. Zfp296 is a novel, pluripotent-specific reprogramming factor.

    PubMed

    Fischedick, Gerrit; Klein, Diana C; Wu, Guangming; Esch, Daniel; Höing, Susanne; Han, Dong Wook; Reinhardt, Peter; Hergarten, Kerstin; Tapia, Natalia; Schöler, Hans R; Sterneckert, Jared L

    2012-01-01

    Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs). However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs), indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296) led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4), and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells. PMID:22485183

  9. Zfp296 Is a Novel, Pluripotent-Specific Reprogramming Factor

    PubMed Central

    Wu, Guangming; Esch, Daniel; Höing, Susanne; Han, Dong Wook; Reinhardt, Peter; Hergarten, Kerstin; Tapia, Natalia; Schöler, Hans R.; Sterneckert, Jared L.

    2012-01-01

    Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs). However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs), indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296) led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell–specific conserved region 4 (CR4), and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells. PMID:22485183

  10. Reprogramming for Cardiac Regeneration-Strategies for Innovation.

    PubMed

    Sanchis-Gomar, Fabian; Galera, Teresa; Lucia, Alejandro; Gallardo, María Esther

    2016-09-01

    It is well-known that the human myocardium has a low capacity for self-regeneration. This fact is especially important after acute myocardial infarction with subsequent heart failure and adverse tissue remodeling. New potential strategies have recently emerged for treating heart diseases, such as the possibility of generating large quantities of cardiomyocytes through genetic iPSC reprogramming, transdifferentiation for in vitro disease modeling, in vivo therapies or telomerase gene reactivation. Approaches based on these techniques may represent the new horizon in cardiology with an appropriate 180-degree turn perspective. J. Cell. Physiol. 231: 1849-1851, 2016. © 2016 Wiley Periodicals, Inc. PMID:27128961

  11. Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded

    PubMed Central

    Cattaneo, Roberto; Miest, Tanner; Shashkova, Elena V.; Barry, Michael A.

    2014-01-01

    Virotherapy is currently undergoing a renaissance, based on our improved understanding of virus biology and genetics and our better knowledge of many different types of cancer. Viruses can be reprogrammed into oncolytic vectors by combining three types of modification: targeting, arming and shielding. Targeting introduces multiple layers of cancer specificity and improves safety and efficacy; arming occurs through the expression of prodrug convertases and cytokines; and coating with polymers and the sequential usage of different envelopes or capsids provides shielding from the host immune response. Virus-based therapeutics are beginning to find their place in cancer clinical practice, in combination with chemotherapy and radiation. PMID:18552863

  12. Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock

    PubMed Central

    Rando, Thomas A.; Chang, Howard Y.

    2012-01-01

    The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges. PMID:22265401

  13. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

    PubMed Central

    Itkonen, Harri M.; Gorad, Saurabh S.; Duveau, Damien Y.; Martin, Sara E.S.; Barkovskaya, Anna; Bathen, Tone F.; Moestue, Siver A.; Mills, Ian G.

    2016-01-01

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

  14. Repair of Dense Connective Tissues via Biomaterial-Mediated Matrix Reprogramming of the Wound Interface

    PubMed Central

    Qu, Feini; Pintauro, Michael P.; Haughan, Joanne; Henning, Elizabeth A.; Esterhai, John L.; Schaer, Thomas P.; Mauck, Robert L.; Fisher, Matthew B.

    2014-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues. PMID:25477175

  15. The Metabolic Reprogramming Evoked by Nitrosative Stress Triggers the Anaerobic Utilization of Citrate in Pseudomonas fluorescens

    PubMed Central

    Auger, Christopher; Lemire, Joseph; Cecchini, Dominic; Bignucolo, Adam; Appanna, Vasu D.

    2011-01-01

    Nitrosative stress is an ongoing challenge that most organisms have to contend with. When nitric oxide (NO) that may be generated either exogenously or endogenously encounters reactive oxygen species (ROS), it produces a set of toxic moieties referred to as reactive nitrogen species (RNS). As these RNS can severely damage essential biomolecules, numerous organisms have evolved elaborate detoxification strategies to nullify RNS. However, the contribution of cellular metabolism in fending off nitrosative stress is poorly understood. Using a variety of functional proteomic and metabolomic analyses, we have identified how the soil microbe Pseudomonas fluorescens reprogrammed its metabolic networks to survive in an environment enriched by sodium nitroprusside (SNP), a generator of nitrosative stress. To combat the RNS-induced ineffective aconitase (ACN) and tricarboxylic acid (TCA) cycle, the microbe invoked the participation of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) to convert citrate, the sole source of carbon into pyruvate and ATP. These enzymes were not evident in the control conditions. This metabolic shift was coupled to the concomitant increase in the activities of such classical RNS detoxifiers as nitrate reductase (NR), nitrite reductase (NIR) and S-nitrosoglutathione reductase (GSNOR). Hence, metabolism may hold the clues to the survival of organisms subjected to nitrosative stress and may provide therapeutic cues against RNS-resistant microbes. PMID:22145048

  16. Reprogramming activity of NANOGP8, a NANOG family member widely expressed in cancer.

    PubMed

    Palla, A R; Piazzolla, D; Abad, M; Li, H; Dominguez, O; Schonthaler, H B; Wagner, E F; Serrano, M

    2014-05-01

    NANOG is a key transcription factor for pluripotency in embryonic stem cells. The analysis of NANOG in human cells is confounded by the presence of multiple and highly similar paralogs. In particular, there are three paralogs encoding full-length proteins, namely, NANOG1, NANOG2 and NANOGP8, and at least eight additional paralogs that do not encode full-length NANOG proteins. Here, we have examined NANOG family expression in human embryonic stem cells (hESCs) and in human cancer cell lines using a multi-NANOG PCR that amplifies the three functional paralogs and most of the non-functional ones. As anticipated, we found that hESCs express large amounts of NANOG1 and, interestingly, they also express NANOG2. In contrast, most human cancer cells tested express NANOGP8 and the non-coding paralogs NANOGP4 and NANOGP5. Notably, in some cancer cell lines, the NANOG protein levels produced by NANOGP8 are comparable to those produced by NANOG1 in pluripotent cells. Finally, we show that NANOGP8 is as active as NANOG1 in the reprogramming of human and murine fibroblasts into induced pluripotent stem cells. These results show that cancer-associated NANOGP8 can contribute to promote de-differentiation and/or cellular plasticity. PMID:23752184

  17. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism.

    PubMed

    Itkonen, Harri M; Gorad, Saurabh S; Duveau, Damien Y; Martin, Sara E S; Barkovskaya, Anna; Bathen, Tone F; Moestue, Siver A; Mills, Ian G

    2016-03-15

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

  18. Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

    PubMed

    Qu, Feini; Pintauro, Michael P; Haughan, Joanne E; Henning, Elizabeth A; Esterhai, John L; Schaer, Thomas P; Mauck, Robert L; Fisher, Matthew B

    2015-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues. PMID:25477175

  19. From Repair to Regeneration: Biomaterials to Reprogram the Meniscus Wound Microenvironment

    PubMed Central

    Mauck, Robert L.; Burdick, Jason A.

    2015-01-01

    When the field of tissue engineering first arose, scaffolds were conceived of as inert 3-dimensional structures whose primary function was to support cellularity and tissue growth. Since then, advances in scaffold and biomaterial design have evolved to not only guide tissue formation, but also to interact dynamically with and manipulate the wound environment. At present, these efforts are being directed towards strategies that directly address limitations in endogenous wound repair, with the goal of reprogramming the local wound environment (and the cells within that locality) from a state that culminates in an inferior tissue repair into a state in which functional regeneration is achieved. This review will address this approach with a focus on recent advances in scaffold design towards the resolution of tears of the knee meniscus as a case example. The inherent limitations to endogenous repair will be discussed, as will specific examples of how biomaterials are being designed to overcome these limitations. Examples will include design of fibrous scaffolds that promote colonization by modulating local ECM density and delivering recruitment factors. Furthermore, we will discuss scaffolds that are themselves modulated by the wound environment to alter porosity and modulate therapeutic release through precise coordination of scaffold degradation. Finally, we will close with emerging concepts in local control of cell mechanics to improve interstitial cell migration and so advance repair. Overall, these examples will illustrate how emergent features within a biomaterial can be tuned to manipulate and harness the local tissue microenvironment in order to promote robust regeneration. PMID:25650096

  20. Characterization of Functional Reprogramming during Osteoclast Development Using Quantitative Proteomics and mRNA Profiling*

    PubMed Central

    An, Eunkyung; Narayanan, Manikandan; Manes, Nathan P.; Nita-Lazar, Aleksandra

    2014-01-01

    In addition to forming macrophages and dendritic cells, monocytes in adult peripheral blood retain the ability to develop into osteoclasts, mature bone-resorbing cells. The extensive morphological and functional transformations that occur during osteoclast differentiation require substantial reprogramming of gene and protein expression. Here we employ -omic-scale technologies to examine in detail the molecular changes at discrete developmental stages in this process (precursor cells, intermediate osteoclasts, and multinuclear osteoclasts), quantitatively comparing their transcriptomes and proteomes. The data have been deposited to the ProteomeXchange with identifier PXD000471. Our analysis identified mitochondrial changes, along with several alterations in signaling pathways, as central to the development of mature osteoclasts, while also confirming changes in pathways previously implicated in osteoclast biology. In particular, changes in the expression of proteins involved in metabolism and redirection of energy flow from basic cellular function toward bone resorption appeared to play a key role in the switch from monocytic immune system function to specialized bone-turnover function. These findings provide new insight into the differentiation program involved in the generation of functional osteoclasts. PMID:25044017

  1. Metformin-induced metabolic reprogramming of chemoresistant ALDHbright breast cancer cells.

    PubMed

    Cioce, Mario; Valerio, MariaCristina; Casadei, Luca; Pulito, Claudio; Sacconi, Andrea; Mori, Federica; Biagioni, Francesca; Manetti, Cesare; Muti, Paola; Strano, Sabrina; Blandino, Giovanni

    2014-06-30

    Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDHbright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDHbright cells exhibited a different metabolic profile as compared to their chemosensitive ALDHlow counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDHbright and ALDHlow cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDHbright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDHbright cells and the chemosensitive ALDHlow cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance. PMID:24980829

  2. Metformin-induced metabolic reprogramming of chemoresistant ALDHbright breast cancer cells

    PubMed Central

    Casadei, Luca; Pulito, Claudio; Sacconi, Andrea; Mori, Federica; Biagioni, Francesca; Manetti, Cesare; Muti, Paola; Strano, Sabrina; Blandino, Giovanni

    2014-01-01

    Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDHbright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDHbright cells exhibited a different metabolic profile as compared to their chemosensitive ALDHlow counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDHbright and ALDHlow cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDHbright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDHbright cells and the chemosensitive ALDHlow cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance. PMID:24980829

  3. Make no bones about it: cells could soon be reprogrammed to grow replacement bones?

    PubMed

    de Peppo, Giuseppe Maria; Marolt, Darja

    2014-01-01

    Recent developments in nuclear reprogramming allow the generation of patient-matched stem cells with broad potential for applications in cell therapies, disease modeling and drug discovery. An increasing body of work is reporting the derivation of lineage-specific progenitors from human-induced pluripotent stem cells (hiPSCs), which could in the near future be used to engineer personalized tissue substitutes, including those for reconstructive therapies of bone. Although the potential clinical impact of such technology is not arguable, significant challenges remain to be addressed before hiPSC-derived progenitors can be employed to engineer bone substitutes of clinical relevance. The most important challenge is indeed the construction of personalized multicellular bone substitutes for the treatment of complex skeletal defects that integrate fast, are immune tolerated and display biofunctionality and long-term safety. As recent studies suggest, the merging of iPSC technology with advanced biomaterials and bioreactor technologies offers a way to generate bone substitutes in a controllable, automated manner with potential to meet the needs for scale-up and requirements for translation into clinical practice. It is only via the use of state-of-the-art cell culture technologies, process automation under GMP-compliant conditions, application of appropriate engineering strategies and compliance with regulatory policies that personalized lab-made bone grafts can start being used to treat human patients. PMID:24053578

  4. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

    PubMed Central

    Howden, Sara E.; Maufort, John P.; Duffin, Bret M.; Elefanty, Andrew G.; Stanley, Edouard G.; Thomson, James A.

    2015-01-01

    Summary The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes. PMID:26584543

  5. Multifactorial Analysis of Conditional Reprogramming of Human Keratinocytes

    PubMed Central

    Ligaba, Segni B.; Khurana, Anikita; Graham, Garrett; Krawczyk, Ewa; Jablonski, Sandra; Petricoin, Emanuel F.; Glazer, Robert I.; Upadhyay, Geeta

    2015-01-01

    Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents. PMID:25714835

  6. Targeted gene therapy and cell reprogramming in Fanconi anemia

    PubMed Central

    Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

    2014-01-01

    Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

  7. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts

    PubMed Central

    Wainger, Brian J.; Buttermore, Elizabeth D.; Oliveira, Julia T.; Mellin, Cassidy; Lee, Seungkyu; Saber, Wardiya Afshar; Wang, Amy; Ichida, Justin K.; Chiu, Isaac M.; Barrett, Lee; Huebner, Eric A.; Bilgin, Canan; Tsujimoto, Naomi; Brenneis, Christian; Kapur, Kush; Rubin, Lee L.; Eggan, Kevin; Woolf, Clifford J.

    2015-01-01

    Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. Here, we identify five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons that recapitulate the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibit TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we show that the technique can reveal novel aspects of human disease phenotypes in vitro. PMID:25420066

  8. Müller glia cell reprogramming and retina regeneration

    PubMed Central

    Goldman, Daniel

    2014-01-01

    Müller glia are the major glial component of the retina. They are one of the last retinal cell types to be born during development and they function to maintain retinal homeostasis and integrity. In mammals, Müller glia respond to retinal injury in a variety of ways that can be either protective or detrimental to retinal function. Although under special circumstances these cells can be coaxed to proliferate and generate neurons, these responses are meager and insufficient for repairing a damaged retina. By contrast, in teleost fish (such as zebrafish) the response of Müller glia to retinal injury involves a reprogramming event that imparts retinal stem cell characteristics and allows them to produce a proliferating population of progenitors that can regenerate all major retinal cell types and restore vision. Recent studies have revealed a number of important mechanisms underlying Müller glia reprogramming and retina regeneration in fish that may lead to new strategies for stimulating retina regeneration in mammals. PMID:24894585

  9. Clinical significance of T cell metabolic reprogramming in cancer.

    PubMed

    Herbel, Christoph; Patsoukis, Nikolaos; Bardhan, Kankana; Seth, Pankaj; Weaver, Jessica D; Boussiotis, Vassiliki A

    2016-12-01

    Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy. PMID:27510264

  10. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.

    PubMed

    Anzalone, Andrew V; Lin, Annie J; Zairis, Sakellarios; Rabadan, Raul; Cornish, Virginia W

    2016-05-01

    Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes. PMID:26999002

  11. Adoptive cellular therapy of cancer: exploring innate and adaptive cellular crosstalk to improve anti-tumor efficacy.

    PubMed

    Payne, Kyle K; Bear, Harry D; Manjili, Masoud H

    2014-08-01

    The mammalian immune system has evolved to produce multi-tiered responses consisting of both innate and adaptive immune cells collaborating to elicit a functional response to a pathogen or neoplasm. Immune cells possess a shared ancestry, suggestive of a degree of coevolution that has resulted in optimal functionality as an orchestrated and highly collaborative unit. Therefore, the development of therapeutic modalities that harness the immune system should consider the crosstalk between cells of the innate and adaptive immune systems in order to elicit the most effective response. In this review, the authors will discuss the success achieved using adoptive cellular therapy in the treatment of cancer, recent trends that focus on purified T cells, T cells with genetically modified T-cell receptors and T cells modified to express chimeric antigen receptors, as well as the use of unfractionated immune cell reprogramming to achieve optimal cellular crosstalk upon infusion for adoptive cellular therapy. PMID:25303057

  12. Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse

    PubMed Central

    Queiroz, Karla C. S.; Milani, Renato; Ruela-de-Sousa, Roberta R.; Fuhler, Gwenny M.; Justo, Giselle Z.; Zambuzzi, Willian F.; Duran, Nelson; Diks, Sander H.; Spek, C. Arnold; Ferreira, Carmen V.; Peppelenbosch, Maikel P.

    2012-01-01

    It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34+/c-Kit+/P-glycoprotein+/MRP1+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells. PMID:23071514

  13. An integrated systems biology approach identifies positive cofactor 4 as a factor that increases reprogramming efficiency

    PubMed Central

    Jo, Junghyun; Hwang, Sohyun; Kim, Hyung Joon; Hong, Soomin; Lee, Jeoung Eun; Lee, Sung-Geum; Baek, Ahmi; Han, Heonjong; Lee, Jin Il; Lee, Insuk; Lee, Dong Ryul

    2016-01-01

    Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes. PMID:26740582

  14. Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

    PubMed

    Prieto, Javier; León, Marian; Ponsoda, Xavier; Sendra, Ramón; Bort, Roque; Ferrer-Lorente, Raquel; Raya, Angel; López-García, Carlos; Torres, Josema

    2016-01-01

    During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency. PMID:27030341

  15. Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

    PubMed Central

    Prieto, Javier; León, Marian; Ponsoda, Xavier; Sendra, Ramón; Bort, Roque; Ferrer-Lorente, Raquel; Raya, Angel; López-García, Carlos; Torres, Josema

    2016-01-01

    During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency. PMID:27030341

  16. Generation of a Drug-inducible Reporter System to Study Cell Reprogramming in Human Cells*

    PubMed Central

    Ruiz, Sergio; Panopoulos, Athanasia D.; Montserrat, Nuria; Multon, Marie-Christine; Daury, Aurélie; Rocher, Corinne; Spanakis, Emmanuel; Batchelder, Erika M.; Orsini, Cécile; Deleuze, Jean-François; Izpisua Belmonte, Juan Carlos

    2012-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years, reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene, driven by the reactivation of endogenous stem cell specific promoters, was used as a reprogramming reporter signal. However, similar reporter systems in human cells have not been generated. Here, we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system, we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency. PMID:23019325

  17. Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

    PubMed

    Abad, María; Mosteiro, Lluc; Pantoja, Cristina; Cañamero, Marta; Rayon, Teresa; Ors, Inmaculada; Graña, Osvaldo; Megías, Diego; Domínguez, Orlando; Martínez, Dolores; Manzanares, Miguel; Ortega, Sagrario; Serrano, Manuel

    2013-10-17

    Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine. PMID:24025773

  18. Reprogramming homing endonuclease specificity through computational design and directed evolution.

    PubMed

    Thyme, Summer B; Boissel, Sandrine J S; Arshiya Quadri, S; Nolan, Tony; Baker, Dean A; Park, Rachel U; Kusak, Lara; Ashworth, Justin; Baker, David

    2014-02-01

    Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally. PMID:24270794

  19. Noncoding RNAs in Regulation of Cancer Metabolic Reprogramming.

    PubMed

    Yang, Dongdong; Sun, Linchong; Li, Zhaoyong; Gao, Ping

    2016-01-01

    Since the description of the Warburg effect 90 years ago, metabolic reprogramming has been gradually recognized as a major hallmark of cancer cells. Mounting evidence now indicates that cancer is a kind of metabolic disease, quite distinct from conventional perception. While metabolic alterations in cancer cells have been extensively observed in glucose, lipid, and amino acid metabolisms, its underlying regulatory mechanisms are still poorly understood. Noncoding RNA, also known as the "dark matter in life," functions through various mechanisms at RNA level regulating different biological pathways. The last two decades have witnessed the booming of noncoding RNA study on microRNA (miRNA), long noncoding RNA (lncRNA), circular RNA (circRNA), PIWI-interacting RNA (piRNA), etc. In this chapter, we will discuss the regulatory roles of noncoding RNAs on cancer metabolism. PMID:27376736

  20. Reprogramming of avian neural crest axial identity and cell fate.

    PubMed

    Simoes-Costa, Marcos; Bronner, Marianne E

    2016-06-24

    Neural crest populations along the embryonic body axis of vertebrates differ in developmental potential and fate, so that only the cranial neural crest can contribute to the craniofacial skeleton in vivo. We explored the regulatory program that imbues the cranial crest with its specialized features. Using axial-level specific enhancers to isolate and perform genome-wide profiling of the cranial versus trunk neural crest in chick embryos, we identified and characterized regulatory relationships between a set of cranial-specific transcription factors. Introducing components of this circuit into neural crest cells of the trunk alters their identity and endows these cells with the ability to give rise to chondroblasts in vivo. Our results demonstrate that gene regulatory circuits that support the formation of particular neural crest derivatives may be used to reprogram specific neural crest-derived cell types. PMID:27339986

  1. A decade of transcription factor-mediated reprogramming to pluripotency.

    PubMed

    Takahashi, Kazutoshi; Yamanaka, Shinya

    2016-03-01

    The past 10 years have seen great advances in our ability to manipulate cell fate, including the induction of pluripotency in vitro to generate induced pluripotent stem cells (iPSCs). This process proved to be remarkably simple from a technical perspective, only needing the host cell and a defined cocktail of transcription factors, with four factors - octamer-binding protein 3/4 (OCT3/4), SOX2, Krüppel-like factor 4 (KLF4) and MYC (collectively referred to as OSKM) - initially used. The mechanisms underlying transcription factor-mediated reprogramming are still poorly understood; however, several mechanistic insights have recently been obtained. Recent years have also brought significant progress in increasing the efficiency of this technique, making it more amenable to applications in the fields of regenerative medicine, disease modelling and drug discovery. PMID:26883003

  2. Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons.

    PubMed

    Lee, Ki Baek; Hou, Chen Yuan; Kim, Chae-Eun; Kim, Dong-Myung; Suga, Hiroaki; Kang, Taek Jin

    2016-07-01

    The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non-proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non-proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA(Arg) in a cell-free protein synthesis system by using a tRNA(Arg) -specific tRNase, colicin D. Then by supplementing the system with tRNACCU , the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids. PMID:27151886

  3. The extended pluripotency protein interactome and its links to reprogramming

    PubMed Central

    Huang, Xin; Wang, Jianlong

    2014-01-01

    A pluripotent state of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is maintained through the combinatorial activity of core transcriptional factors (TFs) such as Oct4, Sox2, and Nanog in conjunction with many other factors including epigenetic regulators. Proteins rarely act alone, and knowledge of protein-protein interaction network (interactome) provides an extraordinary resource about how pluripotency TFs collaborate and crosstalk with epigenetic regulators in ESCs. Recent advances in affinity purification coupled with mass spectrometry (AP-MS) allow for efficient, high-throughput identification of hundreds of interacting protein partners, which can be used to map the pluripotency landscape. Here we review recent publications employing AP-MS to investigate protein interaction networks in ESCs, discuss how protein-protein connections reveal novel pluripotency regulatory circuits and new factors for efficient reprogramming of somatic cells. PMID:25173149

  4. IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism.

    PubMed

    Izquierdo-Garcia, Jose L; Viswanath, Pavithra; Eriksson, Pia; Cai, Larry; Radoul, Marina; Chaumeil, Myriam M; Blough, Michael; Luchman, H Artee; Weiss, Samuel; Cairncross, J Gregory; Phillips, Joanna J; Pieper, Russell O; Ronen, Sabrina M

    2015-08-01

    Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. PMID:26045167

  5. Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors.

    PubMed

    Lalit, Pratik A; Salick, Max R; Nelson, Daryl O; Squirrell, Jayne M; Shafer, Christina M; Patel, Neel G; Saeed, Imaan; Schmuck, Eric G; Markandeya, Yogananda S; Wong, Rachel; Lea, Martin R; Eliceiri, Kevin W; Hacker, Timothy A; Crone, Wendy C; Kyba, Michael; Garry, Daniel J; Stewart, Ron; Thomson, James A; Downs, Karen M; Lyons, Gary E; Kamp, Timothy J

    2016-03-01

    Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy. PMID:26877223

  6. Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells

    PubMed Central

    Biswas, Dhruba; Jiang, Peng

    2016-01-01

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming. PMID:26861316

  7. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    PubMed Central

    Ruiz, Sergio; Lopez-Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Gutierrez-Martinez, Paula; Bua, Sabela; Ramirez, Oscar; Olalde, Iñigo; Rodrigo-Perez, Sara; Li, Han; Marques-Bonet, Tomas; Serrano, Manuel; Blasco, Maria A.; Batada, Nizar N.; Fernandez-Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. PMID:26292731

  8. Mice produced by mitotic reprogramming of sperm injected into haploid parthenogenotes.

    PubMed

    Suzuki, Toru; Asami, Maki; Hoffmann, Martin; Lu, Xin; Gužvić, Miodrag; Klein, Christoph A; Perry, Anthony C F

    2016-01-01

    Sperm are highly differentiated and the activities that reprogram them for embryonic development during fertilization have historically been considered unique to the oocyte. We here challenge this view and demonstrate that mouse embryos in the mitotic cell cycle can also directly reprogram sperm for full-term development. Developmentally incompetent haploid embryos (parthenogenotes) injected with sperm developed to produce healthy offspring at up to 24% of control rates, depending when in the embryonic cell cycle injection took place. This implies that most of the first embryonic cell cycle can be bypassed in sperm genome reprogramming for full development. Remodelling of histones and genomic 5'-methylcytosine and 5'-hydroxymethylcytosine following embryo injection were distinct from remodelling in fertilization and the resulting 2-cell embryos consistently possessed abnormal transcriptomes. These studies demonstrate plasticity in the reprogramming of terminally differentiated sperm nuclei and suggest that different epigenetic pathways or kinetics can establish totipotency. PMID:27623537

  9. Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies.

    PubMed

    Brouwer, Marinka; Zhou, Huiqing; Nadif Kasri, Nael

    2016-02-01

    The ability to generate human induced pluripotent stem cells (iPSCs) from somatic cells provides tremendous promises for regenerative medicine and its use has widely increased over recent years. However, reprogramming efficiencies remain low and chromosomal instability and tumorigenic potential are concerns in the use of iPSCs, especially in clinical settings. Therefore, reprogramming methods have been under development to generate safer iPSCs with higher efficiency and better quality. Developments have mainly focused on the somatic cell source, the cocktail of reprogramming factors, the delivery method used to introduce reprogramming factors and culture conditions to maintain the generated iPSCs. This review discusses the developments on these topics and briefly discusses pros and cons of iPSCs in comparison with human embryonic stem cells generated from somatic cell nuclear transfer. PMID:26424535

  10. Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming.

    PubMed

    Chen, Jun; Chen, Xiaolong; Li, Min; Liu, Xiaoyu; Gao, Yawei; Kou, Xiaochen; Zhao, Yanhong; Zheng, Weisheng; Zhang, Xiaobai; Huo, Yi; Chen, Chuan; Wu, You; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

    2016-02-16

    The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies. PMID:26832419

  11. Environmental Impact on Direct Neuronal Reprogramming In Vivo in the Adult Brain

    PubMed Central

    López-Juárez, Alejandro; Howard, Jennifer; Sakthivel, Bhuvaneswari; Aronow, Bruce; Campbell, Kenneth; Nakafuku, Masato

    2013-01-01

    Direct reprogramming of non-neuronal cells to generate new neurons is a promising approach to repair damaged brains. Impact of the in vivo environment on neuronal reprogramming, however, is poorly understood. Here we show that regional differences and injury conditions have significant influence on the efficacy of reprogramming and subsequent survival of newly generated neurons in the adult rodent brain. A combination of local exposure to growth factors and retrovirus-mediated overexpression of the neurogenic transcription factor Neurogenin2 (Neurog2) can induce new neurons from non-neuronal cells in the adult neocortex and striatum where neuronal turnover is otherwise very limited. These two regions respond to growth factors and Neurog2 differently and instruct new neurons to exhibit distinct molecular phenotypes. Moreover, ischemic insult differentially affects differentiation of new neurons in these regions. These results demonstrate strong environmental impact on direct neuronal reprogramming in vivo. PMID:23974433

  12. Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.

    PubMed

    Hill, Maureen E; MacPherson, Derek J; Wu, Peng; Julien, Olivier; Wells, James A; Hardy, Jeanne A

    2016-06-17

    The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases. PMID:27032039

  13. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

    PubMed Central

    Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

    2013-01-01

    Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

  14. A Highly Optimized Protocol for Reprogramming Cancer Cells to Pluripotency Using Nonviral Plasmid Vectors

    PubMed Central

    Zhao, Hongzhi; Davies, Timothy J.; Ning, Jiaolin; Chang, Yanxu; Sachamitr, Patty; Sattler, Susanne

    2015-01-01

    Abstract In spite of considerable interest in the field, reprogramming induced pluripotent stem cells (iPSCs) directly from cancer cells has encountered considerable challenges, including the extremely low reprogramming efficiency and instability of cancer-derived iPSCs (C-iPSCs). In this study, we aimed to identify the main obstacles that limit cancer cell reprogramming. Through a detailed multidimensional kinetic optimization, a highly optimized protocol is established for reprogramming C-iPSCs using nonviral plasmid vectors. We demonstrated how the initial cancer cell density seeded could be the most critical factor ultimately affecting C-iPSCs reprogramming. We have consistently achieved an unprecedented high C-iPSC reprogramming efficiency, establishing stable colonies with typical iPSC morphology, up to 50% of which express the iPSC phenotypic (Oct3/4, Sox2, Nanog) and enzymatic (alkaline phosphatase) markers. Furthermore, established C-iPSC lines were shown to be capable of forming teratomas in vivo, containing cell types and tissues from each of the embryonic germ layers, fully consistent with their acquisition of pluripotency. This protocol was tested and confirmed in two completely unrelated human lung adenocarcinoma (A549) and mouse melanoma (B16f10) cancer cell lines and thus offers a potentially valuable method for generating effectively virus-free C-iPSCs for future applications. PMID:25549177

  15. Facilitators and Impediments of the Pluripotency Reprogramming Factors’ Initial Engagement with the Genome

    PubMed Central

    Soufi, Abdenour; Donahue, Greg; Zaret, Kenneth S.

    2012-01-01

    SUMMARY The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hours of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include: An unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed. PMID:23159369

  16. Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.

    PubMed

    Fu, Ji-Dong; Stone, Nicole R; Liu, Lei; Spencer, C Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G; Srivastava, Deepak

    2013-01-01

    Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

  17. Transient hypoxia reprograms differentiating adipocytes for enhanced insulin sensitivity and triglyceride accumulation

    PubMed Central

    Lu, Hongyun; Gao, Zhanguo; Zhao, Zhiyun; Weng, Jianping; Ye, Jianping

    2015-01-01

    Objective To investigate the impact of transient (2-4 h) hypoxia on metabolic reprogramming of adipocytes. Methods The impact of transient hypoxia on metabolic reprogramming was investigated in 3T3-L1 cells before and after differentiation. Glucose uptake, fatty acid oxidation, lipolysis, and mitochondria were examined to determine the hypoxia effects. Preadipocytes were exposed to transient hypoxia (4h/day) in the course of differentiation. Insulin sensitivity and TG accumulation was examined in the cells at the end of differentiation to determine the reprogramming effects. AMPK activity and gene expression were determined by quantitative RT-PCR and Western blotting in search for mechanism of the reprogramming. Results In acute response to hypoxia, adipocytes exhibited an increase in insulin-dependent and -independent glucose uptake. Fatty acid β-oxidation and pyruvate dehydrogenase (PDH) activity were decreased. Multiple exposures of differentiating adipocytes to transient hypoxia enhanced insulin signaling, TG accumulation, expression of antioxidant genes in differentiated adipocytes in the absence of hypoxia. The metabolic memory was associated with elevated AMPK activity and gene expression (GLUT1, PGC-1α, PPARγ, SREBP, NRF-1, ESRRα, LPL). The enhanced insulin sensitivity was blocked by an AMPK inhibitor. Conclusions Repeated exposure of differentiating adipocytes to transient hypoxia is able to reprogram the cells for increased TG accumulation and enhanced insulin sensitivity. The metabolic alterations were observed in post-differentiated cells under normoxia. The reprogramming involves AMPK activation and gene expression in the metabolic pathways in cytosol and mitochondria. PMID:26219415

  18. Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

    PubMed Central

    Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

    2013-01-01

    Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

  19. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

    PubMed Central

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X.; Kemp, Sadie E.; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-01-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells. PMID:24371281

  20. Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

    PubMed

    Lee, Jeong-Hwan; Park, Da-Young; Lee, Kyung-Jin; Kim, Young-Kwan; So, Yang-Kang; Ryu, Jae-Sung; Oh, Seung-Han; Han, Yeon-Soo; Ko, Kinarm; Choo, Young-Kug; Park, Sung-Joo; Brodzik, Robert; Lee, Kyoung-Ki; Oh, Doo-Byoung; Hwang, Kyung-A; Koprowski, Hilary; Lee, Yong Seong; Ko, Kisung

    2013-01-01

    Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant. PMID:23967055

  1. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers

    PubMed Central

    Muralidharan, Sujatha; Sasi, Sharath P.; Zuriaga, Maria A.; Hirschi, Karen K.; Porada, Christopher D.; Coleman, Matthew A.; Walsh, Kenneth X.; Yan, Xinhua; Goukassian, David A.

    2015-01-01

    Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long-term damage to hematopoietic cells in the BM niche. We report here that low-dose 1H- and 56Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively) cell numbers in mouse BM over a period of up to 10 months after exposure. Both 1H- and 56Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog, and Oct4 in L-MPPs and 10 months post-IR exposure. We postulate that low doses of 1H- and 56Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype and that IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders, and these findings warrant further mechanistic studies into the effects of low-dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency. PMID:26528440

  2. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers.

    PubMed

    Muralidharan, Sujatha; Sasi, Sharath P; Zuriaga, Maria A; Hirschi, Karen K; Porada, Christopher D; Coleman, Matthew A; Walsh, Kenneth X; Yan, Xinhua; Goukassian, David A

    2015-01-01

    Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long-term damage to hematopoietic cells in the BM niche. We report here that low-dose (1)H- and (56)Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively) cell numbers in mouse BM over a period of up to 10 months after exposure. Both (1)H- and (56)Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog, and Oct4 in L-MPPs and 10 months post-IR exposure. We postulate that low doses of (1)H- and (56)Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype and that IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders, and these findings warrant further mechanistic studies into the effects of low-dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency. PMID:26528440

  3. Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients.

    PubMed

    Esseltine, Jessica L; Shao, Qing; Huang, Tao; Kelly, John J; Sampson, Jacinda; Laird, Dale W

    2015-11-15

    Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5β1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells. PMID:26349540

  4. Exophiala sp. LHL08 reprograms Cucumis sativus to higher growth under abiotic stresses.

    PubMed

    Khan, Abdul L; Hamayun, Muhammad; Ahmad, Nadeem; Waqas, Muhammad; Kang, Sang-Mo; Kim, Yoon-Ha; Lee, In-Jung

    2011-12-01

    Endophytic fungi are potential sources of secondary metabolites; however, they are little known for phytohormones secretion and amelioration of plant growth under abiotic stresses. We isolated a novel endophyte from the roots of Cucumis sativus and identified it as a strain of Exophiala sp. by sequencing internal transcribed spacer/large subunit rDNA and phylogenetic analysis. Prior to identification, culture filtrate (CF) of Exophiala sp. has shown significant growth promotion of Waito-C [a gibberellins (GAs)-deficient mutant cultivar] and Dongjin-byeo (normal GAs biosynthesis cultivar) rice seedlings. CF analysis of Exophiala sp. showed the presence of physiologically active GAs (GA₁, GA₃, GA₄ and GA₇) and inactive GAs (GA₅, GA₈, GA₉, GA₁₂ and GA₂₀). Exophiala sp. had higher GAs in its CF than wild-type strain of Gibberella fujikuroi except GA₃. Influence of Exophiala sp. was assessed on cucumber plant's growth and endogenous abscisic acid (ABA), salicylic acid (SA) and bioactive GAs under salinity and drought stresses. Exophiala sp.-treated plants have shown significantly higher growth and rescued the host plants from stress promulgated water deficit, osmotic and cellular damage. The altered levels of stress-responsive ABA showed low level of stress confined to endophyte-applied plants than control. Elevated levels of SA and bioactive GAs (GA₃ and GA₄) in endophyte-associated plants suggest stress-modulating response toward salinity and drought. In conclusion, symbiotic relations between Exophiala and cucumber have reprogrammed the host plant growth under abiotic stresses, thus indicating a possible threshold role of endophytic fungi in stress alleviation. This study could be extended for improving agricultural productivity under extreme environmental conditions. PMID:21883250

  5. Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways

    PubMed Central

    Ryczko, Michael C.; Pawling, Judy; Chen, Rui; Abdel Rahman, Anas M.; Yau, Kevin; Copeland, Julia K.; Zhang, Cunjie; Surendra, Anu; Guttman, David S.; Figeys, Daniel; Dennis, James W.

    2016-01-01

    De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5−/− mice and in cultured Mgat5−/− hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway. PMID:26972830

  6. Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways.

    PubMed

    Ryczko, Michael C; Pawling, Judy; Chen, Rui; Abdel Rahman, Anas M; Yau, Kevin; Copeland, Julia K; Zhang, Cunjie; Surendra, Anu; Guttman, David S; Figeys, Daniel; Dennis, James W

    2016-01-01

    De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5(-/-) mice and in cultured Mgat5(-/-) hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway. PMID:26972830

  7. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  8. Cochlear microphonic broad tuning curves

    NASA Astrophysics Data System (ADS)

    Ayat, Mohammad; Teal, Paul D.; Searchfield, Grant D.; Razali, Najwani

    2015-12-01

    It is known that the cochlear microphonic voltage exhibits much broader tuning than does the basilar membrane motion. The most commonly used explanation for this is that when an electrode is inserted at a particular point inside the scala media, the microphonic potentials of neighbouring hair cells have different phases, leading to cancelation at the electrodes location. In situ recording of functioning outer hair cells (OHCs) for investigating this hypothesis is exceptionally difficult. Therefore, to investigate the discrepancy between the tuning curves of the basilar membrane and those of the cochlear microphonic, and the effect of phase cancellation of adjacent hair cells on the broadness of the cochlear microphonic tuning curves, we use an electromechanical model of the cochlea to devise an experiment. We explore the effect of adjacent hair cells (i.e., longitudinal phase cancellation) on the broadness of the cochlear microphonic tuning curves in different locations. The results of the experiment indicate that active longitudinal coupling (i.e., coupling with active adjacent outer hair cells) only slightly changes the broadness of the CM tuning curves. The results also demonstrate that there is a π phase difference between the potentials produced by the hair bundle and the soma near the place associated with the characteristic frequency based on place-frequency maps (i.e., the best place). We suggest that the transversal phase cancellation (caused by the phase difference between the hair bundle and the soma) plays a far more important role than longitudinal phase cancellation in the broadness of the cochlear microphonic tuning curves. Moreover, by increasing the modelled longitudinal resistance resulting the cochlear microphonic curves exhibiting sharper tuning. The results of the simulations suggest that the passive network of the organ of Corti determines the phase difference between the hair bundle and soma, and hence determines the sharpness of the

  9. Broad-band beam buncher

    DOEpatents

    Goldberg, David A.; Flood, William S.; Arthur, Allan A.; Voelker, Ferdinand

    1986-01-01

    A broad-band beam buncher is disclosed, comprising an evacuated housing, an electron gun therein for producing an electron beam, a buncher cavity having entrance and exit openings through which the beam is directed, grids across such openings, a source providing a positive DC voltage between the cavity and the electron gun, a drift tube through which the electron beam travels in passing through such cavity, grids across the ends of such drift tube, gaps being provided between the drift tube grids and the entrance and exit grids, a modulator for supplying an ultrahigh frequency modulating signal to the drift tube for producing velocity modulation of the electrons in the beam, a drift space in the housing through which the velocity modulated electron beam travels and in which the beam is bunched, and a discharge opening from such drift tube and having a grid across such opening through which the bunched electron beam is discharged into an accelerator or the like. The buncher cavity and the drift tube may be arranged to constitute an extension of a coaxial transmission line which is employed to deliver the modulating signal from a signal source. The extended transmission line may be terminated in its characteristic impedance to afford a broad-band response and the device as a whole designed to effect broad-band beam coupling, so as to minimize variations of the output across the response band.

  10. Optofluidic Detection for Cellular Phenotyping

    PubMed Central

    Tung, Yi-Chung; Huang, Nien-Tsu; Oh, Bo-Ram; Patra, Bishnubrata; Pan, Chi-Chun; Qiu, Teng; Paul, K. Chu; Zhang, Wenjun; Kurabayashi, Katsuo

    2012-01-01

    Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidic, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection. PMID:22854915

  11. Molecular and Cellular Biophysics

    NASA Astrophysics Data System (ADS)

    Jackson, Meyer B.

    2006-01-01

    Molecular and Cellular Biophysics provides advanced undergraduate and graduate students with a foundation in the basic concepts of biophysics. Students who have taken physical chemistry and calculus courses will find this book an accessible and valuable aid in learning how these concepts can be used in biological research. The text provides a rigorous treatment of the fundamental theories in biophysics and illustrates their application with examples. Conformational transitions of proteins are studied first using thermodynamics, and subsequently with kinetics. Allosteric theory is developed as the synthesis of conformational transitions and association reactions. Basic ideas of thermodynamics and kinetics are applied to topics such as protein folding, enzyme catalysis and ion channel permeation. These concepts are then used as the building blocks in a treatment of membrane excitability. Through these examples, students will gain an understanding of the general importance and broad applicability of biophysical principles to biological problems. Offers a unique synthesis of concepts across a wide range of biophysical topics Provides a rigorous theoretical treatment, alongside applications in biological systems Author has been teaching biophysics for nearly 25 years

  12. Adaptive Mitochondrial Reprogramming and Resistance to PI3K Therapy

    PubMed Central

    Ghosh, Jagadish C.; Siegelin, Markus D.; Vaira, Valentina; Faversani, Alice; Tavecchio, Michele; Chae, Young Chan; Lisanti, Sofia; Rampini, Paolo; Giroda, Massimo; Caino, M. Cecilia; Seo, Jae Ho; Kossenkov, Andrew V.; Michalek, Ryan D.; Schultz, David C.; Bosari, Silvano; Languino, Lucia R.

    2015-01-01

    Background: Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. Methods: We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4–5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. Results: PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). Conclusions: Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel

  13. Induced Pluripotent Stem Cells from Ataxia-Telangiectasia Recapitulate the Cellular Phenotype

    PubMed Central

    Nayler, Sam; Gatei, Magtouf; Kozlov, Sergei; Gatti, Richard; Mar, Jessica C.; Wells, Christine A.

    2012-01-01

    Pluripotent stem cells can differentiate into every cell type of the human body. Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) therefore provides an opportunity to gain insight into the molecular and cellular basis of disease. Because the cellular DNA damage response poses a barrier to reprogramming, generation of iPSCs from patients with chromosomal instability syndromes has thus far proven to be difficult. Here we demonstrate that fibroblasts from patients with ataxia-telangiectasia (A-T), a disorder characterized by chromosomal instability, progressive neurodegeneration, high risk of cancer, and immunodeficiency, can be reprogrammed to bona fide iPSCs, albeit at a reduced efficiency. A-T iPSCs display defective radiation-induced signaling, radiosensitivity, and cell cycle checkpoint defects. Bioinformatic analysis of gene expression in the A-T iPSCs identifies abnormalities in DNA damage signaling pathways, as well as changes in mitochondrial and pentose phosphate pathways. A-T iPSCs can be differentiated into functional neurons and thus represent a suitable model system to investigate A-T-associated neurodegeneration. Collectively, our data show that iPSCs can be generated from a chromosomal instability syndrome and that these cells can be used to discover early developmental consequences of ATM deficiency, such as altered mitochondrial function, that may be relevant to A-T pathogenesis and amenable to therapeutic intervention. PMID:23197857

  14. Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.

    PubMed

    Aksoy, Irene; Jauch, Ralf; Eras, Volker; Chng, Wen-Bin Alfred; Chen, Jiaxuan; Divakar, Ushashree; Ng, Calista Keow Leng; Kolatkar, Prasanna R; Stanton, Lawrence W

    2013-12-01

    The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. PMID:23963638

  15. Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

    PubMed

    Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

    2014-01-01

    Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. PMID:23394081

  16. Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.

    PubMed

    Lopez Juarez, Alejandro; He, Danyang; Richard Lu, Q

    2016-05-01

    Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only). PMID:26546966

  17. Reprogramming with Small Molecules instead of Exogenous Transcription Factors.

    PubMed

    Lin, Tongxiang; Wu, Shouhai

    2015-01-01

    Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs. PMID:25922608

  18. Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming

    PubMed Central

    Li, Heng-Hong; Wang, Yi-wen; Chen, Renxiang; Zhou, Bin; Ashwell, Jonathan D.; Fornace, Albert J.

    2015-01-01

    Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures. PMID:26078715

  19. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome

    PubMed Central

    Wood, Lauren W.; Cox, Nicole I.; Phelps, Cody A.; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1+ lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1+ lung cancer cells (designated EDM-TTF-1+) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1+ conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1+ lung adenocarcinomas. PMID:26912193

  20. Niche adaptation by expansion and reprogramming of general transcription factors

    PubMed Central

    Turkarslan, Serdar; Reiss, David J; Gibbins, Goodwin; Su, Wan Lin; Pan, Min; Bare, J Christopher; Plaisier, Christopher L; Baliga, Nitin S

    2011-01-01

    Numerous lineage-specific expansions of the transcription factor B (TFB) family in archaea suggests an important role for expanded TFBs in encoding environment-specific gene regulatory programs. Given the characteristics of hypersaline lakes, the unusually large numbers of TFBs in halophilic archaea further suggests that they might be especially important in rapid adaptation to the challenges of a dynamically changing environment. Motivated by these observations, we have investigated the implications of TFB expansions by correlating sequence variations, regulation, and physical interactions of all seven TFBs in Halobacterium salinarum NRC-1 to their fitness landscapes, functional hierarchies, and genetic interactions across 2488 experiments covering combinatorial variations in salt, pH, temperature, and Cu stress. This systems analysis has revealed an elegant scheme in which completely novel fitness landscapes are generated by gene conversion events that introduce subtle changes to the regulation or physical interactions of duplicated TFBs. Based on these insights, we have introduced a synthetically redesigned TFB and altered the regulation of existing TFBs to illustrate how archaea can rapidly generate novel phenotypes by simply reprogramming their TFB regulatory network. PMID:22108796

  1. Niche adaptation by expansion and reprogramming of general transcription factors.

    PubMed

    Turkarslan, Serdar; Reiss, David J; Gibbins, Goodwin; Su, Wan Lin; Pan, Min; Bare, J Christopher; Plaisier, Christopher L; Baliga, Nitin S

    2011-01-01

    Numerous lineage-specific expansions of the transcription factor B (TFB) family in archaea suggests an important role for expanded TFBs in encoding environment-specific gene regulatory programs. Given the characteristics of hypersaline lakes, the unusually large numbers of TFBs in halophilic archaea further suggests that they might be especially important in rapid adaptation to the challenges of a dynamically changing environment. Motivated by these observations, we have investigated the implications of TFB expansions by correlating sequence variations, regulation, and physical interactions of all seven TFBs in Halobacterium salinarum NRC-1 to their fitness landscapes, functional hierarchies, and genetic interactions across 2488 experiments covering combinatorial variations in salt, pH, temperature, and Cu stress. This systems analysis has revealed an elegant scheme in which completely novel fitness landscapes are generated by gene conversion events that introduce subtle changes to the regulation or physical interactions of duplicated TFBs. Based on these insights, we have introduced a synthetically redesigned TFB and altered the regulation of existing TFBs to illustrate how archaea can rapidly generate novel phenotypes by simply reprogramming their TFB regulatory network. PMID:22108796

  2. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    PubMed

    Millership, C; Phillips, J J; Main, E R G

    2016-05-01

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch. PMID:26947150

  3. The four reprogramming factors and embryonic development in mice.

    PubMed

    Yan, Xingrong; Yu, Shumin; Lei, Anmin; Hua, Jinlian; Chen, Fulin; Li, Liwen; Xie, Xin; Yang, Xueyi; Geng, Wenxin; Dou, Zhongying

    2010-10-01

    The transcription factors (Oct4, Sox2, c-Myc, and Klf4) play an important role in the generation of induced pluripotent stem cells. These factors are expressed in metaphase II oocytes and embryonic stem cells (ESCs). The mechanisms responsible for the reprogramming of ooplasm during nuclear transfer are expected to be associated with the four factors. Here, we show that different paternal genetic backgrounds are able to influence the in vitro development of parthenogenetic and cloned embryos. Using real- time polymerase chain reaction (PCR) we found that the expression level of Oct4 in oocytes was less than that of ESCs, whereas oocytes from KM x C3H females showed the highest expression level of Sox2 than the other strains tested or in G1 ESCs. c-Myc mRNA levels in oocytes from KM mice were greater than those found in ESCs or oocytes of KM x C3H mice. These data demonstrate that the expression of the four transcription factors was different among the oocytes, which may be a contributing factor for the different efficiencies of parthenogenesis and the development of cloned embryos in vitro. PMID:20936906

  4. Metabolic Reprograming of Mononuclear Phagocytes in Progressive Multiple Sclerosis

    PubMed Central

    Tannahill, Gillian Margaret; Iraci, Nunzio; Gaude, Edoardo; Frezza, Christian; Pluchino, Stefano

    2015-01-01

    Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Accumulation of brain damage in progressive MS is partly the result of mononuclear phagocytes (MPs) attacking myelin sheaths in the CNS. Although there is no cure yet for MS, significant advances have been made in the development of disease modifying agents. Unfortunately, most of these drugs fail to reverse established neurological deficits and can have adverse effects. Recent evidence suggests that MPs polarization is accompanied by profound metabolic changes, whereby pro-inflammatory MPs (M1) switch toward glycolysis, whereas anti-inflammatory MPs (M2) become more oxidative. It is therefore possible that reprograming MPs metabolism could affect their function and repress immune cell activation. This mini review describes the metabolic changes underpinning macrophages polarization and anticipates how metabolic re-education of MPs could be used for the treatment of MS. Key points: Inflammation in progressive MS is mediated primarily by MPs.Cell metabolism regulates the function of MPs.DMAs can re-educate the metabolism of MPs to promote healing. PMID:25814990

  5. Reprogramming the assembly of unmodified DNA with a small molecule

    NASA Astrophysics Data System (ADS)

    Avakyan, Nicole; Greschner, Andrea A.; Aldaye, Faisal; Serpell, Christopher J.; Toader, Violeta; Petitjean, Anne; Sleiman, Hanadi F.

    2016-04-01

    The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA ‘alphabet’ by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.

  6. Reprogramming with Small Molecules instead of Exogenous Transcription Factors

    PubMed Central

    Wu, Shouhai

    2015-01-01

    Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs. PMID:25922608

  7. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

    PubMed

    Wood, Lauren W; Cox, Nicole I; Phelps, Cody A; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas. PMID:26912193

  8. Metabolic reprogramming: a new relevant pathway in adult adrenocortical tumors

    PubMed Central

    Longatto-Filho, Adhemar; Faria, André M.; Fragoso, Maria C. B. V.; Lovisolo, Silvana M.; Lerário, Antonio M.; Almeida, Madson Q.

    2015-01-01

    Adrenocortical carcinomas (ACCs) are complex neoplasias that may present unexpected clinical behavior, being imperative to identify new biological markers that can predict patient prognosis and provide new therapeutic options. The main aim of the present study was to evaluate the prognostic value of metabolism-related key proteins in adrenocortical carcinoma. The immunohistochemical expression of MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX was evaluated in a series of 154 adult patients with adrenocortical neoplasia and associated with patients' clinicopathological parameters. A significant increase in was found for membranous expression of MCT4, GLUT1 and CAIX in carcinomas, when compared to adenomas. Importantly MCT1, GLUT1 and CAIX expressions were significantly associated with poor prognostic variables, including high nuclear grade, high mitotic index, advanced tumor staging, presence of metastasis, as well as shorter overall and disease free survival. In opposition, MCT2 membranous expression was associated with favorable prognostic parameters. Importantly, cytoplasmic expression of CD147 was identified as an independent predictor of longer overall survival and cytoplasmic expression of CAIX as an independent predictor of longer disease-free survival. We provide evidence for a metabolic reprogramming in adrenocortical malignant tumors towards the hyperglycolytic and acid-resistant phenotype, which was associated with poor prognosis. PMID:26587828

  9. Reprogramming the assembly of unmodified DNA with a small molecule.

    PubMed

    Avakyan, Nicole; Greschner, Andrea A; Aldaye, Faisal; Serpell, Christopher J; Toader, Violeta; Petitjean, Anne; Sleiman, Hanadi F

    2016-04-01

    The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA 'alphabet' by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials. PMID:27001733

  10. Broad-Spectrum Antiviral Therapeutics

    PubMed Central

    Rider, Todd H.; Zook, Christina E.; Boettcher, Tara L.; Wick, Scott T.; Pancoast, Jennifer S.; Zusman, Benjamin D.

    2011-01-01

    Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered. PMID:21818340

  11. Broad-band beam buncher

    DOEpatents

    Goldberg, D.A.; Flood, W.S.; Arthur, A.A.; Voelker, F.

    1984-03-20

    A broad-band beam bunther is disclosed, comprising an evacuated housing, an electron gun therein for producing an electron beam, a buncher cavity having entrance and exit openings through which the beam is directed, grids across such openings, a source providing a positive DC voltage between the cavity and the electron gun, a drift tube through which the electron beam travels in passing through such cavity, grids across the ends of such drift tube, gaps being provided between the drift tube grids and the entrance and exit grids, a modulator for supplying an ultrahigh frequency modulating signal to the drift tube for producing velocity modulation of the electrons in the beam, a drift space in the housing through which the velocity modulated electron beam travels and in which the beam is bunched, and a discharge opening from such drift tube and having a grid across such opening through which the bunched electron beam is discharged into an accelerator or the like. The buncher cavity and the drift tube may be arranged to constitute an extension of a coaxial transmission line which is employed to deliver the modulating signal from a signal source. The extended transmission line may be terminated in its characteristic impedance to afford a broad-

  12. The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up-regulation.

    PubMed

    Liu, Dong; Zhang, Yongqing; Gharavi, Robert; Park, Hee Ra; Lee, Jaewon; Siddiqui, Sana; Telljohann, Richard; Nassar, Matthew R; Cutler, Roy G; Becker, Kevin G; Mattson, Mark P

    2015-08-01

    Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the

  13. The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival.

    PubMed

    Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

    2016-01-01

    Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na(+)/H(+) exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

  14. The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival

    PubMed Central

    Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

    2016-01-01

    Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na+/H+ exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

  15. Broad-Band Activatable White-Opsin

    PubMed Central

    Batabyal, Subrata; Cervenka, Gregory; Ha, Ji Hee; Kim, Young-tae; Mohanty, Samarendra

    2015-01-01

    Currently, the use of optogenetic sensitization of retinal cells combined with activation/inhibition has the potential to be an alternative to retinal implants that would require electrodes inside every single neuron for high visual resolution. However, clinical translation of optogenetic activation for restoration of vision suffers from the drawback that the narrow spectral sensitivity of an opsin requires active stimulation by a blue laser or a light emitting diode with much higher intensities than ambient light. In order to allow an ambient light-based stimulation paradigm, we report the development of a ‘white-opsin’ that has broad spectral excitability in the visible spectrum. The cells sensitized with white-opsin showed excitability at an order of magnitude higher with white light compared to using only narrow-band light components. Further, cells sensitized with white-opsin produced a photocurrent that was five times higher than Channelrhodopsin-2 under similar photo-excitation conditions. The use of fast white-opsin may allow opsin-sensitized neurons in a degenerated retina to exhibit a higher sensitivity to ambient white light. This property, therefore, significantly lowers the activation threshold in contrast to conventional approaches that use intense narrow-band opsins and light to activate cellular stimulation. PMID:26360377

  16. Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

    PubMed

    Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

    2015-06-01

    The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons. PMID:25790823

  17. Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming

    PubMed Central

    Ashwin, S. S.; Sasai, Masaki

    2015-01-01

    Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming. PMID:26581803

  18. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases.

    PubMed

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz I Altaba, Ariel

    2016-04-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/- cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2(high) and AXIN2(low), which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  19. Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors.

    PubMed

    Sanges, Daniela; Simonte, Giacoma; Di Vicino, Umberto; Romo, Neus; Pinilla, Isabel; Nicolás, Marta; Cosma, Maria Pia

    2016-08-01

    Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration. PMID:27427986

  20. Epigenetic Aberrations Are Not Specific to Transcription Factor-Mediated Reprogramming.

    PubMed

    Tiemann, Ulf; Wu, Guangming; Marthaler, Adele Gabriele; Schöler, Hans Robert; Tapia, Natalia

    2016-01-12

    Somatic cells can be reprogrammed to pluripotency using different methods. In comparison with pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here, we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under defined culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptional and epigenetic level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic abnormalities detected in iPSCs are not specific to transcription factor-mediated reprogramming. PMID:26711876

  1. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases

    PubMed Central

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz i Altaba, Ariel

    2016-01-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/− cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2high and AXIN2low, which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  2. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

    PubMed Central

    Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

    2014-01-01

    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

  3. Histone variant macroH2A confers resistance to nuclear reprogramming

    PubMed Central

    Pasque, Vincent; Gillich, Astrid; Garrett, Nigel; Gurdon, John B

    2011-01-01

    How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes. PMID:21552206

  4. Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells.

    PubMed

    Benchetrit, Hana; Herman, Shay; van Wietmarschen, Niek; Wu, Tao; Makedonski, Kirill; Maoz, Noam; Yom Tov, Nataly; Stave, Danielle; Lasry, Rachel; Zayat, Valery; Xiao, Andrew; Lansdorp, Peter M; Sebban, Shulamit; Buganim, Yosef

    2015-11-01

    Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency. PMID:26412562

  5. Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming

    NASA Astrophysics Data System (ADS)

    Ashwin, S. S.; Sasai, Masaki

    2015-11-01

    Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming.

  6. Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

    PubMed

    Jin, Wensong; Wang, Lei; Zhu, Fei; Tan, Weiqi; Lin, Wei; Chen, Dahua; Sun, Qinmiao; Xia, Zongping

    2016-01-01

    The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming. PMID:26877091

  7. Selenium Augments microRNA Directed Reprogramming of Fibroblasts to Cardiomyocytes via Nanog

    PubMed Central

    Wang, Xiaowen; Hodgkinson, Conrad P; Lu, Kefeng; Payne, Alan J; Pratt, Richard E; Dzau, Victor J

    2016-01-01

    We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. However, direct reprogramming strategies are inefficient and slow. Moving towards the eventual goal of clinical application it is necessary to develop new methodologies to overcome these limitations. Here, we report the identification of a specific media composition, reprogramming media (RM), which augmented the effect of miR combo by 5–15-fold depending upon the cardiac marker tested. RM alone was sufficient to strongly induce cardiac gene and protein expression in neonatal tail-tip as well as cardiac fibroblasts. Expression of pluripotency markers Nanog, Oct4, Sox2, and Klf4 was significantly enhanced by RM, with miR combo augmenting the effect further. Knockdown of Nanog by siRNA inhibited the effect of RM on cardiac gene expression. Removal of insulin-transferrin-selenium completely inhibited the effect of reprogramming media upon cardiac gene expression and the addition of selenium to standard culture media recapitulated the effects of RM. Moreover, selenium enhanced the reprogramming efficiency of miR combo. PMID:26975336

  8. RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming.

    PubMed

    Toh, Cheng-Xu Delon; Chan, Jun-Wei; Chong, Zheng-Shan; Wang, Hao Fei; Guo, Hong Chao; Satapathy, Sandeep; Ma, Dongrui; Goh, Germaine Yen Lin; Khattar, Ekta; Yang, Lin; Tergaonkar, Vinay; Chang, Young-Tae; Collins, James J; Daley, George Q; Wee, Keng Boon; Farran, Chadi A El; Li, Hu; Lim, Yoon-Pin; Bard, Frederic A; Loh, Yuin-Han

    2016-06-21

    Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries. PMID:27292646

  9. Cellular Stress Responses and Monitored Cellular Activities.

    PubMed

    Sawa, Teiji; Naito, Yoshifumi; Kato, Hideya; Amaya, Fumimasa

    2016-08-01

    To survive, organisms require mechanisms that enable them to sense changes in the outside environment, introduce necessary responses, and resist unfavorable distortion. Consequently, through evolutionary adaptation, cells have become equipped with the apparatus required to monitor their fundamental intracellular processes and the mechanisms needed to try to offset malfunction without receiving any direct signals from the outside environment. It has been shown recently that eukaryotic cells are equipped with a special mechanism that monitors their fundamental cellular functions and that some pathogenic proteobacteria can override this monitoring mechanism to cause harm. The monitored cellular activities involved in the stressed intracellular response have been researched extensively in Caenorhabditis elegans, where discovery of an association between key mitochondrial activities and innate immune responses was named "cellular associated detoxification and defenses (cSADD)." This cellular surveillance pathway (cSADD) oversees core cellular activities such as mitochondrial respiration and protein transport into mitochondria, detects xenobiotics and invading pathogens, and activates the endocrine pathways controlling behavior, detoxification, and immunity. The cSADD pathway is probably associated with cellular responses to stress in human inflammatory diseases. In the critical care field, the pathogenesis of lethal inflammatory syndromes (e.g., respiratory distress syndromes and sepsis) involves the disturbance of mitochondrial respiration leading to cell death. Up-to-date knowledge about monitored cellular activities and cSADD, especially focusing on mitochondrial involvement, can probably help fill a knowledge gap regarding the pathogenesis of lethal inflammatory syndromes in the critical care field. PMID:26954943

  10. Broad interband semiconductor laser diodes

    NASA Astrophysics Data System (ADS)

    Tan, Chee Loon

    A semiconductor laser is a diode device that emits light via stimulated emission. Conventionally, light emitted from a semiconductor laser is spatially coherent or narrowband. The fundamental mechanism of stimulated emission process in general leads only to a single wavelength emission. However, there are some lasers emit light with a broad spectrum or different distinct wavelength subjected to various operating conditions such as external grating configuration with semiconductor laser, diode-pumped self-Q-switch fiber laser, ultrashort pulse excitation, photonic crystal fiber, ultrabroadband solid-state lasers, semiconductor optical amplifier-based multiwavelength tunable fiber lasers, nonlinear crystal, broadband semiconductor laser etc. This type of broadband laser is vital in many practical applications such as optical telecommunications, spectroscopy measurement, imaging technology, etc. Recently, an ultra-broadband semiconductor laser that utilizes intersubband optical transitions via quantum cascade configuration has been realized. Laser action with a Fabry-Perot spectrum covering all wavelengths from 6 to 8 microm simultaneously is demonstrated with this approach. More recently, broadband emission results from interband optical transitions via quantum-dot/dash nanostructures have been demonstrated in a simple p-i-n laser diode structure. To date, this latest approach offers the simplest design by proper engineering of quantized energy states as well as utilizing the high inhomogeneity of the dot/dash nanostructures, which is inherent from self-assembled growth technology. In this dissertation, modeling of semiconductor InGaAs/GaAs quantum-dot broadband laser utilizing the properties of inhomogeneous and homogeneous broadening effects on lasing spectral will be discussed, followed by a detail analysis of another type of broad interband semiconductor laser, which is InAs/InGaAlAs quantum-dash broadband laser. Based on the device characterization results

  11. Broad-Application Test Reactor

    SciTech Connect

    Motloch, C.G.

    1992-05-01

    This report is about a new, safe, and operationally efficient DOE reactor of nuclear research and testing proposed for the early to mid- 21st Century. Dubbed the Broad-Application Test Reactor (BATR), the proposed facility incorporates a multiple-application, multiple-mission design to support DOE programs such as naval reactors and space power and propulsion, as well as research in medical, science, isotope, and electronics arenas. DOE research reactors are aging, and implementing major replacement projects requires long lead times. Primary design drivers include safety, low risk, minimum operation cost, mission flexibility, waste minimization, and long life. Scientists and engineers at the Idaho National Engineering Laboratory are evaluating possible fuel forms, structural materials, reactor geometries, coolants, and moderators.

  12. Cellular Phone Towers

    MedlinePlus

    ... the call. How are people exposed to the energy from cellular phone towers? As people use cell ... where people can be exposed to them. The energy from a cellular phone tower antenna, like that ...

  13. Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

    PubMed Central

    2014-01-01

    Background Aberrant energy metabolism is a hallmark of cancer. To fulfill the increased energy requirements, tumor cells secrete cytokines/factors inducing muscle and fat degradation in cancer patients, a condition known as cancer cachexia. It accounts for nearly 20% of all cancer-related deaths. However, the mechanistic basis of cancer cachexia and therapies targeting cancer cachexia thus far remain elusive. A ketogenic diet, a high-fat and low-carbohydrate diet that elevates circulating levels of ketone bodies (i.e., acetoacetate, β-hydroxybutyrate, and acetone), serves as an alternative energy source. It has also been proposed that a ketogenic diet leads to systemic metabolic changes. Keeping in view the significant role of metabolic alterations in cancer, we hypothesized that a ketogenic diet may diminish glycolytic flux in tumor cells to alleviate cachexia syndrome and, hence, may provide an efficient therapeutic strategy. Results We observed reduced glycolytic flux in tumor cells upon treatment with ketone bodies. Ketone bodies also diminished glutamine uptake, overall ATP content, and survival in multiple pancreatic cancer cell lines, while inducing apoptosis. A decrease in levels of c-Myc, a metabolic master regulator, and its recruitment on glycolytic gene promoters, was in part responsible for the metabolic phenotype in tumor cells. Ketone body-induced intracellular metabolomic reprogramming in pancreatic cancer cells also leads to a significantly diminished cachexia in cell line models. Our mouse orthotopic xenograft models further confirmed the effect of a ketogenic diet in diminishing tumor growth and cachexia. Conclusions Thus, our studies demonstrate that the cachectic phenotype is in part due to metabolic alterations in tumor cells, which can be reverted by a ketogenic diet, causing reduced tumor growth and inhibition of muscle and body weight loss. PMID:25228990

  14. Mitochondrial DNA copy number and replication in reprogramming and differentiation.

    PubMed

    St John, Justin C

    2016-04-01

    Until recently, it was thought that the role of the mitochondrial genome was confined to encoding key proteins that generate ATP through the process of oxidative phosphorylation in the electron transfer chain. However, with increasing new evidence, it is apparent that the mitochondrial genome has a major role to play in a number of diseases and phenotypes. For example, mitochondrial variants and copy number have been implicated in the processes of fertilisation outcome and development and the onset of tumorigenesis. On the other hand, mitochondrial DNA (mtDNA) haplotypes have been implicated in a variety of diseases and most likely account for the adaptation that our ancestors achieved in order that they were fit for their environments. The mechanisms, which enable the mitochondrial genome to either protect or promote the disease phenotype, require further elucidation. However, there appears to be significant 'crosstalk' between the chromosomal and mitochondrial genomes that enable this to take place. One such mechanism is the regulation of DNA methylation by mitochondrial DNA, which is often perturbed in reprogrammed cells that have undergone dedifferentiation and affects mitochondrial DNA copy number. Furthermore, it appears that the mitochondrial genome interacts with the chromosomal genome to regulate the transcription of key genes at certain stages during development. Additionally, the mitochondrial genome can accumulate a series of mtDNA variants, which can lead to diseases such as cancer. It is likely that a combination of certain mitochondrial variants and aberrant patterns of mtDNA copy number could indeed account for many diseases that have previously been unaccounted for. This review focuses on the role that the mitochondrial genome plays especially during early stages of development and in cancer. PMID:26827792

  15. Primary cancer cell culture: mammary-optimized vs conditional reprogramming.

    PubMed

    Alamri, Ahmad M; Kang, Keunsoo; Groeneveld, Svenja; Wang, Weisheng; Zhong, Xiaogang; Kallakury, Bhaskar; Hennighausen, Lothar; Liu, Xuefeng; Furth, Priscilla A

    2016-07-01

    The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53 Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial-mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments. PMID:27267121

  16. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-01-01

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young's modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  17. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-12-31

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young`s modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  18. HMGB1 Facilitated Macrophage Reprogramming towards a Proinflammatory M1-like Phenotype in Experimental Autoimmune Myocarditis Development

    PubMed Central

    Su, Zhaoliang; Zhang, Pan; Yu, Ying; Lu, Hongxiang; Liu, Yanfang; Ni, Ping; Su, Xiaolian; Wang, Dan; Liu, Yueqin; Wang, Jia; Shen, Huiling; Xu, Wenlin; Xu, Huaxi

    2016-01-01

    Macrophages can be reprogramming, such as the classical activated macrophage, M1 or alternative activated macrophages, M2 phenotype following the milieu danger signals, especially inflammatory factors. Macrophage reprogramming is now considered as a key determinant of disease development and/or regression. Experimental autoimmune myocarditis (EAM) is characterized by monocytes/macrophage infiltration, Th17 cells activation and inflammatory factors producing such as high mobility group box 1 (HMGB1). Whether infiltrated macrophages could be reprogramming in EAM? HMGB1 was associated with macrophage reprogramming? Our results clearly demonstrated that infiltrated macrophage was reprogrammed towards a proinflammatory M1-like phenotype and cardiac protection by monocytes/macrophages depletion or HMGB1 blockade in EAM; in vitro, HMGB1 facilitated macrophage reprogramming towards M1-like phenotype dependent on TLR4-PI3Kγ-Erk1/2 pathway; furthermore, the reprogramming M1-like macrophage promoted Th17 expansion. Therefore, we speculated that HMGB1 contributed EAM development via facilitating macrophage reprogramming towards M1-like phenotype except for directly modulating Th17 cells expansion. PMID:26899795

  19. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    PubMed Central

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Summary Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  20. Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

    PubMed Central

    Baeyens, Luc; Lemper, Marie; Leuckx, Gunter; De Groef, Sofie; Bonfanti, Paola; Stangé, Geert; Shemer, Ruth; Nord, Christoffer; Scheel, David W.; Pan, Fong C.; Ahlgren, Ulf; Gu, Guoqiang; Stoffers, Doris A.; Dor, Yuval; Ferrer, Jorge; Gradwohl, Gerard; Wright, Christopher VE; Van de Casteele, Mark; German, Michael S.; Bouwens, Luc; Heimberg, Harry

    2014-01-01

    Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose-responsive, and reinstate normal glycemic control for up to 248 days. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3) expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta cell reprogramming through transient cytokine exposure rather than genetic modification. PMID:24240391

  1. Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

    PubMed Central

    Lepikhov, Konstantin; Zakhartchenko, Valeri; Hao, Ru; Yang, Feikun; Wrenzycki, Christine; Niemann, Heiner; Wolf, Eckhard; Walter, Joern

    2008-01-01

    Background In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. Results When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. Conclusion Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species. PMID:19014417

  2. Physiological, pathological, and engineered cell identity reprogramming in the central nervous system.

    PubMed

    Smith, Derek K; Wang, Lei-Lei; Zhang, Chun-Li

    2016-07-01

    Multipotent neural stem cells persist in restricted regions of the adult mammalian central nervous system. These proliferative cells differentiate into diverse neuron subtypes to maintain neural homeostasis. This endogenous process can be reprogrammed as a compensatory response to physiological cues, traumatic injury, and neurodegeneration. In addition to innate neurogenesis, recent research has demonstrated that new neurons can be engineered via cell identity reprogramming in non-neurogenic regions of the adult central nervous system. A comprehensive understanding of these reprogramming mechanisms will be essential to the development of therapeutic neural regeneration strategies that aim to improve functional recovery after injury and neurodegeneration. WIREs Dev Biol 2016, 5:499-517. doi: 10.1002/wdev.234 For further resources related to this article, please visit the WIREs website. PMID:27258392

  3. Generation of Induced Pluripotent Stem (iPS) Cells by Nuclear Reprogramming

    PubMed Central

    Dey, Dilip; Evans, Gregory R. D.

    2011-01-01

    During embryonic development pluripotency is progressively lost irreversibly by cell division, differentiation, migration and organ formation. Terminally differentiated cells do not generate other kinds of cells. Pluripotent stem cells are a great source of varying cell types that are used for tissue regeneration or repair of damaged tissue. The pluripotent stem cells can be derived from inner cell mass of blastocyte but its application is limited due to ethical concerns. The recent discovery of iPS with defined reprogramming factors has initiated a flurry of works on stem cell in various laboratories. The pluripotent cells can be derived from various differentiated adult cells as well as from adult stem cells by nuclear reprogramming, somatic cell nuclear transfer etc. In this review article, different aspects of nuclear reprogramming are discussed. PMID:22007240

  4. Reprogramming within hours following nuclear transfer into mouse but not human zygotes.

    PubMed

    Egli, Dieter; Chen, Alice E; Saphier, Genevieve; Ichida, Justin; Fitzgerald, Claire; Go, Kathryn J; Acevedo, Nicole; Patel, Jay; Baetscher, Manfred; Kearns, William G; Goland, Robin; Leibel, Rudolph L; Melton, Douglas A; Eggan, Kevin

    2011-01-01

    Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation. PMID:21971503

  5. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  6. In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells.

    PubMed

    Li, Hedong; Chen, Gong

    2016-08-17

    Neuroregeneration in the CNS has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this Perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart, and liver and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient's own internal cells for tissue repair. PMID:27537482

  7. Direct Reprogramming for Pancreatic Beta-Cells Using Key Developmental Genes

    PubMed Central

    Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann, Marianne; Yamada, Takatsugu; Bonner-Weir, Susan; Weir, Gordon

    2015-01-01

    Direct reprogramming is a promising approach for regenerative medicine whereby one cell type is directly converted into another without going through a multipotent or pluripotent stage. This reprogramming approach has been extensively explored for the generation of functional insulin-secreting cells from non-beta-cells with the aim of developing novel cell therapies for the treatment of people with diabetes lacking sufficient endogenous beta-cells. A common approach for such conversion studies is the introduction of key regulators that are important in controlling beta-cell development and maintenance. In this review, we will summarize the recent advances in the field of beta-cell reprogramming and discuss the challenges of creating functional and long-lasting beta-cells. PMID:26998407

  8. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

    PubMed Central

    Liu, Xinjian; Li, Fang; Stubblefield, Elizabeth A; Blanchard, Barbara; Richards, Toni L; Larson, Gaynor A; He, Yujun; Huang, Qian; Tan, Aik-Choon; Zhang, Dabing; Benke, Timothy A; Sladek, John R; Zahniser, Nancy R; Li, Chuan-Yuan

    2012-01-01

    Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD. PMID:22105488

  9. Inhibition of Transforming Growth Factor β (TGF-β) Signaling can Substitute for Oct4 Protein in Reprogramming and Maintain Pluripotency*

    PubMed Central

    Tan, Fangzhi; Qian, Cheng; Tang, Ke; Abd-Allah, Saber Mohamed; Jing, Naihe

    2015-01-01

    Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/β-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-β signaling in this process remains elusive. Here we show that inhibiting TGF-β signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-β signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-β signaling inhibition in the generation and maintenance of PSCs. PMID:25548277

  10. Cell-free production of transducible transcription factors for nuclear reprogramming.

    PubMed

    Yang, William C; Patel, Kedar G; Lee, Jieun; Ghebremariam, Yohannes T; Wong, H Edward; Cooke, John P; Swartz, James R

    2009-12-15

    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. PMID:19718703

  11. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

    PubMed

    Doege, Claudia A; Inoue, Keiichi; Yamashita, Toru; Rhee, David B; Travis, Skylar; Fujita, Ryousuke; Guarnieri, Paolo; Bhagat, Govind; Vanti, William B; Shih, Alan; Levine, Ross L; Nik, Sara; Chen, Emily I; Abeliovich, Asa

    2012-08-30

    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming. PMID:22902501

  12. Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

    PubMed

    Bueno, C; Sardina, J L; Di Stefano, B; Romero-Moya, D; Muñoz-López, A; Ariza, L; Chillón, M C; Balanzategui, A; Castaño, J; Herreros, A; Fraga, M F; Fernández, A; Granada, I; Quintana-Bustamante, O; Segovia, J C; Nishimura, K; Ohtaka, M; Nakanishi, M; Graf, T; Menendez, P

    2016-03-01

    B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies. PMID:26500142

  13. The distribution of genomic variations in human iPSCs is related to replication timing reorganization during reprogramming

    PubMed Central

    Lu, Junjie; Li, Hu; Hu, Ming; Sasaki, Takayo; Baccei, Anna; Gilber, David M.; Liu, Jun S.; Collins, James J.; Lerou, Paul H.

    2014-01-01

    SUMMARY Cell fate change involves significant genome reorganization, including change in replication timing, but how these changes are related to genetic variation has not been examined. To study how change in replication timing that occurs during reprogramming impacts the copy number variation (CNV) landscape, we generated genome-wide replication timing profiles of induced pluripotent stem cells (iPSCs) and their parental fibroblasts. A significant portion of the genome changes replication timing as a result of reprogramming, indicative of overall genome reorganization. We found that early and late replicating domains in iPSCs are differentially affected by copy number gains and losses, and that in particular CNV gains accumulate in regions of the genome that change to earlier replication during the reprogramming process. This differential relationship was present irrespective of reprogramming method. Overall, our findings reveal a functional association between reorganization of replication timing and the CNV landscape that emerges during reprogramming. PMID:24685138

  14. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    PubMed

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated. PMID:26895068

  15. TGFβ signaling regulates the choice between pluripotent and neural fates during reprogramming of human urine derived cells

    PubMed Central

    Wang, Lihui; Li, Xirui; Huang, Wenhao; Zhou, Tiancheng; Wang, Haitao; Lin, Aiping; Hutchins, Andrew Paul; Su, Zhenghui; Chen, Qianyu; Pei, Duanqing; Pan, Guangjin

    2016-01-01

    Human urine cells (HUCs) can be reprogrammed into neural progenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) with defined factors and a small molecule cocktail, but the underlying fate choice remains unresolved. Here, through sequential removal of individual compound from small molecule cocktail, we showed that A8301, a TGFβ signaling inhibitor, is sufficient to switch the cell fate from iPSCs into NPCs in OSKM-mediated HUCs reprogramming. However, TGFβ exposure at early stage inhibits HUCs reprogramming by promoting EMT. Base on these data, we developed an optimized approach for generation of NPCs or iPSCs from HUCs with significantly improved efficiency by regulating TGFβ activity at different reprogramming stages. This approach provides a simplified and improved way for HUCs reprogramming, thus would be valuable for banking human iPSCs or NPCs from people with different genetic background. PMID:26935433

  16. Broadly tunable picosecond ir source

    DOEpatents

    Campillo, A.J.; Hyer, R.C.; Shapiro, S.L.

    1980-04-23

    A picosecond traveling-wave parametric device capable of controlled spectral bandwidth and wavelength in the infrared is reported. Intense 1.064 ..mu..m picosecond pulses (1) pass through a 4.5 cm long LiNbO/sub 3/ optical parametric oscillator crystal (2) set at its degeneracy angle. A broad band emerges, and a simple grating (3) and mirror (4) arrangement is used to inject a selected narrow-band into a 2 cm long LiNbO/sub 3/ optical parametric amplifier crystal (5) along a second pump line. Typical input energies at 1.064 ..mu..m along both pump lines are 6 to 8 mJ for the oscillator and 10 mJ for the amplifier. This yields 1 mJ of tunable output in the range 1.98 to 2.38 ..mu..m which when down-converted in a 1 cm long CdSe crystal mixer (6) gives 2 ..mu..J of tunable radiation over the 14.8 to 18.5 ..mu..m region. The bandwidth and wavelength of both the 2 and 16 ..mu..m radiation output are controlled solely by the diffraction grating.

  17. Ghost imaging with broad distance

    NASA Astrophysics Data System (ADS)

    Duan, De-Yang; Zhang, Lu; Du, Shao-Jiang; Xia, Yun-Jie

    2015-10-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is. Project supported by the National Natural Science Foundation of China (Grant Nos. 61178012, 11204156, 11304179, and 11247240), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant Nos. 20133705110001 and 20123705120002), the Scientific Research Foundation for Outstanding Young Scientists of Shandong Province, China (Grant No. BS2013DX034), and the Natural Science Foundation of Shandong Province, China (Grant No. ZR2012FQ024).

  18. Broadly tunable picosecond IR source

    DOEpatents

    Campillo, Anthony J.; Hyer, Ronald C.; Shapiro, Stanley J.

    1982-01-01

    A picosecond traveling-wave parametric device capable of controlled spectral bandwidth and wavelength in the infrared is reported. Intense 1.064 .mu.m picosecond pulses (1) pass through a 4.5 cm long LiNbO.sub.3 optical parametric oscillator crystal (2) set at its degeneracy angle. A broad band emerges, and a simple grating (3) and mirror (4) arrangement is used to inject a selected narrow-band into a 2 cm long LiNbO.sub.3 optical parametric amplifier crystal (5) along a second pump line. Typical input energies at 1.064 .mu.m along both pump lines are 6-8 mJ for the oscillator and 10 mJ for the amplifier. This yields 1 mJ of tunable output in the range 1.98 to 2.38 .mu.m which when down-converted in a 1 cm long CdSe crystal mixer (6) gives 2 .mu.J of tunable radiation over the 14.8 to 18.5 .mu.m region. The bandwidth and wavelength of both the 2 and 16 .mu.m radiation output are controlled solely by the diffraction grating.

  19. A "Hit and Run" Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells.

    PubMed

    Poulou, Maria; Mandalos, Nikolaos P; Karnavas, Theodoros; Saridaki, Marannia; McKay, Ronald D G; Remboutsika, Eumorphia

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel "hit and run" inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2(FLAG) under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine. PMID:27148066

  20. Antagonistic role of natural compounds in mTOR-mediated metabolic reprogramming.

    PubMed

    Cerella, Claudia; Gaigneaux, Anthoula; Dicato, Mario; Diederich, Marc

    2015-01-28

    Cells reprogram their metabolism very early during carcinogenesis; this event is critical for the establishment of other cancer hallmarks. Many oncogenes and tumor suppressor genes control metabolism by interplaying with the existing nutrient-sensing intracellular pathways. Mammalian target of rapamycin, mTOR, is emerging as a collector and sorter of a metabolic network controlling upstream and downstream modulation of these same genes. Natural compounds represent a source of anti-cancer molecules with chemopreventive and therapeutic properties. This review describes selected pathways and genes orchestrating the metabolic reprogramming and discusses the potential of natural compounds to target oncogenic metabolic aberrations. PMID:24530513

  1. MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

    PubMed Central

    Tomasetti, Marco; Amati, Monica; Santarelli, Lory; Neuzil, Jiri

    2016-01-01

    The process of metabolic re-programing is linked to the activation of oncogenes and/or suppression of tumour suppressor genes, which are regulated by microRNAs (miRNAs). The interplay between oncogenic transformation-driven metabolic re-programming and modulation of aberrant miRNAs further established their critical role in the initiation, promotion and progression of cancer by creating a tumorigenesis-prone microenvironment, thus orchestrating processes of evasion to apoptosis, angiogenesis and invasion/migration, as well metastasis. Given the involvement of miRNAs in tumour development and their global deregulation, they may be perceived as biomarkers in cancer of therapeutic relevance. PMID:27213336

  2. MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis.

    PubMed

    Tomasetti, Marco; Amati, Monica; Santarelli, Lory; Neuzil, Jiri

    2016-01-01

    The process of metabolic re-programing is linked to the activation of oncogenes and/or suppression of tumour suppressor genes, which are regulated by microRNAs (miRNAs). The interplay between oncogenic transformation-driven metabolic re-programming and modulation of aberrant miRNAs further established their critical role in the initiation, promotion and progression of cancer by creating a tumorigenesis-prone microenvironment, thus orchestrating processes of evasion to apoptosis, angiogenesis and invasion/migration, as well metastasis. Given the involvement of miRNAs in tumour development and their global deregulation, they may be perceived as biomarkers in cancer of therapeutic relevance. PMID:27213336

  3. Metabolic Reprogramming and Dependencies Associated with Epithelial Cancer Stem Cells Independent of the Epithelial-Mesenchymal Transition Program.

    PubMed

    Aguilar, Esther; Marin de Mas, Igor; Zodda, Erika; Marin, Silvia; Morrish, Fionnuala; Selivanov, Vitaly; Meca-Cortés, Óscar; Delowar, Hossain; Pons, Mònica; Izquierdo, Inés; Celià-Terrassa, Toni; de Atauri, Pedro; Centelles, Josep J; Hockenbery, David; Thomson, Timothy M; Cascante, Marta

    2016-05-01

    In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial-mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e-CSCs) from non-CSCs expressing a stable EMT. We have found that the e-CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e-CSCs consistent with the metabolomics and fluxomics analyses that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics, and transcriptomics analysis allowed us to identify key metabolic players regulated at the post-transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis. Stem Cells 2016;34:1163-1176. PMID:27146024

  4. The Function of the MEF2 Family of Transcription Factors in Cardiac Development, Cardiogenomics, and Direct Reprogramming

    PubMed Central

    Desjardins, Cody A.; Naya, Francisco J.

    2016-01-01

    Proper formation of the mammalian heart requires precise spatiotemporal transcriptional regulation of gene programs in cardiomyocytes. Sophisticated regulatory networks have evolved to not only integrate the activities of distinct transcription factors to control tissue-specific gene programs but also, in many instances, to incorporate multiple members within these transcription factor families to ensure accuracy and specificity in the system. Unsurprisingly, perturbations in this elaborate transcriptional circuitry can lead to severe cardiac abnormalities. Myocyte enhancer factor–2 (MEF2) transcription factor belongs to the evolutionarily conserved cardiac gene regulatory network. Given its central role in muscle gene regulation and its evolutionary conservation, MEF2 is considered one of only a few core cardiac transcription factors. In addition to its firmly established role as a differentiation factor, MEF2 regulates wide variety of, sometimes antagonistic, cellular processes such as cell survival and death. Vertebrate genomes encode multiple MEF2 family members thereby expanding the transcriptional potential of this core transcription factor in the heart. This review highlights the requirement of the MEF2 family and their orthologs in cardiac development in diverse animal model systems. Furthermore, we describe the recently characterized role of MEF2 in direct reprogramming and genome-wide cardiomyocyte gene regulation. A thorough understanding of the regulatory functions of the MEF2 family in cardiac development and cardiogenomics is required in order to develop effective therapeutic strategies to repair the diseased heart.

  5. Transcriptional Reprogramming of CD11b+Esamhi Dendritic Cell Identity and Function by Loss of Runx3

    PubMed Central

    Dicken, Joseph; Mildner, Alexander; Leshkowitz, Dena; Touw, Ivo P.; Hantisteanu, Shay; Jung, Steffen; Groner, Yoram

    2013-01-01

    Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esamhi DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4+/CD11b+ DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b+Esamhi DC. Mechanistically, loss of Runx3 alters Esamhi DC gene expression to a signature characteristic of WT Esamlow DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3-/- Esamhi DC capacity to prime CD4+ T cells, attesting to the significant role of Runx3 in specifying Esamhi DC identity and function. PMID:24204843

  6. Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma

    PubMed Central

    Gilmore, Sarah A.; Voorhies, Mark; Gebhart, Dana; Sil, Anita

    2015-01-01

    Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature. PMID:26177267

  7. 5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation

    PubMed Central

    Hon, Gary C.; Song, Chun-Xiao; Du, Tingting; Jin, Fulai; Selvaraj, Siddarth; Lee, Ah Young; Yen, Chia-an; Ye, Zhen; Mao, Shi-Qing; Wang, Bang-An; Kuan, Samantha; Edsall, Lee E.; Zhao, Boxuan Simen; Xu, Guo-Liang; He, Chuan; Ren, Bing

    2014-01-01

    Summary In mammals, cytosine methylation (5mC) is widely distributed throughout the genome, but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation. PMID:25263596

  8. Jasmonate signaling involves the abscisic acid receptor PYL4 to regulate metabolic reprogramming in Arabidopsis and tobacco

    PubMed Central

    Lackman, Petri; González-Guzmán, Miguel; Tilleman, Sofie; Carqueijeiro, Inês; Pérez, Amparo Cuéllar; Moses, Tessa; Seo, Mitsunori; Kanno, Yuri; Häkkinen, Suvi T.; Van Montagu, Marc C. E.; Thevelein, Johan M.; Maaheimo, Hannu; Oksman-Caldentey, Kirsi-Marja; Rodriguez, Pedro L.; Rischer, Heiko; Goossens, Alain

    2011-01-01

    The phytohormones jasmonates (JAs) constitute an important class of elicitors for many plant secondary metabolic pathways. However, JAs do not act independently but operate in complex networks with crosstalk to several other phytohormonal signaling pathways. Here, crosstalk was detected between the JA and abscisic acid (ABA) signaling pathways in the regulation of tobacco (Nicotiana tabacum) alkaloid biosynthesis. A tobacco gene from the PYR/PYL/RCAR family, NtPYL4, the expression of which is regulated by JAs, was found to encode a functional ABA receptor. NtPYL4 inhibited the type-2C protein phosphatases known to be key negative regulators of ABA signaling in an ABA-dependent manner. Overexpression of NtPYL4 in tobacco hairy roots caused a reprogramming of the cellular metabolism that resulted in a decreased alkaloid accumulation and conferred ABA sensitivity to the production of alkaloids. In contrast, the alkaloid biosynthetic pathway was not responsive to ABA in control tobacco roots. Functional analysis of the Arabidopsis (Arabidopsis thaliana) homologs of NtPYL4, PYL4 and PYL5, indicated that also in Arabidopsis altered PYL expression affected the JA response, both in terms of biomass and anthocyanin production. These findings define a connection between a component of the core ABA signaling pathway and the JA responses and contribute to the understanding of the role of JAs in balancing tradeoffs between growth and defense. PMID:21436041

  9. Alterations in the Vaginal Microbiome by Maternal Stress Are Associated With Metabolic Reprogramming of the Offspring Gut and Brain.

    PubMed

    Jašarević, Eldin; Howerton, Christopher L; Howard, Christopher D; Bale, Tracy L

    2015-09-01

    The neonate is exposed to the maternal vaginal microbiota during parturition, providing the primary source for normal gut colonization, host immune maturation, and metabolism. These early interactions between the host and microbiota occur during a critical window of neurodevelopment, suggesting early life as an important period of cross talk between the developing gut and brain. Because perturbations in the prenatal environment such as maternal stress increase neurodevelopmental disease risk, disruptions to the vaginal ecosystem could be a contributing factor in significant and long-term consequences for the offspring. Therefore, to examine the hypothesis that changes in the vaginal microbiome are associated with effects on the offspring gut microbiota and on the developing brain, we used genomic, proteomic and metabolomic technologies to examine outcomes in our mouse model of early prenatal stress. Multivariate modeling identified broad proteomic changes to the maternal vaginal environment that influence offspring microbiota composition and metabolic processes essential for normal neurodevelopment. Maternal stress altered proteins related to vaginal immunity and abundance of Lactobacillus, the prominent taxa in the maternal vagina. Loss of maternal vaginal Lactobacillus resulted in decreased transmission of this bacterium to offspring. Further, altered microbiota composition in the neonate gut corresponded with changes in metabolite profiles involved in energy balance, and with region- and sex-specific disruptions of amino acid profiles in the developing brain. Taken together, these results identify the vaginal microbiota as a novel factor by which maternal stress may contribute to reprogramming of the developing brain that may predispose individuals to neurodevelopmental disorders. PMID:26079804

  10. Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis.

    PubMed

    Sishc, Brock J; Nelson, Christopher B; McKenna, Miles J; Battaglia, Christine L R; Herndon, Andrea; Idate, Rupa; Liber, Howard L; Bailey, Susan M

    2015-01-01

    Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore, telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles, telomeres and telomerase play in the response of human cells to IRs of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET, high charge, high energy (HZE) particles, delivered either acutely or at low dose rates. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprograming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the

  11. Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis

    PubMed Central

    Sishc, Brock J.; Nelson, Christopher B.; McKenna, Miles J.; Battaglia, Christine L. R.; Herndon, Andrea; Idate, Rupa; Liber, Howard L.; Bailey, Susan M.

    2015-01-01

    Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore, telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles, telomeres and telomerase play in the response of human cells to IRs of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET, high charge, high energy (HZE) particles, delivered either acutely or at low dose rates. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprograming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the

  12. The 2006 Broad Prize for Urban Education

    ERIC Educational Resources Information Center

    Broad Foundation, 2006

    2006-01-01

    The $1 million Broad Prize in Urban Education is the nation's largest award in K-12 public education. The Broad Prize is given annually by The Broad Foundation in the form of scholarships to urban school districts that demonstrate the greatest overall performance and improvement in student achievement while reducing achievement gaps among ethnic…

  13. 77 FR 50144 - Broad Stakeholder Survey

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-20

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until... across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  14. 78 FR 20119 - Broad Stakeholder Survey

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-03

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 30-day... soliciting comments concerning the Broad Stakeholder Survey. DHS previously published this ICR in the Federal... responders across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  15. Jupiter's Temperatures--Broad Latitude

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This is one of the highest resolution images ever recorded of Jupiter's temperature field. It was obtained by NASA's Galileo mission, with its Photopolarimeter-Radiometer (PPR) experiment, during the seventh of its 10 orbits around Jupiter to date. This map, shown in the left panel, indicates the forces powering Jovian winds, and differentiates between areas of strongest upwelling and downwelling winds in the upper part of the atmosphere. A Hubble Space Telescope Planetary Camera color composite of this same region, taken within 10 hours of the PPR map, is shown in the right panel for the same region, as a reference to the visual clouds. An outline of the region mapped by the PPR is also shown.

    This atmospheric observation covered a broad latitude region, and it shows that the visually dark regions generally have warmer temperatures than the visually light ones, indicating that they are regions of downwelling, dry air which clear out cloud condensate particles. The 'little red spot' at the northernmost part of this image is colder than its surroundings, consistent with it being a region of upwelling and cooling gas. The smaller spots to its southeast (lower right) and other lighter spots in the HST image are all colder than their surroundings, consistent with regions of upwelling and cooling gas. The northern half of the brightest band in the map is brighter than the southern half, and it reveals some detailed structure, down to the 1900- kilometer (1200-mile) resolution of the PPR, which is not always readily correlated with variations of the visual cloud field.

    One surprise of this temperature map involved temperatures near the dark blue-gray feature in the map, an area like the one into which the Probe descended. While large regions of downwelling wind heat the local area elsewhere in Jupiter, this region of vigorous downwelling appears close to being thermally neutral. The drying, downwelling winds may be deeper in the atmosphere than sensed by the PPR

  16. NF-κB activation impairs somatic cell reprogramming in ageing.

    PubMed

    Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

    2015-08-01

    Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies. PMID:26214134

  17. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    PubMed Central

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  18. Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

    PubMed

    Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

    2013-07-01

    The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports. PMID:23660742

  19. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

    PubMed

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. PMID:27435961

  20. Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming.

    PubMed

    Cantone, Irene; Bagci, Hakan; Dormann, Dirk; Dharmalingam, Gopuraja; Nesterova, Tatyana; Brockdorff, Neil; Rougeulle, Claire; Vallot, Celine; Heard, Edith; Chaligne, Ronan; Merkenschlager, Matthias; Fisher, Amanda G

    2016-01-01

    Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. PMID:27507283

  1. ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency

    PubMed Central

    Kida, Yasuyuki S.; Kawamura, Teruhisa; Wei, Zong; Sogo, Takahiro; Jacinto, Sandra; Shigeno-Kamitsuji, Asako; Yoshihara, Eiji; Liddle, Christopher; Ecker, Joseph R.; Yu, Ruth T.; Atkins, Annette R.; Downes, Michael; Evans, Ronald M.

    2015-01-01

    Summary Cell metabolism is adaptive to extrinsic demands, however the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. While glycolysis increases throughout the reprogramming process, we show that the estrogen related nuclear receptors (ERRα and γ) and their partnered co-factors PGC-1α and β, are transiently induced at an early stage resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Up-regulation of ERRα or γ is required for both the OXPHOS burst in human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1−/CD34− sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1β positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency. PMID:25865501

  2. Reprogramming of mouse and human somatic cells by high-performance engineered factors

    PubMed Central

    Wang, Yang; Chen, Jiekai; Hu, Jia-Lei; Wei, Xi-Xiao; Qin, Dajiang; Gao, Juan; Zhang, Lei; Jiang, Jing; Li, Jin-Song; Liu, Jing; Lai, Ke-Yu; Kuang, Xia; Zhang, Jian; Pei, Duanqing; Xu, Guo-Liang

    2011-01-01

    Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with enhanced efficiency and accelerated kinetics. Remarkably, Oct4–VP16 alone could efficiently reprogramme mouse embryonic fibroblasts (MEFs) into germline-competent iPSCs. Furthermore, episomally delivered synthetic factors could reproducibly generate integration-free iPSCs from MEFs with enhanced efficiency. Our results not only demonstrate the feasibility of engineering more potent reprogramming factors, but also suggest that transcriptional reactivation of OCT4 target genes might be a rate-limiting step in the conversion of somatic cells to pluripotent cells. Synthetic factor-based reprogramming might lead to a paradigm shift in reprogramming research. PMID:21399616

  3. Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

    PubMed Central

    Cantone, Irene; Bagci, Hakan; Dormann, Dirk; Dharmalingam, Gopuraja; Nesterova, Tatyana; Brockdorff, Neil; Rougeulle, Claire; Vallot, Celine; Heard, Edith; Chaligne, Ronan; Merkenschlager, Matthias; Fisher, Amanda G.

    2016-01-01

    Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. PMID:27507283

  4. High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

    PubMed

    Zhao, Yuanbiao; Londono, Pilar; Cao, Yingqiong; Sharpe, Emily J; Proenza, Catherine; O'Rourke, Rebecca; Jones, Kenneth L; Jeong, Mark Y; Walker, Lori A; Buttrick, Peter M; McKinsey, Timothy A; Song, Kunhua

    2015-01-01

    Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-β or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications. PMID:26354680

  5. Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

    PubMed Central

    Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

    2016-01-01

    Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

  6. Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells

    PubMed Central

    Mahalingam, Dashayini; Kong, Chiou Mee; Lai, Jason; Tay, Ling Lee; Yang, Henry; Wang, Xueying

    2012-01-01

    Recent reports on direct reprogramming of cancer cells (iPCs) which results in reduced tumorigenic potential has attributed the importance of epigenetics in tumorigenesis, but lacked genome-wide analysis. Here we describe successful generation of iPCs from non-small cell lung cancer (NSCLC) cell lines. Following reprogramming, they resembled embryonic stem and induced pluripotent stem cells in pluripotency markers expression, gene expression patterns and in vitro differentiation ability. Genome-wide methylation analysis revealed that aberrantly methylated promoters which were mostly developmental-associated genes and tumor suppressors; as well as commonly upregulated genes in NSCLC i.e. KRT19 and S100P were reversed in iPCs upon reprogramming. Also, the reversal of oncogenes and tumor suppressors status were partially explainable by DNA methylation. These findings suggest that DNA methylation patterns explain the downstream transcriptional effects, which potentially caused the reduced tumorigenicity in iPCs, thus providing evidence that reprogramming reverses the aberrantly dysregulated genes in NSCLC both epigenetically and transcriptionally. PMID:22912920

  7. High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling

    PubMed Central

    Zhao, Yuanbiao; Londono, Pilar; Cao, Yingqiong; Sharpe, Emily J.; Proenza, Catherine; O'Rourke, Rebecca; Jones, Kenneth L.; Jeong, Mark Y.; Walker, Lori A.; Buttrick, Peter M.; McKinsey, Timothy A.; Song, Kunhua

    2015-01-01

    Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-β or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications. PMID:26354680

  8. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

    PubMed

    Zhao, Chunli; Farruggio, Alfonso P; Bjornson, Christopher R R; Chavez, Christopher L; Geisinger, Jonathan M; Neal, Tawny L; Karow, Marisa; Calos, Michele P

    2014-01-01

    A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies. PMID:24781921

  9. Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.

    PubMed

    Liu, Jing; Brzeszczynska, Joanna; Samuel, Kay; Black, Jim; Palakkan, Anwar; Anderson, Richard A; Gallagher, Ronald; Ross, James A

    2015-11-01

    The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function. PMID:26256888

  10. ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency.

    PubMed

    Kida, Yasuyuki S; Kawamura, Teruhisa; Wei, Zong; Sogo, Takahiro; Jacinto, Sandra; Shigeno, Asako; Kushige, Hiroko; Yoshihara, Eiji; Liddle, Christopher; Ecker, Joseph R; Yu, Ruth T; Atkins, Annette R; Downes, Michael; Evans, Ronald M

    2015-05-01

    Cell metabolism is adaptive to extrinsic demands; however, the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. Although glycolysis increases throughout the reprogramming process, we show that the estrogen-related nuclear receptors (ERRα and ERRγ) and their partnered co-factors PGC-1α and PGC-1β are transiently induced at an early stage, resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Upregulation of ERRα or ERRγ is required for the OXPHOS burst in both human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1(-)/CD34(-) sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1β positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency. PMID:25865501

  11. Metabolic reprogramming of myeloid-derived suppressor cells (MDSC) in cancer.

    PubMed

    Al-Khami, Amir A; Rodriguez, Paulo C; Ochoa, Augusto C

    2016-08-01

    MDSC undergo metabolic reprogramming in the tumor resulting in an increased fatty acid β oxidation that supports their immunosuppressive functions. Fatty acid oxidation inhibitors, used to treat coronary disease, significantly delayed tumor growth and had a significantly increased antitumor effect when combined with adoptive cell therapy or low dose chemotherapy. PMID:27622069

  12. Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells

    PubMed Central

    Tian, Zuojun; Guo, Fuzheng; Biswas, Sangita; Deng, Wenbin

    2016-01-01

    Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs) was discovered in 2006. Later, induced neural progenitor cells (iNPCs) were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM) have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs), making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs. PMID:27104529

  13. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

    SciTech Connect

    Zhai, Yingying; Chen, Xi; Yu, Dehai; Li, Tao; Cui, Jiuwei; Wang, Guanjun; Hu, Ji-Fan; Li, Wei

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  14. Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

    PubMed Central

    Bermejo-Álvarez, P.; Ramos-Ibeas, P.; Park, K.E.; Powell, A. P.; Vansandt, L.; Derek, Bickhart; Ramirez, M. A.; Gutiérrez-Adán, A.; Telugu, B. P.

    2015-01-01

    Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after in vivo differentiation into teratomas. Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus. PMID:26328763

  15. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

    PubMed

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs. PMID:26938987

  16. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts

    PubMed Central

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs. PMID:26938987

  17. Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors.

    PubMed

    Hermann, Andreas; Kim, Jeong Beom; Srimasorn, Sumitra; Zaehres, Holm; Reinhardt, Peter; Schöler, Hans R; Storch, Alexander

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies. PMID:26977154

  18. Risk of cellular phone interference with an implantable loop recorder.

    PubMed

    Trigano, Alexandre; Blandeau, Olivier; Dale, Christian; Wong, Man-Faï; Wiart, Joe

    2007-03-01

    This study examined the risk of cellular phone ringing interference with implantable loop recorders (ILR). The technical manual of ILR warns of potential interference by cellular phone in close proximity to the implanted device, corrupting the data stored in memory or causing inappropriate device operation. The ringing phase of a digital Global System for Mobile Communication (GSM) or Personal Communication Services (PCS) cellular phone includes a brief burst of peak emitted power. To obviate the risk of dysfunction in recipients of implanted ILRs, the testing was performed with externally applied devices. The ILR was positioned in the left parasternal region and the telemetry wand removed after regular programming. Digital cellular telephones were placed over the device at a 1-cm distance and calls were placed. The phone systems tested were single- or dual-band receivers. The GSM used a maximal power output of 2 W, operating on a 900 MHz carrier frequency, and the PCS a maximal output of 1 W, operating on a 1800 MHz carrier frequency. The device activator was used to store the episodes encompassing the tests. Sixty nine tests were performed in 45 patients. In 61 tests, high-frequency polymorphic artifacts were visible on manually activated recordings, beginning a few seconds before the first audible ringing tone and persisting throughout the ringing phase. Cellular phone ringing in close proximity to an externally applied ILR caused bursts of high-frequency signals during electrocardiogram monitoring, without causing permanent device dysfunction or reprogramming. Cellular telephones are a potential source of electrocardiographic artifacts on ILR recordings. PMID:16839630

  19. Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells

    PubMed Central

    Gunji, Yoko; Iwasawa, Shunichiro; Nelson, Amy; Farid, Maha; Ikari, Jun; Liu, Xiangde; Wang, Xingqi; Michalski, Joel; Smith, Lynette; Iqbal, Javeed; Behery, Radwa El; West, William; Yelamanchili, Sowmya; Rennard, Deborah; Holz, Olaf; Mueller, Kai-Christian; Magnussen, Helgo; Rabe, Klaus; Castaldi, Peter J; Rennard, Stephen I.

    2014-01-01

    Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs. PMID

  20. C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells.

    PubMed

    Di Stefano, Bruno; Sardina, Jose Luis; van Oevelen, Chris; Collombet, Samuel; Kallin, Eric M; Vicent, Guillermo P; Lu, Jun; Thieffry, Denis; Beato, Miguel; Graf, Thomas

    2014-02-13

    CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications. PMID:24336202

  1. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    PubMed Central

    Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

    2009-01-01

    Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

  2. Reprogramming of human cancer cells to pluripotency for models of cancer progression

    PubMed Central

    Kim, Jungsun; Zaret, Kenneth S

    2015-01-01

    The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

  3. The Power and the Promise of Cell Reprogramming: Personalized Autologous Body Organ and Cell Transplantation

    PubMed Central

    Alvarez Palomo, Ana Belen; Lucas, Michaela; Dilley, Rodney J.; McLenachan, Samuel; Chen, Fred Kuanfu; Requena, Jordi; Sal, Marti Farrera; Lucas, Andrew; Alvarez, Inaki; Jaraquemada, Dolores; Edel, Michael J.

    2014-01-01

    Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids), bone, optic vesicle-like structures (eye), cardiac muscle tissue (heart), primitive pancreas islet cells, a tooth-like structure (teeth), and functional liver buds (liver). Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1) such transplants will stimulate host immune responses; and (2) whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by i

  4. Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

    PubMed

    Ebrahimi, Behnam

    2016-01-01

    Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. PMID:27056282

  5. Modelling cellular behaviour

    NASA Astrophysics Data System (ADS)

    Endy, Drew; Brent, Roger

    2001-01-01

    Representations of cellular processes that can be used to compute their future behaviour would be of general scientific and practical value. But past attempts to construct such representations have been disappointing. This is now changing. Increases in biological understanding combined with advances in computational methods and in computer power make it possible to foresee construction of useful and predictive simulations of cellular processes.

  6. Using RNA as Molecular Code for Programming Cellular Function.

    PubMed

    Kushwaha, Manish; Rostain, William; Prakash, Satya; Duncan, John N; Jaramillo, Alfonso

    2016-08-19

    RNA is involved in a wide-range of important molecular processes in the cell, serving diverse functions: regulatory, enzymatic, and structural. Together with its ease and predictability of design, these properties can lead RNA to become a useful handle for biological engineers with which to control the cellular machinery. By modifying the many RNA links in cellular processes, it is possible to reprogram cells toward specific design goals. We propose that RNA can be viewed as a molecular programming language that, together with protein-based execution platforms, can be used to rewrite wide ranging aspects of cellular function. In this review, we catalogue developments in the use of RNA parts, methods, and associated computational models that have contributed to the programmability of biology. We discuss how RNA part repertoires have been combined to build complex genetic circuits, and review recent applications of RNA-based parts and circuitry. We explore the future potential of RNA engineering and posit that RNA programmability is an important resource for firmly establishing an era of rationally designed synthetic biology. PMID:26999422

  7. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

    PubMed Central

    Zulak, Katherine G; Weljie, Aalim M; Vogel, Hans J; Facchini, Peter J

    2008-01-01

    Background Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. Results Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary

  8. Hallmarks of progeroid syndromes: lessons from mice and reprogrammed cells

    PubMed Central

    López-Otín, Carlos

    2016-01-01

    ABSTRACT Ageing is a process that inevitably affects most living organisms and involves the accumulation of macromolecular damage, genomic instability and loss of heterochromatin. Together, these alterations lead to a decline in stem cell function and to a reduced capability to regenerate tissue. In recent years, several genetic pathways and biochemical mechanisms that contribute to physiological ageing have been described, but further research is needed to better characterize this complex biological process. Because premature ageing (progeroid) syndromes, including progeria, mimic many of the characteristics of human ageing, research into these conditions has proven to be very useful not only to identify the underlying causal mechanisms and identify treatments for these pathologies, but also for the study of physiological ageing. In this Review, we summarize the main cellular and animal models used in progeria research, with an emphasis on patient-derived induced pluripotent stem cell models, and define a series of molecular and cellular hallmarks that characterize progeroid syndromes and parallel physiological ageing. Finally, we describe the therapeutic strategies being investigated for the treatment of progeroid syndromes, and their main limitations. PMID:27482812

  9. Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing.

    PubMed

    Savva, Yiannis A; Rezaei, Ali; St Laurent, Georges; Reenan, Robert A

    2016-01-01

    Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. PMID:27458478

  10. Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing

    PubMed Central

    Savva, Yiannis A.; Rezaei, Ali; St. Laurent, Georges; Reenan, Robert A.

    2016-01-01

    Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. PMID:27458478

  11. Viral Reprogramming of the Daxx Histone H3.3 Chaperone during Early Epstein-Barr Virus Infection

    PubMed Central

    Tsai, Kevin; Chan, Lilian; Gibeault, Rebecca; Conn, Kristen; Dheekollu, Jayaraju; Domsic, John; Marmorstein, Ronen; Schang, Luis M.

    2014-01-01

    ABSTRACT Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive cochaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using coimmunoprecipitation and size-exclusion chromatography with highly purified components. FRAP (fluorescence recovery after photobleaching) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence combined with fluorescence in situ hybridization that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression. IMPORTANCE Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters

  12. Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    ScienceCinema

    Gallivan, Justin [Emory University

    2013-01-22

    Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, Calif

  13. Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    SciTech Connect

    Gallivan, Justin

    2012-03-21

    Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, Calif

  14. Cellular Reflectarray Antenna

    NASA Technical Reports Server (NTRS)

    Romanofsky, Robert R.

    2010-01-01

    The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

  15. Postnatal epigenetic reprogramming in the germline of a marsupial, the tammar wallaby

    PubMed Central

    2013-01-01

    Background Epigenetic reprogramming is essential to restore totipotency and to reset genomic imprints during mammalian germ cell development and gamete formation. The dynamic DNA methylation change at DMRs (differentially methylated regions) within imprinted domains and of retrotransposons is characteristic of this process. Both marsupials and eutherian mammals have genomic imprinting but these two subgroups have been evolving separately for up to 160 million years. Marsupials have a unique reproductive strategy and deliver tiny, altricial young that complete their development within their mother's pouch. Germ cell proliferation in the genital ridge continues after birth in the tammar wallaby (Macropus eugenii), and it is only after 25 days postpartum that female germ cells begin to enter meiosis and male germ cells begin to enter mitotic arrest. At least two marsupial imprinted loci (PEG10 and H19) also have DMRs. To investigate the evolution of epigenetic reprogramming in the marsupial germline, here we collected germ cells from male pouch young of the tammar wallaby and analysed the methylation status of PEG10 and H19 DMR, an LTR (long terminal repeat) and a non-LTR retrotransposons. Results Demethylation of the H19 DMR was almost completed by 14 days postpartum and de-novo methylation started from 34 days postpartum. These stages correspond to 14 days after the completion of primordial germ cell migration into genital ridge (demethylation) and 9 days after the first detection of mitotic arrest (re-methylation) in the male germ cells. Interestingly, the PEG10 DMR was already unmethylated at 7 days postpartum, suggesting that the timing of epigenetic reprogramming is not the same at all genomic loci. Retrotransposon methylation was not completely removed after the demethylation event in the germ cells, similar to the situation in the mouse. Conclusions Thus, despite the postnatal occurrence of epigenetic reprogramming and the persistence of genome

  16. SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

    PubMed Central

    Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

    2014-01-01

    The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network

  17. SCL, LMO1 and Notch1 reprogram thymocytes into self-renewing cells.

    PubMed

    Gerby, Bastien; Tremblay, Cedric S; Tremblay, Mathieu; Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F T; Hoang, Trang

    2014-12-01

    The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature t