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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

    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

  4. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  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. 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

  5. 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

  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

    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

  10. 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

  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. 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

  8. 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

  9. 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

  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. 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

  17. 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

  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. 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

  10. 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.

  11. 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

  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 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

  5. 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

  6. 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

  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. 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

  5. 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

  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. 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

  15. 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

  16. 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

  17. 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

  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. 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

  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 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  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. 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

  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

    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 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

  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