Shifting behaviour: epigenetic reprogramming in eusocial insects.

PubMed

Patalano, Solenn; Hore, Timothy A; Reik, Wolf; Sumner, Seirian

2012-06-01

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interplay between Oxidative Stress and Nutrient Sensing Signaling in the Developmental Origins of Cardiovascular Disease

PubMed Central

Tain, You-Lin; Hsu, Chien-Ning

2017-01-01

Cardiovascular disease (CVD) presents a global health burden, despite recent advances in management. CVD can originate from early life by so-called “developmental origins of health and disease” (DOHaD). Epidemiological and experimental evidence supports that early-life insults can induce programming of later CVD. Underlying the DOHaD concept, early intervention may offset programming process to prevent the development of CVD, namely reprogramming. Oxidative stress and nutrient sensing signals have been considered to be major mechanisms of cardiovascular programming, while the interplay between these two mechanisms have not been examined in detail. This review summarizes current evidence that supports the link between oxidative stress and nutrient sensing signaling to cardiovascular programming, with an emphasis on the l-arginine–asymmetric dimethylarginine (ADMA)–nitric oxide (NO) pathway. This review provides an overview of evidence from human studies supporting fetal programming of CVD, insight from animal models of cardiovascular programming and oxidative stress, impact of the l-arginine–ADMA–NO pathway in cardiovascular programming, the crosstalk between l-arginine metabolism and nutrient sensing signals, and application of reprogramming interventions to prevent the programming of CVD. A greater understanding of the mechanisms underlying cardiovascular programming is essential to developing early reprogramming interventions to combat the globally growing epidemic of CVD. PMID:28420139

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

PubMed Central

Miyamoto, Kei; Nagai, Kouhei; Kitamura, Naoya; Nishikawa, Tomoaki; Ikegami, Haruka; Binh, Nguyen T.; Tsukamoto, Satoshi; Matsumoto, Mai; Tsukiyama, Tomoyuki; Minami, Naojiro; Yamada, Masayasu; Ariga, Hiroyoshi; Miyake, Masashi; Kawarasaki, Tatsuo; Matsumoto, Kazuya; Imai, Hiroshi

2011-01-01

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti–DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer. PMID:21482765

Induction of pluripotency by defined factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okita, Keisuke, E-mail: okita@cira.kyoto-u.ac.jp; Yamanaka, Shinya; Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507

2010-10-01

Somatic cells can be reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors. The induced pluripotent stem (iPS) cells from a patient's somatic cells could be useful source of cells for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into host genomes and may increase the risk of tumor formation. Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells. On the other hand, patient-specificmore » iPS cells have already been established and used for recapitulating disease pathology.« less

Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?

PubMed

Yilmazer, Açelya; de Lázaro, Irene; Taheri, Hadiseh

2015-12-01

Chromatin dynamics have been the major focus of many physiological and pathological processes over the past 20 years. Epigenetic mechanisms have been shown to be reshaped during both cellular reprogramming and tumorigenesis. For this reason, cancer cell reprogramming can provide a powerful tool to better understand both regenerative and cancer-fate processes, with a potential to develop novel therapeutic approaches. Recent studies showed that cancer cells can be reprogrammed to a pluripotent state by the overexpression of reprogramming transcription factors. Activation of transcription factors and modification of chromatin regulators may result in the remodeling of epigenetic status and refueling of tumorigenicity in these reprogrammed cancer cells. However, studies focusing on cancer cell reprogramming are contradictory; some studies reported increased tumor progression whereas others showed that cellular reprogramming has a treatment potential for cancer. In this review, first, the current knowledge on the epigenetic mechanisms involved during cancer development and cellular reprogramming will be presented. Later, different reports and key factors about pluripotency-based reprogramming of cancer cells will be reviewed in detail. New insights will be provided on cancer biology and therapy in the light of cellular reprogramming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications.

PubMed

Omole, Adekunle Ebenezer; Fakoya, Adegbenro Omotuyi John

2018-01-01

The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling, drug discovery and regenerative medicine. Additionally, this study appraises the role of genomic editing technology in the generation of healthy iPSCs.

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications

PubMed Central

2018-01-01

The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling, drug discovery and regenerative medicine. Additionally, this study appraises the role of genomic editing technology in the generation of healthy iPSCs. PMID:29770269

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

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

PubMed

Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

2017-10-01

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

PubMed Central

Malyshev, Igor; Malyshev, Yuri

2015-01-01

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

Boosters and barriers for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Zonooz, Elmira Rezaei; Baharvand, Hossein

2017-06-01

Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFβ signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming. Copyright © 2017 Elsevier Inc. All rights reserved.

A Cellular Game of Telephone: Trans Tissue Reprogramming of Responses to Toxic Stimuli

EPA Science Inventory

Exposure to air pollution is a leading cause of cardiopulmonary morbidity and mortality; however, while these effects outside the lung have been associated with aberrant oxidative stress and inflammation, the underlying molecular mechanisms are poorly understood. We hypothesized ...

Glucose enhances tilapia against Edwardsiella tarda infection through metabolome reprogramming.

PubMed

Zeng, Zao-hai; Du, Chao-Chao; Liu, Shi-Rao; Li, Hui; Peng, Xuan-Xian; Peng, Bo

2017-02-01

We have recently reported that the survival of tilapia, Oreochromis niloticus, during Edwardsiella tarda infection is tightly associated with their metabolome, where the survived O. niloticus has distinct metabolomic profile to dying O. niloticus. Glucose is the key metabolite to distinguish the survival- and dying-metabolome. More importantly, exogenous administration of glucose to the fish greatly enhances their survival for the infection, indicating the functional roles of glucose in metabolome repurposing, known as reprogramming metabolomics. However, the underlying information for the reprogramming is not yet available. Here, GC/MS based metabolomics is used to understand the mechanisms by which how exogenous glucose elevates O. niloticus, anti-infectious ability to E. tarda. Results showed that exogenous glucose promotes stearic acid and palmitic acid biosynthesis but attenuates TCA cycle to potentiate O. niloticus against bacterial infection, which is confirmed by the fact that exogenous stearic acid increases immune protection in O. niloticus against E. tarda infection in a manner of Mx protein. These results indicate that exogenous glucose reprograms O. niloticus anti-infective metabolome that characterizes elevation of stearic acid and palmitic acid and attenuation of the TCA cycle. Therefore, our results proposed a novel mechanism that glucose promotes unsaturated fatty acid biosynthesis to cope with infection, thereby highlighting a potential way of enhancing fish immunity in aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

PubMed Central

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Cerebellar Granule Cell Replenishment Post-Injury by Adaptive Reprogramming of Nestin+ Progenitors

PubMed Central

Wojcinski, Alexandre; Lawton, Andrew K.; Bayin, N Sumru.; Lao, Zhimin; Stephen, Daniel N.; Joyner, Alexandra L.

2017-01-01

Regeneration of several organs involves adaptive reprogramming of progenitors, however, the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. In this study, we discovered that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors (NEPs) specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog-signaling in two NEP populations is crucial not only for the compensatory replenishment of granule neurons but also to scale interneuron and astrocyte numbers. Thus we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum, and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration. PMID:28805814

Epigenomic Reprogramming of the Developing Reproductive Tract and Disease Susceptibility in Adulthood

PubMed Central

Walker, Cheryl Lyn

2014-01-01

During development, epigenetic programs are “installed” on the genome that direct differentiation and normal tissue and organ function in adulthood. Consequently, development is also a period of susceptibility to reprogramming of the epigenome. Developmental reprogramming occurs when an adverse stimulus or insult interrupts the proper “install” of epigenetic programs during development, reprogramming normal physiological responses in such a way as to promote disease later in life. Some of the best examples of developmental reprogramming involve the reproductive tract, where early life exposures to environmental estrogens can increase susceptibility to benign and malignant tumors in adulthood including leiomyoma (fibroids), endometrial and prostate cancer. Although specific mechanism(s) by which environmental estrogens reprogram the developing epigenome were unknown, both DNA and histone methylation were considered likely targets for epigenetic reprogramming. We have now identified a mechanism by which developmental exposures to environmental estrogens reprogram the epigenome by inducing inappropriate activation of nongenomic estrogen receptor (ER) signaling. Activation of non-genomic ER signaling via the PI3K pathway activates the kinase AKT/PKB in the developing reproductive tract, which phosphorylates the histone lysine methyltransferase (HKMT) EZH2, the key “installer” of epigenetic histone H3 lysine 27 trimethylation (H3K27me3). AKT phosphorylation inactivates EZH2, decreasing levels of H3K27 methylation, a repressive mark that inhibits gene expression, in the developing uterus. As a result of this developmental reprogramming, many estrogen-responsive genes become hypersensitive to estrogen in adulthood, exhibiting elevated expression throughout the estrus cycle, and resulting in a “hyper-estrogenized” phenotype in the adult uterus that promotes development of hormone dependent tumors. PMID:21656660

Berberine Regulated Lipid Metabolism in the Presence of C75, Compound C, and TOFA in Breast Cancer Cell Line MCF-7.

PubMed

Tan, Wen; Zhong, Zhangfeng; Wang, Shengpeng; Suo, Zhanwei; Yang, Xian; Hu, Xiaodong; Wang, Yitao

2015-01-01

Berberine interfering with cancer reprogramming metabolism was confirmed in our previous study. Lipid metabolism and mitochondrial function were also the core parts in reprogramming metabolism. In the presence of some energy-related inhibitors, including C75, compound C, and TOFA, the discrete roles of berberine in lipid metabolism and mitochondrial function were elucidated. An altered lipid metabolism induced by berberine was observed under the inhibition of FASN, AMPK, and ACC in breast cancer cell MCF-7. And the reversion of berberine-induced lipid suppression indicated that ACC inhibition might be involved in that process instead of FASN inhibition. A robust apoptosis induced by berberine even under the inhibition of AMPK and lipid synthesis was also indicated. Finally, mitochondrial function regulation under the inhibition of AMPK and ACC might be in an ACL-independent manner. Undoubtedly, the detailed mechanisms of berberine interfering with lipid metabolism and mitochondrial function combined with energy-related inhibitors need further investigation, including the potential compensatory mechanisms for ATP production and the upregulation of ACL.

Berberine Regulated Lipid Metabolism in the Presence of C75, Compound C, and TOFA in Breast Cancer Cell Line MCF-7

PubMed Central

Tan, Wen; Zhong, Zhangfeng; Suo, Zhanwei; Yang, Xian; Hu, Xiaodong; Wang, Yitao

2015-01-01

Berberine interfering with cancer reprogramming metabolism was confirmed in our previous study. Lipid metabolism and mitochondrial function were also the core parts in reprogramming metabolism. In the presence of some energy-related inhibitors, including C75, compound C, and TOFA, the discrete roles of berberine in lipid metabolism and mitochondrial function were elucidated. An altered lipid metabolism induced by berberine was observed under the inhibition of FASN, AMPK, and ACC in breast cancer cell MCF-7. And the reversion of berberine-induced lipid suppression indicated that ACC inhibition might be involved in that process instead of FASN inhibition. A robust apoptosis induced by berberine even under the inhibition of AMPK and lipid synthesis was also indicated. Finally, mitochondrial function regulation under the inhibition of AMPK and ACC might be in an ACL-independent manner. Undoubtedly, the detailed mechanisms of berberine interfering with lipid metabolism and mitochondrial function combined with energy-related inhibitors need further investigation, including the potential compensatory mechanisms for ATP production and the upregulation of ACL. PMID:26351511

Discovery and progress of direct cardiac reprogramming.

PubMed

Kojima, Hidenori; Ieda, Masaki

2017-06-01

Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

PubMed

Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

2016-12-01

The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

Developmental Programming of Renal Function and Re-Programming Approaches.

PubMed

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

PubMed Central

Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

2015-01-01

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

PubMed

Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

2018-05-04

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

Epigenetic regulation leading to induced pluripotency drives cancer development in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohnishi, Kotaro; Department of Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194; Semi, Katsunori

Highlights: • Epigenetic regulation of failed reprogramming-associated cancer cells is discussed. • Similarity between pediatric cancer and reprogramming-associated cancer is discussed. • Concept for epigenetic cancer is discussed. - Abstract: Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotencymore » in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.« less

Role of human oocyte-enriched factors in somatic cell reprograming.

PubMed

El-Gammal, Zaynab; AlOkda, Abdelrahman; El-Badri, Nagwa

2018-06-08

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming.

PubMed

Gascón, Sergio; Murenu, Elisa; Masserdotti, Giacomo; Ortega, Felipe; Russo, Gianluca L; Petrik, David; Deshpande, Aditi; Heinrich, Christophe; Karow, Marisa; Robertson, Stephen P; Schroeder, Timm; Beckers, Johannes; Irmler, Martin; Berndt, Carsten; Angeli, José P Friedmann; Conrad, Marcus; Berninger, Benedikt; Götz, Magdalena

2016-03-03

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Conversion of partially reprogrammed cells to fully pluripotent stem cells is associated with further activation of stem cell maintenance- and gamete generation-related genes.

PubMed

Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Seo, Han Geuk; Moon, Sung-Hwan; Chung, Hyung-Min; Do, Jeong Tae

2014-11-01

Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.

Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation

PubMed Central

Rodgers, Ali B.; Morgan, Christopher P.; Bronson, Stefanie L.; Revello, Sonia; Bale, Tracy L.

2013-01-01

Neuropsychiatric disease frequently presents with an underlying hypo- or hyper- reactivity of the HPA stress axis, suggesting an exceptional vulnerability of this circuitry to external perturbations. Parental lifetime exposures to environmental challenges are associated with increased offspring neuropsychiatric disease risk, and likely contribute to stress dysregulation. While maternal influences have been extensively examined, much less is known regarding the specific role of paternal factors. To investigate the potential mechanisms by which paternal stress may contribute to offspring hypothalamic-pituitary-adrenal (HPA) axis dysregulation, we exposed mice to six weeks of chronic stress prior to breeding. As epidemiological studies support variation in paternal germ cell susceptibility to reprogramming across the lifespan, male stress exposure occurred either throughout puberty or in adulthood. Remarkably, offspring of sires from both paternal stress groups displayed significantly reduced HPA axis stress responsivity. Gene set enrichment analyses in offspring stress regulating brain regions, the paraventricular nucleus (PVN) and the bed nucleus of stria terminalis (BNST), revealed global pattern changes in transcription suggestive of epigenetic reprogramming and consistent with altered offspring stress responsivity, including increased expression of glucocorticoid-responsive genes in the PVN. In examining potential epigenetic mechanisms of germ cell transmission, we found robust changes in sperm miRNA (miR) content, where nine specific miRs were significantly increased in both paternal stress groups. Overall, these results demonstrate that paternal experience across the lifespan can induce germ cell epigenetic reprogramming and impact offspring HPA stress axis regulation, and may therefore offer novel insight into factors influencing neuropsychiatric disease risk. PMID:23699511

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

PubMed

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

2014-01-01

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

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

PubMed Central

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

PubMed

Matrka, Marie C; Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F; Lane, Andrew N; Romick-Rosendale, Lindsey E; Wells, Susanne I

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

PubMed

Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor*

PubMed Central

Dai, Xiangpeng; Hao, Jie; Hou, Xiao-jun; Hai, Tang; Fan, Yong; Yu, Yang; Jouneau, Alice; Wang, Liu; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming. PMID:20566633

Somatic cell reprogramming informed by the oocyte.

PubMed

Gonzalez-Munoz, Elena; Cibelli, Jose B

2018-05-08

The successful production of animals and embryonic stem cells (ESCs) using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency.

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B

PubMed Central

Lee, Chih-Yung Sean; Lu, Tu

2017-01-01

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin. PMID:29111977

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.

PubMed

Lee, Chih-Yung Sean; Lu, Tu; Seydoux, Geraldine

2017-11-07

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

Robotics in the Construction Industry

DTIC Science & Technology

1990-06-01

accomplished through reprogramming and the attachment of different end effectors. 2.1.2.3 Manipulator I This is the mechanism for moving objects in the...other 3 types of robots), limited repeatability (ability to "hit" the same point in space time after time without reprogramming or adjustment by the... Reprogramming for a different 3 sequence of steps is generally difficult and time- consuming, as the stops must be relocated and I calibrated for the new sequence

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

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells.

PubMed

Miyoshi, Kazuchika; Rzucidlo, S Jacek; Pratt, Scott L; Stice, Steven L

2003-04-01

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.

Transgenerational response to stress in plants and its application for breeding.

PubMed

Bilichak, Andriy; Kovalchuk, Igor

2016-03-01

A growing number of reports indicate that plants possess the ability to maintain a memory of stress exposure throughout their ontogenesis and even transmit it faithfully to the following generation. Some of the features of transgenerational memory include elevated genome instability, a higher tolerance to stress experienced by parents, and a cross-tolerance. Although the underlying molecular mechanisms of this phenomenon are not clear, a likely contributing factor is the absence of full-scale reprogramming of the epigenetic landscape during gametogenesis; therefore, epigenetic marks can occasionally escape the reprogramming process and can be passed on to the progeny. To date, it is not entirely clear which part of the epigenetic landscape is more likely to escape the reprogramming events, and whether such a process is random or directed and sequence specific. The identification of specific epigenetic marks associated with specific stressors would allow generation of stress-tolerant plants through the recently discovered techniques for precision epigenome engineering. The engineered DNA-binding domains (e.g. ZF, TALE, and dCas9) fused to particular chromatin modifiers would make it possible to target epigenetic modifications to the selected loci, probably allowing stress tolerance to be achieved in the progeny. This approach, termed epigenetic breeding, along with other methods has great potential to be used for both the assessment of the propagation of epigenetic marks across generations and trait improvement in plants. In this communication, we provide a short overview of recent reports demonstrating a transgenerational response to stress in plants, and discuss the underlying potential molecular mechanisms of this phenomenon and its use for plant biotechnology applications. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways.

PubMed

Li, Jibin; Huang, Qichao; Long, Xiaoyu; Zhang, Jing; Huang, Xiaojun; Aa, Jiye; Yang, Hushan; Chen, Zhinan; Xing, Jinliang

2015-12-01

CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive. We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms. Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells. Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Fatty acid metabolic reprogramming via mTOR-mediated inductions of PPARγ directs early activation of T cells

PubMed Central

Angela, Mulki; Endo, Yusuke; Asou, Hikari K.; Yamamoto, Takeshi; Tumes, Damon J.; Tokuyama, Hirotake; Yokote, Koutaro; Nakayama, Toshinori

2016-01-01

To fulfil the bioenergetic requirements for increased cell size and clonal expansion, activated T cells reprogramme their metabolic signatures from energetically quiescent to activated. However, the molecular mechanisms and essential components controlling metabolic reprogramming in T cells are not well understood. Here, we show that the mTORC1–PPARγ pathway is crucial for the fatty acid uptake programme in activated CD4+ T cells. This pathway is required for full activation and rapid proliferation of naive and memory CD4+ T cells. PPARγ directly binds and induces genes associated with fatty acid uptake in CD4+ T cells in both mice and humans. The PPARγ-dependent fatty acid uptake programme is critical for metabolic reprogramming. Thus, we provide important mechanistic insights into the metabolic reprogramming mechanisms that govern the expression of key enzymes, fatty acid metabolism and the acquisition of an activated phenotype during CD4+ T cell activation. PMID:27901044

Moving Toward the Ground State.

PubMed

Kumar, Ishan; Ivanova, Natalia

2015-10-01

Transferring mouse ESCs to a media supplemented with Mek and Gsk3β inhibitors (2i) provokes marked transcriptional and epigenetic changes, embodying a shift toward ground-state pluripotency. In this issue of Cell Stem Cell, Kolodziejczyk et al. (2015) examine population structures of ESCs while Galonska et al. (2015) unravel the mechanisms underlying regulatory network rewiring during 2i-mediated reprogramming. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

PubMed

Umei, Tomohiko C; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-08-19

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

PubMed Central

Umei, Tomohiko C.; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-01-01

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming. PMID:28825623

Hydrogen Sulfide in Hypertension and Kidney Disease of Developmental Origins.

PubMed

Hsu, Chien-Ning; Tain, You-Lin

2018-05-11

Adverse environments occurring during kidney development may produce long-term programming effects, namely renal programming, to create increased vulnerability to the development of later-life hypertension and kidney disease. Conversely, reprogramming is a strategy aimed at reversing the programming processes in early life, even before the onset of clinical symptoms, which may counter the rising epidemic of hypertension and kidney disease. Hydrogen sulfide (H₂S), the third gasotransmitter, plays a key role in blood pressure regulation and renal physiology. This review will first present the role of H₂S in the renal system and provide evidence for the links between H₂S signaling and the underlying mechanisms of renal programming, including the renin⁻angiotensin system, oxidative stress, nutrient-sensing signals, sodium transporters, and epigenetic regulation. This will be followed by potential H₂S treatment modalities that may serve as reprogramming strategies to prevent hypertension and kidney disease of developmental origins. These H₂S treatment modalities include precursors for H₂S synthesis, H₂S donors, and natural plant-derived compounds. Despite emerging evidence from experimental studies in support of reprogramming strategies targeting the H₂S signaling pathway to protect against hypertension and kidney disease of developmental origins, these results need further clinical translation.

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

Fundamentals of cancer metabolism

PubMed Central

DeBerardinis, Ralph J.; Chandel, Navdeep S.

2016-01-01

Tumors reprogram pathways of nutrient acquisition and metabolism to meet the bioenergetic, biosynthetic, and redox demands of malignant cells. These reprogrammed activities are now recognized as hallmarks of cancer, and recent work has uncovered remarkable flexibility in the specific pathways activated by tumor cells to support these key functions. In this perspective, we provide a conceptual framework to understand how and why metabolic reprogramming occurs in tumor cells, and the mechanisms linking altered metabolism to tumorigenesis and metastasis. Understanding these concepts will progressively support the development of new strategies to treat human cancer. PMID:27386546

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

PubMed Central

Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

2017-01-01

The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage. PMID:28328977

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5.

PubMed

Wang, Li; Liu, Ziqing; Yin, Chaoying; Zhou, Yang; Liu, Jiandong; Qian, Li

2015-11-13

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

Developmental Programming of Renal Function and Re-Programming Approaches

PubMed Central

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T.; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health. PMID:29535992

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

PubMed Central

Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

2013-01-01

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

PubMed Central

Kretsovali, Androniki; Hadjimichael, Christiana; Charmpilas, Nikolaos

2012-01-01

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. PMID:22550500

Combining Patient-Reprogrammed Neural Cells and Proteomics as a Model to Study Psychiatric Disorders.

PubMed

Zuccoli, Giuliana S; Martins-de-Souza, Daniel; Guest, Paul C; Rehen, Stevens K; Nascimento, Juliana Minardi

2017-01-01

The mechanisms underlying the pathophysiology of psychiatric disorders are still poorly known. Most of the studies about these disorders have been conducted on postmortem tissue or in limited preclinical models. The development of human induced pluripotent stem cells (iPSCs) has helped to increase the translational capacity of molecular profiling studies of psychiatric disorders through provision of human neuronal-like tissue. This approach consists of generation of pluripotent cells by genetically reprogramming somatic cells to produce the multiple neural cell types as observed within the nervous tissue. The finding that iPSCs can recapitulate the phenotype of the donor also affords the possibility of using this approach to study both the disease and control states in a given medical area. Here, we present a protocol for differentiation of human pluripotent stem cells to neural progenitor cells followed by subcellular fractionation which allows the study of specific cellular organelles and proteomic analysis.

AMP-Activated Protein Kinase as a Reprogramming Strategy for Hypertension and Kidney Disease of Developmental Origin.

PubMed

Tain, You-Lin; Hsu, Chien-Ning

2018-06-12

Suboptimal early-life conditions affect the developing kidney, resulting in long-term programming effects, namely renal programming. Adverse renal programming increases the risk for developing hypertension and kidney disease in adulthood. Conversely, reprogramming is a strategy aimed at reversing the programming processes in early life. AMP-activated protein kinase (AMPK) plays a key role in normal renal physiology and the pathogenesis of hypertension and kidney disease. This review discusses the regulation of AMPK in the kidney and provides hypothetical mechanisms linking AMPK to renal programming. This will be followed by studies targeting AMPK activators like metformin, resveratrol, thiazolidinediones, and polyphenols as reprogramming strategies to prevent hypertension and kidney disease. Further studies that broaden our understanding of AMPK isoform- and tissue-specific effects on renal programming are needed to ultimately develop reprogramming strategies. Despite the fact that animal models have provided interesting results with regard to reprogramming strategies targeting AMPK signaling to protect against hypertension and kidney disease with developmental origins, these results await further clinical translation.

NF-κB activation impairs somatic cell reprogramming in ageing.

PubMed

Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

2015-08-01

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

PubMed

Biswas, Dhruba; Jiang, Peng

2016-02-06

The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

PubMed Central

Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

2016-01-01

Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

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

Epigenetics studies of fetal alcohol spectrum disorder: where are we now?

PubMed

Lussier, Alexandre A; Weinberg, Joanne; Kobor, Michael S

2017-03-01

Adverse in utero events can alter the development and function of numerous physiological systems, giving rise to lasting neurodevelopmental deficits. In particular, data have shown that prenatal alcohol exposure can reprogram neurobiological systems, altering developmental trajectories and resulting in increased vulnerability to adverse neurobiological, behavioral and health outcomes. Increasing evidence suggests that epigenetic mechanisms are potential mediators for the reprogramming of neurobiological systems, as they may provide a link between the genome, environmental conditions and neurodevelopmental outcomes. This review outlines the current state of epigenetic research in fetal alcohol spectrum disorder, highlighting the role of epigenetic mechanisms in the reprogramming of neurobiological systems by alcohol and as potential diagnostic tools for fetal alcohol spectrum disorder. We also present an assessment of the current limitations in studies of prenatal alcohol exposure, and highlight the future steps needed in the field.

Nitrogen economics of root foraging: Transitive closure of the nitrate–cytokinin relay and distinct systemic signaling for N supply vs. demand

PubMed Central

Ruffel, Sandrine; Krouk, Gabriel; Ristova, Daniela; Shasha, Dennis; Birnbaum, Kenneth D.; Coruzzi, Gloria M.

2011-01-01

As sessile organisms, root plasticity enables plants to forage for and acquire nutrients in a fluctuating underground environment. Here, we use genetic and genomic approaches in a “split-root” framework—in which physically isolated root systems of the same plant are challenged with different nitrogen (N) environments—to investigate how systemic signaling affects genome-wide reprogramming and root development. The integration of transcriptome and root phenotypes enables us to identify distinct mechanisms underlying “N economy” (i.e., N supply and demand) of plants as a system. Under nitrate-limited conditions, plant roots adopt an “active-foraging strategy”, characterized by lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate deprivation. By contrast, in nitrate-replete conditions, plant roots adopt a “dormant strategy”, characterized by a repression of lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate supply. Sentinel genes responding to systemic N signaling identified by genome-wide comparisons of heterogeneous vs. homogeneous split-root N treatments were used to probe systemic N responses in Arabidopsis mutants impaired in nitrate reduction and hormone synthesis and also in decapitated plants. This combined analysis identified genetically distinct systemic signaling underlying plant N economy: (i) N supply, corresponding to a long-distance systemic signaling triggered by nitrate sensing; and (ii) N demand, experimental support for the transitive closure of a previously inferred nitrate–cytokinin shoot–root relay system that reports the nitrate demand of the whole plant, promoting a compensatory root growth in nitrate-rich patches of heterogeneous soil. PMID:22025711

Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

PubMed

Malinowski, Andrzej R; Fisher, Amanda G

2016-01-01

Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

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

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

PubMed

El Khatib, Moustafa M; Ohmine, Seiga; Jacobus, Egon J; Tonne, Jason M; Morsy, Salma G; Holditch, Sara J; Schreiber, Claire A; Uetsuka, Koji; Fusaki, Noemi; Wigle, Dennis A; Terzic, Andre; Kudva, Yogish C; Ikeda, Yasuhiro

2016-05-01

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature β cell phenotype would lead to safe islet replacement therapy for diabetes. ©AlphaMed Press.

Epigenetics studies of fetal alcohol spectrum disorder: where are we now?

PubMed Central

Lussier, Alexandre A; Weinberg, Joanne; Kobor, Michael S

2017-01-01

Adverse in utero events can alter the development and function of numerous physiological systems, giving rise to lasting neurodevelopmental deficits. In particular, data have shown that prenatal alcohol exposure can reprogram neurobiological systems, altering developmental trajectories and resulting in increased vulnerability to adverse neurobiological, behavioral and health outcomes. Increasing evidence suggests that epigenetic mechanisms are potential mediators for the reprogramming of neurobiological systems, as they may provide a link between the genome, environmental conditions and neurodevelopmental outcomes. This review outlines the current state of epigenetic research in fetal alcohol spectrum disorder, highlighting the role of epigenetic mechanisms in the reprogramming of neurobiological systems by alcohol and as potential diagnostic tools for fetal alcohol spectrum disorder. We also present an assessment of the current limitations in studies of prenatal alcohol exposure, and highlight the future steps needed in the field. PMID:28234026

Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

PubMed Central

Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

2016-01-01

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism

NASA Astrophysics Data System (ADS)

Ke, Zhang-Hong; Pan, Jie-Xue; Jin, Lu-Yang; Xu, Hai-Yan; Yu, Tian-Tian; Ullah, Kamran; Rahman, Tanzil Ur; Ren, Jun; Cheng, Yi; Dong, Xin-Yan; Sheng, Jian-Zhong; Huang, He-Feng

2016-08-01

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns.

Liquid chromatography-mass spectrometry-based metabolomics and lipidomics reveal toxicological mechanisms of bisphenol F in breast cancer xenografts.

PubMed

Zhao, Chao; Xie, Peisi; Wang, Hailin; Cai, Zongwei

2018-05-05

Bisphenol F (BPF) is a major alternative to bisphenol (BPA) and has been widely used. Although BPA exposure is known to generate various toxic effects, toxicity of BPF remains under-explored. A comprehensive method involving mass spectrometry (MS)-based global lipidomics and metabolomics, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI)- MS imaging (MSI) was used to study toxic effects of BPF and the underlying mechanisms on tumor metastasis-related tissues (liver and kidney) in breast cancer xenografts. Our results demonstrated that BPF exposure disturbed the metabolome and lipidome of liver and kidney. Exposure induced reprogramming of the glutathione (GSH) biosynthesis and glycolytic metabolism by activating glycine, serine, cysteine, glutamine, lactate and pyruvate in liver and kidney tissues. It also perturbed the biosynthesis and degradation of glycerophospholipids (GPs) and glycerolipids (GLs), resulting in abnormality of membrane homeostasis and cellular functions in kidney tissues. Moreover, spatial distribution and profile of metabolites changed across renal cortex and medulla regions after BPF treatment. Levels of phosphatidylethanolamines (PE) and triacylglycerols (TAG) increased in renal medulla and pelvis, while the levels of phosphatidylcholines (PC) and phosphatidylinositols (PI) increased in cortex and pelvis. These observations offer a deeper understanding of critical role of metabolites and lipid reprogramming in BPF-induced biological effects. Copyright © 2018 Elsevier B.V. All rights reserved.

Bisphenol A Exposure May Induce Hepatic Lipid Accumulation via Reprogramming the DNA Methylation Patterns of Genes Involved in Lipid Metabolism

PubMed Central

Ke, Zhang-Hong; Pan, Jie-Xue; Jin, Lu-Yang; Xu, Hai-Yan; Yu, Tian-Tian; Ullah, Kamran; Rahman, Tanzil Ur; Ren, Jun; Cheng, Yi; Dong, Xin-Yan; Sheng, Jian-Zhong; Huang, He-Feng

2016-01-01

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns. PMID:27502578

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

PubMed

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-07-17

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. © 2014 The Authors.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

PubMed Central

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-01-01

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

PubMed Central

Li, Chunhe; Wang, Jin

2013-01-01

Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

Metabolic Reprogramming in Leaf Lettuce Grown Under Different Light Quality and Intensity Conditions Using Narrow-Band LEDs.

PubMed

Kitazaki, Kazuyoshi; Fukushima, Atsushi; Nakabayashi, Ryo; Okazaki, Yozo; Kobayashi, Makoto; Mori, Tetsuya; Nishizawa, Tomoko; Reyes-Chin-Wo, Sebastian; Michelmore, Richard W; Saito, Kazuki; Shoji, Kazuhiro; Kusano, Miyako

2018-05-21

Light-emitting diodes (LEDs) are an artificial light source used in closed-type plant factories and provide a promising solution for a year-round supply of green leafy vegetables, such as lettuce (Lactuca sativa L.). Obtaining high-quality seedlings using controlled irradiation from LEDs is critical, as the seedling health affects the growth and yield of leaf lettuce after transplantation. Because key molecular pathways underlying plant responses to a specific light quality and intensity remain poorly characterised, we used a multi-omics-based approach to evaluate the metabolic and transcriptional reprogramming of leaf lettuce seedlings grown under narrow-band LED lighting. Four types of monochromatic LEDs (one blue, two green and one red) and white fluorescent light (control) were used at low and high intensities (100 and 300 μmol·m -2 ·s -1 , respectively). Multi-platform mass spectrometry-based metabolomics and RNA-Seq were used to determine changes in the metabolome and transcriptome of lettuce plants in response to different light qualities and intensities. Metabolic pathway analysis revealed distinct regulatory mechanisms involved in flavonoid and phenylpropanoid biosynthetic pathways under blue and green wavelengths. Taken together, these data suggest that the energy transmitted by green light is effective in creating a balance between biomass production and the production of secondary metabolites involved in plant defence.

Advances in generation of functional β-cells from induced pluripotent stem cells as a cure for diabetes mellitus.

PubMed

Kalra, Kunal; Chandrabose, Srijaya Thekkeparambil; Ramasamy, Thamil Selvee; Kasim, Noor Hayaty Binti Abu

2018-06-04

Diabetes mellitus is one of the leading cause for death worldwide. Loss and functional failure of pancreatic β-cells, the parenchyma cells in the islets of Langerhans onsets and progresses diabetes mellitus. The increasing incidence of this metabolic disorder necessitates efficient strategies to produce functional β-cells for treating diabetes mellitus. Human induced pluripotent stem cells (hiPSC), holds potential for treating diabetes owning to their self-renewal capacity and ability to differentiate into β-cells. iPSC technology also provides unlimited starting material to generate differentiated cells for regenerative applications. Progress has also been made in establishing in-vitro culture protocols to yield definitive endoderm, pancreatic endoderm progenitor cells and β-cells via different reprogramming strategies and growth factor supplementation. However, these generated β-cells are still immature, lack functional characteristics and exhibit lower capability in reversing the diseases conditions. Current methods employed to generate mature and functional β-cells include; use of small and large molecules to enhance the reprogramming and differentiation efficiency, 3D culture systems to improve the functional properties and heterogeneity of differentiated cells. This review details recent advancements in the generation of mature β-cells by reprogramming stem cells into iPSCs that is further programmed to β-cells. It also provides deeper insight of current reprogramming protocols and their efficacy, focusing on the underlying mechanism of chemical based approach to generate iPSCs. Furthermore, we have highlighted the recent differentiation strategies both in-vitro and in-vivo to date and the future prospects in generation of mature β-cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Rule-Based Policy-Level Model of Nonsuperpower Behavior in Strategic Conflicts.

DTIC Science & Technology

1982-12-01

a mechanism. The human mind tends to work linearly and to focus implicitly on a few variables. Experience results in subconscious models with far...which is slower. Alternatives to the current ROSIE implementation include reprogramming Scenario Agent in the C language (the language used for the Red...perception, opportunity perception, opportunity response, and assertiveness. As rules are refined, maintenance and reprogramming of the model will be required

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

NASA Astrophysics Data System (ADS)

Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

2015-02-01

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Dissecting genomic imprinting and genetic conflict from a game theory prospective. Comment on: ;Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition; by Qian Wang et al.

NASA Astrophysics Data System (ADS)

Cui, Yuehua; Yang, Haitao

2017-03-01

Epigenetics typically refers to changes in the structure of a chromosome that affect gene activity and expression. Genomic imprinting is a special type of epigenetic phenomenon in which the expression of an allele depends on its parental origin. When an allele inherited from the mother (or father) is imprinted (i.e., silent), it is termed as maternal (or paternal) imprinting. Imprinting is often resulted from DNA methylation and tends to cluster together in the genome [1]. It has been shown to play a key role in many genetic disorders in humans [2]. Imprinting is heritable and undergoes a reprogramming process in gametes before and after fertilization [1]. Sometimes the reprogramming process is not reversible, leading to the loss of imprinting [3]. Although efforts have been made to experimentally or computationally infer imprinting genes, the underlying molecular mechanism that leads to unbalanced allelic expression is still largely unknown.

Chemotherapeutic tumor microparticles combining low-dose irradiation reprogram tumor-promoting macrophages through a tumor-repopulating cell-curtailing pathway

PubMed Central

Sun, Yanling; Zheng, Zu'an; Zhang, Huafeng; Yu, Yuandong; Ma, Jingwei; Tang, Ke; Xu, Pingwei; Ji, Tiantian; Liang, Xiaoyu; Chen, Degao; Jin, Xun; Zhang, Tianzhen; Long, Zhixiong; Liu, Yuying; Huang, Bo

2017-01-01

ABSTRACT Stem cell-like tumor-repopulating cells (TRCs) have a critical role in establishing a tumor immunosuppressive microenvironment. However, means to enhance antitumor immunity by disrupting TRCs are absent. Our previous studies have shown that tumor cell-derived microparticles (T-MPs) preferentially abrogate TRCs by delivering antitumor drugs into nuclei of TRCs. Here, we show that low dose irradiation (LDI) enhances the effect of cisplatin-packaging T-MPs (Cis-MPs) on TRCs, leading to inhibiting tumor growth in different tumor models. This antitumor effect is not due to the direct killing of tumor cells but is T cell-dependent and relies on macrophages for their efficacy. The underlying mechanism is involved in therapeutic reprograming macrophages from tumor-promotion to tumor-inhibition by disrupting TRCs and curtailing their vicious education on macrophages. These findings provide a novel strategy to reset macrophage polarization and confer their function more like M1 than M2 types with highly promising potential clinical applications. PMID:28680743

The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival

PubMed Central

Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

2016-01-01

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na+/H+ exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

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

Epigenetic Research of Neurodegenerative Disorders Using Patient iPSC-Based Models

PubMed Central

2016-01-01

Epigenetic mechanisms play a role in human disease but their involvement in pathologies from the central nervous system has been hampered by the complexity of the brain together with its unique cellular architecture and diversity. Until recently, disease targeted neural types were only available as postmortem materials after many years of disease evolution. Current in vitro systems of induced pluripotent stem cells (iPSCs) generated by cell reprogramming of somatic cells from patients have provided valuable disease models recapitulating key pathological molecular events. Yet whether cell reprogramming on itself implies a truly epigenetic reprogramming, the epigenetic mechanisms governing this process are only partially understood. Moreover, elucidating epigenetic regulation using patient-specific iPSC-derived neural models is expected to have a great impact to unravel the pathophysiology of neurodegenerative diseases and to hopefully expand future therapeutic possibilities. Here we will critically review current knowledge of epigenetic involvement in neurodegenerative disorders focusing on the potential of iPSCs as a promising tool for epigenetic research of these diseases. PMID:26697081

Gadd45a opens up the promoter regions of miR-295 facilitating pluripotency induction

PubMed Central

Li, Linpeng; Chen, Keshi; Wu, Yi; Long, Qi; Zhao, Danyun; Ma, Bochao; Pei, Duanqing; Liu, Xingguo

2017-01-01

MicroRNAs (miRNAs) play crucial roles in the establishment of pluripotent state by controlling pluripotent network. However, the molecular mechanisms controlling miRNAs during somatic cell reprogramming remain obscure. In this study, we show Gadd45a (growth arrest and DNA-damage-inducible protein 45a) enhances reprogramming by activating miR-295. Furthermore, we show that Gadd45a binds the promoter regions of miR-295. Nuclease accessibility assay indicates that Gadd45a opens the promoter regions of miR-295. Levels of H3K9Ac and H3K27Ac on the promoter regions of miR-295 were also increased. In conclusion, our results indicate that Gadd45a relaxes the promoter regions of miR-295 and promotes the expression of miR-295 during reprogramming, implying a concise mechanism of Gadd45a and miR-290 cluster cooperation in cell-fate determination. PMID:29022923

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

Design principles for nuclease-deficient CRISPR-based transcriptional regulators.

PubMed

Jensen, Michael K

2018-06-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies.

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Biophysical regulation of epigenetic state and cell reprogramming

NASA Astrophysics Data System (ADS)

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

2013-12-01

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

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

3T magnetic resonance imaging testing of externally programmable shunt valves

PubMed Central

Zabramski, Joseph M.; Preul, Mark C.; Debbins, Josef; McCusker, Daniel J.

2012-01-01

Background: Exposure of externally programmable shunt-valves (EPS-valves) to magnetic resonance imaging (MRI) may lead to unexpected changes in shunt settings, or affect the ability to reprogram the valve. We undertook this study to examine the effect of exposure to a 3T MRI on a group of widely used EPS-valves. Methods: Evaluations were performed on first generation EPS-valves (those without a locking mechanism to prevent changes in shunt settings by external magnets other than the programmer) and second generation EPS-valves (those with a locking mechanisms). Fifteen new shunt-valves were divided into five groups of three identical valves each, and then exposed to a series of six simulated MRI scans. After each of the exposures, the valves were evaluated to determine if the valve settings had changed, and whether the valves could be reprogrammed. The study produced 18 evaluations for each line of shunt-valves. Results: Exposure of the first generation EPS-valves to a 3T magnetic field resulted in frequent changes in the valve settings; however, all valves retained their ability to be reprogrammed. Repeated exposure of the second generation EPS-valves has no effect on shunt valve settings, and all valves retained their ability to be interrogated and reprogrammed. Conclusions: Second generation EPS-valves with locking mechanisms can be safely exposed to repeated 3T MRI systems, without evidence that shunt settings will change. The exposure of the first generation EPS-valves to 3T MRI results in frequent changes in shunt settings that necessitate re-evaluation soon after MRI to avoid complications. PMID:22937481

Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engendersmore » parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.« less

Distinct physiological and metabolic reprogramming by highbush blueberry (Vaccinium corymbosum) cultivars revealed during long-term UV-B radiation.

PubMed

Luengo Escobar, Ana; Alberdi, Miren; Acevedo, Patricio; Machado, Mariana; Nunes-Nesi, Adriano; Inostroza-Blancheteau, Claudio; Reyes-Díaz, Marjorie

2017-05-01

Despite the Montreal protocol and the eventual recovery of the ozone layer over Antarctica, there are still concerns about increased levels of ultraviolet-B (UV-B) radiation in the Southern Hemisphere. UV-B induces physiological, biochemical and morphological stress responses in plants, which are species-specific and different even for closely related cultivars. In woody plant species, understanding of long-term mechanisms to cope with UV-B-induced stress is limited. Therefore, a greenhouse UV-B daily course simulation was performed for 21 days with two blueberry cultivars (Legacy and Bluegold) under UV-B BE irradiance doses of 0, 0.07 and 0.19 W m -2 . Morphological changes, photosynthetic performance, antioxidants, lipid peroxidation and metabolic features were evaluated. We found that both cultivars behaved differently under UV-B exposure, with Legacy being a UV-B-resistant cultivar. Interestingly, Legacy used a combined strategy: initially, in the first week of exposure its photoprotective compounds increased, coping with the intake of UV-B radiation (avoidance strategy), and then, increasing its antioxidant capacity. These strategies proved to be UV-B radiation dose dependent. The avoidance strategy is triggered early under high UV-B radiation in Legacy. Moreover, the rapid metabolic reprogramming capacity of this cultivar, in contrast to Bluegold, seems to be the most relevant contribution to its UV-B stress-coping strategy. © 2016 Scandinavian Plant Physiology Society.

Direct Cardiac Reprogramming as a Novel Therapeutic Strategy for Treatment of Myocardial Infarction.

PubMed

Ma, Hong; Wang, Li; Liu, Jiandong; Qian, Li

2017-01-01

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a novel therapy for the treatment of heart failure, a common and morbid disease that is usually caused by irreversible loss of functional cardiomyocytes (CMs). Recently, we and others showed that in a murine model of acute myocardial infarction, delivery of three transcription factors, Gata4, Mef2c, and Tbx5 converted endogenous cardiac fibroblasts into functional iCMs. These iCMs integrated electrically and mechanically with surrounding myocardium, resulting in a reduction in scar size and an improvement in heart function. Our findings suggest that iCM reprogramming may be a means of regenerating functional CMs in vivo for patients with heart disease. However, because relatively little is known about the factors that regulate iCM reprogramming, the applicability of iCM reprogramming is currently limited to the experimental settings in which it has been attempted. Specific hurdles include the relatively low conversion rate of iCMs and the need for reprogramming to occur in the context of acute injury. Therefore, before this treatment can become a viable therapy for human heart disease, the optimal condition for efficient iCM generation must be determined. Here, we provide a detailed protocol for both in vitro and in vivo iCM generation that has been optimized so far in our lab. We hope that this protocol will lay a foundation for future further improvement of iCM generation and provide a platform for mechanistic studies.

Epigenetic alterations mediate iPSC normalization of DNA-repair expression and TNR stability in Huntington's disease.

PubMed

Mollica, Peter A; Zamponi, Martina; Reid, John A; Sharma, Deepak K; White, Alyson E; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

2018-06-13

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction during pluripotency and neurodevelopment, are poorly understood. Here we tested the hypothesis that hypomethylation during cellular reprogramming leads to up-regulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early-stage HD-affected neural stem cells (NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced during pluripotency, and maintained in HD-NSCs. We also identify up-regulation of 5-hmC catalyzing ten-eleven translocation (TET1/2) proteins, and show their knockdown leads to a corresponding decrease in select DNA repair gene expression. We further confirm decreased expression of TET regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms involved in pluripotency recover the selected DNA repair gene expression and stabilizes pathogenic TNRs in HD. © 2018. Published by The Company of Biologists Ltd.

Non-self recognition, transcriptional reprogramming, and secondary metabolite accumulation during plant/pathogen interactions

PubMed Central

Hahlbrock, Klaus; Bednarek, Pawel; Ciolkowski, Ingo; Hamberger, Björn; Heise, Andreas; Liedgens, Hiltrud; Logemann, Elke; Nürnberger, Thorsten; Schmelzer, Elmon; Somssich, Imre E.; Tan, Jianwen

2003-01-01

Disease resistance of plants involves two distinct forms of chemical communication with the pathogen: recognition and defense. Both are essential components of a highly complex, multifaceted defense response, which begins with non-self recognition through the perception of pathogen-derived signal molecules and results in the production, inter alia, of antibiotically active compounds (phytoalexins) and cell wall-reinforcing material around the infection site. To elucidate the molecular details and the genomic basis of the underlying chains of events, we used two different experimental systems: suspension-cultured cells of Petroselinum crispum (parsley) and wild-type as well as mutant plants of Arabidopsis thaliana. Particular emphasis was placed on the structural and functional identification of signal and defense molecules, and on the mechanisms of signal perception, intracellular signal transduction and transcriptional reprogramming, including the structural and functional characterization of the responsible cis-acting gene promoter elements and transacting regulatory proteins. Comparing P. crispum and A. thaliana allows us to distinguish species-specific defense mechanisms from more universal responses, and furthermore provides general insights into the nature of the interactions. Despite the complexity of the pathogen defense response, it is experimentally tractable, and knowledge gained so far has opened up a new realm of gene technology-assisted strategies for resistance breeding of crop plants. PMID:12704242

Mitochondrial-Associated Cell Death Mechanisms Are Reset to an Embryonic-Like State in Aged Donor-Derived iPS Cells Harboring Chromosomal Aberrations

PubMed Central

Prigione, Alessandro; Hossini, Amir M.; Lichtner, Björn; Serin, Akdes; Fauler, Beatrix; Megges, Matthias; Lurz, Rudi; Lehrach, Hans; Zouboulis, Christos C.

2011-01-01

Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic alterations. Nevertheless, we believe it will be essential to develop reprogramming protocols capable of safeguarding the integrity of the genome of aged somatic cells, before employing iPSC-based therapy for age-associated disorders. PMID:22110631

PubMed

Sarman, Ihsan; Rangmar, Jenny

2017-08-03

Fetal alcohol syndrome is not the only consequence of prenatal alcohol exposure  The prevalence of fetal alcohol syndrome and fetal alcohol spectrum disorders in larger communities in USA is now updated to 0.4 % and 4.8 % respectively. Affected individuals bear witness to disease symptoms from many organ systems in addition to the brain and behavioural dysfunctions. In the light of modern epigenetic research, early alcohol exposure appears to play a hidden role in fetal reprogramming. The underlying mechanisms explain the »developmental origin of health and disease«, which has an impact on complex interactions between genome, environment and epigenetics.

Mitochondria on the move: emerging paradigms of organelle trafficking in tumour plasticity and metastasis.

PubMed

Altieri, Dario C

2017-07-25

There is now a resurgent interest in the role of mitochondria in cancer. Long considered controversial or outright unimportant, mitochondrial biology is now increasingly recognised as an important tumour driver. The underlying mechanisms remain to be fully elucidated. But recent studies have uncovered a complex landscape where reprogramming of mitochondrial homoeostasis, including organelle dynamics, metabolic output, apoptosis control and redox status converge to promote tumour adaptation to an unfavourable microenvironment and inject new traits of aggressive disease. In particular, mechanisms of subcellular mitochondrial trafficking have unexpectedly emerged as central regulators of metastatic competence in disparate tumours. Some of these pathways are druggable, opening fresh therapeutic opportunities for advanced and disseminated disease.

The PluriNetWork: An Electronic Representation of the Network Underlying Pluripotency in Mouse, and Its Applications

PubMed Central

Greber, Boris; Siatkowski, Marcin; Paudel, Yogesh; Warsow, Gregor; Cap, Clemens; Schöler, Hans; Fuellen, Georg

2010-01-01

Background Analysis of the mechanisms underlying pluripotency and reprogramming would benefit substantially from easy access to an electronic network of genes, proteins and mechanisms. Moreover, interpreting gene expression data needs to move beyond just the identification of the up-/downregulation of key genes and of overrepresented processes and pathways, towards clarifying the essential effects of the experiment in molecular terms. Methodology/Principal Findings We have assembled a network of 574 molecular interactions, stimulations and inhibitions, based on a collection of research data from 177 publications until June 2010, involving 274 mouse genes/proteins, all in a standard electronic format, enabling analyses by readily available software such as Cytoscape and its plugins. The network includes the core circuit of Oct4 (Pou5f1), Sox2 and Nanog, its periphery (such as Stat3, Klf4, Esrrb, and c-Myc), connections to upstream signaling pathways (such as Activin, WNT, FGF, BMP, Insulin, Notch and LIF), and epigenetic regulators as well as some other relevant genes/proteins, such as proteins involved in nuclear import/export. We describe the general properties of the network, as well as a Gene Ontology analysis of the genes included. We use several expression data sets to condense the network to a set of network links that are affected in the course of an experiment, yielding hypotheses about the underlying mechanisms. Conclusions/Significance We have initiated an electronic data repository that will be useful to understand pluripotency and to facilitate the interpretation of high-throughput data. To keep up with the growth of knowledge on the fundamental processes of pluripotency and reprogramming, we suggest to combine Wiki and social networking software towards a community curation system that is easy to use and flexible, and tailored to provide a benefit for the scientist, and to improve communication and exchange of research results. A PluriNetWork tutorial is available at http://www.ibima.med.uni-rostock.de/IBIMA/PluriNetWork/. PMID:21179244

A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency

NASA Astrophysics Data System (ADS)

Artyomov, Maxim; Meissner, Alex; Chakraborty, Arup

2010-03-01

Most cells in an organism have the same DNA. Yet, different cell types express different proteins and carry out different functions. This is because of epigenetic differences; i.e., DNA in different cell types is packaged distinctly, making it hard to express certain genes while facilitating the expression of others. During development, upon receipt of appropriate cues, pluripotent embryonic stem cells differentiate into diverse cell types that make up the organism (e.g., a human). There has long been an effort to make this process go backward -- i.e., reprogram a differentiated cell (e.g., a skin cell) to pluripotent status. Recently, this has been achieved by transfecting certain transcription factors into differentiated cells. This method does not use embryonic material and promises the development of patient-specific regenerative medicine, but it is inefficient. The mechanisms that make reprogramming rare, or even possible, are poorly understood. We have developed the first computational model of transcription factor-induced reprogramming. Results obtained from the model are consistent with diverse observations, and identify the rare pathways that allow reprogramming to occur. If validated, our model could be further developed to design optimal strategies for reprogramming and shed light on basic questions in biology.

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

PubMed Central

Jensen, Michael K

2018-01-01

Abstract The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies. PMID:29726937

Biochemical basis for pharmacological intervention as a reprogramming strategy against hypertension and kidney disease of developmental origin.

PubMed

Tain, You-Lin; Chan, Samuel H H; Chan, Julie Y H

2018-07-01

The concept of "developmental origins of health and disease" (DOHaD) stipulates that both hypertension and kidney disease may take origin from early-life insults. The DOHaD concept also offers reprogramming strategies aiming at shifting therapeutic interventions from adulthood to early life, even before clinical symptoms are evident. Based on those two concepts, this review will present the evidence for the existence of, and the programming mechanisms in, kidney developmental programming that may lead to hypertension and kidney disease. This will be followed by potential pharmacological interventions that may serve as a reprogramming strategy to counter the rising epidemic of hypertension and kidney disease. We point out that before patients could benefit from this strategy, the most pressing issue is for the growing body of evidence from animal studies in support of pharmacological intervention as a reprogramming strategy to long-term protect against hypertension and kidney disease of developmental origins to be validated clinically and the critical window, drug dose, dosing regimen, and therapeutic duration identified. Copyright © 2018 Elsevier Inc. All rights reserved.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Stem cell fusion as an ultimate line of defense against xenobiotics.

PubMed

Padron Velazquez, Julio Lazaro

2006-01-01

There are several indications that the potential of stem cells to fuse with somatic cells is extremely high and, what's more exciting, in some instances goes as far as reprogramming and/or rescuing altered cells. It remains unclear, however, how frequent this mechanism is and what patho-physiological role it might play in nature. A plausible hypothesis, discussed in this paper, suggests that stem cell niches might provide a safeguard for the intact genome and epigenome. By fusing with somatic de-differentiated cells, stem cells might consent epigenetic reprogramming and/or genetic recovery of genes which otherwise could drive altered cells to malignancy. If the many sophisticated mechanisms of metabolism, cell repair, programmed cell death and tissue regeneration should fail, stem cells might represent a final attempt to recover dedifferentiated cells to avoid inflowing in cancer. In the current reappraisal of the different mechanisms of defense against xenobiotics, even the incidence of cancer itself is considered an evolving mechanism which, through a kind of programmed death of individuals exhibiting defective mutations, favors advancement of the phenotypes which adapt best. Additionally, with regard to the mechanisms of transmitting somatic mutations, based on stem cells' capacity to migrate and to fuse, here it is speculated that stem cells might be capable of carrying acquired somatic mutations from peripheral tissues to the gonads, and transmit that information into the germinal line. If appropriately demonstrated, these mechanisms might delineate a novel therapeutic area to be explored. The use of stem cells to reprogram/recover irreversibly damaged cells or to transmit beneficial mutations might be a valuable therapeutic approach in the future.

The long noncoding RNA Chaer defines an epigenetic checkpoint in cardiac hypertrophy.

PubMed

Wang, Zhihua; Zhang, Xiao-Jing; Ji, Yan-Xiao; Zhang, Peng; Deng, Ke-Qiong; Gong, Jun; Ren, Shuxun; Wang, Xinghua; Chen, Iris; Wang, He; Gao, Chen; Yokota, Tomohiro; Ang, Yen Sin; Li, Shen; Cass, Ashley; Vondriska, Thomas M; Li, Guangping; Deb, Arjun; Srivastava, Deepak; Yang, Huang-Tian; Xiao, Xinshu; Li, Hongliang; Wang, Yibin

2016-10-01

Epigenetic reprogramming is a critical process of pathological gene induction during cardiac hypertrophy and remodeling, but the underlying regulatory mechanisms remain to be elucidated. Here we identified a heart-enriched long noncoding (lnc)RNA, named cardiac-hypertrophy-associated epigenetic regulator (Chaer), which is necessary for the development of cardiac hypertrophy. Mechanistically, Chaer directly interacts with the catalytic subunit of polycomb repressor complex 2 (PRC2). This interaction, which is mediated by a 66-mer motif in Chaer, interferes with PRC2 targeting to genomic loci, thereby inhibiting histone H3 lysine 27 methylation at the promoter regions of genes involved in cardiac hypertrophy. The interaction between Chaer and PRC2 is transiently induced after hormone or stress stimulation in a process involving mammalian target of rapamycin complex 1, and this interaction is a prerequisite for epigenetic reprogramming and induction of genes involved in hypertrophy. Inhibition of Chaer expression in the heart before, but not after, the onset of pressure overload substantially attenuates cardiac hypertrophy and dysfunction. Our study reveals that stress-induced pathological gene activation in the heart requires a previously uncharacterized lncRNA-dependent epigenetic checkpoint.

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

PubMed Central

Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

2009-01-01

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

PubMed Central

Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

2011-01-01

Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

PubMed

Inoue, Kimiko; Kohda, Takashi; Sugimoto, Michihiko; Sado, Takashi; Ogonuki, Narumi; Matoba, Shogo; Shiura, Hirosuke; Ikeda, Rieko; Mochida, Keiji; Fujii, Takashi; Sawai, Ken; Otte, Arie P; Tian, X Cindy; Yang, Xiangzhong; Ishino, Fumitoshi; Abe, Kuniya; Ogura, Atsuo

2010-10-22

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

PubMed

Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

2013-07-01

The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports.

Molecular Mechanisms of Induced Pluripotency

PubMed Central

Muchkaeva, I.A.; Dashinimaev, E.B.; Terskikh, V.V.; Sukhanov, Y.V.; Vasiliev, A.V.

2012-01-01

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered. PMID:22708059

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity

PubMed Central

Thakurela, Sudhir; Sahu, Sanjeeb Kumar; Garding, Angela; Tiwari, Vijay K.

2015-01-01

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity. PMID:26170447

PKM2-dependent metabolic reprogramming in CD4+ T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis.

PubMed

Lü, Silin; Deng, Jiacheng; Liu, Huiying; Liu, Bo; Yang, Juan; Miao, Yutong; Li, Jing; Wang, Nan; Jiang, Changtao; Xu, Qingbo; Wang, Xian; Feng, Juan

2018-06-01

Inflammation mediated by activated T cells plays an important role in the initiation and progression of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis in ApoE -/- mice. Homocysteine (Hcy) activates T cells to secrete proinflammatory cytokines, especially interferon (IFN)-γ; however, the precise mechanisms remain unclear. Metabolic reprogramming is critical for T cell inflammatory activation and effector functions. Our previous study demonstrated that Hcy regulates T cell mitochondrial reprogramming by enhancing endoplasmic reticulum (ER)-mitochondria coupling. In this study, we further explored the important role of glycolysis-mediated metabolic reprogramming in Hcy-activated CD4 + T cells. Mechanistically, Hcy-activated CD4 + T cell increased the protein expression and activity of pyruvate kinase muscle isozyme 2 (PKM2), the final rate-limiting enzyme in glycolysis, via the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin signaling pathway. Knockdown of PKM2 by small interfering RNA reduced Hcy-induced CD4 + T cell IFN-γ secretion. Furthermore, we generated T cell-specific PKM2 knockout mice by crossing LckCre transgenic mice with PKM2 fl/fl mice and observed that Hcy-induced glycolysis and oxidative phosphorylation were both diminished in PKM2-deficient CD4 + T cells with reduced glucose and lipid metabolites, and subsequently reduced IFN-γ secretion. T cell-depleted apolipoprotein E-deficient (ApoE -/- ) mice adoptively transferred with PKM2-deficient CD4 + T cells, compared to mice transferred with control cells, showed significantly decreased HHcy-accelerated early atherosclerotic lesion formation. In conclusion, this work indicates that the PKM2-dependent glycolytic-lipogenic axis, a novel mechanism of metabolic regulation, is crucial for HHcy-induced CD4 + T cell activation to accelerate early atherosclerosis in ApoE -/- mice. Metabolic reprogramming is crucial for Hcy-induced CD4 + T cell inflammatory activation. Hcy activates the glycolytic-lipogenic pathway in CD4 + T cells via PKM2. Targeting PKM2 attenuated HHcy-accelerated early atherosclerosis in ApoE -/- mice in vivo.

Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

PubMed Central

Miyamoto, Tadashi; Furusawa, Chikara; Kaneko, Kunihiko

2015-01-01

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development. PMID:26308610

Lineage plasticity-mediated therapy resistance in prostate cancer.

PubMed

Blee, Alexandra M; Huang, Haojie

2018-06-12

Therapy resistance is a significant challenge for prostate cancer treatment in clinic. Although targeted therapies such as androgen deprivation and androgen receptor (AR) inhibition are effective initially, tumor cells eventually evade these strategies through multiple mechanisms. Lineage reprogramming in response to hormone therapy represents a key mechanism that is increasingly observed. The studies in this area have revealed specific combinations of alterations present in adenocarcinomas that provide cells with the ability to transdifferentiate and perpetuate AR-independent tumor growth after androgen-based therapies. Interestingly, several master regulators have been identified that drive plasticity, some of which also play key roles during development and differentiation of the cell lineages in the normal prostate. Thus, further study of each AR-independent tumor type and understanding underlying mechanisms are warranted to develop combinational therapies that combat lineage plasticity in prostate cancer.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation*

PubMed Central

Bernard, Karen; Logsdon, Naomi J.; Ravi, Saranya; Xie, Na; Persons, Benjamin P.; Rangarajan, Sunad; Zmijewski, Jaroslaw W.; Mitra, Kasturi; Liu, Gang; Darley-Usmar, Victor M.; Thannickal, Victor J.

2015-01-01

Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation. PMID:26318453

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

Spermatogenesis disruption by dioxins: Epigenetic reprograming and windows of susceptibility.

PubMed

Pilsner, J Richard; Parker, Mikhail; Sergeyev, Oleg; Suvorov, Alexander

2017-04-01

Dioxins are a group of highly persistent chemicals that are generated as by-products of industrial and natural processes. Reduction in sperm counts is among the most sensitive endpoints of dioxin toxicity. The exact mechanism by which dioxins reduce sperm counts is not known. Recent data implicate the role of epididymal factors rather than disruption of spermatogenesis. Studies reviewed here demonstrate that dioxins induce the transfer of environmental conditions to the next generation via male germline following exposures during the window of epigenetic reprogramming of primordial germ cells. Increased incidence of birth defects in offspring of male veterans exposed to dioxin containing, Agent Orange, suggest that dioxins may induce epigenomic changes in male germ cells of adults during spermatogenesis. This is supported by recent animal data that show that environmental conditions can cause epigenetic dysregulation in sperm in the context of specific windows of epigenetic reprogramming during spermatogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Adaptation of the symbiotic Mesorhizobium-chickpea relationship to phosphate deficiency relies on reprogramming of whole-plant metabolism.

PubMed

Nasr Esfahani, Maryam; Kusano, Miyako; Nguyen, Kien Huu; Watanabe, Yasuko; Ha, Chien Van; Saito, Kazuki; Sulieman, Saad; Herrera-Estrella, Luis; Tran, L S

2016-08-09

Low inorganic phosphate (Pi) availability is a major constraint for efficient nitrogen fixation in legumes, including chickpea. To elucidate the mechanisms involved in nodule acclimation to low Pi availability, two Mesorhizobium-chickpea associations exhibiting differential symbiotic performances, Mesorhizobium ciceri CP-31 (McCP-31)-chickpea and Mesorhizobium mediterranum SWRI9 (MmSWRI9)-chickpea, were comprehensively studied under both control and low Pi conditions. MmSWRI9-chickpea showed a lower symbiotic efficiency under low Pi availability than McCP-31-chickpea as evidenced by reduced growth parameters and down-regulation of nifD and nifK These differences can be attributed to decline in Pi level in MmSWRI9-induced nodules under low Pi stress, which coincided with up-regulation of several key Pi starvation-responsive genes, and accumulation of asparagine in nodules and the levels of identified amino acids in Pi-deficient leaves of MmSWRI9-inoculated plants exceeding the shoot nitrogen requirement during Pi starvation, indicative of nitrogen feedback inhibition. Conversely, Pi levels increased in nodules of Pi-stressed McCP-31-inoculated plants, because these plants evolved various metabolic and biochemical strategies to maintain nodular Pi homeostasis under Pi deficiency. These adaptations involve the activation of alternative pathways of carbon metabolism, enhanced production and exudation of organic acids from roots into the rhizosphere, and the ability to protect nodule metabolism against Pi deficiency-induced oxidative stress. Collectively, the adaptation of symbiotic efficiency under Pi deficiency resulted from highly coordinated processes with an extensive reprogramming of whole-plant metabolism. The findings of this study will enable us to design effective breeding and genetic engineering strategies to enhance symbiotic efficiency in legume crops.

Interspecies Somatic Cell Nuclear Transfer: Advancements and Problems

PubMed Central

Lagutina, Irina; Fulka, Helena; Lazzari, Giovanna

2013-01-01

Abstract Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the “Dolly experiment,” the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus–cytoplasmic incompatibility. PMID:24033141

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts

PubMed Central

Doi, Akiko; Park, In-Hyun; Wen, Bo; Murakami, Peter; Aryee, Martin J; Irizarry, Rafael; Herb, Brian; Ladd-Acosta, Christine; Rho, Junsung; Loewer, Sabine; Miller, Justine; Schlaeger, Thorsten; Daley, George Q; Feinberg, Andrew P

2010-01-01

Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10−4) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10−4). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer. PMID:19881528

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Matula, Pavel; Stejskal, Stanislav; Hampl, Ales; Koutna, Irena

2017-03-21

Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells.

PubMed

Yannarelli, Gustavo; Pacienza, Natalia; Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment.

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells

PubMed Central

Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment. PMID:29216265

Concise Review: Methods and Cell Types Used to Generate Down Syndrome Induced Pluripotent Stem Cells

PubMed Central

Hibaoui, Youssef; Feki, Anis

2015-01-01

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Several models have been used to investigate the mechanisms by which the extra copy of chromosome 21 leads to the DS phenotype. In the last five years, several laboratories have been successful in reprogramming patient cells carrying the trisomy 21 anomaly into induced pluripotent stem cells, i.e., T21-iPSCs. In this review, we summarize the different T21-iPSCs that have been generated with a particular interest in the technical procedures and the somatic cell types used for the reprogramming. PMID:26239351

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy.

PubMed

Kehler, James; Greco, Marianna; Martino, Valentina; Pachiappan, Manickam; Yokoe, Hiroko; Chen, Alice; Yang, Miranda; Auerbach, Jonathan; Jessee, Joel; Gotte, Martin; Milanesi, Luciano; Albertini, Alberto; Bellipanni, Gianfranco; Zucchi, Ileana; Reinbold, Rolland A; Giordano, Antonio

2017-06-01

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Small molecule-induced cellular fate reprogramming: promising road leading to Rome.

PubMed

Li, Xiang; Xu, Jun; Deng, Hongkui

2018-05-29

Cellular fate reprogramming holds great promise to generate functional cell types for replenishing new cells and restoring functional loss. Inspired by transcription factor-induced reprogramming, the field of cellular reprogramming has greatly advanced and developed into divergent streams of reprogramming approaches. Remarkably, increasing studies have shown the power and advantages of small molecule-based approaches for cellular fate reprogramming, which could overcome the limitations of conventional transgenic-based reprogramming. In this concise review, we discuss these findings and highlight the future potentiality with particular focus on this new trend of chemical reprogramming. Copyright © 2018 Elsevier Ltd. All rights reserved.

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

PubMed Central

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells. PMID:28796841

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes.

PubMed

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant; Fu, Ji-Dong

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.

Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation--Methods and Protocols.

PubMed

Graversen, Veronica Kon; Chavala, Sai H

2016-01-01

Reprogramming fibroblasts into induced pluripotent stem cells (iPSC) remains a promising technique for cell replacement therapy. Diverse populations of somatic cells have been examined for their reprogramming potential. Recently, ocular ciliary body epithelial cells (CECs) have been reprogrammed with high reprogramming efficiency and single transcription factor reprogramming, making them an exciting candidate for cellular reprogramming strategies.

DNA double-strand break response in stem cells: mechanisms to maintain genomic integrity.

PubMed

Nagaria, Pratik; Robert, Carine; Rassool, Feyruz V

2013-02-01

Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease. Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells. ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin. The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Reprogramming of human cancer cells to pluripotency for models of cancer progression

PubMed Central

Kim, Jungsun; Zaret, Kenneth S

2015-01-01

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

Self-Organizing Global Gene Expression Regulated through Criticality: Mechanism of the Cell-Fate Change

PubMed Central

Tsuchiya, Masa; Giuliani, Alessandro; Hashimoto, Midori; Erenpreisa, Jekaterina; Yoshikawa, Kenichi

2016-01-01

Background A fundamental issue in bioscience is to understand the mechanism that underlies the dynamic control of genome-wide expression through the complex temporal-spatial self-organization of the genome to regulate the change in cell fate. We address this issue by elucidating a physically motivated mechanism of self-organization. Principal Findings Building upon transcriptome experimental data for seven distinct cell fates, including early embryonic development, we demonstrate that self-organized criticality (SOC) plays an essential role in the dynamic control of global gene expression regulation at both the population and single-cell levels. The novel findings are as follows: i) Mechanism of cell-fate changes: A sandpile-type critical transition self-organizes overall expression into a few transcription response domains (critical states). A cell-fate change occurs by means of a dissipative pulse-like global perturbation in self-organization through the erasure of initial-state critical behaviors (criticality). Most notably, the reprogramming of early embryo cells destroys the zygote SOC control to initiate self-organization in the new embryonal genome, which passes through a stochastic overall expression pattern. ii) Mechanism of perturbation of SOC controls: Global perturbations in self-organization involve the temporal regulation of critical states. Quantitative evaluation of this perturbation in terminal cell fates reveals that dynamic interactions between critical states determine the critical-state coherent regulation. The occurrence of a temporal change in criticality perturbs this between-states interaction, which directly affects the entire genomic system. Surprisingly, a sub-critical state, corresponding to an ensemble of genes that shows only marginal changes in expression and consequently are considered to be devoid of any interest, plays an essential role in generating a global perturbation in self-organization directed toward the cell-fate change. Conclusion and Significance ‘Whole-genome’ regulation of gene expression through self-regulatory SOC control complements gene-by-gene fine tuning and represents a still largely unexplored non-equilibrium statistical mechanism that is responsible for the massive reprogramming of genome expression. PMID:27997556

Epigenetic Regulation in Prostate Cancer Progression.

PubMed

Ruggero, Katia; Farran-Matas, Sonia; Martinez-Tebar, Adrian; Aytes, Alvaro

2018-01-01

An important number of newly identified molecular alterations in prostate cancer affect gene encoding master regulators of chromatin biology epigenetic regulation. This review will provide an updated view of the key epigenetic mechanisms underlying prostate cancer progression, therapy resistance, and potential actionable mechanisms and biomarkers. Key players in chromatin biology and epigenetic master regulators has been recently described to be crucially altered in metastatic CRPC and tumors that progress to AR independency. As such, epigenetic dysregulation represents a driving mechanism in the reprograming of prostate cancer cells as they lose AR-imposed identity. Chromatin integrity and accessibility for transcriptional regulation are key features altered in cancer progression, and particularly relevant in nuclear hormone receptor-driven tumors like prostate cancer. Understanding how chromatin remodeling dictates prostate development and how its deregulation contributes to prostate cancer onset and progression may improve risk stratification and treatment selection for prostate cancer patients.

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

PubMed Central

Rando, Thomas A.; Chang, Howard Y.

2012-01-01

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

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator

PubMed Central

SAZONOVA, I. Y.; MCNAMEE, R. A.; HOUNG, A. K.; KING, S. M.; HEDSTROM, L.; REED, G. L.

2013-01-01

Summary Background: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA’s fibrin-targeted properties that focus plasminogen activation on the fibrin surface. Objective: We examined whether re-programming SK’s mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. Methods and Results: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKΔ1 and SKΔ59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKΔ59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by α2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel ‘humanized’ fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKΔ1 and SKΔ59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2–3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. Conclusion: These experiments suggest that reprogramming SK’s mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency. PMID:19566545

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

PubMed

Sazonova, I Y; McNamee, R A; Houng, A K; King, S M; Hedstrom, L; Reed, G L

2009-08-01

Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta

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.more » 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.« less

Mitochondrial Sirtuin 4 Resolves Immune Tolerance in Monocytes by Rebalancing Glycolysis and Glucose Oxidation Homeostasis

PubMed Central

Tao, Jie; Zhang, Jingpu; Ling, Yun; McCall, Charles E.; Liu, Tie Fu

2018-01-01

The goal of this investigation was to define the molecular mechanism underlying physiologic conversion of immune tolerance to resolution of the acute inflammatory response, which is unknown. An example of this knowledge gap and its clinical importance is the broad-based energy deficit and immunometabolic paralysis in blood monocytes from non-survivors of human and mouse sepsis that precludes sepsis resolution. This immunometabolic dysregulation is biomarked by ex vivo endotoxin tolerance to increased glycolysis and TNF-α expression. To investigate how tolerance switches to resolution, we adapted our previously documented models associated with acute inflammatory, immune, and metabolic reprogramming that induces endotoxin tolerance as a model of sepsis in human monocytes. We report here that mitochondrial sirtuin 4 (SIRT4) physiologically breaks tolerance and resolves acute inflammation in human monocytes by coordinately reprogramming of metabolism and bioenergetics. We find that increased SIRT4 mRNA and protein expression during immune tolerance counters the increase in pyruvate dehydrogenase kinase 1 (PDK1) and SIRT1 that promote tolerance by switching glucose-dependent support of immune resistance to fatty acid oxidation support of immune tolerance. By decreasing PDK1, pyruvate dehydrogenase complex reactivation rebalances mitochondrial respiration, and by decreasing SIRT1, SIRT4 represses fatty acid oxidation. The precise mechanism for the mitochondrial SIRT4 nuclear feedback is unclear. Our findings are consistent with a new concept in which mitochondrial SIRT4 directs the axis that controls anabolic and catabolic energy sources. PMID:29593712

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells

PubMed Central

Merling, Randall K.; Sweeney, Colin L.; Choi, Uimook; De Ravin, Suk See; Myers, Timothy G.; Otaizo-Carrasquero, Francisco; Pan, Jason; Linton, Gilda; Chen, Lifeng; Koontz, Sherry; Theobald, Narda L.; Malech, Harry L.

2013-01-01

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34+ hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34+ HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60+, yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60+), with variable yield (6 to >500 TRA-1-60+ iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP–reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34+ HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14+ monocytes do not yield iPSC colonies under these reprogramming conditions. PMID:23386128

Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

PubMed

Ho, Lin; Hsu, Shan-Hui

2018-04-01

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be reprogrammed into neural crest-like stem cells by 3D bioprinting with the gel, and the reprogrammed cells underwent neural differentiation in the printed structure after induction. The neural-like tissue engineering constructs fabricated by 3D bioprinting from human fibroblasts may be applied for neuroregeneration or further developed as mini-brain for basic research and drug screening. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Stress-responsive microRNAs are involved in re-programming of metabolic functions in hibernators.

PubMed

Arfat, Yasir; Chang, Hui; Gao, Yunfang

2018-04-01

Mammalian hibernation includes re-programing of metabolic capacities, partially, encouraged by microRNAs (miRNAs). Albeit much is known about the functions of miRNAs, we need learning on low temperature miRNAs target determination. As hibernators can withstand low body temperatures (TB) for a long time without anguish tissue damage, understanding the means and mechanisms that empower them to do as such are of restorative intrigue. Nonetheless, these mechanisms by which miRNAs and the hibernators react to stressful conditions are not much clear. It is evident from recent data that the gene expression and the translation of mRNA to protein are controlled by miRNAs. The miRNAs also influence regulation of major cellular processes. As the significance of miRNAs in stress conditions adaptation are getting clearer, this audit article abridges the key alterations in miRNA expression and the mechanism that facilitates stress survival. © 2017 Wiley Periodicals, Inc.

MYC-induced reprogramming of glutamine catabolism supports optimal virus replication

PubMed Central

Thai, Minh; Thaker, Shivani K.; Feng, Jun; Du, Yushen; Hu, Hailiang; Ting Wu, Ting; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2015-01-01

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses. PMID:26561297

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

PubMed Central

Hämäläinen, Riikka H.; Ahlqvist, Kati J.; Ellonen, Pekka; Lepistö, Maija; Logan, Angela; Otonkoski, Timo; Murphy, Michael P.; Suomalainen, Anu

2015-01-01

Summary mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. PMID:26027936

DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).

PubMed

Canovas, Sebastian; Ross, Pablo J; Kelsey, Gavin; Coy, Pilar

2017-11-01

DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children. © 2017 WILEY Periodicals, Inc.

Delayed transition to new cell fates during cellular reprogramming

PubMed Central

Cheng, Xianrui; Lyons, Deirdre C.; Socolar, Joshua E. S.; McClay, David R.

2014-01-01

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues. PMID:24780626

The Potential of iPSCs for the Treatment of Premature Aging Disorders

PubMed Central

Compagnucci, Claudia; Bertini, Enrico

2017-01-01

Premature aging disorders including Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome, are a group of rare monogenic diseases leading to reduced lifespan of the patients. Importantly, these disorders mimic several features of physiological aging. Despite the interest on the study of these diseases, the underlying biological mechanisms remain unknown and no treatment is available. Recent studies on HGPS (due to mutations of the LMNA gene encoding for the nucleoskeletal proteins lamin A/C) have reported disruptions in cellular and molecular mechanisms modulating genomic stability and stem cell populations, thus giving the nuclear lamina a relevant function in nuclear organization, epigenetic regulation and in the maintenance of the stem cell pool. In this context, modeling premature aging with induced pluripotent stem cells (iPSCs) offers the possibility to study these disorders during self-renewal and differentiation into relevant cell types. iPSCs generated by cellular reprogramming from adult somatic cells allows researchers to understand pathophysiological mechanisms and enables the performance of drug screenings. Moreover, the recent development of precision genome editing offers the possibility to study the complex mechanisms underlying senescence and the possibility to correct disease phenotypes, paving the way for future therapeutic interventions. PMID:29112121

Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells

PubMed Central

Sharma, Bal Krishan; Kolhe, Ravindra; Black, Stephen M.; Keller, Jonathan R.; Mivechi, Nahid F.; Satyanarayana, Ande

2016-01-01

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50–70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction of c-Myc and Id1 (∼70%), suggesting that Hif1α suppresses Id1 and c-Myc under anaerobic conditions via Mxi1. Together, our findings indicate a prominent novel role for Id1 in liver cancer cell metabolic adaptation.—Sharma, B. K., Kolhe, R., Black, S. M., Keller, J. R., Mivechi, N. F., Satyanarayana, A. Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells. PMID:26330493

Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

PubMed

Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

2008-06-10

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

PubMed

Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique

PubMed Central

Yunusova, Anastasia M.; Fishman, Veniamin S.; Vasiliev, Gennady V.

2017-01-01

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells. High-throughput sequencing of barcodes from successfully reprogrammed cells revealed a significant number of barcodes from related cells. We developed a computer model, according to which a probability of synchronous reprogramming of sister cells equals 10–30%. We conclude that the reprogramming success is pre-established in some particular cells and, being a heritable trait, can be maintained through cell division. Thus, reprogramming progresses in a deterministic manner, at least at the level of cell lineages. PMID:28446707

Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function[OPEN

PubMed Central

2018-01-01

Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

PubMed

Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.

PubMed

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-08-15

Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues-niche mimicking factors, (in)activation of transcription factors, to name a few-enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc

PubMed Central

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-01-01

Abstract Motivation Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues—niche mimicking factors, (in)activation of transcription factors, to name a few—enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. Results We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. Availability and Implementation eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. Contact christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com Supplementary information Supplementary data are available at Bioinformatics online. PMID:28398503

Reprogramming cancer cells: overview & current progress.

PubMed

Lim, Kian Lam; Teoh, Hoon Koon; Choong, Pei Feng; Teh, Hui Xin; Cheong, Soon Keng; Kamarul, Tunku

2016-07-01

Cancer is a disease with genetic and epigenetic origins, and the possible effects of reprogramming cancer cells using the defined sets of transcription factors remain largely uninvestigated. In the handful of publications available so far, findings have shown that reprogramming cancer cells changed the characteristics of the cells to differ from the parental cancer cells. These findings indicated the possibility of utilizing reprogramming technology to create a disease model in the laboratory to be used in studying the molecular pathogenesis or for drug screening of a particular cancer model. Despite numerous methods employed in generating induced pluripotent stem cells (iPSCs) from cancer cells only a few studies have successfully reprogrammed malignant human cells. In this review we will provide an overview on i) methods to reprogram cancer cells, ii) characterization of the reprogrammed cancer cells, and iii) the differential effects of reprogramming on malignancy, epigenetics and response of the cancer cells to chemotherapeutic agents. Continued technical progress in cancer cell reprogramming technology will be instrumental for more refined in vitro disease models and ultimately for the development of directed and personalized therapy for cancer patients in the future.

75 FR 54526 - Defense Federal Acquisition Regulation Supplement; Additional Requirements Applicable to...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-08

... Procurement of Major Systems of the Department of Defense.'' DATES: Effective Date: September 8, 2010. FOR... 217 Government procurement. Ynette R. Shelkin, Editor, Defense Acquisition Regulations System. 0... funding or reprogramming for procurement of a major system under a multiyear contract authorized under...

Using somatic-cell nuclear transfer to study aging.

PubMed

Kishigami, Satoshi; Lee, Ah Reum; Wakayama, Teruhiko

2013-01-01

In mammals, a diploid genome following fertilization of haploid cells, an egg, and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual's inevitable demise. Since it was first reported in 1997 that Dolly the sheep had been cloned, many mammalian species have been cloned successfully using somatic-cell nuclear transfer (SCNT). The success of SCNT in mammals enables us not only to reproduce offspring without germ cells, that is, to "passage" a unique diploid genome, but also to address valuable biological questions on development, nuclear reprogramming, and epigenetic memory. Successful cloning can also support epigenetic reprogramming where the aging clock is reset or reversed. Recent work using iPS cell technology has explored the practicality and led to the recapitulation of premature aging with iPSCs from progeroid laminopathies. As a result, reprogramming tools are also expected to contribute to studying biological age. However, the efficiency of animal cloning is still low in most cases and the mechanism of reprogramming in cloned embryos is still largely unclear. Here, based on recent advances, we describe an improved, more efficient mouse cloning protocol using histone deacetylase inhibitors (HDACis) and latrunculin A, which increases the success rates of producing cloned mice or establishing ES cells fivefold. This improved method of cloning will provide a strong tool to address many issues including biological aging more easily and with lower cost.

Perspectives on Systems Modeling of Human Peripheral Blood Mononuclear Cells

PubMed Central

Sen, Partho; Kemppainen, Esko; Orešič, Matej

2018-01-01

Human peripheral blood mononuclear cells (PBMCs) are the key drivers of the immune responses. These cells undergo activation, proliferation and differentiation into various subsets. During these processes they initiate metabolic reprogramming, which is coordinated by specific gene and protein activities. PBMCs as a model system have been widely used to study metabolic and autoimmune diseases. Herein we review various omics and systems-based approaches such as transcriptomics, epigenomics, proteomics, and metabolomics as applied to PBMCs, particularly T helper subsets, that unveiled disease markers and the underlying mechanisms. We also discuss and emphasize several aspects of T cell metabolic modeling in healthy and disease states using genome-scale metabolic models. PMID:29376056

Establishment of induced pluripotent stem cell (iPSC) line from 55-year old male patient with hemorrhagic Moyamoya disease.

PubMed

Cardano, Marina; Marsoner, Fabio; Marcatili, Matteo; Karnavas, Thodoris; Zasso, Jacopo; Lanterna, Luigi Andrea; Conti, Luciano

2016-11-01

Peripheral blood mononuclear cells (PBMCs) were collected from 55-year old male patient with a confirmed diagnosis of hemorrhagic Moyamoya disease (MMD). PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. A footprint-free hiPSC line was characterized by the expression of pluripotency markers and a normal karyotype. These cells were able to give rise to Embryoid Bodies and to a progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a suitable tool for modelling in vitro MMD disease to investigate the cellular mechanisms underlying the occurrence of this pathology. Copyright © 2016. Published by Elsevier B.V.

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.

Root Type-Specific Reprogramming of Maize Pericycle Transcriptomes by Local High Nitrate Results in Disparate Lateral Root Branching Patterns1[OPEN

PubMed Central

Lithio, Andrew

2016-01-01

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching patterns of lateral roots upon local high nitrate stimulation. In particular, brace roots displayed an exceptional architectural plasticity compared to other root types. Transcriptome profiling revealed root type-specific transcriptomic reprogramming of pericycle cells upon local high nitrate stimulation. The alteration of the transcriptomic landscape of brace root pericycle cells in response to local high nitrate stimulation was most significant. Root type-specific transcriptome diversity in response to local high nitrate highlighted differences in the functional adaptability and systemic shoot nitrogen starvation response during development. Integration of morphological, anatomical, and transcriptomic data resulted in a framework underscoring similarity and diversity among root types grown in heterogeneous nitrate environments. PMID:26811190

Metabolic Flux Redirection and Transcriptomic Reprogramming in the Albino Tea Cultivar ‘Yu-Jin-Xiang’ with an Emphasis on Catechin Production

PubMed Central

Liu, Guo-Feng; Han, Zhuo-Xiao; Feng, Lin; Gao, Li-Ping; Gao, Ming-Jun; Gruber, Margaret Y.; Zhang, Zhao-Liang; Xia, Tao; Wan, Xiao-Chun; Wei, Shu

2017-01-01

In this study, shade-induced conversion from a young pale/yellow leaf phenotype to a green leaf phenotype was studied using metabolic and transcriptomic profiling and the albino cultivar ‘Yu-Jin-Xiang’ (‘YJX’) of Camellia sinensis for a better understanding of mechanisms underlying the phenotype shift and the altered catechin and theanine production. Shaded leaf greening resulted from an increase in leaf chlorophyll and carotenoid abundance and chloroplast development. A total of 1,196 differentially expressed genes (DEGs) were identified between the ‘YJX’ pale and shaded green leaves, and these DEGs affected ‘chloroplast organization’ and ‘response to high light’ besides many other biological processes and pathways. Metabolic flux redirection and transcriptomic reprogramming were found in flavonoid and carotenoid pathways of the ‘YJX’ pale leaves and shaded green leaves to different extents compared to the green cultivar ‘Shu-Cha-Zao’. Enhanced production of the antioxidant quercetin rather than catechin biosynthesis was correlated positively with the enhanced transcription of FLAVONOL SYNTHASE and FLAVANONE/FLAVONOL HYDROXYLASES leading to quercetin accumulation and negatively correlated to suppressed LEUCOANTHOCYANIDIN REDUCTASE, ANTHOCYANIDIN REDUCTASE and SYNTHASE leading to catechin biosynthesis. The altered levels of quercetin and catechins in ‘YJX’ will impact on its tea flavor and health benefits. PMID:28332598

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

PubMed

Quintana-Bustamante, Oscar; Segovia, Jose C

2016-01-01

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

Targeted Cell Fusion Facilitates Stable Heterokaryon Generation In Vitro and In Vivo

PubMed Central

Long, Michael A.; Rossi, Fabio M. V.

2011-01-01

Induced cell fusion has enabled several important discoveries, including the phenomenon of nuclear reprogramming and may yet be applied as a novel therapy for degenerative diseases. However, existing fusogens lack the efficiency required to enable investigation of the epigenetic modifications underlying nuclear reprogramming and the specificity required for clinical application. Here we present a chimeric measles hemagglutinin, Hα7, which specifically and efficiently mediates the fusion of diverse cell types with skeletal muscle both in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a substantial increase in heterokaryon yield and exhibited insignificant levels of toxicity. Moreover, this increased fusion efficiency enabled detection of chromatin modifications associated with nuclear reprogramming following Hα7-mediated fusion of human fibroblasts and mouse myotubes. Finally, Hα7 was also capable of increasing the contribution of transplanted fibroblasts to skeletal muscle repair in vivo, suggesting that this strategy could be used for therapeutic gene delivery. PMID:22039476

(Re-)programming of subtype specific cardiomyocytes.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

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

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

Coordination of m6A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming

PubMed Central

Aguilo, Francesca; Zhang, Fan; Sancho, Ana; Fidalgo, Miguel; Di Cecilia, Serena; Vashisht, Ajay; Lee, Dung-Fang; Chen, Chih-Hung; Rengasamy, Madhumitha; Andino, Blanca; Jahouh, Farid; Roman, Angel; Krig, Sheryl R.; Wang, Rong; Zhang, Weijia; Wohlschlegel, James A.; Wang, Jianlong; Walsh, Martin J.

2015-01-01

SUMMARY Epigenetic and epitranscriptomic networks have important functions in maintaining pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However the mechanisms integrating the actions of these distinct networks are only partially understood. Here, we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates transcription of key pluripotency genes, and modulates N6-methyladenosine (m6A) deposition on their transcripts by sequestering the enzyme m6A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m6A RNA levels, and enhances m6A modification of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3-associated, and is enriched at promoters of m6A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m6A RNA modification to insure ESC identity. PMID:26526723

Protein kinase C and calcineurin cooperatively mediate cell survival under compressive mechanical stress.

PubMed

Mishra, Ranjan; van Drogen, Frank; Dechant, Reinhard; Oh, Soojung; Jeon, Noo Li; Lee, Sung Sik; Peter, Matthias

2017-12-19

Cells experience compressive stress while growing in limited space or migrating through narrow constrictions. To survive such stress, cells reprogram their intracellular organization to acquire appropriate mechanical properties. However, the mechanosensors and downstream signaling networks mediating these changes remain largely unknown. Here, we have established a microfluidic platform to specifically trigger compressive stress, and to quantitatively monitor single-cell responses of budding yeast in situ. We found that yeast senses compressive stress via the cell surface protein Mid2 and the calcium channel proteins Mid1 and Cch1, which then activate the Pkc1/Mpk1 MAP kinase pathway and calcium signaling, respectively. Genetic analysis revealed that these pathways work in parallel to mediate cell survival. Mid2 contains a short intracellular tail and a serine-threonine-rich extracellular domain with spring-like properties, and both domains are required for mechanosignaling. Mid2-dependent spatial activation of the Pkc1/Mpk1 pathway depolarizes the actin cytoskeleton in budding or shmooing cells, thereby antagonizing polarized growth to protect cells under compressive stress conditions. Together, these results identify a conserved signaling network responding to compressive mechanical stress, which, in higher eukaryotes, may ensure cell survival in confined environments.

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

PubMed

von Meyenn, Ferdinand; Iurlaro, Mario; Habibi, Ehsan; Liu, Ning Qing; Salehzadeh-Yazdi, Ali; Santos, Fátima; Petrini, Edoardo; Milagre, Inês; Yu, Miao; Xie, Zhenqing; Kroeze, Leonie I; Nesterova, Tatyana B; Jansen, Joop H; Xie, Hehuang; He, Chuan; Reik, Wolf; Stunnenberg, Hendrik G

2016-06-16

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Mechanisms Underlying Latent Disease Risk Associated with Early-Life Arsenic Exposure: Current Research Trends and Scientific Gaps

PubMed Central

Bailey, Kathryn A.; Smith, Allan H.; Tokar, Erik J.; Graziano, Joseph H.; Kim, Kyoung-Woong; Navasumrit, Panida; Ruchirawat, Mathuros; Thiantanawat, Apinya; Suk, William A.; Fry, Rebecca C.

2015-01-01

Background Millions of individuals worldwide, particularly those living in rural and developing areas, are exposed to harmful levels of inorganic arsenic (iAs) in their drinking water. Inorganic As exposure during key developmental periods is associated with a variety of adverse health effects, including those that are evident in adulthood. There is considerable interest in identifying the molecular mechanisms that relate early-life iAs exposure to the development of these latent diseases, particularly in relationship to cancer. Objectives This work summarizes research on the molecular mechanisms that underlie the increased risk of cancer development in adulthood that is associated with early-life iAs exposure. Discussion Epigenetic reprogramming that imparts functional changes in gene expression, the development of cancer stem cells, and immunomodulation are plausible underlying mechanisms by which early-life iAs exposure elicits latent carcinogenic effects. Conclusions Evidence is mounting that relates early-life iAs exposure and cancer development later in life. Future research should include animal studies that address mechanistic hypotheses and studies of human populations that integrate early-life exposure, molecular alterations, and latent disease outcomes. Citation Bailey KA, Smith AH, Tokar EJ, Graziano JH, Kim KW, Navasumrit P, Ruchirawat M, Thiantanawat A, Suk WA, Fry RC. 2016. Mechanisms underlying latent disease risk associated with early-life arsenic exposure: current research trends and scientific gaps. Environ Health Perspect 124:170–175; http://dx.doi.org/10.1289/ehp.1409360 PMID:26115410

Adolescent Alcohol Exposure: Burden of Epigenetic Reprogramming, Synaptic Remodeling, and Adult Psychopathology

PubMed Central

Kyzar, Evan J.; Floreani, Christina; Teppen, Tara L.; Pandey, Subhash C.

2016-01-01

Adolescence represents a crucial phase of synaptic maturation characterized by molecular changes in the developing brain that shape normal behavioral patterns. Epigenetic mechanisms play an important role in these neuromaturation processes. Perturbations of normal epigenetic programming during adolescence by ethanol can disrupt these molecular events, leading to synaptic remodeling and abnormal adult behaviors. Repeated exposure to binge levels of alcohol increases the risk for alcohol use disorder (AUD) and comorbid psychopathology including anxiety in adulthood. Recent studies in the field clearly suggest that adolescent alcohol exposure causes widespread and persistent changes in epigenetic, neurotrophic, and neuroimmune pathways in the brain. These changes are manifested by altered synaptic remodeling and neurogenesis in key brain regions leading to adult psychopathology such as anxiety and alcoholism. This review details the molecular mechanisms underlying adolescent alcohol exposure-induced changes in synaptic plasticity and the development of alcohol addiction-related phenotypes in adulthood. PMID:27303256

Direct reprogramming and biomaterials for controlling cell fate.

PubMed

Kim, Eunsol; Tae, Giyoong

2016-01-01

Direct reprogramming which changes the fate of matured cell is a very useful technique with a great interest recently. This approach can eliminate the drawbacks of direct usage of stem cells and allow the patient specific treatment in regenerative medicine. Overexpression of diverse factors such as general reprogramming factors or lineage specific transcription factors can change the fate of already differentiated cells. On the other hand, biomaterials can provide physical and topographical cues or biochemical cues on cells, which can dictate or significantly affect the differentiation of stem cells. The role of biomaterials on direct reprogramming has not been elucidated much, but will be potentially significant to improve the efficiency or specificity of direct reprogramming. In this review, the strategies for general direct reprogramming and biomaterials-guided stem cell differentiation are summarized with the addition of the up-to-date progress on biomaterials for direct reprogramming.

Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos.

PubMed

Sadeesh, E M; Fozia, Shah; Meena, Kataria

2017-04-01

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P < 0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P < 0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P < 0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P < 0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

From Understanding the Development Landscape of the Canonical Fate-Switch Pair to Constructing a Dynamic Landscape for Two-Step Neural Differentiation

PubMed Central

2012-01-01

Abstract Recent progress in stem cell biology, notably cell fate conversion, calls for novel theoretical understanding for cell differentiation. The existing qualitative concept of Waddington’s “epigenetic landscape” has attracted particular attention because it captures subsequent fate decision points, thus manifesting the hierarchical (“tree-like”) nature of cell fate diversification. Here, we generalized a recent work and explored such a developmental landscape for a two-gene fate decision circuit by integrating the underlying probability landscapes with different parameters (corresponding to distinct developmental stages). The change of entropy production rate along the parameter changes indicates which parameter changes can represent a normal developmental process while other parameters’ change can not. The transdifferentiation paths over the landscape under certain conditions reveal the possibility of a direct and reversible phenotypic conversion. As the intensity of noise increases, we found that the landscape becomes flatter and the dominant paths more straight, implying the importance of biological noise processing mechanism in development and reprogramming. We further extended the landscape of the one-step fate decision to that for two-step decisions in central nervous system (CNS) differentiation. A minimal network and dynamic model for CNS differentiation was firstly constructed where two three-gene motifs are coupled. We then implemented the SDEs (Stochastic Differentiation Equations) simulation for the validity of the network and model. By integrating the two landscapes for the two switch gene pairs, we constructed the two-step development landscape for CNS differentiation. Our work provides new insights into cellular differentiation and important clues for better reprogramming strategies. PMID:23300518

Major Transcriptome Reprogramming Underlies Floral Mimicry Induced by the Rust Fungus Puccinia monoica in Boechera stricta

PubMed Central

Haugen, Riston H.; Saunders, Diane G. O.; Leonelli, Lauriebeth; MacLean, Dan; Hogenhout, Saskia A.; Kamoun, Sophien

2013-01-01

Pucciniamonoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boechera stricta . Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. Although much ecological research has been performed on P . monoica -induced pseudoflowers, this system has yet to be investigated at the molecular or genomic level. To date, the molecular alterations underlying the development of pseudoflowers and the genes involved have not been described. To address this, we performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated. These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers. This work illustrates the complex perturbations induced by rust pathogens in their host plants, and provides a starting point for understanding the molecular mechanisms of pathogen-induced floral mimicry. PMID:24069397

Major transcriptome reprogramming underlies floral mimicry induced by the rust fungus Puccinia monoica in Boechera stricta.

PubMed

Cano, Liliana M; Raffaele, Sylvain; Haugen, Riston H; Saunders, Diane G O; Leonelli, Lauriebeth; MacLean, Dan; Hogenhout, Saskia A; Kamoun, Sophien

2013-01-01

Pucciniamonoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boecherastricta. Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. Although much ecological research has been performed on P. monoica-induced pseudoflowers, this system has yet to be investigated at the molecular or genomic level. To date, the molecular alterations underlying the development of pseudoflowers and the genes involved have not been described. To address this, we performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated. These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers. This work illustrates the complex perturbations induced by rust pathogens in their host plants, and provides a starting point for understanding the molecular mechanisms of pathogen-induced floral mimicry.

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

Stress-mediated translational control in cancer cells.

PubMed

Leprivier, Gabriel; Rotblat, Barak; Khan, Debjit; Jan, Eric; Sorensen, Poul H

2015-07-01

Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

PubMed

Xue, Gang; Ren, Zhenxin; Grabham, Peter W; Chen, Yaxiong; Zhu, Jiayun; Du, Yarong; Pan, Dong; Li, Xiaoman; Hu, Burong

2015-07-01

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Spin glass model for cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Castillo, Horacio E.

2014-03-01

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 state to another attractor state. We use a simple model based on spin glass theory that can construct a simulated epigenetic landscape starting from the experimental genomic data. We modify the model to incorporate experimental reprogramming protocols. Our simulations successfully reproduce several reprogramming experiments. We probe the robustness of the results against random changes in the model, explore the importance of asymmetric interactions between transcription factors and study the importance of histone modification errors in reprogramming.

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

PubMed Central

2014-01-01

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

Tumoral stem cell reprogramming as a driver of cancer: Theory, biological models, implications in cancer therapy.

PubMed

Vicente-Dueñas, Carolina; Hauer, Julia; Ruiz-Roca, Lucía; Ingenhag, Deborah; Rodríguez-Meira, Alba; Auer, Franziska; Borkhardt, Arndt; Sánchez-García, Isidro

2015-06-01

Cancer is a clonal malignant disease originated in a single cell and characterized by the accumulation of partially differentiated cells that are phenotypically reminiscent of normal stages of differentiation. According to current models, therapeutic strategies that block oncogene activity are likely to selectively target tumor cells. However, recent evidences have revealed that cancer stem cells could arise through a tumor stem cell reprogramming mechanism, suggesting that genetic lesions that initiate the cancer process might be dispensable for tumor progression and maintenance. This review addresses the impact of these results toward a better understanding of cancer development and proposes new approaches to treat cancer in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

The role of metabolic reprogramming in γ-herpesvirus-associated oncogenesis.

PubMed

Lo, Angela Kwok-Fung; Dawson, Christopher W; Young, Lawrence S; Lo, Kwok-Wai

2017-10-15

The γ-herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ-herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ-herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ-herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ-herpesviruses alter metabolic phenotypes as a means to support viral infection and long-term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ-herpesviruses provide new opportunities for therapeutic intervention. © 2017 UICC.

Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors.

PubMed

Saera-Vila, Alfonso; Louie, Ke'ale W; Sha, Cuilee; Kelly, Ryan M; Kish, Phillip E; Kahana, Alon

2018-01-01

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira

2012-01-13

Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain largemore » amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.« less

In vivo reprogramming for heart regeneration: A glance at efficiency, environmental impacts, challenges and future directions.

PubMed

Ebrahimi, Behnam

2017-07-01

Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

PubMed

Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function.

PubMed

Kourelis, Jiorgos; van der Hoorn, Renier A L

2018-02-01

Plants have many, highly variable resistance ( R ) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize ( Zea mays ) Hm1 , was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. © 2018 American Society of Plant Biologists. All rights reserved.

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice

PubMed Central

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C.; Zhou, Qiao

2016-01-01

Aims/hypothesis Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Methods Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. Results We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue’s response to clear the pancreas from insufficiently reprogrammed cells. Conclusions/interpretation Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications. PMID:26693711

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice.

PubMed

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C; Zhou, Qiao

2016-03-01

Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue's response to clear the pancreas from insufficiently reprogrammed cells. Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications.

Deterministic direct reprogramming of somatic cells to pluripotency.

PubMed

Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

2013-10-03

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

PubMed Central

Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

2013-01-01

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

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

PubMed Central

Asuelime, Grace E.; Shi, Yanhong

2012-01-01

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

A Blueprint for a Synthetic Genetic Feedback Controller to Reprogram Cell Fate.

PubMed

Del Vecchio, Domitilla; Abdallah, Hussein; Qian, Yili; Collins, James J

2017-01-25

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae

2014-01-17

Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cellsmore » under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.« less

Reprogrammed Glucose Metabolic Pathways of Inhibitor-Tolerant Yeast

USDA-ARS?s Scientific Manuscript database

Representative inhibitory compounds such as furfural and 5-hydroxymethylfurfural generated from lignocellulosic biomass pretreatment inhibit yeast growth and interfere with the subsequent ethanol fermentation. Evolutionary engineering under laboratory settings is a powerful tool that can be used to ...

Reprogrammed glucose metabolic pathways of inhibitor-tolerant yeast

USDA-ARS?s Scientific Manuscript database

Representative inhibitory compounds such as furfural and 5-hydroxymethylfurfural generated from lignocellulosic biomass pretreatment inhibit yeast growth and interfere with the subsequent ethanol fermentation. Evolutionary engineering under laboratory settings is a powerful tool that can be used to...

Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes

PubMed Central

Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Durai, Prasannavenkatesh; Shah, Masaud; Choi, Sangdun

2016-01-01

The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming. PMID:26790000

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis[OPEN

PubMed Central

Ohnuma, Mariko; Kurata, Tetsuya; Nakata, Masaru; Ohme-Takagi, Masaru

2017-01-01

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana. The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants. PMID:28011694

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

PubMed

Dormiani, Kianoush; Mir Mohammad Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Forouzanfar, Mahboobeh; Baharvand, Hossein; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2017-01-01

Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells.

PubMed

Kamath, Anant; Ternes, Sara; McGowan, Stephen; English, Anthony; Mallampalli, Rama; Moy, Alan B

2017-08-01

Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28 . We describe a robust Myc and Lin28 -free IPSC reprogramming approach using reprogramming molecules. IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28 -free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28 . This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

PubMed

Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

2016-05-17

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

PubMed

Gallego-Perez, Daniel; Otero, Jose J; Czeisler, Catherine; Ma, Junyu; Ortiz, Cristina; Gygli, Patrick; Catacutan, Fay Patsy; Gokozan, Hamza Numan; Cowgill, Aaron; Sherwood, Thomas; Ghatak, Subhadip; Malkoc, Veysi; Zhao, Xi; Liao, Wei-Ching; Gnyawali, Surya; Wang, Xinmei; Adler, Andrew F; Leong, Kam; Wulff, Brian; Wilgus, Traci A; Askwith, Candice; Khanna, Savita; Rink, Cameron; Sen, Chandan K; Lee, L James

2016-02-01

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs. Copyright © 2016 Elsevier Inc. All rights reserved.

Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation.

PubMed

Marwick, John A; Mills, Ross; Kay, Oliver; Michail, Kyriakos; Stephen, Jillian; Rossi, Adriano G; Dransfield, Ian; Hirani, Nikhil

2018-06-04

Apoptotic cells modulate the function of macrophages to control and resolve inflammation. Here, we show that neutrophils induce a rapid and sustained suppression of NF-κB signalling in the macrophage through a unique regulatory relationship which is independent of apoptosis. The reduction of macrophage NF-κB activation occurs through a blockade in transforming growth factor β-activated kinase 1 (TAK1) and IKKβ activation. As a consequence, NF-κB (p65) phosphorylation is reduced, its translocation to the nucleus is inhibited and NF-κB-mediated inflammatory cytokine transcription is suppressed. Gene Set Enrichment Analysis reveals that this suppression of NF-κB activation is not restricted to post-translational modifications of the canonical NF-κB pathway, but is also imprinted at the transcriptional level. Thus neutrophils exert a sustained anti-inflammatory phenotypic reprogramming of the macrophage, which is reflected by the sustained reduction in the release of pro- but not anti- inflammatory cytokines from the macrophage. Together, our findings identify a novel apoptosis-independent mechanism by which neutrophils regulate the mediator profile and reprogramming of monocytes/macrophages, representing an important nodal point for inflammatory control.

Dynamic DNA methylation reconfiguration during seed development and germination.

PubMed

Kawakatsu, Taiji; Nery, Joseph R; Castanon, Rosa; Ecker, Joseph R

2017-09-15

Unlike animals, plants can pause their life cycle as dormant seeds. In both plants and animals, DNA methylation is involved in the regulation of gene expression and genome integrity. In animals, reprogramming erases and re-establishes DNA methylation during development. However, knowledge of reprogramming or reconfiguration in plants has been limited to pollen and the central cell. To better understand epigenetic reconfiguration in the embryo, which forms the plant body, we compared time-series methylomes of dry and germinating seeds to publicly available seed development methylomes. Time-series whole genome bisulfite sequencing reveals extensive gain of CHH methylation during seed development and drastic loss of CHH methylation during germination. These dynamic changes in methylation mainly occur within transposable elements. Active DNA methylation during seed development depends on both RNA-directed DNA methylation and heterochromatin formation pathways, whereas global demethylation during germination occurs in a passive manner. However, an active DNA demethylation pathway is initiated during late seed development. This study provides new insights into dynamic DNA methylation reprogramming events during seed development and germination and suggests possible mechanisms of regulation. The observed sequential methylation/demethylation cycle suggests an important role of DNA methylation in seed dormancy.

Cancer drug addiction is relayed by an ERK2-dependent phenotype switch.

PubMed

Kong, Xiangjun; Kuilman, Thomas; Shahrabi, Aida; Boshuizen, Julia; Kemper, Kristel; Song, Ji-Ying; Niessen, Hans W M; Rozeman, Elisa A; Geukes Foppen, Marnix H; Blank, Christian U; Peeper, Daniel S

2017-10-12

Observations from cultured cells, animal models and patients raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR-Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for 'addiction genes'. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial-mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

Transplantation of Reprogrammed Autologous Stem Cells for Chronic Pain and Drug Abuse

DTIC Science & Technology

2012-10-01

Hyperalgesia and Mechanical Allodynia by Curcumin in a Rat Model of Partial Thickness Thermal Injury Clifford, John Evaluation Of A Non-Invasive System Of...Thermal Injury Garza, Thomas Biochemical Correlates for the Analgesic Action Of Curcumin : A Naturally occurring Anti-inflammatory Agent Greer, Angie

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

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans.

PubMed

Fassnacht, Christina; Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos; Stadler, Michael B; Ciosk, Rafal

2018-03-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans

PubMed Central

Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos

2018-01-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition. PMID:29579041

Modulation of host cell biology by plant pathogenic microbes.

PubMed

Le Fevre, Ruth; Evangelisti, Edouard; Rey, Thomas; Schornack, Sebastian

2015-01-01

Plant-pathogen interactions can result in dramatic visual changes in the host, such as galls, phyllody, pseudoflowers, and altered root-system architecture, indicating that the invading microbe has perturbed normal plant growth and development. These effects occur on a cellular level but range up to the organ scale, and they commonly involve attenuation of hormone homeostasis and deployment of effector proteins with varying activities to modify host cell processes. This review focuses on the cellular-reprogramming mechanisms of filamentous and bacterial plant pathogens that exhibit a biotrophic lifestyle for part, if not all, of their lifecycle in association with the host. We also highlight strategies for exploiting our growing knowledge of microbial host reprogramming to study plant processes other than immunity and to explore alternative strategies for durable plant resistance.

Left dorsal premotor cortex and supramarginal gyrus complement each other during rapid action reprogramming

PubMed Central

Hartwigsen, Gesa; Bestmann, Sven; Ward, Nick S.; Woerbel, Saskia; Mastroeni, Claudia; Granert, Oliver; Siebner, Hartwig R.

2013-01-01

The ability to discard a prepared action plan in favor of an alternative action is critical when facing sudden environmental changes. We tested whether the functional contribution of left supramarginal gyrus (SMG) during action reprogramming depends on the functional integrity of left dorsal premotor cortex (PMd). Adopting a dual-site repetitive transcranial magnetic stimulation (rTMS) strategy, we first transiently disrupted PMd with “offline” 1Hz rTMS and then applied focal “online” rTMS to SMG whilst human subjects performed a spatially-precued reaction time task. Effective online rTMS of SMG but not sham rTMS of SMG increased errors when subjects had to reprogram their action in response to an invalid precue regardless of the type of preceding offline rTMS. This suggests that left SMG primarily contributes to the online updating of actions by suppressing invalidly prepared responses. Online rTMS of SMG additionally increased reaction times for correct responses in invalidly-precued trials, but only after offline rTMS of PMd. We infer that offline rTMS caused an additional dysfunction of PMd which increased the functional relevance of SMG for rapid activation of the correct response, and sensitized SMG to the disruptive effects of online rTMS. These results not only provide causal evidence that left PMd and SMG jointly contribute to action reprogramming, but also that the respective functional weight of these areas can be rapidly redistributed. This mechanism might constitute a generic feature of functional networks that allows for rapid functional compensation in response to focal dysfunctions. PMID:23152600

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Understanding direct neuronal reprogramming-from pioneer factors to 3D chromatin.

PubMed

Ninkovic, Jovica; Götz, Magdalena

2018-06-14

Cell replacement therapies aim at reestablishment of neuronal circuits after brain injury, stroke or neurodegeneration. Recently, direct reprogramming of resident glial cells into the affected neuronal subtypes has become a feasible and promising option for central nervous system regeneration. Direct reprogramming relies on the implementation of a new transcriptional program defining the desired neuronal identity in fully differentiated glial cells implying the more or less complete down-regulation of the program for the former identity of the glial cell. Despite the enormous progress achieved in this regard with highly efficient in vivo reprogramming after injury, a number of hurdles still need to be resolved. One way to further improve direct neuronal reprogramming is to understand the molecular hurdles which we discuss with the focus on chromatin states of the starting versus the reprogrammed cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Temporal-Spatial Interaction between Reactive Oxygen Species and Abscisic Acid Regulates Rapid Systemic Acclimation in Plants[W][OPEN

PubMed Central

Suzuki, Nobuhiro; Miller, Gad; Salazar, Carolina; Mondal, Hossain A.; Shulaev, Elena; Cortes, Diego F.; Shuman, Joel L.; Luo, Xiaozhong; Shah, Jyoti; Schlauch, Karen; Shulaev, Vladimir; Mittler, Ron

2013-01-01

Being sessile organisms, plants evolved sophisticated acclimation mechanisms to cope with abiotic challenges in their environment. These are activated at the initial site of exposure to stress, as well as in systemic tissues that have not been subjected to stress (termed systemic acquired acclimation [SAA]). Although SAA is thought to play a key role in plant survival during stress, little is known about the signaling mechanisms underlying it. Here, we report that SAA in plants requires at least two different signals: an autopropagating wave of reactive oxygen species (ROS) that rapidly spreads from the initial site of exposure to the entire plant and a stress-specific signal that conveys abiotic stress specificity. We further demonstrate that SAA is stress specific and that a temporal–spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. In addition, we demonstrate that the rapid ROS signal is associated with the propagation of electric signals in Arabidopsis thaliana. Our findings unravel some of the basic signaling mechanisms underlying SAA in plants and reveal that signaling events and transcriptome and metabolome reprogramming of systemic tissues in response to abiotic stress occur at a much faster rate than previously envisioned. PMID:24038652

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer.

PubMed

Shao, Yaping; Ye, Guozhu; Ren, Shancheng; Piao, Hai-Long; Zhao, Xinjie; Lu, Xin; Wang, Fubo; Ma, Wang; Li, Jia; Yin, Peiyuan; Xia, Tian; Xu, Chuanliang; Yu, Jane J; Sun, Yinghao; Xu, Guowang

2018-07-15

Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies. © 2018 UICC.

The impact of Polycomb group (PcG) and Trithorax group (TrxG) epigenetic factors in plant plasticity.

PubMed

de la Paz Sanchez, Maria; Aceves-García, Pamela; Petrone, Emilio; Steckenborn, Stefan; Vega-León, Rosario; Álvarez-Buylla, Elena R; Garay-Arroyo, Adriana; García-Ponce, Berenice

2015-11-01

Current advances indicate that epigenetic mechanisms play important roles in the regulatory networks involved in plant developmental responses to environmental conditions. Hence, understanding the role of such components becomes crucial to understanding the mechanisms underlying the plasticity and variability of plant traits, and thus the ecology and evolution of plant development. We now know that important components of phenotypic variation may result from heritable and reversible epigenetic mechanisms without genetic alterations. The epigenetic factors Polycomb group (PcG) and Trithorax group (TrxG) are involved in developmental processes that respond to environmental signals, playing important roles in plant plasticity. In this review, we discuss current knowledge of TrxG and PcG functions in different developmental processes in response to internal and environmental cues and we also integrate the emerging evidence concerning their function in plant plasticity. Many such plastic responses rely on meristematic cell behavior, including stem cell niche maintenance, cellular reprogramming, flowering and dormancy as well as stress memory. This information will help to determine how to integrate the role of epigenetic regulation into models of gene regulatory networks, which have mostly included transcriptional interactions underlying various aspects of plant development and its plastic response to environmental conditions. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

PubMed Central

Li, Dong; Secher, Jan O.; Mashayekhi, Kaveh; Nielsen, Troels T.; Hyttel, Poul; Freude, Kristine K.

2017-01-01

ABSTRACT Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig. PMID:28426281

Development of Defective and Persistent Sendai Virus Vector

PubMed Central

Nishimura, Ken; Sano, Masayuki; Ohtaka, Manami; Furuta, Birei; Umemura, Yoko; Nakajima, Yoshiro; Ikehara, Yuzuru; Kobayashi, Toshihiro; Segawa, Hiroaki; Takayasu, Satoko; Sato, Hideyuki; Motomura, Kaori; Uchida, Eriko; Kanayasu-Toyoda, Toshie; Asashima, Makoto; Nakauchi, Hiromitsu; Yamaguchi, Teruhide; Nakanishi, Mahito

2011-01-01

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. PMID:21138846

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

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 cellularmore » 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.« less

Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways

PubMed Central

Ma, Menggen; Song, Mingzhou

2010-01-01

Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)+ for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress. PMID:19517136

From skin biopsy to neurons through a pluripotent intermediate under Good Manufacturing Practice protocols.

PubMed

Karumbayaram, Saravanan; Lee, Peiyee; Azghadi, Soheila F; Cooper, Aaron R; Patterson, Michaela; Kohn, Donald B; Pyle, April; Clark, Amander; Byrne, James; Zack, Jerome A; Plath, Kathrin; Lowry, William E

2012-01-01

The clinical application of human-induced pluripotent stem cells (hiPSCs) requires not only the production of Good Manufacturing Practice-grade (GMP-grade) hiPSCs but also the derivation of specified cell types for transplantation under GMP conditions. Previous reports have suggested that hiPSCs can be produced in the absence of animal-derived reagents (xenobiotics) to ease the transition to production under GMP standards. However, to facilitate the use of hiPSCs in cell-based therapeutics, their progeny should be produced not only in the absence of xenobiotics but also under GMP conditions requiring extensive standardization of protocols, documentation, and reproducibility of methods and product. Here, we present a successful framework to produce GMP-grade derivatives of hiPSCs that are free of xenobiotic exposure from the collection of patient fibroblasts, through reprogramming, maintenance of hiPSCs, identification of reprogramming vector integration sites (nrLAM-PCR), and finally specification and terminal differentiation of clinically relevant cells. Furthermore, we developed a primary set of Standard Operating Procedures for the GMP-grade derivation and differentiation of these cells as a resource to facilitate widespread adoption of these practices.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

PubMed

Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.

PubMed

Mohamed, Tamer M A; Stone, Nicole R; Berry, Emily C; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N; Srivastava, Deepak

2017-03-07

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin. Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. © 2016 American Heart Association, Inc.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming

PubMed Central

Mohamed, Tamer M. A.; Stone, Nicole R.; Berry, Emily C.; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N.; Srivastava, Deepak

2017-01-01

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Methods We screened 5,500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. Results We found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-β and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Conclusions Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. PMID:27834668

"Nutrient-sensing" and self-renewal: O-GlcNAc in a new role.

PubMed

Sharma, Nikita S; Saluja, Ashok K; Banerjee, Sulagna

2018-06-01

Whether embryonic, hematopoietic or cancer stem cells, this metabolic reprogramming is dependent on the nutrient-status and bioenergetic pathways that is influenced by the micro-environmental niches like hypoxia. Thus, the microenvironment plays a vital role in determining the stem cell fate by inducing metabolic reprogramming. Under the influence of the microenvironment, like hypoxia, the stem cells have increased glucose and glutamine uptake which result in activation of hexosamine biosynthesis pathway (HBP) and increased O-GlcNAc Transferase (OGT). The current review is focused on understanding how HBP, a nutrient-sensing pathway (that leads to increased OGT activity) is instrumental in regulating self-renewal not only in embryonic and hematopoietic stem cells (ESC/HSC) but also in cancer stem cells.

Synchrotron Radiation X-Ray Microfluorescence Reveals Polarized Distribution of Atomic Elements during Differentiation of Pluripotent Stem Cells

PubMed Central

Paulsen, Bruna S.; Rehen, Stevens K.

2011-01-01

The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work, we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB) derived from embryonic and induced pluripotent stem (ES and iPS) cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed, indicating that neural differentiation and elemental polarization are strongly correlated. PMID:22195032

Induction of pluripotent stem cells from fibroblast cultures.

PubMed

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

PubMed

Qian, Li; Berry, Emily C; Fu, Ji-dong; Ieda, Masaki; Srivastava, Deepak

2013-06-01

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

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.

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

PubMed

Yoo, Junsang; Chang, Yujung; Kim, Hongwon; Baek, Soonbong; Choi, Hwan; Jeong, Gun-Jae; Shin, Jaein; Kim, Hongnam; Kim, Byung-Soo; Kim, Jongpil

2017-03-01

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.

Role of SIRT6 in Metabolic Reprogramming During Colorectal Carcinoma

DTIC Science & Technology

2015-09-01

Merlos-Suarez, A., Barriga, F. M., Jung , P., Iglesias, M., Cespedes, M. V., Rossell, D., Sevillano, M., Hernando-Momblona, X., da Silva-Diz, V...2011;39:1–13. [129] Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D. Investigating the mechanism for AMP activation of the AMP-activated

Infectious Agents as Stimuli of Trained Innate Immunity.

PubMed

Rusek, Paulina; Wala, Mateusz; Druszczyńska, Magdalena; Fol, Marek

2018-02-03

The discoveries made over the past few years have modified the current immunological paradigm. It turns out that innate immunity cells can mount some kind of immunological memory, similar to that observed in the acquired immunity and corresponding to the defense mechanisms of lower organisms, which increases their resistance to reinfection. This phenomenon is termed trained innate immunity. It is based on epigenetic changes in innate immune cells (monocytes/macrophages, NK cells) after their stimulation with various infectious or non-infectious agents. Many infectious stimuli, including bacterial or fungal cells and their components (LPS, β-glucan, chitin) as well as viruses or even parasites are considered potent inducers of innate immune memory. Epigenetic cell reprogramming occurring at the heart of the phenomenon may provide a useful basis for designing novel prophylactic and therapeutic strategies to prevent and protect against multiple diseases. In this article, we present the current state of art on trained innate immunity occurring as a result of infectious agent induction. Additionally, we discuss the mechanisms of cell reprogramming and the implications for immune response stimulation/manipulation.

Insect-induced effects on plants and possible effectors used by galling and leaf-mining insects to manipulate their host-plant.

PubMed

Giron, David; Huguet, Elisabeth; Stone, Graham N; Body, Mélanie

2016-01-01

Gall-inducing insects are iconic examples in the manipulation and reprogramming of plant development, inducing spectacular morphological and physiological changes of host-plant tissues within which the insect feeds and grows. Despite decades of research, effectors involved in gall induction and basic mechanisms of gall formation remain unknown. Recent research suggests that some aspects of the plant manipulation shown by gall-inducers may be shared with other insect herbivorous life histories. Here, we illustrate similarities and contrasts by reviewing current knowledge of metabolic and morphological effects induced on plants by gall-inducing and leaf-mining insects, and ask whether leaf-miners can also be considered to be plant reprogrammers. We review key plant functions targeted by various plant reprogrammers, including plant-manipulating insects and nematodes, and functionally characterize insect herbivore-derived effectors to provide a broader understanding of possible mechanisms used in host-plant manipulation. Consequences of plant reprogramming in terms of ecology, coevolution and diversification of plant-manipulating insects are also discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging.

PubMed

Sato, Shogo; Solanas, Guiomar; Peixoto, Francisca Oliveira; Bee, Leonardo; Symeonidi, Aikaterini; Schmidt, Mark S; Brenner, Charles; Masri, Selma; Benitah, Salvador Aznar; Sassone-Corsi, Paolo

2017-08-10

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD + -related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging. Copyright © 2017 Elsevier Inc. All rights reserved.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

PubMed

Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

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

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

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A water-responsive shape memory ionomer with permanent shape reconfiguration ability

NASA Astrophysics Data System (ADS)

Bai, Yongkang; Zhang, Jiwen; Tian, Ran; Chen, Xin

2018-04-01

In this work, a water-responsive shape memory ionomer with high toughness was fabricated by cross-linking hyaluronic acid sodium (HAS) and polyvinyl alcohol (PVA) through coordination interactions. The strong Fe3+-carboxyl (from HAS) coordination interactions served as main physical cross-linking points for the performance of water-responsive shape memory, which associated with the flexibility of PVA chain producing excellent mechanical properties of this ionomer. The optimized ionomer was not only able to recover to its original shape within just 22 s by exposing to water, but exhibited high tensile strength up to 35.4 MPa and 4 times higher tractility than the ionomer without PVA. Moreover, the ionomers can be repeatedly programed to various new permanent shapes on demand due to the reversible physical interactions, which still performed complete and fast geometric recovery under stimuli even after 4 cycles of reprograming with 3 different shapes. The excellent shape memory and strong mechanical behaviors make our ionomers significant and promising smart materials for variety of applications.

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

PubMed

Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

2016-04-01

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases. ©AlphaMed Press.

Reprogramming cells with synthetic proteins

PubMed Central

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 vivo counterparts 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

Targeted gene therapy and cell reprogramming in Fanconi anemia.

PubMed

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

2014-06-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications.

PubMed

Di Baldassarre, Angela; Cimetta, Elisa; Bollini, Sveva; Gaggi, Giulia; Ghinassi, Barbara

2018-05-25

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications.

Reprogramming of Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors

PubMed Central

Li, Yang; Cang, Ming; Lee, Andrew Stephen; Zhang, Kehua; Liu, Dongjun

2011-01-01

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes. PMID:21253598

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

PubMed

Cerdá-Esteban, Nuria; Spagnoli, Francesca M

2014-01-01

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

Derivation and Characterization of Induced Pluripotent Stem Cells from Equine Fibroblasts

PubMed Central

Breton, Amandine; Sharma, Ruchi; Diaz, Andrea Catalina; Parham, Alea Gillian; Graham, Audrey; Neil, Claire; Whitelaw, Christopher Bruce; Milne, Elspeth

2013-01-01

Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine. PMID:22897112

The specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.

PubMed

Samson, Mark; Jow, Margaret M; Wong, Catherine C L; Fitzpatrick, Colin; Aslanian, Aaron; Saucedo, Israel; Estrada, Rodrigo; Ito, Takashi; Park, Sung-kyu Robin; Yates, John R; Chu, Diana S

2014-10-01

In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Epigenetic regulation of cell fate reprogramming in aging and disease: A predictive computational model.

PubMed

Folguera-Blasco, Núria; Cuyàs, Elisabet; Menéndez, Javier A; Alarcón, Tomás

2018-03-01

Understanding the control of epigenetic regulation is key to explain and modify the aging process. Because histone-modifying enzymes are sensitive to shifts in availability of cofactors (e.g. metabolites), cellular epigenetic states may be tied to changing conditions associated with cofactor variability. The aim of this study is to analyse the relationships between cofactor fluctuations, epigenetic landscapes, and cell state transitions. Using Approximate Bayesian Computation, we generate an ensemble of epigenetic regulation (ER) systems whose heterogeneity reflects variability in cofactor pools used by histone modifiers. The heterogeneity of epigenetic metabolites, which operates as regulator of the kinetic parameters promoting/preventing histone modifications, stochastically drives phenotypic variability. The ensemble of ER configurations reveals the occurrence of distinct epi-states within the ensemble. Whereas resilient states maintain large epigenetic barriers refractory to reprogramming cellular identity, plastic states lower these barriers, and increase the sensitivity to reprogramming. Moreover, fine-tuning of cofactor levels redirects plastic epigenetic states to re-enter epigenetic resilience, and vice versa. Our ensemble model agrees with a model of metabolism-responsive loss of epigenetic resilience as a cellular aging mechanism. Our findings support the notion that cellular aging, and its reversal, might result from stochastic translation of metabolic inputs into resilient/plastic cell states via ER systems.

Developmental reprogramming of rat GLUT-5 requires de novo mRNA and protein synthesis.

PubMed

Jiang, L; Ferraris, R P

2001-01-01

Fructose transporter (GLUT-5) expression is low in mid-weaning rat small intestine, increases normally after weaning is completed, and can be precociously induced by premature consumption of a high-fructose (HF) diet. In this study, an in vivo perfusion model was used to determine the mechanisms regulating this substrate-induced reprogramming of GLUT-5 development. HF (100 mM) but not high-glucose (HG) perfusion increased GLUT-5 activity and mRNA abundance. In contrast, HF and HG perfusion had no effect on Na(+)-dependent glucose transporter (SGLT-1) expression but increased c-fos and c-jun expression. Intraperitoneal injection of actinomycin D before intestinal perfusion blocked the HF-induced increase in fructose uptake rate and GLUT-5 mRNA abundance. Actinomycin D also prevented the perfusion-induced increase in c-fos and c-jun mRNA abundance but did not affect glucose uptake rate and SGLT-1 mRNA abundance. Cycloheximide blocked the HF-induced increase in fructose uptake rate but not the increase in GLUT-5 mRNA abundance and had no effect on glucose uptake rate and SGLT-1 mRNA abundance. In neonatal rats, the substrate-induced reprogramming of intestinal fructose transport is likely to involve transcription and translation of the GLUT-5 gene.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

24 CFR 1003.701 - Corrective and remedial action.

Code of Federal Regulations, 2011 CFR

2011-04-01

... regulations, including the environmental responsibilities assumed under section 104(g) of title I of the Act... environmental review deficiencies and housing assistance deficiencies), describing the corrective actions to be..., discontinue, or not incur costs for the affected activity; (4) Advise the grantee to reprogram funds from...

24 CFR 1003.701 - Corrective and remedial action.

Code of Federal Regulations, 2010 CFR

2010-04-01

... regulations, including the environmental responsibilities assumed under section 104(g) of title I of the Act... environmental review deficiencies and housing assistance deficiencies), describing the corrective actions to be..., discontinue, or not incur costs for the affected activity; (4) Advise the grantee to reprogram funds from...

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

PubMed

D'Antonio, Matteo; Woodruff, Grace; Nathanson, Jason L; D'Antonio-Chronowska, Agnieszka; Arias, Angelo; Matsui, Hiroko; Williams, Roy; Herrera, Cheryl; Reyna, Sol M; Yeo, Gene W; Goldstein, Lawrence S B; Panopoulos, Athanasia D; Frazer, Kelly A

2017-04-11

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost. Published by Elsevier Inc.

Self-identity reprogrammed by a single residue switch in a cell surface receptor of a social bacterium.

PubMed

Cao, Pengbo; Wall, Daniel

2017-04-04

The ability to recognize close kin confers survival benefits on single-celled microbes that live in complex and changing environments. Microbial kinship detection relies on perceptible cues that reflect relatedness between individuals, although the mechanisms underlying recognition in natural populations remain poorly understood. In myxobacteria, cells identify related individuals through a polymorphic cell surface receptor, TraA. Recognition of compatible receptors leads to outer membrane exchange among clonemates and fitness consequences. Here, we investigated how a single receptor creates a diversity in recognition across myxobacterial populations. We first show that TraA requires its partner protein TraB to function in cell-cell adhesion. Recognition is shown to be traA allele-specific, where polymorphisms within TraA dictate binding selectivity. We reveal the malleability of TraA recognition, and seemingly minor changes to its variable region reprogram recognition outcomes. Strikingly, we identify a single residue (A/P205) as a molecular switch for TraA recognition. Substitutions at this position change the specificity of a diverse panel of environmental TraA receptors. In addition, we engineered a receptor with unique specificity by simply creating an A205P substitution, suggesting that modest changes in TraA can lead to diversification of new recognition groups in nature. We hypothesize that the malleable property of TraA has allowed it to evolve and create social barriers between myxobacterial populations and in turn avoid adverse interactions with relatives.

Glycogen metabolism has a key role in the cancer microenvironment and provides new targets for cancer therapy.

PubMed

Zois, Christos E; Harris, Adrian L

2016-02-01

Metabolic reprogramming is a hallmark of cancer cells and contributes to their adaption within the tumour microenvironment and resistance to anticancer therapies. Recently, glycogen metabolism has become a recognised feature of cancer cells since it is upregulated in many tumour types, suggesting that it is an important aspect of cancer cell pathophysiology. Here, we provide an overview of glycogen metabolism and its regulation, with a focus on its role in metabolic reprogramming of cancer cells under stress conditions such as hypoxia, glucose deprivation and anticancer treatment. The various methods to detect glycogen in tumours in vivo as well as pharmacological modulators of glycogen metabolism are also reviewed. Finally, we discuss the therapeutic value of targeting glycogen metabolism as a strategy for combinational approaches in cancer treatment.

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

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:26712746

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

PubMed

Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

PubMed

Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

2015-06-01

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons. © 2015 by the Society for Experimental Biology and Medicine.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

PubMed

Miyamoto, Kazutaka; Akiyama, Mizuha; Tamura, Fumiya; Isomi, Mari; Yamakawa, Hiroyuki; Sadahiro, Taketaro; Muraoka, Naoto; Kojima, Hidenori; Haginiwa, Sho; Kurotsu, Shota; Tani, Hidenori; Wang, Li; Qian, Li; Inoue, Makoto; Ide, Yoshinori; Kurokawa, Junko; Yamamoto, Tsunehisa; Seki, Tomohisa; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2018-01-04

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-Cell RNA-Seq Reveals Dynamic Early Embryonic-like Programs during Chemical Reprogramming.

PubMed

Zhao, Ting; Fu, Yao; Zhu, Jialiang; Liu, Yifang; Zhang, Qian; Yi, Zexuan; Chen, Shi; Jiao, Zhonggang; Xu, Xiaochan; Xu, Junquan; Duo, Shuguang; Bai, Yun; Tang, Chao; Li, Cheng; Deng, Hongkui

2018-06-12

Chemical reprogramming provides a powerful platform for exploring the molecular dynamics that lead to pluripotency. Although previous studies have uncovered an intermediate extraembryonic endoderm (XEN)-like state during this process, the molecular underpinnings of pluripotency acquisition remain largely undefined. Here, we profile 36,199 single-cell transcriptomes at multiple time points throughout a highly efficient chemical reprogramming system using RNA-sequencing and reconstruct their progression trajectories. Through identifying sequential molecular events, we reveal that the dynamic early embryonic-like programs are key aspects of successful reprogramming from XEN-like state to pluripotency, including the concomitant transcriptomic signatures of two-cell (2C) embryonic-like and early pluripotency programs and the epigenetic signature of notable genome-wide DNA demethylation. Moreover, via enhancing the 2C-like program by fine-tuning chemical treatment, the reprogramming process is remarkably accelerated. Collectively, our findings offer a high-resolution dissection of cell fate dynamics during chemical reprogramming and shed light on mechanistic insights into the nature of induced pluripotency. Copyright © 2018 Elsevier Inc. All rights reserved.

The Histone Acetyltransferase MOF Promotes Induces Generation of Pluripotent Stem Cells.

PubMed

Mu, Xupeng; Yan, Shaohua; Fu, Changhao; Wei, Anhui

2015-08-01

Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

Altered Cellular Metabolism Drives Trained Immunity.

PubMed

Sohrabi, Yahya; Godfrey, Rinesh; Findeisen, Hannes M

2018-04-04

Exposing innate immune cells to an initial insult induces a long-term proinflammatory response due to metabolic and epigenetic alterations which encompass an emerging new concept called trained immunity. Recent studies provide novel insights into mechanisms centered on metabolic reprogramming which induce innate immune memory in hematopoietic stem cells and monocytes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ready, Set, Go: EGFR at the pancreatic cancer starting line

PubMed Central

Perera, Rushika M.; Bardeesy, Nabeel

2012-01-01

Acinar-to-ductal metaplasia (ADM) results from pancreatic injury or KRAS activation, and is an early step in pancreatic cancer progression. In this Cancer Cell issue, Ardito et al. and Navas et al. demonstrate that ADM and KRAS-driven pancreatic cancer require EGFR signaling, revealing a mechanism for developmental reprogramming that primes tumorigenesis. PMID:22975369

Retinoic Acid Inducible Gene 1 Protein (RIG1)-like Receptor Pathway is Required for Efficient Nuclear Reprogramming

PubMed Central

Sayed, Nazish; Ospino, Frank; Himmati, Farhan; Lee, Jieun; Chanda, Palas; Mocarski, Edward S.; Cooke, John P.

2017-01-01

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. PMID:28276156

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

PubMed

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PubMed Central

Winiecka-Klimek, Marta; Smolarz, Maciej; Walczak, Maciej P.; Zieba, Jolanta; Hulas-Bigoszewska, Krystyna; Kmieciak, Blazej; Piaskowski, Sylwester; Rieske, Piotr; Grzela, Dawid P.; Stoczynska-Fidelus, Ewelina

2015-01-01

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols. PMID:26535892

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

PubMed Central

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

2015-01-01

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

Fractional Factorial Design to Investigate Stromal Cell Regulation of Macrophage Plasticity

PubMed Central

Barminko, Jeffrey A.; Nativ, Nir I.; Schloss, Rene; Yarmush, Martin L.

2018-01-01

Understanding the regulatory networks which control specific macrophage phenotypes is essential in identifying novel targets to correct macrophage mediated clinical disorders, often accompanied by inflammatory events. Since mesenchymal stromal cells (MSCs) have been shown to play key roles in regulating immune functions predominantly via a large number of secreted products, we used a fractional factorial approach to streamline experimental evaluation of MSC mediated inflammatory macrophage regulation. Our macrophage reprogramming metrics, human bone marrow MSC attenuation of macrophage pro-inflammatory M1 TNFα secretion and simultaneous enhanced expression of the M2 macrophage marker, CD206, were used as analysis endpoints. Objective evaluation of a panel of MSC secreted mediators indicated that PGE2 alone was sufficient in facilitating macrophage reprogramming, while IL4 only provided partial reprogramming. Inhibiting stromal cell PGE2 secretion with Indomethacin, reversed the macrophage reprogramming effect. PGE2 reprogramming was mediated through the EP4 receptor and indirectly through the CREB signaling pathway as GSK3 specific inhibitors induced M1 macrophages to express CD206. This reprogramming pathway functioned independently from the M1 suppression pathway, as neither CREB nor GSK3 inhibition reversed PGE2 TNF-α secretion attenuation. In conclusion, fractional factorial experimental design identified stromal derived PGE2 as the factor most important in facilitating macrophage reprogramming, albeit via two unique pathways. PMID:24891120

Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

PubMed

Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

2017-09-01

Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

Apparatus, Systems, and Methods for Reconfigurable Robotic Manipulator and Coupling

NASA Technical Reports Server (NTRS)

Chu, Mars Wei (Inventor); Wolfe, Bryn Tyler (Inventor); Burridge, Robert Raven (Inventor)

2016-01-01

A robotic manipulator arm is disclosed. The arm includes joints that are attachable and detachable in a tool-free manner via a universal mating adapter. The universal mating adapter includes a built-in electrical interface for an operative electrical connection upon mechanical coupling of the adapter portions. The universal mating adapter includes mechanisms and the ability to store and communicate parameter configurations such that the joints can be rearranged for immediate operation of the arm without further reprogramming, recompiling, or other software intervention.

Five classic articles in somatic cell reprogramming.

PubMed

Park, In-Hyun

2010-09-01

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.

Lipid Metabolism and Lipid Droplets in Pancreatic Cancer and Stellate Cells

PubMed Central

Sunami, Yoshiaki; Rebelo, Artur; Kleeff, Jörg

2017-01-01

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second deadliest cancer by 2030, and the overall 5-year survival rate is currently less than 7%. Cancer cells frequently exhibit reprogramming of their metabolic activity. It is increasingly recognized that aberrant de novo lipid synthesis and reprogrammed lipid metabolism are both associated with the development and progression of various cancers, including pancreatic cancer. In this review, the current knowledge about lipid metabolism and lipid droplets in pancreatic cancer is discussed. In the first part, molecular mechanisms of lipid metabolism and roles of enzymes involved in lipid metabolism which are relevant for pancreatic cancer research are presented. Further, preclinical studies and clinical trials with drugs/inhibitors targeting cancer metabolic systems in cancer are summarized. An increase of our knowledge in lipid metabolism in pancreatic cancer cells and in tumor stroma is important for developing novel strategies of future individualized therapies of pancreatic cancer. PMID:29295482

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

PubMed

Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes.

PubMed

Li, Qiuhong; Hutchins, Andrew P; Chen, Yong; Li, Shengbiao; Shan, Yongli; Liao, Baojian; Zheng, Dejin; Shi, Xi; Li, Yinxiong; Chan, Wai-Yee; Pan, Guangjin; Wei, Shicheng; Shu, Xiaodong; Pei, Duanqing

2017-05-03

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

Dynamically Cross-linked Elastomer Hybrids with Light-Induced Rapid and Efficient Self-Healing Ability and Reprogrammable Shape Memory Behavior.

PubMed

Bai, Jing; Shi, Zixing

2017-08-16

Pristine carbon nanotubes (CNTs) were activated to exhibit Diels-Alder (DA) reactivity in a polymer matrix, which was modified with monomers containing furan groups. The DA-active polymer matrix was transferred into a dynamic reversible cross-linked inorganic-organic network via a Diels-Alder reaction with CNTs, where pristine CNTs were used as dienophile chemicals and furan-modified SBS acted as the macromolecular diene. In this system, the mechanical properties as well as resilience and solvent resistance were greatly improved even with the presence of only 1 wt % CNTs. Meanwhile, the hybrids retained recyclability and exhibited some smart behaviors, including self-healing and reprogrammable shape memory properties. Furthermore, due to the photothermal effect of CNTs, a retro-Diels-Alder (rDA) reaction was activated under laser irradiation, and healing of a crack on the hybrid surface was demonstrated in approximately 10 s with almost complete recovery of the mechanical properties. Such fast and efficient self-healing performance provides a new concept in designing self-healing nanocomposites with tunable structures and mechanical properties. Furthermore, the DA and rDA reactions could be combined to reprogram the shape memory behavior under laser irradiation or thermal treatment, wherein the temporary shape of the sample could be transferred to a permanent shape via the rDA reaction at high temperature.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Cardiac Biomarkers: a Focus on Cardiac Regeneration

PubMed Central

Forough, Reza; Scarcello, Catherine; Perkins, Matthew

2011-01-01

Historically, biomarkers have been used in two major ways to maintain and improve better health status: first, for diagnostic purposes, and second, as specific targets to treat various diseases. A new era in treatment and even cure for the some diseases using reprograming of somatic cells is about to be born. In this approach, scientists are successfully taking human skin cells (previously considered terminally-differentiated cells) and re-programming them into functional cardiac myocytes and other cell types in vitro. A cell reprograming approach for treatment of cardiovascular diseases will revolutionize the field of medicine and significantly expand the human lifetime. Availability of a comprehensive catalogue for cardiac biomarkers is necessary for developing cell reprograming modalities to treat cardiac diseases, as well as for determining the progress of reprogrammed cells as they become cardiac cells. In this review, we present a comprehensive survey of the cardiac biomarkers currently known. PMID:23074366

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

Marine Corps Budgetary Reprogramming Effectiveness

DTIC Science & Technology

2015-03-01

infrastructure (Appropriations Act of Congress, 2008). The environmental restoration is a transfer account controlled by the DOD. Usually in the case of...at an average just over 11 percent and the Marine Corps encircle the backend of the DOD portion of reprogramming with the Marine Corps reprogramming...blue force tracker (BFT), radio systems, high mobility multipurpose wheeled vehicle (HMMWV), medium tactical vehicle replacement (MTVR), and

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.

Muscle tissue engineering and regeneration through epigenetic reprogramming and scaffold manipulation

PubMed Central

Tan, S.J.; Fang, J.Y.; Wu, Y.; Yang, Z.; Liang, G.; Han, B.

2015-01-01

Efficiency of cell-based tissue engineering and regenerative medicine has been limited by inadequate cellular responses to injury because of aging and poor controllability of cellular interactions. Since cell progression is under a tight epigenetic regulation, epigenetic modulators such as 5-azacytidine (5-Aza-CR) have been utilized to facilitate reprogramming and development of somatic cells in 2-dimensional (2-D) settings. Nonetheless, progression of a specific tissue lineage toward the terminal phenotype is dependent not only on the genomic potential, but also on the microenvironment cues that are beyond the capability of 2-D approaches. In this study, we investigated the combined effects of matrices of variable rigidities and the treatment with the epigenetic modulator 5-Aza-CR on reprogramming adipose-derived stromal cells (ADSCs) into myoblast-like cells by utilizing tunable transglutaminase cross-linked gelatin (Col-Tgel) in vitro and in vivo. Our experiments demonstrated that cellular plasticity and trans-differentiation were significantly enhanced when ADSCs were treated with an effective dose of 5-Aza-CR (1.25 to 12.5 ng) in the optimal myogenic matrix (15 ± 5 kPa Col-Tgel). Our findings suggest that both physical signals and chemical milieu are critical for the regulation of cellular responses. PMID:26548559

Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors.

PubMed

Du, Xuguang; Feng, Tao; Yu, Dawei; Wu, Yuanyuan; Zou, Huiying; Ma, Shuangyu; Feng, Chong; Huang, Yongye; Ouyang, Hongsheng; Hu, Xiaoxiang; Pan, Dengke; Li, Ning; Wu, Sen

2015-11-01

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets. © 2015 AlphaMed Press.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

PubMed

Torres, AnnMarie; Luke, Joanna D; Kullas, Amy L; Kapilashrami, Kanishk; Botbol, Yair; Koller, Antonius; Tonge, Peter J; Chen, Emily I; Macian, Fernando; van der Velden, Adrianus W M

2016-02-01

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming. © Society for Leukocyte Biology.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

PubMed

Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

2014-05-06

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Developmental Origins of Chronic Kidney Disease: Should We Focus on Early Life?

PubMed Central

Tain, You-Lin; Hsu, Chien-Ning

2017-01-01

Chronic kidney disease (CKD) is becoming a global burden, despite recent advances in management. CKD can begin in early life by so-called “developmental programming” or “developmental origins of health and disease” (DOHaD). Early-life insults cause structural and functional changes in the developing kidney, which is called renal programming. Epidemiological and experimental evidence supports the proposition that early-life adverse events lead to renal programming and make subjects vulnerable to developing CKD and its comorbidities in later life. In addition to low nephron endowment, several mechanisms have been proposed for renal programming. The DOHaD concept opens a new window to offset the programming process in early life to prevent the development of adult kidney disease, namely reprogramming. Here, we review the key themes on the developmental origins of CKD. We have particularly focused on the following areas: evidence from human studies support fetal programming of kidney disease; insight from animal models of renal programming; hypothetical mechanisms of renal programming; alterations of renal transcriptome in response to early-life insults; and the application of reprogramming interventions to prevent the programming of kidney disease. PMID:28208659

Patient-Specific Pluripotent Stem Cells in Neurological Diseases

PubMed Central

Durnaoglu, Serpen; Genc, Sermin; Genc, Kursad

2011-01-01

Many human neurological diseases are not currently curable and result in devastating neurologic sequelae. The increasing availability of induced pluripotent stem cells (iPSCs) derived from adult human somatic cells provides new prospects for cellreplacement strategies and disease-related basic research in a broad spectrum of human neurologic diseases. Patient-specific iPSC-based modeling of neurogenetic and neurodegenerative diseases is an emerging efficient tool for in vitro modeling to understand disease and to screen for genes and drugs that modify the disease process. With the exponential increase in iPSC research in recent years, human iPSCs have been successfully derived with different technologies and from various cell types. Although there remain a great deal to learn about patient-specific iPSC safety, the reprogramming mechanisms, better ways to direct a specific reprogramming, ideal cell source for cellular grafts, and the mechanisms by which transplanted stem cells lead to an enhanced functional recovery and structural reorganization, the discovery of the therapeutic potential of iPSCs offers new opportunities for the treatment of incurable neurologic diseases. However, iPSC-based therapeutic strategies need to be thoroughly evaluated in preclinical animal models of neurological diseases before they can be applied in a clinical setting. PMID:21776279

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

PubMed

Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

2016-11-22

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Production of cloned mice from somatic cells, ES cells, and frozen bodies.

PubMed

Wakayama, Sayaka; Mizutani, Eiji; Wakayama, Teruhiko

2010-01-01

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach" and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold. Copyright (c) 2010 Elsevier Inc. All rights reserved.

A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

PubMed

Liu, Hua; Nonomura, Ken-Ichi

2016-10-01

The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis. © 2016. Published by The Company of Biologists Ltd.

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

PubMed Central

2015-01-01

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro “domain swapped” NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis. PMID:24815013

Reprogramming to developmental plasticity in cancer stem cells.

PubMed

O'Brien-Ball, Caitlin; Biddle, Adrian

2017-10-15

During development and throughout adult life, sub-populations of cells exist that exhibit phenotypic plasticity - the ability to differentiate into multiple lineages. This behaviour is important in embryogenesis, is exhibited in a more limited context by adult stem cells, and can be re-activated in cancer cells to drive important processes underlying tumour progression. A well-studied mechanism of phenotypic plasticity is the epithelial-to-mesenchymal transition (EMT), a process which has been observed in both normal and cancerous cells. The epigenetic and metabolic modifications necessary to facilitate phenotypic plasticity are first seen in development and can be re-activated both in normal regeneration and in cancer. In cancer, the re-activation of these mechanisms enables tumour cells to acquire a cancer stem cell (CSC) phenotype with enhanced ability to survive in hostile environments, resist therapeutic interventions, and undergo metastasis. However, recent research has suggested that plasticity may also expose weaknesses in cancer cells that could be exploited for future therapeutic development. More research is needed to identify developmental mechanisms that are active in cancer, so that these may be targeted to reduce tumour growth and metastasis and overcome therapeutic resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis

PubMed Central

Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang

2016-01-01

The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation. PMID:27532329

Bioenergetic reprogramming plasticity under nitrogen depletion by the unicellular green alga Scenedesmus obliquus.

PubMed

Papazi, Aikaterini; Korelidou, Anna; Andronis, Efthimios; Parasyri, Athina; Stamatis, Nikolaos; Kotzabasis, Kiriakos

2018-03-01

Simultaneous nitrogen depletion and 3,4-dichlorophenol addition induce a bioenergetic microalgal reprogramming, through strong Cyt b 6 f synthesis, that quench excess electrons from dichlorophenol's biodegradation to an overactivated photosynthetic electron flow and H 2 -productivity. Cellular energy management includes "rational" planning and operation of energy production and energy consumption units. Microalgae seem to have the ability to calculate their energy reserves and select the most profitable bioenergetic pathways. Under oxygenic mixotrophic conditions, microalgae invest the exogenously supplied carbon source (glucose) to biomass increase. If 3,4-dichlorophenol is added in the culture medium, then glucose is invested more to biodegradation rather than to growth. The biodegradation yield is enhanced in nitrogen-depleted conditions, because of an increase in the starch accumulation and a delay in the establishment of oxygen-depleted conditions in a closed system. In nitrogen-depleted conditions, starch cannot be invested in PSII-dependent and PSII-independent pathways for H 2 -production, mainly because of a strong decrease of the cytochrome b 6 f complex of the photosynthetic electron flow. For this reason, it seems more profitable for the microalga under these conditions to direct the metabolism to the synthesis of lipids as cellular energy reserves. Nitrogen-depleted conditions with exogenously supplied 3,4-dichlorophenol induce reprogramming of the microalgal bioenergetic strategy. Cytochrome b 6 f is strongly synthesized (mainly through catabolism of polyamines) to manage the electron bypass from the dichlorophenol biodegradation procedure to the photosynthetic electron flow (at the level of PQ pool) and consequently through cytochrome b 6 f and PSI to hydrogenase and H 2 -production. All the above showed that the selection of the appropriate cultivation conditions is the key for the manipulation of microalgal bioenergetic strategy that leads to different metabolic products and paves the way for a future microalgal "smart" biotechnology.

Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.

PubMed

Ben Jehuda, Ronen; Shemer, Yuval; Binah, Ofer

2018-06-01

The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.

Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants.

PubMed

Zhang, Zhaoliang; Liao, Hong; Lucas, William J

2014-03-01

As an essential plant macronutrient, the low availability of phosphorus (P) in most soils imposes serious limitation on crop production. Plants have evolved complex responsive and adaptive mechanisms for acquisition, remobilization and recycling of phosphate (Pi) to maintain P homeostasis. Spatio-temporal molecular, physiological, and biochemical Pi deficiency responses developed by plants are the consequence of local and systemic sensing and signaling pathways. Pi deficiency is sensed locally by the root system where hormones serve as important signaling components in terms of developmental reprogramming, leading to changes in root system architecture. Root-to-shoot and shoot-to-root signals, delivered through the xylem and phloem, respectively, involving Pi itself, hormones, miRNAs, mRNAs, and sucrose, serve to coordinate Pi deficiency responses at the whole-plant level. A combination of chromatin remodeling, transcriptional and posttranslational events contribute to globally regulating a wide range of Pi deficiency responses. In this review, recent advances are evaluated in terms of progress toward developing a comprehensive understanding of the molecular events underlying control over P homeostasis. Application of this knowledge, in terms of developing crop plants having enhanced attributes for P use efficiency, is discussed from the perspective of agricultural sustainability in the face of diminishing global P supplies. © 2014 Institute of Botany, Chinese Academy of Sciences.

Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

PubMed

Powell, Anne E; Anderson, Eric C; Davies, Paige S; Silk, Alain D; Pelz, Carl; Impey, Soren; Wong, Melissa H

2011-02-15

The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells. ©2011 AACR.

CD24 tracks divergent pluripotent states in mouse and human cells

PubMed Central

Shakiba, Nika; White, Carl A.; Lipsitz, Yonatan Y.; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M. I.; Puri, Mira C.; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M.; Hanna, Jacob H.; Heck, Albert J. R.; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W

2015-01-01

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835

X Chromosome of female cells shows dynamic changes in status during human somatic cell reprogramming.

PubMed

Kim, Kun-Yong; Hysolli, Eriona; Tanaka, Yoshiaki; Wang, Brandon; Jung, Yong-Wook; Pan, Xinghua; Weissman, Sherman Morton; Park, In-Hyun

2014-06-03

Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.

Reprogramming Human Retinal Pigmented Epithelial Cells to Neurons Using Recombinant Proteins

PubMed Central

Hu, Qirui; Chen, Renwei; Teesalu, Tambet; Ruoslahti, Erkki

2014-01-01

Somatic cells can be reprogrammed to an altered lineage by overexpressing specific transcription factors. To avoid introducing exogenous genetic material into the genome of host cells, cell-penetrating peptides can be used to deliver transcription factors into cells for reprogramming. Position-dependent C-end rule (CendR) cell- and tissue-penetrating peptides provide an alternative to the conventional cell-penetrating peptides, such as polyarginine. In this study, we used a prototypic, already active CendR peptide, RPARPAR, to deliver the transcription factor SOX2 to retinal pigmented epithelial (RPE) cells. We demonstrated that RPE cells can be directly reprogrammed to a neuronal fate by introduction of SOX2. Resulting neuronal cells expressed neuronal marker mRNAs and proteins and downregulated expression of RPE markers. Cells produced extensive neurites and developed synaptic machinery capable of dye uptake after depolarization with potassium. The RPARPAR-mediated delivery of SOX2 alone was sufficient to allow cell lineage reprogramming of both fetal and stem cell-derived RPE cells to become functional neurons. PMID:25298373

Local epigenetic reprograming induced by G-quadruplex ligands

PubMed Central

Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-01-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here we demonstrate that impeding replication by small molecule-mediated stabilisation of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterisation of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps, first loss of H3K4me3 and subsequently DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming. PMID:29064488

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

Local epigenetic reprogramming induced by G-quadruplex ligands

NASA Astrophysics Data System (ADS)

Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-11-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

Ecophysiological responses to excess iron in lowland and upland rice cultivars.

PubMed

Müller, Caroline; Silveira, Solange Ferreira da Silveira; Daloso, Danilo de Menezes; Mendes, Giselle Camargo; Merchant, Andrew; Kuki, Kacilda Naomi; Oliva, Marco Antonio; Loureiro, Marcelo Ehlers; Almeida, Andréa Miyasaka

2017-12-01

Iron (Fe) is an essential nutrient for plants but under high concentrations, such as that found naturally in clay and waterlogged soils, its toxic effect can limit production. This study aimed to investigate the stress tolerance responses exhibited by different rice cultivars. Both lowland and upland cultivars were grown under excess Fe and hypoxic conditions. Lowland cultivars showed higher Fe accumulation in roots compared with upland cultivars suggesting the use of different strategies to tolerate excess Fe. The upland Canastra cultivar displayed a mechanism to limit iron translocation from roots to the shoots, minimizing leaf oxidative stress induced by excess Fe. Conversely, the cultivar Curinga invested in the increase of R1/A, as an alternative drain of electrons. However, the higher iron accumulation in the leaves, was not necessarily related to high toxicity. Nutrient uptake and/or utilization mechanisms in rice plants are in accordance with their needs, which may be defined in relation to crop environments. Alterations in the biochemical parameters of photosynthesis suggest that photosynthesis in rice under excess Fe is primarily limited by biochemical processes rather than by diffusional limitations, particularly in the upland cultivars. The electron transport rate, carboxylation efficiency and electron excess dissipation by photorespiration demonstrate to be good indicators of iron tolerance. Altogether, these chemical and molecular patterns suggests that rice plants grown under excess Fe exhibit gene expression reprogramming in response to the Fe excess per se and in response to changes in photosynthesis and nutrient levels to maintain growth under stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

Does gastric bypass surgery change body weight set point?

PubMed Central

Hao, Z; Mumphrey, M B; Morrison, C D; Münzberg, H; Ye, J; Berthoud, H R

2016-01-01

The relatively stable body weight during adulthood is attributed to a homeostatic regulatory mechanism residing in the brain which uses feedback from the body to control energy intake and expenditure. This mechanism guarantees that if perturbed up or down by design, body weight will return to pre-perturbation levels, defined as the defended level or set point. The fact that weight re-gain is common after dieting suggests that obese subjects defend a higher level of body weight. Thus, the set point for body weight is flexible and likely determined by the complex interaction of genetic, epigenetic and environmental factors. Unlike dieting, bariatric surgery does a much better job in producing sustained suppression of food intake and body weight, and an intensive search for the underlying mechanisms has started. Although one explanation for this lasting effect of particularly Roux-en-Y gastric bypass surgery (RYGB) is simple physical restriction due to the invasive surgery, a more exciting explanation is that the surgery physiologically reprograms the body weight defense mechanism. In this non-systematic review, we present behavioral evidence from our own and other studies that defended body weight is lowered after RYGB and sleeve gastrectomy. After these surgeries, rodents return to their preferred lower body weight if over- or underfed for a period of time, and the ability to drastically increase food intake during the anabolic phase strongly argues against the physical restriction hypothesis. However, the underlying mechanisms remain obscure. Although the mechanism involves central leptin and melanocortin signaling pathways, other peripheral signals such as gut hormones and their neural effector pathways likely contribute. Future research using both targeted and non-targeted ‘omics’ techniques in both humans and rodents as well as modern, genetically targeted, neuronal manipulation techniques in rodents will be necessary. PMID:28685029

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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

PubMed Central

Kelton, William; Waindok, Ann Cathrin; Pesch, Theresa; Pogson, Mark; Ford, Kyle; Parola, Cristina; Reddy, Sai T.

2017-01-01

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation. PMID:28374766

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

The AP-1 transcription factor FOSL1 causes melanocyte reprogramming and transformation.

PubMed

Maurus, K; Hufnagel, A; Geiger, F; Graf, S; Berking, C; Heinemann, A; Paschen, A; Kneitz, S; Stigloher, C; Geissinger, E; Otto, C; Bosserhoff, A; Schartl, M; Meierjohann, S

2017-09-07

The MAPK pathway is activated in the majority of melanomas and is the target of therapeutic approaches. Under normal conditions, it initiates the so-called immediate early response, which encompasses the transient transcription of several genes belonging to the AP-1 transcription factor family. Under pathological conditions, such as continuous MAPK pathway overactivation due to oncogenic alterations occurring in melanoma, these genes are constitutively expressed. The consequences of a permanent expression of these genes are largely unknown. Here, we show that FOSL1 is the main immediate early AP-1 member induced by melanoma oncogenes. We first examined its role in established melanoma cells. We found that FOSL1 is involved in melanoma cell migration as well as cell proliferation and anoikis-independent growth, which is mediated by the gene product of its target gene HMGA1, encoding a multipotent chromatin modifier. As FOSL1 expression is increased in patient melanoma samples compared to nevi, we investigated the effect of enhanced FOSL1 expression on melanocytes. Intriguingly, we found that FOSL1 acts oncogenic and transforms melanocytes, enabling subcutaneous tumor growth in vivo. During the process of transformation, FOSL1 reprogrammed the melanocytes and downregulated MITF in a HMGA1-dependent manner. At the same time, AXL was upregulated, leading to a shift in the MITF/AXL balance. Furthermore, FOSL1 re-enforced pro-tumorigenic transcription factors MYC, E2F3 and AP-1. Together, this led to the enhancement of several growth-promoting processes, such as ribosome biogenesis, cellular detachment and pyrimidine metabolism. Overall, we demonstrate that FOSL1 is a novel reprogramming factor for melanocytes with potent tumor transformation potential.

Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.

PubMed

Lopez Juarez, Alejandro; He, Danyang; Richard Lu, Q

2016-05-01

Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only). Copyright © 2015 Elsevier B.V. All rights reserved.

PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration.

PubMed

Kang, Hojin; Jo, Areum; Kim, Hyein; Khang, Rin; Lee, Ji-Yeong; Kim, Hanna; Park, Chi-Hu; Choi, Jeong-Yun; Lee, Yunjong; Shin, Joo-Ho

2018-01-22

Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H 2 O 2 , and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS. Copyright © 2017 Elsevier Inc. All rights reserved.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

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

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

PubMed Central

Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

2012-01-01

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC. PMID:22905176

MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

PubMed

Singh, Vivek P; Mathison, Megumi; Patel, Vivekkumar; Sanagasetti, Deepthi; Gibson, Brian W; Yang, Jianchang; Rosengart, Todd K

2016-11-10

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

Hallmarks of progeroid syndromes: lessons from mice and reprogrammed cells

PubMed Central

López-Otín, Carlos

2016-01-01

ABSTRACT Ageing is a process that inevitably affects most living organisms and involves the accumulation of macromolecular damage, genomic instability and loss of heterochromatin. Together, these alterations lead to a decline in stem cell function and to a reduced capability to regenerate tissue. In recent years, several genetic pathways and biochemical mechanisms that contribute to physiological ageing have been described, but further research is needed to better characterize this complex biological process. Because premature ageing (progeroid) syndromes, including progeria, mimic many of the characteristics of human ageing, research into these conditions has proven to be very useful not only to identify the underlying causal mechanisms and identify treatments for these pathologies, but also for the study of physiological ageing. In this Review, we summarize the main cellular and animal models used in progeria research, with an emphasis on patient-derived induced pluripotent stem cell models, and define a series of molecular and cellular hallmarks that characterize progeroid syndromes and parallel physiological ageing. Finally, we describe the therapeutic strategies being investigated for the treatment of progeroid syndromes, and their main limitations. PMID:27482812

Epigenetic Therapeutics: A New Weapon in the War Against Cancer.

PubMed

Ahuja, Nita; Sharma, Anup R; Baylin, Stephen B

2016-01-01

The past 15 years have seen an explosion of discoveries related to the cellular regulation of phenotypes through epigenetic mechanisms. This regulation provides a software that packages DNA, without changing the primary base sequence, to establish heritable patterns of gene expression. In cancer, many aspects of the epigenome, controlled by DNA methylation, chromatin, and nucleosome positioning, are altered as one means by which tumor cells maintain abnormal states of self-renewal at the expense of normal maturation. Epigenetic and genetic abnormalities thus collaborate in cancer initiation and progression, as exemplified by frequent mutations in genes encoding proteins that control the epigenome. There is growing emphasis on using epigenetic therapies to reprogram neoplastic cells toward a normal state. Many agents targeting epigenetic regulation are under development and entering clinical trials. This review highlights the promise that epigenetic therapy, often in combination with other therapies, will become a potent tool for cancer management over the next decade.

Synthesis and materialization of a reaction-diffusion French flag pattern

NASA Astrophysics Data System (ADS)

Zadorin, Anton S.; Rondelez, Yannick; Gines, Guillaume; Dilhas, Vadim; Urtel, Georg; Zambrano, Adrian; Galas, Jean-Christophe; Estevez-Torres, André

2017-10-01

During embryo development, patterns of protein concentration appear in response to morphogen gradients. These patterns provide spatial and chemical information that directs the fate of the underlying cells. Here, we emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material. First, we use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chemically distinct zones with sharp borders whose synthetic analogue has remained elusive. A bistable network within a shallow concentration gradient creates an immobile, sharp and long-lasting concentration front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Second, these concentration patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This experimental framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental programme.

Enzyme-free nucleic acid dynamical systems.

PubMed

Srinivas, Niranjan; Parkin, James; Seelig, Georg; Winfree, Erik; Soloveichik, David

2017-12-15

Chemistries exhibiting complex dynamics-from inorganic oscillators to gene regulatory networks-have been long known but either cannot be reprogrammed at will or rely on the sophisticated enzyme chemistry underlying the central dogma. Can simpler molecular mechanisms, designed from scratch, exhibit the same range of behaviors? Abstract chemical reaction networks have been proposed as a programming language for complex dynamics, along with their systematic implementation using short synthetic DNA molecules. We developed this technology for dynamical systems by identifying critical design principles and codifying them into a compiler automating the design process. Using this approach, we built an oscillator containing only DNA components, establishing that Watson-Crick base-pairing interactions alone suffice for complex chemical dynamics and that autonomous molecular systems can be designed via molecular programming languages. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Lethal Dysregulation of Energy Metabolism During Embryonic Vitamin E Deficiency

PubMed Central

McDougall, Melissa; Choi, Jaewoo; Kim, Hye-Kyeong; Bobe, Gerd; Stevens, J. Frederik; Cadenas, Enrique; Tanguay, Robert; Traber, Maret G.

2017-01-01

Vitamin E (α-tocopherol, VitE) was discovered in 1922 for its role in preventing embryonic mortality. We investigated the underlying mechanisms causing lethality using targeted metabolomics analyses of zebrafish VitE-deficient embryos over five days of development, which coincided with their increased morbidity and mortality. VitE deficiency resulted in peroxidation of docosahexaenoic acid (DHA), depleting DHA-containing phospholipids, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation also increased NADPH oxidation, which depleted glucose by shunting it to the pentose phosphate pathway. VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. Thus, embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis with lethal outcomes. PMID:28095320

Development and Function of the Drosophila Tracheal System.

PubMed

Hayashi, Shigeo; Kondo, Takefumi

2018-06-01

The tracheal system of insects is a network of epithelial tubules that functions as a respiratory organ to supply oxygen to various target organs. Target-derived signaling inputs regulate stereotyped modes of cell specification, branching morphogenesis, and collective cell migration in the embryonic stage. In the postembryonic stages, the same set of signaling pathways controls highly plastic regulation of size increase and pattern elaboration during larval stages, and cell proliferation and reprograming during metamorphosis. Tracheal tube morphogenesis is also regulated by physicochemical interaction of the cell and apical extracellular matrix to regulate optimal geometry suitable for air flow. The trachea system senses both the external oxygen level and the metabolic activity of internal organs, and helps organismal adaptation to changes in environmental oxygen level. Cellular and molecular mechanisms underlying the high plasticity of tracheal development and physiology uncovered through research on Drosophila are discussed. Copyright © 2018 by the Genetics Society of America.

Mitochondrial Ion Channels in Cancer Transformation

PubMed Central

Madamba, Stephen M.; Damri, Kevin N.; Dejean, Laurent M.; Peixoto, Pablo M.

2015-01-01

Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process. PMID:26090338

Analysis of Garment Production Methods. Part 1: Comparison of Cost and Production Between a Traditional Bundle System and a Unit Production System Installation

DTIC Science & Technology

1992-01-01

fixed sequence. What made the Eton System different was that I one could very quickly, via a mechanical keypad, reprogram the sequencing of each work...possible was that this group of five workers subconsciously decided that they would themselves fullfil the needs of the increased output because they did

Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells.

PubMed

Wei, Chao; Li, Xia; Zhang, Pengfei; Zhang, Yu; Liu, Tong; Jiang, Shaoshuai; Han, Fei; Zhang, Yunhai

2015-05-01

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos. © 2015 Society for Reproduction and Fertility.

Identification of Sleep-Modulated Pathways Involved in Neuroprotection from Stroke.

PubMed

Pace, Marta; Baracchi, Francesca; Gao, Bo; Bassetti, Claudio

2015-11-01

Sleep deprivation (SDp) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood, we used DNA oligonucleotide microarray analysis to identify the genes and the gene-pathways underlying SDp preconditioning effects. Gene expression was analyzed 3 days after stroke in 4 experimental groups: (i) SDp performed before focal cerebral ischemia (IS) induction; (ii) SDp performed before sham surgery; (iii) IS without SDp; and (iv) sham surgery without SDp. SDp was performed by gentle handling during the last 6 h of the light period, and ischemia was induced immediately after. Basic sleep research laboratory. Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SDp induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover, an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-α-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. Our data indicate that sleep deprivation before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment, whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms. © 2015 Associated Professional Sleep Societies, LLC.

Inducing pluripotency using in vivo gene therapy.

PubMed

Gardlik, Roman

2012-08-01

Since the original study of Takahashi and Yamanaka in 2006 [1], the field of induced pluripotent stem (iPS) cells has made a great progress. Since then, a number of different cell types have been successfully brought to a state of pluripotency and a different set of transcription factors have been reported to be sufficient to reprogram mouse and human somatic cells. Although still with low efficiency of reprogramming, the patient- and disease-specific therapy represents the most valuable outcome of the whole area of iPS cells. Herein we hypothesize that inducing pluripotency in vivo might be an interesting alternative to the standard ex vivo methods. In vivo reprogramming would benefit from the direct administration of the DNA encoding the reprogramming factors into the target tissue/organ of an individual. The target cells that are to be reprogrammed would be transduced in their natural environment that can provide all the necessary molecular and spatial factors that could be missing during ex vivo reprogramming. However, since no available data exist on in vivo induced pluripotency, it is difficult to predict if testing the hypothesis will provide any promising results. On the way to this point, a number of pilot experiments have to be performed to overcome many limitations and pitfalls that are arising from such a risky concept. Safety issues, such as the risk of somatic tumor formation, will likely be the crucial point to focus on during the process of proving the validity of the hypothesis. However, initial data from the study on inflammatory bowel disease suggest that there might be some beneficial effect of in vivo gene therapy based on reprogramming the target cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

PubMed

Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P; Schöler, Hans R; Mitalipov, Shoukhrat

2014-05-01

Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.

CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models

PubMed Central

Zhu, Yu; Knolhoff, Brett L.; Meyer, Melissa A.; Nywening, Timothy M.; West, Brian L.; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C.; DeNardo, David G.

2014-01-01

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC), which not only mediate immune suppression but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive anti-tumor T cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. PMID:25082815

CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models.

PubMed

Zhu, Yu; Knolhoff, Brett L; Meyer, Melissa A; Nywening, Timothy M; West, Brian L; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C; DeNardo, David G

2014-09-15

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages and myeloid-derived suppressor cells, which not only mediate immune suppression, but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive antitumor T-cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T-cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. ©2014 American Association for Cancer Research.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

PubMed

Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming

PubMed Central

Mitani, Yasuyuki; Vagnozzi, Ronald J.; Millay, Douglas P.

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non–muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle–specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.—Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. PMID:27825107

Binary colloidal crystals (BCCs) as a feeder-free system to generate human induced pluripotent stem cells (hiPSCs)

PubMed Central

Wang, Peng-Yuan; Hung, Sandy Shen-Chi; Thissen, Helmut; Kingshott, Peter; Wong, Raymond Ching-Bong

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into any cell type and provide significant advances to cell therapy and regenerative medicine. However, the current protocol for hiPSC generation is relatively inefficient and often results in many partially reprogrammed colonies, which increases the cost and reduces the applicability of hiPSCs. Biophysical stimulation, in particular from tuning cell-surface interactions, can trigger specific cellular responses that could in turn promote the reprogramming process. In this study, human fibroblasts were reprogrammed into hiPSCs using a feeder-free system and episomal vectors using novel substrates based on binary colloidal crystals (BCCs). BCCs are made from two different spherical particle materials (Si and PMMA) ranging in size from nanometers to micrometers that self-assemble into hexagonal close-packed arrays. Our results show that the BCCs, particularly those made from a crystal of 2 μm Si and 0.11 μm PMMA particles (2SiPM) facilitate the reprogramming process and increase the proportion of fully reprogrammed hiPSC colonies, even without a vitronectin coating. Subsequent isolation of clonal hiPSC lines demonstrates that they express pluripotent markers (OCT4 and TRA-1-60). This proof-of-concept study demonstrates that cell reprogramming can be improved on substrates where surface properties are tailored to the application. PMID:27833126

Genome-Wide Transcriptome Analysis Reveals Conserved and Distinct Molecular Mechanisms of Al Resistance in Buckwheat (Fagopyrum esculentum Moench) Leaves

PubMed Central

Chen, Wei Wei; Xu, Jia Meng; Jin, Jian Feng; Lou, He Qiang; Fan, Wei

2017-01-01

Being an Al-accumulating crop, buckwheat detoxifies and tolerates Al not only in roots but also in leaves. While much progress has recently been made toward Al toxicity and resistance mechanisms in roots, little is known about the molecular basis responsible for detoxification and tolerance processes in leaves. Here, we carried out transcriptome analysis of buckwheat leaves in response to Al stress (20 µM, 24 h). We obtained 33,931 unigenes with 26,300 unigenes annotated in the NCBI database, and identified 1063 upregulated and 944 downregulated genes under Al stress. Functional category analysis revealed that genes related to protein translation, processing, degradation and metabolism comprised the biological processes most affected by Al, suggesting that buckwheat leaves maintain flexibility under Al stress by rapidly reprogramming their physiology and metabolism. Analysis of genes related to transcription regulation revealed that a large proportion of chromatin-regulation genes are specifically downregulated by Al stress, whereas transcription factor genes are overwhelmingly upregulated. Furthermore, we identified 78 upregulated and 22 downregulated genes that encode transporters. Intriguingly, only a few genes were overlapped with root Al-regulated transporter genes, which include homologs of AtMATE, ALS1, STAR1, ALS3 and a divalent ion symporter. In addition, we identified a subset of genes involved in development, in which genes associated with flowering regulation were important. Based on these data, it is proposed that buckwheat leaves develop conserved and distinct mechanisms to cope with Al toxicity. PMID:28846612

Genome-Wide Transcriptome Analysis Reveals Conserved and Distinct Molecular Mechanisms of Al Resistance in Buckwheat (Fagopyrum esculentum Moench) Leaves.

PubMed

Chen, Wei Wei; Xu, Jia Meng; Jin, Jian Feng; Lou, He Qiang; Fan, Wei; Yang, Jian Li

2017-08-27

Being an Al-accumulating crop, buckwheat detoxifies and tolerates Al not only in roots but also in leaves. While much progress has recently been made toward Al toxicity and resistance mechanisms in roots, little is known about the molecular basis responsible for detoxification and tolerance processes in leaves. Here, we carried out transcriptome analysis of buckwheat leaves in response to Al stress (20 µM, 24 h). We obtained 33,931 unigenes with 26,300 unigenes annotated in the NCBI database, and identified 1063 upregulated and 944 downregulated genes under Al stress. Functional category analysis revealed that genes related to protein translation, processing, degradation and metabolism comprised the biological processes most affected by Al, suggesting that buckwheat leaves maintain flexibility under Al stress by rapidly reprogramming their physiology and metabolism. Analysis of genes related to transcription regulation revealed that a large proportion of chromatin-regulation genes are specifically downregulated by Al stress, whereas transcription factor genes are overwhelmingly upregulated. Furthermore, we identified 78 upregulated and 22 downregulated genes that encode transporters. Intriguingly, only a few genes were overlapped with root Al-regulated transporter genes, which include homologs of AtMATE , ALS1 , STAR1 , ALS3 and a divalent ion symporter. In addition, we identified a subset of genes involved in development, in which genes associated with flowering regulation were important. Based on these data, it is proposed that buckwheat leaves develop conserved and distinct mechanisms to cope with Al toxicity.

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

PubMed

Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S; Benigni, Ariela

2015-04-14

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

PubMed Central

Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

PubMed

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-04-08

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

PubMed Central

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-01-01

Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments

PubMed Central

Liu, Wei; Glunde, Kristine; Bhujwalla, Zaver M.; Raman, Venu; Sharma, Anit; Phang, James M.

2012-01-01

Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation co-localized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low-glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase (AMPK) activation, but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics. PMID:22609800

Go in for the kill: How plants deploy effector-triggered immunity to combat pathogens. [Corrected].

PubMed

Wu, Liang; Chen, Huan; Curtis, Chad; Fu, Zheng Qing

2014-01-01

Plant resistance (R) proteins perceive specific pathogen effectors from diverse plant pathogens to initiate defense responses, designated effector-triggered immunity (ETI). Plant R proteins are mostly nucleotide binding-leucine rich repeat (NB-LRR) proteins, which recognize pathogen effectors directly or indirectly through sophisticated mechanisms. Upon activation by effector proteins, R proteins elicit robust defense responses, including a rapid burst of reactive oxygen species (ROS), induced biosynthesis and accumulation of salicylic acid (SA), a rapid programmed cell death (PCD) called hypersensitive response (HR) at the infection sites, and increased expression of pathogenesis-related (PR) genes. Initiation of ETI is correlated with a complex network of defense signaling pathways, resulting in defensive cellular responses and large-scale transcriptional reprogramming events. In this review, we highlight important recent advances on the recognition of effectors, regulation and activation of plant R proteins, dynamic intracellular trafficking of R proteins, induction of cell death, and transcriptional reprogramming associated with ETI. Current knowledge gaps and future research directions are also discussed in this review.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

NASA Astrophysics Data System (ADS)

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-11-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria--which models tuberculous granulomas--are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

PubMed

Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

2016-06-10

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

Go in for the kill

PubMed Central

Wu, Liang; Chen, Huan; Curtis, Chad; Fu, Zheng Qing

2014-01-01

Plant resistance (R) proteins perceive specific pathogen effectors from diverse plant pathogens to initiate defense responses, designated effector-triggered immunity (ETI). Plant R proteins are mostly nucleotide binding-leucine rich repeat (NB-LRR) proteins, which recognize pathogen effectors directly or indirectly through sophisticated mechanisms. Upon activation by effector proteins, R proteins elicit robust defense responses, including a rapid burst of reactive oxygen species (ROS), induced biosynthesis and accumulation of salicylic acid (SA), a rapid programmed cell death (PCD) called hypersensitive response (HR) at the infection sites, and increased expression of pathogenesis-related (PR) genes. Initiation of ETI is correlated with a complex network of defense signaling pathways, resulting in defensive cellular responses and large-scale transcriptional reprogramming events. In this review, we highlight important recent advances on the recognition of effectors, regulation and activation of plant R proteins, dynamic intracellular trafficking of R proteins, induction of cell death, and transcriptional reprogramming associated with ETI. Current knowledge gaps and future research directions are also discussed in this review. PMID:25513772

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

PubMed Central

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-01-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria. PMID:27834374

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

PubMed Central

Patsoukis, Nikolaos; Bardhan, Kankana; Chatterjee, Pranam; Sari, Duygu; Liu, Bianling; Bell, Lauren N.; Karoly, Edward D.; Freeman, Gordon J.; Petkova, Victoria; Seth, Pankaj; Li, Lequn; Boussiotis, Vassiliki A.

2015-01-01

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade. PMID:25809635

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis

PubMed Central

Liu, Fabao; Ma, Fengfei; Wang, Yuyuan; Hao, Ling; Zeng, Hao; Jia, Chenxi; Wang, Yidan; Liu, Peng; Ong, Irene M; Li, Baobin; Chen, Guojun; Jiang, Jiaoyang; Gong, Shaoqin; Li, Lingjun; Xu, Wei

2017-01-01

Metabolic reprogramming is a hallmark of cancer. Herein we discovered that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondria calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol 1, 4, 5-trisphosphate receptors (IP3Rs), methylated PKM2 inhibits the influx of calcium from endoplasmic reticulum (ER) to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticle perturbs metabolic energy balance in cancer cells, leading to decrease of cell proliferation, migration, and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to IP3R-dependent mitochondrial functions. PMID:29058718

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

PubMed

Liu, Fabao; Ma, Fengfei; Wang, Yuyuan; Hao, Ling; Zeng, Hao; Jia, Chenxi; Wang, Yidan; Liu, Peng; Ong, Irene M; Li, Baobin; Chen, Guojun; Jiang, Jiaoyang; Gong, Shaoqin; Li, Lingjun; Xu, Wei

2017-11-01

Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP 3 Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP 3 R-dependent mitochondrial functions.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication

PubMed Central

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K.; McCormick, Frank; Graeber, Thomas G.; Christofk, Heather R.

2014-01-01

SUMMARY Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. While recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. PMID:24703700

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

PubMed

Hannemann, Sebastian; Galán, Jorge E

2017-07-01

Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma

PubMed Central

Gilmore, Sarah A.; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-01-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature. PMID:26177267

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma.

PubMed

Gilmore, Sarah A; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-07-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5' leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities

PubMed Central

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behaviour and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatio-temporal gradients of these endogenous transmembrane voltage potentials (Vmem) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells’ programs towards the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in non-neural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. PMID:23897652

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities.

PubMed

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behavior and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatiotemporal gradients of these endogenous transmembrane voltage potentials (Vmem ) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells' programs toward the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in nonneural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. Copyright © 2013 Wiley Periodicals, Inc.

Recent advances in cancer metabolism: a technological perspective.

PubMed

Kang, Yun Pyo; Ward, Nathan P; DeNicola, Gina M

2018-04-16

Cancer cells are highly dependent on metabolic pathways to sustain both their proliferation and adaption to harsh microenvironments. Thus, understanding the metabolic reprogramming that occurs in tumors can provide critical insights for the development of therapies targeting metabolism. In this review, we will discuss recent advancements in metabolomics and other multidisciplinary techniques that have led to the discovery of novel metabolic pathways and mechanisms in diverse cancer types.

Efficacy of Lysine-Specific Demethylase 1 Inhibition in PCa

DTIC Science & Technology

2016-08-01

specific demethylase 1 (LSD1) forms a complex with CoREST and has been well-characterized as an epigenetic regulator that mediates transcriptional...castration-resistant prostate cancer (CRPC), where AR activity persists and its function may be altered by epigenetic mechanisms. Specifically, we...hypothesized that LSD1 activity in PCa may allow tumor cells to epigenetically reprogram the AR cistrome by closing AR binding sites through which AR

Transcriptional control of the autophagy-lysosome system in pancreatic cancer

PubMed Central

Perera, Rushika M.; Stoykova, Svetlana; Nicolay, Brandon N.; Ross, Kenneth N.; Fitamant, Julien; Boukhali, Myriam; Lengrand, Justine; Deshpande, Vikram; Selig, Martin K.; Ferrone, Cristina R.; Settleman, Jeff; Stephanopoulos, Gregory; Dyson, Nicholas J.; Zoncu, Roberto; Ramaswamy, Sridhar; Haas, Wilhelm; Bardeesy, Nabeel

2016-01-01

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers1. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy2–4, a conserved self-degradative process5. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. We now show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family transcription factors. In PDA cells, the MiT/TFE proteins6 – MITF, TFE3 and TFEB – are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular amino acid (AA) pools. These results identify the MiT/TFE transcription factors as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate activation of clearance pathways converging on the lysosome as a novel hallmark of aggressive malignancy. PMID:26168401

Deubiquitylating enzymes as cancer stem cell therapeutics.

PubMed

Haq, Saba; Suresh, Bharathi; Ramakrishna, Suresh

2018-01-01

The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis.

PubMed

Han, Ying; Yue, Lanzhu; Wei, Max; Ren, Xiubao; Shao, Zonghong; Zhang, Ling; Levine, Ross L; Epling-Burnette, Pearlie K

2017-01-01

Myelofibrosis is an indicator of poor prognosis in myeloproliferative neoplasms (MPNs), but the precise mechanism(s) contributing to extracellular matrix remodeling and collagen deposition in the bone marrow (BM) niche remains unanswered. In this study, we isolated mesenchymal stromal cells (MSCs) from mice transplanted with wild-type thrombopoietin receptor (MPLWT) and MPLW515L retroviral-transduced bone marrow. Using MSCs derived from MPLW515-transplant recipients, excessive collagen deposition was maintained in the absence of the virus and neoplastic hematopoietic cells suggested that the MSCs were reprogrammed in vivo. TGFβ production by malignant megakaryocytes plays a definitive role promoting myelofibrosis in MPNs. However, TGFβ was equally expressed by MSCs derived from MPLWT and MPLW515L expressing mice and the addition of neutralizing anti-TGFβ antibody only partially reduced collagen secretion in vitro. Interestingly, profibrotic MSCs displayed increased levels of pSmad3 and pSTAT3 suggesting that inflammatory mediators cooperating with the TGFβ-receptor signaling may maintain the aberrant phenotype ex vivo. FGFb is a known suppressor of TGFβ signaling. Reduced collagen deposition by FGFb-treated MSCs derived from MPLW515L mice suggests that the activating pathway is vulnerable to this suppressive mediator. Therefore, our findings have implications for the future investigation of therapies to reverse fibrosis in MPNs.

Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis

PubMed Central

Wei, Max; Ren, Xiubao; Shao, Zonghong; Zhang, Ling; Levine, Ross L.; Epling-Burnette, Pearlie K.

2017-01-01

Myelofibrosis is an indicator of poor prognosis in myeloproliferative neoplasms (MPNs), but the precise mechanism(s) contributing to extracellular matrix remodeling and collagen deposition in the bone marrow (BM) niche remains unanswered. In this study, we isolated mesenchymal stromal cells (MSCs) from mice transplanted with wild-type thrombopoietin receptor (MPLWT) and MPLW515L retroviral-transduced bone marrow. Using MSCs derived from MPLW515-transplant recipients, excessive collagen deposition was maintained in the absence of the virus and neoplastic hematopoietic cells suggested that the MSCs were reprogrammed in vivo. TGFβ production by malignant megakaryocytes plays a definitive role promoting myelofibrosis in MPNs. However, TGFβ was equally expressed by MSCs derived from MPLWT and MPLW515L expressing mice and the addition of neutralizing anti-TGFβ antibody only partially reduced collagen secretion in vitro. Interestingly, profibrotic MSCs displayed increased levels of pSmad3 and pSTAT3 suggesting that inflammatory mediators cooperating with the TGFβ-receptor signaling may maintain the aberrant phenotype ex vivo. FGFb is a known suppressor of TGFβ signaling. Reduced collagen deposition by FGFb-treated MSCs derived from MPLW515L mice suggests that the activating pathway is vulnerable to this suppressive mediator. Therefore, our findings have implications for the future investigation of therapies to reverse fibrosis in MPNs. PMID:28135282

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.

Budget Execution Exercise for Use in the Financial Management in the Armed Forces Course at the Naval Postgraduate School.

DTIC Science & Technology

1985-12-01

provide additional funding immediately; the program had to be initiated within existing budget constraints by reprogramming funds at the local level. Some...comptroller could recall only one instance during the past two fiscal years wherein COMNAVAIRLANT rejected a request for reprogramming in excess of $50...lists. The first contains items which must be fully funded in the course of the year, even if reprogramming authority must . be requested from the sponsor

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate

PubMed Central

Utsumi, Yoshinori; Utsumi, Chikako; Tanaka, Maho; Ha, Vu The; Matsui, Akihiro; Takahashi, Satoshi; Seki, Motoaki

2017-01-01

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava. PMID:28806727

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

Non-Viral Generation of Marmoset Monkey iPS Cells by a Six-Factor-in-One-Vector Approach

PubMed Central

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings. PMID:25785453

Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

PubMed

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Defined three-dimensional microenvironments boost induction of pluripotency

NASA Astrophysics Data System (ADS)

Caiazzo, Massimiliano; Okawa, Yuya; Ranga, Adrian; Piersigilli, Alessandra; Tabata, Yoji; Lutolf, Matthias P.

2016-03-01

Since the discovery of induced pluripotent stem cells (iPSCs), numerous approaches have been explored to improve the original protocol, which is based on a two-dimensional (2D) cell-culture system. Surprisingly, nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here, we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness, degradability and biochemical composition, we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp.

PubMed

Fuchino, Katsuya; Flärdh, Klas; Dyson, Paul; Ausmees, Nora

2017-01-01

Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genus Streptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth. Polar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension of Streptomyces hyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influences Streptomyces tip growth. We report here that severe hyperosmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip. Copyright © 2016 American Society for Microbiology.

Cloning from stem cells: different lineages, different species, same story.

PubMed

Oback, Björn

2009-01-01

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

PubMed

Hysolli, Eriona; Tanaka, Yoshiaki; Su, Juan; Kim, Kun-Yong; Zhong, Tianyu; Janknecht, Ralf; Zhou, Xiao-Ling; Geng, Lin; Qiu, Caihong; Pan, Xinghua; Jung, Yong-Wook; Cheng, Jijun; Lu, Jun; Zhong, Mei; Weissman, Sherman M; Park, In-Hyun

2016-07-12

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

PubMed

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

PubMed Central

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

2012-01-01

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

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

PubMed

Al-Adsani, Amani; Burke, Zoë D; Eberhard, Daniel; Lawrence, Katherine L; Shen, Chia-Ning; Rustgi, Anil K; Sakaue, Hiroshi; Farrant, J Mark; Tosh, David

2010-10-27

The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

Animal cloning: problems and prospects.

PubMed

Wells, D N

2005-04-01

An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer acceptance. Future improvements in animal cloning will largely arise from a greater understanding of the molecular mechanisms of reprogramming.

Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

PubMed Central

Diaz Perez, Silvia V.; Kim, Rachel; Li, Ziwei; Marquez, Victor E.; Patel, Sanjeet; Plath, Kathrin; Clark, Amander T.

2012-01-01

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. PMID:22058289

Attractor Signaling Models for Discovery of Combinatorial Therapies

DTIC Science & Technology

2013-09-01

year!survival!rate!for!this! disease ! less!than!15%.!Over!the!years,!many!specific!mechanisms!associated!with!drug!resistance!in!lung!cancer! have!been...reprogramming of pluripotent stem cells [4]. More- over, it has been suggested that a biological system in a chronic or therapy-resistant disease state can...designing new therapeutic methods for complex diseases such as can- cer. Even if our knowledge of biological networks is in- complete, fast progress

Programmable shunts and headphones: Are they safe together?

PubMed

Spader, Heather S; Ratanaprasatporn, Linda; Morrison, John F; Grossberg, Jonathan A; Cosgrove, G Rees

2015-10-01

Programmable shunts have a valuable role in the treatment of patients with hydrocephalus, but because a magnet is used to change valve settings, interactions with external magnets may reprogram these shunts. Previous studies have demonstrated the ability of magnetic toys and iPads to erroneously reprogram shunts. Headphones are even more ubiquitous, and they contain an electromagnet for sound projection that sits on the head very close to the shunt valve. This study is the first to look at the magnetic field emissions of headphones and their effect on reprogrammable shunt valves to ascertain whether headphones are safe for patients with these shunts to wear. In this in vitro study of the magnetic properties of headphones and their interactions with 3 different programmable shunts, the authors evaluated Apple earbuds, Beats by Dr. Dre, and Bose QuietComfort Acoustic Noise Cancelling headphones. Each headphone was tested for electromagnetic field emissions using a direct current gaussmeter. The following valves were evaluated: Codman Hakim programmable valve, Medtronic Strata II valve, and Aesculap proGAV. Each valve was tested at distances of 0 to 50 mm (in 5-mm increments) from each headphone. The exposure time at each distance was 1 minute, and 3 trials were performed to confirm results at each valve setting and distance. All 3 headphones generated magnetic fields greater than the respective shunt manufacturer's recommended strength of exposure, but these fields did not persist beyond 5 mm. By 2 cm, the fields levels were below 20 G, well below the Medtronic recommendation of 90 G and the Codman recommendation of 80 G. Because the mechanism for the proGAV is different, there is no recommended gauss level. There was no change in gauss-level emissions by the headphones with changes in frequency and amplitude. Both the Strata and Codman-Hakim valves were reprogrammed by direct contact (distance 0 mm) with the Bose headphones. When a rotation component was added, all 3 headphones reprogrammed the Strata and Codman-Hakim valves at 0 mm. At all distances above 0 mm, the headphones did not affect the shunts. The proGAV valve was not affected by headphones at any distance. Although all the headphones studied generated significant gauss fields at distances less than 5 mm, the programmable valve settings only changed at a distance of 0 mm (i.e., with direct contact). Given the subcutaneous location of the valve, the authors conclude that is highly unlikely that commercially available or customary headphones can contribute to the reprogramming of shunts.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

PubMed

Cieślar-Pobuda, Artur; Knoflach, Viktoria; Ringh, Mikael V; Stark, Joachim; Likus, Wirginia; Siemianowicz, Krzysztof; Ghavami, Saeid; Hudecki, Andrzej; Green, Jason L; Łos, Marek J

2017-07-01

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

PubMed Central

Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

2016-01-01

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

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.

In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells

PubMed Central

Li, Hedong; Chen, Gong

2017-01-01

Neuroregeneration in the central nervous system (CNS) has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart and liver, and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient’s own internal cells for tissue repair. PMID:27537482

A comparison of non-integrating reprogramming methods

PubMed Central

Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

2015-01-01

Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

Coactivator SRC-2–dependent metabolic reprogramming mediates prostate cancer survival and metastasis

PubMed Central

Dasgupta, Subhamoy; Putluri, Nagireddy; Long, Weiwen; Zhang, Bin; Wang, Jianghua; Kaushik, Akash K.; Arnold, James M.; Bhowmik, Salil K.; Stashi, Erin; Brennan, Christine A.; Rajapakshe, Kimal; Coarfa, Cristian; Mitsiades, Nicholas; Ittmann, Michael M.; Chinnaiyan, Arul M.; Sreekumar, Arun; O’Malley, Bert W.

2015-01-01

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2–driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer. PMID:25664849

Specific Cell (Re-)Programming: Approaches and Perspectives.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; David, Robert

2018-01-01

Many disorders are manifested by dysfunction of key cell types or their disturbed integration in complex organs. Thereby, adult organ systems often bear restricted self-renewal potential and are incapable of achieving functional regeneration. This underlies the need for novel strategies in the field of cell (re-)programming-based regenerative medicine as well as for drug development in vitro. The regenerative field has been hampered by restricted availability of adult stem cells and the potentially hazardous features of pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Moreover, ethical concerns and legal restrictions regarding the generation and use of ESCs still exist. The establishment of direct reprogramming protocols for various therapeutically valuable somatic cell types has overcome some of these limitations. Meanwhile, new perspectives for safe and efficient generation of different specified somatic cell types have emerged from numerous approaches relying on exogenous expression of lineage-specific transcription factors, coding and noncoding RNAs, and chemical compounds.It should be of highest priority to develop protocols for the production of mature and physiologically functional cells with properties ideally matching those of their endogenous counterparts. Their availability can bring together basic research, drug screening, safety testing, and ultimately clinical trials. Here, we highlight the remarkable successes in cellular (re-)programming, which have greatly advanced the field of regenerative medicine in recent years. In particular, we review recent progress on the generation of cardiomyocyte subtypes, with a focus on cardiac pacemaker cells. Graphical Abstract.

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

PubMed Central

Zhang, Wei; Wang, Hanning; Zhang, Shaopeng; Zhong, Liang; Wang, Yanliang; Pei, Yangli; Cao, Suying

2018-01-01

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation. PMID:29419748

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.

Investigation of Rett syndrome using pluripotent stem cells.

PubMed

Dajani, Rana; Koo, Sung-Eun; Sullivan, Gareth J; Park, In-Hyun

2013-11-01

Rett syndrome (RTT) is one of most prevalent female neurodevelopmental disorders. De novo mutations in X-linked MECP2 are mostly responsible for RTT. Since the identification of MeCP2 as the underlying cause of RTT, murine models have contributed to understanding the pathophysiology of RTT and function of MeCP2. Reprogramming is a procedure to produce induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors. iPSCs obtain similar features as embryonic stem cells and are capable of self-renewing and differentiating into cells of all three layers. iPSCs have been utilized in modeling human diseases in vitro. Neurons differentiated from RTT-iPSCs showed the recapitulation of RTT phenotypes. Despite the early success, genetic and epigenetic instability upon reprogramming and ensuing maintenance of iPSCs raise concerns in using RTT-iPSCs as an accurate in vitro model. Here, we update the current iPSC-based RTT modeling, and its concerns and challenges. © 2013 Wiley Periodicals, Inc.

Targeting Unique Metabolic Properties of Breast Tumor Initiating Cells

PubMed Central

Feng, Weiguo; Gentles, Andrew; Nair, Ramesh V.; Huang, Min; Lin, Yuan; Lee, Cleo Y.; Cai, Shang; Scheeren, Ferenc A.; Kuo, Angera H.; Diehn, Maximilian

2014-01-01

Normal stem cells from a variety of tissues display unique metabolic properties compared to their more differentiated progeny. However, relatively little is known about heterogeneity of metabolic properties cancer stem cells, also called tumor initiating cells (TICs). In this study we show that, analogous to some normal stem cells, breast TICs have distinct metabolic properties compared to non-tumorigenic cancer cells (NTCs). Transcriptome profiling using RNA-Seq revealed TICs under-express genes involved in mitochondrial biology and mitochondrial oxidative phosphorylation and metabolic analyses revealed TICs preferentially perform glycolysis over oxidative phosphorylation compared to NTCs. Mechanistic analyses demonstrated that decreased expression and activity of pyruvate dehydrogenase (Pdh), a key regulator of oxidative phosphorylation, play a critical role in promoting the pro-glycolytic phenotype of TICs. Metabolic reprogramming via forced activation of Pdh preferentially eliminates TICs both in vitro and in vivo. Our findings reveal unique metabolic properties of TICs and demonstrate that metabolic reprogramming represents a promising strategy for targeting these cells. PMID:24497069

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

PubMed

Michlits, Georg; Hubmann, Maria; Wu, Szu-Hsien; Vainorius, Gintautas; Budusan, Elena; Zhuk, Sergei; Burkard, Thomas R; Novatchkova, Maria; Aichinger, Martin; Lu, Yiqing; Reece-Hoyes, John; Nitsch, Roberto; Schramek, Daniel; Hoepfner, Dominic; Elling, Ulrich

2017-12-01

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

Response (re-)programming in aging: a kinematic analysis.

PubMed

Bellgrove, M A; Phillips, J G; Bradshaw, J L; Gallucci, R M

1998-05-01

Age-related motor slowing may reflect either motor programming deficits, poorer movement execution, or mere strategic preferences for online guidance of movement. We controlled such preferences, limiting the extent to which movements could be programmed. Twenty-four young and 24 older adults performed a line drawing task that allowed movements to be prepared in advance in one case (i.e., cue initially available indicating target location) and not in another (i.e., no cue initially available as to target location). Participants connected large or small targets illuminated by light-emitting diodes upon a graphics tablet that sampled pen tip position at 200 Hz. Older adults had a disproportionate difficulty initiating movement when prevented from programming in advance. Older adults produced slower, less efficient movements, particularly when prevented from programming under greater precision requirements. The slower movements of older adults do not simply reflect a preference for online control, as older adults have less efficient movements when forced to reprogram their movements. Age-related motor slowing kinematically resembles that seen in patients with cerebellar dysfunction.

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

PubMed

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

PubMed

Martinovich, Kelly M; Iosifidis, Thomas; Buckley, Alysia G; Looi, Kevin; Ling, Kak-Ming; Sutanto, Erika N; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Montgomery, Samuel; Lannigan, Francis J; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2017-12-21

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

PubMed

Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

PubMed

Clayton, Z E; Sadeghipour, S; Patel, S

2015-10-15

Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

De novo assembly of plant body plan: a step ahead of Deadpool.

PubMed

Kareem, Abdul; Radhakrishnan, Dhanya; Sondhi, Yash; Aiyaz, Mohammed; Roy, Merin V; Sugimoto, Kaoru; Prasad, Kalika

2016-08-01

While in the movie Deadpool it is possible for a human to recreate an arm from scratch, in reality plants can even surpass that. Not only can they regenerate lost parts, but also the whole plant body can be reborn from a few existing cells. Despite the decades old realization that plant cells possess the ability to regenerate a complete shoot and root system, it is only now that the underlying mechanisms are being unraveled. De novo plant regeneration involves the initiation of regenerative mass, acquisition of the pluripotent state, reconstitution of stem cells and assembly of regulatory interactions. Recent studies have furthered our understanding on the making of a complete plant system in the absence of embryonic positional cues. We review the recent studies probing the molecular mechanisms of de novo plant regeneration in response to external inductive cues and our current knowledge of direct reprogramming of root to shoot and vice versa. We further discuss how de novo regeneration can be exploited to meet the demands of green culture industries and to serve as a general model to address the fundamental questions of regeneration across the plant kingdom.

The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast

PubMed Central

Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José M.; Arroyo, Javier

2017-01-01

Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI) pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies. PMID:29371494

De novo assembly of plant body plan: a step ahead of Deadpool

PubMed Central

Kareem, Abdul; Radhakrishnan, Dhanya; Sondhi, Yash; Aiyaz, Mohammed; Roy, Merin V.; Sugimoto, Kaoru

2016-01-01

Abstract While in the movie Deadpool it is possible for a human to recreate an arm from scratch, in reality plants can even surpass that. Not only can they regenerate lost parts, but also the whole plant body can be reborn from a few existing cells. Despite the decades old realization that plant cells possess the ability to regenerate a complete shoot and root system, it is only now that the underlying mechanisms are being unraveled. De novo plant regeneration involves the initiation of regenerative mass, acquisition of the pluripotent state, reconstitution of stem cells and assembly of regulatory interactions. Recent studies have furthered our understanding on the making of a complete plant system in the absence of embryonic positional cues. We review the recent studies probing the molecular mechanisms of de novo plant regeneration in response to external inductive cues and our current knowledge of direct reprogramming of root to shoot and vice versa. We further discuss how de novo regeneration can be exploited to meet the demands of green culture industries and to serve as a general model to address the fundamental questions of regeneration across the plant kingdom. PMID:27800169

Dnd1-mediated epigenetic control of teratoma formation in mouse

PubMed Central

Gu, Wei; Mochizuki, Kentaro; Otsuka, Kei; Hamada, Ryohei; Takehara, Asuka

2018-01-01

ABSTRACT Spontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1. In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation. PMID:29378702

Conversion of one cell type into another: implications for understanding organ development, pathogenesis of cancer and generating cells for therapy.

PubMed

Corbett, James L; Tosh, David

2014-06-01

Metaplasia is the irreversible conversion of one differentiated cell or tissue type into another. Metaplasia usually occurs in tissues that undergo regeneration, and may, in a pathological context, predispose to an increased risk of disease. Studying the conditions leading to the development of metaplasia is therefore of significant clinical interest. In contrast, transdifferentiation (or cellular reprogramming) is a subset of metaplasia that describes the permanent conversion of one differentiated cell type into another, and generally occurs between cells that arise from neighbouring regions of the same germ layer. Transdifferentiation, although rare, has been shown to occur in Nature. New insights into the signalling pathways involved in normal tissue development may be obtained by investigating the cellular and molecular mechanisms in metaplasia and transdifferentiation, and additional identification of key molecular regulators in transdifferentiation and metaplasia could provide new targets for therapeutic treatment of diseases such as cancer, as well as generating cells for transplantation into patients with degenerative disorders. In the present review, we focus on the transdifferentiation of pancreatic cells into hepatocyte-like cells, the development of Barrett's metaplasia in the oesophagus, and the cellular and molecular mechanisms underlying both processes.

An atlas of the human kinome reveals the mutational landscape underlying dysregulated phosphorylation cascades in cancer

PubMed Central

Olow, Aleksandra; Chen, Zhongzhong; Niedner, R. Hannes; Wolf, Denise M.; Yau, Christina; Pankov, Aleksandr; Lee, Evelyn Pei Rong; Brown-Swigart, Lamorna; van’t Veer, Laura J.; Coppé, Jean-Philippe

2016-01-01

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues, also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular sub-networks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks which drive tumorigenesis. PMID:26921330

Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery

PubMed Central

Singh, Vimal K.; Kalsan, Manisha; Kumar, Neeraj; Saini, Abhishek; Chandra, Ramesh

2015-01-01

Recent progresses in the field of Induced Pluripotent Stem Cells (iPSCs) have opened up many gateways for the research in therapeutics. iPSCs are the cells which are reprogrammed from somatic cells using different transcription factors. iPSCs possess unique properties of self renewal and differentiation to many types of cell lineage. Hence could replace the use of embryonic stem cells (ESC), and may overcome the various ethical issues regarding the use of embryos in research and clinics. Overwhelming responses prompted worldwide by a large number of researchers about the use of iPSCs evoked a large number of peple to establish more authentic methods for iPSC generation. This would require understanding the underlying mechanism in a detailed manner. There have been a large number of reports showing potential role of different molecules as putative regulators of iPSC generating methods. The molecular mechanisms that play role in reprogramming to generate iPSCs from different types of somatic cell sources involves a plethora of molecules including miRNAs, DNA modifying agents (viz. DNA methyl transferases), NANOG, etc. While promising a number of important roles in various clinical/research studies, iPSCs could also be of great use in studying molecular mechanism of many diseases. There are various diseases that have been modeled by uing iPSCs for better understanding of their etiology which maybe further utilized for developing putative treatments for these diseases. In addition, iPSCs are used for the production of patient-specific cells which can be transplanted to the site of injury or the site of tissue degeneration due to various disease conditions. The use of iPSCs may eliminate the chances of immune rejection as patient specific cells may be used for transplantation in various engraftment processes. Moreover, iPSC technology has been employed in various diseases for disease modeling and gene therapy. The technique offers benefits over other similar techniques such as animal models. Many toxic compounds (different chemical compounds, pharmaceutical drugs, other hazardous chemicals, or environmental conditions) which are encountered by humans and newly designed drugs may be evaluated for toxicity and effects by using iPSCs. Thus, the applications of iPSCs in regenerative medicine, disease modeling, and drug discovery are enormous and should be explored in a more comprehensive manner. PMID:25699255

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

PubMed

Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Epigenetic Principles and Mechanisms Underlying Nervous System Functions in Health and Disease

PubMed Central

Mehler, Mark F.

2009-01-01

Epigenetics and epigenomic medicine encompass a new science of brain and behavior that are already providing unique insights into the mechanisms underlying brain development, evolution, neuronal and network plasticity and homeostasis, senescence, the etiology of diverse neurological diseases and neural regenerative processes. Epigenetic mechanisms include DNA methylation, histone modifications, nucleosome repositioning, higher-order chromatin remodeling, non-coding RNAs, and RNA and DNA editing. RNA is centrally involved in directing these processes, implying that the transcriptional state of the cell is the primary determinant of epigenetic memory. This transcriptional state can be modified by internal and external cues affecting gene expression and post-transcriptional processing, but also by RNA and DNA editing through activity-dependent intracellular transport and modulation of RNAs and RNA regulatory supercomplexes, and through trans-neuronal and systemic trafficking of functional RNA subclasses. These integrated processes promote dynamic reorganization of nuclear architecture and the genomic landscape to modulate functional gene and neural networks with complex temporal and spatial trajectories. Epigenetics represents the long sought after molecular interface mediating gene-environmental interactions during critical periods throughout the lifecycle. The discipline of environmental epigenomics has begun to identify combinatorial profiles of environmental stressors modulating the latency, initiation and progression of specific neurological disorders, and more selective disease biomarkers and graded molecular responses to emerging therapeutic interventions. Pharmacoepigenomic therapies will promote accelerated recovery of impaired and seemingly irrevocably lost cognitive, behavioral, sensorimotor functions through epigenetic reprogramming of endogenous regional neural stem cell fate decisions, targeted tissue remodeling and restoration of neural network integrity, plasticity and connectivity. PMID:18940229

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Cell reprogramming: Therapeutic potential and the promise of rejuvenation for the aging brain.

PubMed

López-León, Micaela; Outeiro, Tiago F; Goya, Rodolfo G

2017-11-01

Aging is associated with a progressive increase in the incidence of neurodegenerative diseases, with Alzheimer's (AD) and Parkinson's (PD) disease being the most conspicuous examples. Within this context, the absence of efficacious therapies for most age-related brain pathologies has increased the interest in regenerative medicine. In particular, cell reprogramming technologies have ushered in the era of personalized therapies that not only show a significant potential for the treatment of neurodegenerative diseases but also promise to make biological rejuvenation feasible. We will first review recent evidence supporting the emerging view that aging is a reversible epigenetic phenomenon. Next, we will describe novel reprogramming approaches that overcome some of the intrinsic limitations of conventional induced-pluripotent-stem-cell technology. One of the alternative approaches, lineage reprogramming, consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific transcription factors (TF). Another strategy, termed pluripotency factor-mediated direct reprogramming, uses universal TF to generate epigenetically unstable intermediates able to differentiate into somatic cell types in response to specific differentiation factors. In the third part we will review studies showing the potential relevance of the above approaches for the treatment of AD and PD. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Bershteyn, Marina; Hayashi, Yohei; Desachy, Guillaume; Hsiao, Edward C.; Sami, Salma; Tsang, Kathryn M.; Weiss, Lauren A.; Kriegstein, Arnold R.; Yamanaka, Shinya; Wynshaw-Boris, Anthony

2014-03-01

Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of `chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

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

PubMed Central

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

2012-01-01

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

Utilizing induced pluripotent stem cells (iPSCs) to understand the actions of estrogens in human neurons

PubMed Central

Shum, Carole; Macedo, Sara C.; Warre-Cornish, Katherine; Cocks, Graham; Price, Jack; Srivastava, Deepak P.

2015-01-01

This article is part of a Special Issue “Estradiol and Cognition”. Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic “makeup” of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics. PMID:26143621

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

PubMed Central

Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

Somatic Cell Nuclear Transfer in the Mouse

NASA Astrophysics Data System (ADS)

Kishigami, Satoshi; Wakayama, Teruhiko

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells

PubMed Central

Myers, Samuel A; Peddada, Sailaja; Chatterjee, Nilanjana; Friedrich, Tara; Tomoda, Kiichrio; Krings, Gregor; Thomas, Sean; Maynard, Jason; Broeker, Michael; Thomson, Matthew; Pollard, Katherine; Yamanaka, Shinya; Burlingame, Alma L; Panning, Barbara

2016-01-01

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. DOI: http://dx.doi.org/10.7554/eLife.10647.001 PMID:26949256

Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing.

PubMed

Tidball, Andrew M; Dang, Louis T; Glenn, Trevor W; Kilbane, Emma G; Klarr, Daniel J; Margolis, Joshua L; Uhler, Michael D; Parent, Jack M

2017-09-12

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

PubMed Central

Burkhardt, Matthew F; Martinez, Fernando J; Wright, Sarah; Ramos, Carla; Volfson, Dmitri; Mason, Michael; Garnes, Jeff; Dang, Vu; Lievers, Jeffery; Shoukat-Mumtaz, Uzma; Martinez, Rita; Gai, Hui; Blake, Robert; Vaisberg, Eugeni; Grskovic, Marica; Johnson, Charles; Irion, Stefan; Bright, Jessica; Cooper, Bonnie; Nguyen, Leane; Griswold-Prenner, Irene; Javaherian, Ashkan

2016-01-01

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients’ fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modelling for drug screening. PMID:23891805

YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression.

PubMed

Fitamant, Julien; Kottakis, Filippos; Benhamouche, Samira; Tian, Helen S; Chuvin, Nicolas; Parachoniak, Christine A; Nagle, Julia M; Perera, Rushika M; Lapouge, Marjorie; Deshpande, Vikram; Zhu, Andrew X; Lai, Albert; Min, Bosun; Hoshida, Yujin; Avruch, Joseph; Sia, Daniela; Campreciós, Genís; McClatchey, Andrea I; Llovet, Josep M; Morrissey, David; Raj, Lakshmi; Bardeesy, Nabeel

2015-03-10

Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Emerging Role of Epigenetics in Stroke

PubMed Central

Qureshi, Irfan A.; Mehler, Mark F.

2013-01-01

The transplantation of exogenous stem cells and the activation of endogenous neural stem and progenitor cells (NSPCs) are promising treatments for stroke. These cells can modulate intrinsic responses to ischemic injury and may even integrate directly into damaged neural networks. However, the neuroprotective and neural regenerative effects that can be mediated by these cells are limited and may even be deleterious. Epigenetic reprogramming represents a novel strategy for enhancing the intrinsic potential of the brain to protect and repair itself by modulating pathologic neural gene expression and promoting the recapitulation of seminal neural developmental processes. In fact, recent evidence suggests that emerging epigenetic mechanisms are critical for orchestrating nearly every aspect of neural development and homeostasis, including brain patterning, neural stem cell maintenance, neurogenesis and gliogenesis, neural subtype specification, and synaptic and neural network connectivity and plasticity. In this review, we survey the therapeutic potential of exogenous stem cells and endogenous NSPCs and highlight innovative technological approaches for designing, developing, and delivering epigenetic therapies for targeted reprogramming of endogenous pools of NSPCs, neural cells at risk, and dysfunctional neural networks to rescue and restore neurologic function in the ischemic brain. PMID:21403016

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers.

PubMed

Loft, Anne; Forss, Isabel; Siersbæk, Majken Storm; Schmidt, Søren Fisker; Larsen, Ann-Sofie Bøgh; Madsen, Jesper Grud Skat; Pisani, Didier F; Nielsen, Ronni; Aagaard, Mads Malik; Mathison, Angela; Neville, Matt J; Urrutia, Raul; Karpe, Fredrik; Amri, Ez-Zoubir; Mandrup, Susanne

2015-01-01

Long-term exposure to peroxisome proliferator-activated receptor γ (PPARγ) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARγ binding, leading to the formation of PPARγ "superenhancers" that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARγ and appears to cooperate with PPARγ in a feed-forward manner to activate and maintain the brite-selective gene program. © 2015 Loft et al.; Published by Cold Spring Harbor Laboratory Press.

The function of the Mediator complex in plant immunity.

PubMed

An, Chuanfu; Mou, Zhonglin

2013-03-01

Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.

PubMed

Thai, Minh; Graham, Nicholas A; Braas, Daniel; Nehil, Michael; Komisopoulou, Evangelia; Kurdistani, Siavash K; McCormick, Frank; Graeber, Thomas G; Christofk, Heather R

2014-04-01

Virus infections trigger metabolic changes in host cells that support the bioenergetic and biosynthetic demands of viral replication. Although recent studies have characterized virus-induced changes in host cell metabolism (Munger et al., 2008; Terry et al., 2012), the molecular mechanisms by which viruses reprogram cellular metabolism have remained elusive. Here, we show that the gene product of adenovirus E4ORF1 is necessary for adenovirus-induced upregulation of host cell glucose metabolism and sufficient to promote enhanced glycolysis in cultured epithelial cells by activation of MYC. E4ORF1 localizes to the nucleus, binds to MYC, and enhances MYC binding to glycolytic target genes, resulting in elevated expression of specific glycolytic enzymes. E4ORF1 activation of MYC promotes increased nucleotide biosynthesis from glucose intermediates and enables optimal adenovirus replication in primary lung epithelial cells. Our findings show how a viral protein exploits host cell machinery to reprogram cellular metabolism and promote optimal progeny virion generation. Copyright © 2014 Elsevier Inc. All rights reserved.

Partners in crime: VEGF and IL-4 conscript tumour-promoting macrophages.

PubMed

De Palma, Michele

2012-05-01

Tumour-associated macrophages (TAMs) foster tumour progression by several mechanisms, including the promotion of angiogenesis, tissue remodelling, and immunosuppression. Such pro-tumoural activities are thought to be executed by TAM subtypes that harbour features of alternatively activated (or M2-polarized) macrophages. However, the molecular signals in tumours that induce recruitment and differentiation of M2-like macrophages are not fully defined. In this issue of The Journal of Pathology, Linde et al investigate the role of the tumour-derived cytokines, VEGF and IL-4, in the recruitment and polarization of macrophages in a mouse model of skin cancer. The authors report that while VEGF-A recruits monocytes from the peripheral circulation, IL-4 induces their differentiation into tumour-promoting, M2-like macrophages. IL-4 signalling blockade was sufficient to reprogram TAMs away from the M2-like phenotype and inhibited tumour angiogenesis and growth. This study attests to the potential of reprogramming TAMs to abate their pro-angiogenic and pro-tumoural functions in tumours. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma

PubMed Central

Krah, Nathan M; De La O, Jean-Paul; Swift, Galvin H; Hoang, Chinh Q; Willet, Spencer G; Chen Pan, Fong; Cash, Gabriela M; Bronner, Mary P; Wright, Christopher VE; MacDonald, Raymond J; Murtaugh, L Charles

2015-01-01

Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas. DOI: http://dx.doi.org/10.7554/eLife.07125.001 PMID:26151762

Highly Predictive Reprogramming of tRNA Modifications Is Linked to Selective Expression of Codon-Biased Genes

PubMed Central

2016-01-01

Cells respond to stress by controlling gene expression at several levels, with little known about the role of translation. Here, we demonstrate a coordinated translational stress response system involving stress-specific reprogramming of tRNA wobble modifications that leads to selective translation of codon-biased mRNAs representing different classes of critical response proteins. In budding yeast exposed to four oxidants and five alkylating agents, tRNA modification patterns accurately distinguished among chemically similar stressors, with 14 modified ribonucleosides forming the basis for a data-driven model that predicts toxicant chemistry with >80% sensitivity and specificity. tRNA modification subpatterns also distinguish SN1 from SN2 alkylating agents, with SN2-induced increases in m3C in tRNA mechanistically linked to selective translation of threonine-rich membrane proteins from genes enriched with ACC and ACT degenerate codons for threonine. These results establish tRNA modifications as predictive biomarkers of exposure and illustrate a novel regulatory mechanism for translational control of cell stress response. PMID:25772370

Dax1 and Nanog act in parallel to stabilize mouse embryonic stem cells and induced pluripotency

PubMed Central

Zhang, Junlei; Liu, Gaoke; Ruan, Yan; Wang, Jiali; Zhao, Ke; Wan, Ying; Liu, Bing; Zheng, Hongting; Peng, Tao; Wu, Wei; He, Ping; Hu, Fu-Quan; Jian, Rui

2014-01-01

Nanog expression is heterogeneous and dynamic in embryonic stem cells (ESCs). However, the mechanism for stabilizing pluripotency during the transitions between Nanoghigh and Nanoglow states is not well understood. Here we report that Dax1 acts in parallel with Nanog to regulate mouse ESC (mESCs) identity. Dax1 stable knockdown mESCs are predisposed towards differentiation but do not lose pluripotency, whereas Dax1 overexpression supports LIF-independent self-renewal. Although partially complementary, Dax1 and Nanog function independently and cannot replace one another. They are both required for full reprogramming to induce pluripotency. Importantly, Dax1 is indispensable for self-renewal of Nanoglow mESCs. Moreover, we report that Dax1 prevents extra-embryonic endoderm (ExEn) commitment by directly repressing Gata6 transcription. Dax1 may also mediate inhibition of trophectoderm differentiation independent or as a downstream effector of Oct4. These findings establish a basal role of Dax1 in maintaining pluripotency during the state transition of mESCs and somatic cell reprogramming. PMID:25284313

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation

PubMed Central

Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

2016-01-01

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size. DOI: http://dx.doi.org/10.7554/eLife.13374.001 PMID:27282387

Lactobacillus johnsonii Supplementation Attenuates Respiratory Viral Infection via Metabolic Reprogramming and Immune Cell Modulation

PubMed Central

Fonseca, Wendy; Lucey, Kaitlyn; Jang, Sihyug; Fujimura, Kei E.; Rasky, Andrew; Ting, Hung-An; Petersen, Julia; Johnson, Christine C.; Boushey, Homer A.; Zoratti, Edward; Ownby, Dennis R.; Levine, Albert M.; Bobbit, Kevin R.

2017-01-01

Summary Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii-supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii-supplementation reduced airway Th2 cytokines, dendritic cell function, increased T-regulatory cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone-marrow derived dendritic cells (BMDC) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice, or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice, or with wild-type derived BMDCs pre-treated with plasma from L. johnsonii-supplemented mice, reduced airway pathologic responses to infection in recipient animals. Thus, L. johnsonii-supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function. PMID:28295020

Lactobacillus johnsonii supplementation attenuates respiratory viral infection via metabolic reprogramming and immune cell modulation.

PubMed

Fonseca, W; Lucey, K; Jang, S; Fujimura, K E; Rasky, A; Ting, H-A; Petersen, J; Johnson, C C; Boushey, H A; Zoratti, E; Ownby, D R; Levine, A M; Bobbit, K R; Lynch, S V; Lukacs, N W

2017-11-01

Regulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii supplementation reduced airway T helper type 2 cytokines and dendritic cell (DC) function, increased regulatory T cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone marrow-derived DCs (BMDCs) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T-cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice or with wild-type derived BMDCs pretreated with plasma from L. johnsonii-supplemented mice reduced airway pathological responses to infection in recipient animals. Thus L. johnsonii supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function.

In High-Light-Acclimated Coffee Plants the Metabolic Machinery Is Adjusted to Avoid Oxidative Stress Rather than to Benefit from Extra Light Enhancement in Photosynthetic Yield

PubMed Central

Martins, Samuel C. V.; Araújo, Wagner L.; Tohge, Takayuki; Fernie, Alisdair R.; DaMatta, Fábio M.

2014-01-01

Coffee (Coffea arabica L.) has been traditionally considered as shade-demanding, although it performs well without shade and even out-yields shaded coffee. Here we investigated how coffee plants adjust their metabolic machinery to varying light supply and whether these adjustments are supported by a reprogramming of the primary and secondary metabolism. We demonstrate that coffee plants are able to adjust its metabolic machinery to high light conditions through marked increases in its antioxidant capacity associated with enhanced consumption of reducing equivalents. Photorespiration and alternative pathways are suggested to be key players in reductant-consumption under high light conditions. We also demonstrate that both primary and secondary metabolism undergo extensive reprogramming under high light supply, including depression of the levels of intermediates of the tricarboxylic acid cycle that were accompanied by an up-regulation of a range of amino acids, sugars and sugar alcohols, polyamines and flavonoids such as kaempferol and quercetin derivatives. When taken together, the entire dataset is consistent with these metabolic alterations being primarily associated with oxidative stress avoidance rather than representing adjustments in order to facilitate the plants from utilizing the additional light to improve their photosynthetic performance. PMID:24733284

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

PubMed Central

Subba Rao, Mekala; Sasikala, Mitnala; Reddy, D Nageshwar

2013-01-01

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications. PMID:23801830

Thinking outside the liver: induced pluripotent stem cells for hepatic applications.

PubMed

Subba Rao, Mekala; Sasikala, Mitnala; Nageshwar Reddy, D

2013-06-14

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

Tumor-derived exosomes and their role in cancer progression

PubMed Central

Whiteside, Theresa L

2017-01-01

Tumor cells actively produce, release and utilize exosomes to promote tumor growth. Mechanisms through which tumor-derived exosomes subserve the tumor are under intense investigation. These exosomes are information carriers, conveying molecular and genetic messages from tumor cells to normal or other abnormal cells residing at close or distant sites. Tumor-derived exosomes are found in all body fluids. Upon the contact with target cells, they alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to angiogenesis, thrombosis, metastasis and immunosuppression. Exosomes produced by tumors carry cargos that in part mimic contents of parent cells and are of potential interest as non-invasive biomarkers of cancer. Their role in inhibiting the host antitumor responses and in mediating drug resistance is important for cancer therapy. Tumor-derived exosomes may interfere with cancer immunotherapy, but they also could serve as adjuvants and antigenic components of antitumor vaccines. Their biological roles in cancer development or progression as well as cancer therapy suggest that tumor-derived exosomes are critical components of oncogenic transformation. PMID:27117662

Roles of p53, MYC and HIF-1 in regulating glycolysis - the seventh hallmark of cancer.

PubMed

Yeung, S J; Pan, J; Lee, M-H

2008-12-01

Despite diversity in genetic events in oncogenesis, cancer cells exhibit a common set of functional characteristics. Otto Warburg discovered that cancer cells have consistently higher rates of glycolysis than normal cells. The underlying mechanisms leading to the Warburg phenomenon include mitochondrial changes, upregulation of rate-limiting enzymes/proteins in glycolysis and intracellular pH regulation, hypoxia-induced switch to anaerobic metabolism, and metabolic reprogramming after loss of p53 function. The regulation of energy metabolism can be traced to a "triad" of transcription factors: c-MYC, HIF-1 and p53. Oncogenetic changes involve a nonrandom set of gene deletions, amplifications and mutations, and many oncogenes and tumor suppressor genes cluster along the signaling pathways that regulate c-MYC, HIF-1 and p53. Glycolysis in cancer cells has clinical implications in cancer diagnosis, treatment and interaction with diabetes mellitus. Many drugs targeting energy metabolism are in development. Future advances in technology may bring about transcriptome and metabolome-guided chemotherapy.

Developmental reprogramming by UV-B radiation in plants.

PubMed

Dotto, Marcela; Casati, Paula

2017-11-01

Plants are extremely plastic organisms with the ability to adapt and respond to the changing environmental conditions surrounding them. Sunlight is one of the main resources for plants, both as a primary energy source for photosynthesis and as a stimulus that regulates different aspects of their growth and development. UV-B comprises wavelengths that correspond to a high energy region of the solar spectrum capable of reaching the biosphere, influencing plant growth. It is currently believed that plants are able to acclimate when growing under the influence of this radiation and perceive it as a signal, without stress signs. Nonetheless, many UV-B induced changes are elicited after DNA damage occurs as a consequence of exposure. In this review we focus on the influence of UV-B on leaf, flower and root development and emphasize the limited understanding of the molecular mechanisms for most of this developmental processes affected by UV-B documented over the years of research in this area. Copyright © 2017 Elsevier B.V. All rights reserved.

Epidemiological-molecular evidence of metabolic reprogramming on proliferation, autophagy and cell signaling in pancreas cancer.

PubMed

Søreide, Kjetil; Sund, Malin

2015-01-28

Pancreatic cancer remains one of the deadliest human cancers with little progress made in survival over the past decades, and 5-year survival usually below 5%. Despite this dismal scenario, progresses have been made in understanding of the underlying tumor biology through among other definition of precursor lesions, delineation of molecular pathways, and advances in genome-wide technology. Further, exploring the relationship between epidemiological risk factors involving metabolic features to that of an altered cancer metabolism may provide the foundation for new therapies. Here we explore how nutrients and caloric intake may influence the KRAS-driven ductal carcinogenesis through mediators of metabolic stress, including autophagy in presence of TP53, advanced glycation end products (AGE) and the receptors (RAGE) and ligands (HMGB1), as well as glutamine pathways, among others. Effective understanding the cancer metabolism mechanisms in pancreatic cancer may propose new ways of prevention and treatment. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Molecular Mechanisms Underlying Cardiac Adaptation to Exercise

PubMed Central

Vega, Rick B.; Konhilas, John P.; Kelly, Daniel P.; Leinwand, Leslie A.

2017-01-01

Exercise elicits coordinated multi-organ responses including skeletal muscle, vasculature, heart and lung. In the short term, the output of the heart increases to meet the demand of strenuous exercise. Long term exercise instigates remodeling of the heart including growth and adaptive molecular and cellular re-programming. Signaling pathways such as the insulin-like growth factor 1/PI3K/Akt pathway mediate many of these responses. Exercise-induced, or physiologic, cardiac growth contrasts with growth elicited by pathological stimuli such as hypertension. Comparing the molecular and cellular underpinnings of physiologic and pathologic cardiac growth has unveiled phenotype-specific signaling pathways and transcriptional regulatory programs. Studies suggest that exercise pathways likely antagonize pathological pathways, and exercise training is often recommended for patients with chronic stable heart failure or following myocardial infarction. Herein, we summarize the current understanding of the structural and functional cardiac responses to exercise as well as signaling pathways and downstream effector molecules responsible for these adaptations. PMID:28467921

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.

PubMed

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-02-27

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

PubMed Central

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-01-01

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001 PMID:26920219

Contrasting Transcriptional Programs Control Postharvest Development of Apples (Malus x domestica Borkh.) Submitted to Cold Storage and Ethylene Blockage.

PubMed

Storch, Tatiane Timm; Finatto, Taciane; Bruneau, Maryline; Orsel-Baldwin, Mathilde; Renou, Jean-Pierre; Rombaldi, Cesar Valmor; Quecini, Vera; Laurens, François; Girardi, César Luis

2017-09-06

Apple is commercially important worldwide. Favorable genomic contexts and postharvest technologies allow year-round availability. Although ripening is considered a unidirectional developmental process toward senescence, storage at low temperatures, alone or in combination with ethylene blockage, is effective in preserving apple properties. Quality traits and genome wide expression were integrated to investigate the mechanisms underlying postharvest changes. Development and conservation techniques were responsible for transcriptional reprogramming and distinct programs associated with quality traits. A large portion of the differentially regulated genes constitutes a program involved in ripening and senescence, whereas a smaller module consists of genes associated with reestablishment and maintenance of juvenile traits after harvest. Ethylene inhibition was associated with a reversal of ripening by transcriptional induction of anabolic pathways. Our results demonstrate that the blockage of ethylene perception and signaling leads to upregulation of genes in anabolic pathways. We also associated complex phenotypes to subsets of differentially regulated genes.

[Posture and aging. Current fundamental studies and management concepts].

PubMed

Mourey, F; Camus, A; Pfitzenmeyer, P

2000-02-19

FUNDAMENTAL IMPORTANCE OF POSTURE: In the elderly subject, preservation of posture is fundamental to maintaining functional independence. In recent years, there has been much progress in our understanding of the mechanisms underlying strategies used to control equilibrium in the upright position. Physiological aging, associated with diverse disease states, dangerously alters the postural function, particularly anticipated adjustments which allow an adaptation of posture to movement. CLINICAL ASSESSMENT OF POSTURE: Several tests have been developed to assess posture in the elderly subject, particularly the time it takes to start walking. We selected certain tests which can be used in everyday practice to predict falls: the stance test, the improved Romberg test, the "timed get up and go test", measurement of walking cadence, assessment of balance reactions, sitting-standing and standing-sitting movements and capacity to get up off the floor. PATIENT CARE: Elderly patients with equilibrium disorders can benefit from specific personalized rehabilitation protocols. Different techniques have been developed for multiple afferential stimulation, reprogramming postural strategies, and correcting for deficient motor automatisms.

Environmental Estrogens Differentially Engage the Histone Methyltransferase EZH2 to Increase Risk of Uterine Tumorigenesis

PubMed Central

Greathouse, K. Leigh; Bredfeldt, Tiffany; Everitt, Jeffrey I.; Lin, Kevin; Berry, Tia; Kannan, Kurunthachalam; Mittelstadt, Megan L.; Ho, Shuk-mei; Walker, Cheryl L.

2013-01-01

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol (DES) during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyper-responsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein (GEN), a soy phytoestrogen, and the plasticizer bisphenol A (BPA), differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. While both GEN and BPA induce genomic estrogen receptor (ER) signaling in the developing uterus, only GEN induced PI3K/AKT non-genomic ER signaling to the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2). As a result, this “pre-genomic” signaling phosphorylates and represses EZH2, and reduces levels of H3K27 repressive mark in chromatin. Furthermore, only GEN caused estrogen-responsive genes in the adult myometrium to become hyper-responsive to hormone; estrogen-responsive genes were repressed in BPA exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by GEN promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, while BPA did not. These data demonstrate that environmental estrogens have distinct non-genomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis. PMID:22504913

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

PubMed

Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

2016-01-01

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

PubMed

Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

2014-12-21

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions

PubMed Central

KATAYAMA, Masafumi; HIRAYAMA, Takashi; KIYONO, Tohru; ONUMA, Manabu; TANI, Tetsuya; TAKEDA, Satoru; NISHIMORI, Katsuhiko; FUKUDA, Tomokazu

2017-01-01

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment. PMID:28331164

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Kiyono, Tohru; Onuma, Manabu; Tani, Tetsuya; Takeda, Satoru; Nishimori, Katsuhiko; Fukuda, Tomokazu

2017-06-21

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

PubMed

Ebrahimi, Behnam

2016-01-01

Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

PubMed

West, Jason A; Cook, April; Alver, Burak H; Stadtfeld, Matthias; Deaton, Aimee M; Hochedlinger, Konrad; Park, Peter J; Tolstorukov, Michael Y; Kingston, Robert E

2014-08-27

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive Pluripotency.

PubMed

Adachi, Kenjiro; Kopp, Wolfgang; Wu, Guangming; Heising, Sandra; Greber, Boris; Stehling, Martin; Araúzo-Bravo, Marcos J; Boerno, Stefan T; Timmermann, Bernd; Vingron, Martin; Schöler, Hans R

2018-06-11

Transcription factor (TF)-mediated reprogramming to pluripotency is a slow and inefficient process, because most pluripotency TFs fail to access relevant target sites in a refractory chromatin environment. It is still unclear how TFs actually orchestrate the opening of repressive chromatin during the long latency period of reprogramming. Here, we show that the orphan nuclear receptor Esrrb plays a pioneering role in recruiting the core pluripotency factors Oct4, Sox2, and Nanog to inactive enhancers in closed chromatin during the reprogramming of epiblast stem cells. Esrrb binds to silenced enhancers containing stable nucleosomes and hypermethylated DNA, which are inaccessible to the core factors. Esrrb binding is accompanied by local loss of DNA methylation, LIF-dependent engagement of p300, and nucleosome displacement, leading to the recruitment of core factors within approximately 2 days. These results suggest that TFs can drive rapid remodeling of the local chromatin structure, highlighting the remarkable plasticity of stable epigenetic information. Copyright © 2018 Elsevier Inc. All rights reserved.

Reprogramming Glia Into Neurons in the Peripheral Auditory System as a Solution for Sensorineural Hearing Loss: Lessons From the Central Nervous System

PubMed Central

Meas, Steven J.; Zhang, Chun-Li; Dabdoub, Alain

2018-01-01

Disabling hearing loss affects over 5% of the world’s population and impacts the lives of individuals from all age groups. Within the next three decades, the worldwide incidence of hearing impairment is expected to double. Since a leading cause of hearing loss is the degeneration of primary auditory neurons (PANs), the sensory neurons of the auditory system that receive input from mechanosensory hair cells in the cochlea, it may be possible to restore hearing by regenerating PANs. A direct reprogramming approach can be used to convert the resident spiral ganglion glial cells into induced neurons to restore hearing. This review summarizes recent advances in reprogramming glia in the CNS to suggest future steps for regenerating the peripheral auditory system. In the coming years, direct reprogramming of spiral ganglion glial cells has the potential to become one of the leading biological strategies to treat hearing impairment. PMID:29593497

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

PubMed

Couldrey, Christine; Wells, David N

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.

DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

PubMed Central

Couldrey, Christine; Wells, David N.

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low. PMID:23383311

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

PubMed Central

Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

2014-01-01

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL. PMID:25522233

MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

PubMed Central

Tomasetti, Marco; Amati, Monica; Santarelli, Lory; Neuzil, Jiri

2016-01-01

The process of metabolic re-programing is linked to the activation of oncogenes and/or suppression of tumour suppressor genes, which are regulated by microRNAs (miRNAs). The interplay between oncogenic transformation-driven metabolic re-programming and modulation of aberrant miRNAs further established their critical role in the initiation, promotion and progression of cancer by creating a tumorigenesis-prone microenvironment, thus orchestrating processes of evasion to apoptosis, angiogenesis and invasion/migration, as well metastasis. Given the involvement of miRNAs in tumour development and their global deregulation, they may be perceived as biomarkers in cancer of therapeutic relevance. PMID:27213336

Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

2015-11-01

We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.

Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

PubMed

Burns, David; Blau, Helen M

2014-07-01

Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Metabolic reprogramming in glioblastoma: the influence of cancer metabolism on epigenetics and unanswered questions

PubMed Central

Agnihotri, Sameer; Zadeh, Gelareh

2016-01-01

A defining hallmark of glioblastoma is altered tumor metabolism. The metabolic shift towards aerobic glycolysis with reprogramming of mitochondrial oxidative phosphorylation, regardless of oxygen availability, is a phenomenon known as the Warburg effect. In addition to the Warburg effect, glioblastoma tumor cells also utilize the tricarboxylic acid cycle/oxidative phosphorylation in a different capacity than normal tissue. Altered metabolic enzymes and their metabolites are oncogenic and not simply a product of tumor proliferation. Here we highlight the advantages of why tumor cells, including glioblastoma cells, require metabolic reprogramming and how tumor metabolism can converge on tumor epigenetics and unanswered questions in the field. PMID:26180081

Adaptive mitochondrial reprogramming and resistance to PI3K therapy.

PubMed

Ghosh, Jagadish C; Siegelin, Markus D; Vaira, Valentina; Faversani, Alice; Tavecchio, Michele; Chae, Young Chan; Lisanti, Sofia; Rampini, Paolo; Giroda, Massimo; Caino, M Cecilia; Seo, Jae Ho; Kossenkov, Andrew V; Michalek, Ryan D; Schultz, David C; Bosari, Silvano; Languino, Lucia R; Altieri, Dario C

2015-03-01

Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4-5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial adaptation can dramatically improve on the efficacy of PI3K therapy in the clinic. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Induced pluripotent stem cells as a cellular model for studying Down Syndrome

PubMed Central

Brigida, Anna Lisa; Siniscalco, Dario

2016-01-01

Down Syndrome (DS), or Trisomy 21 Syndrome, is one of the most common genetic diseases. It is a chromosomal abnormality caused by a duplication of chromosome 21. DS patients show the presence of a third copy (or a partial third copy) of chromosome 21 (trisomy), as result of meiotic errors. These patients suffer of many health problems, such as intellectual disability, congenital heart disease, duodenal stenosis, Alzheimer’s disease, leukemia, immune system deficiencies, muscle hypotonia and motor disorders. About one in 1000 babies born each year are affected by DS. Alterations in the dosage of genes located on chromosome 21 (also called HSA21) are responsible for the DS phenotype. However, the molecular pathogenic mechanisms of DS triggering are still not understood; newest evidences suggest the involvement of epigenetic mechanisms. For obvious ethical reasons, studies performed on DS patients, as well as on human trisomic tissues are limited. Some authors have proposed mouse models of this syndrome. However, not all the features of the syndrome are represented. Stem cells are considered the future of molecular and regenerative medicine. Several types of stem cells could provide a valid approach to offer a potential treatment for some untreatable human diseases. Stem cells also represent a valid system to develop new cell-based drugs and/or a model to study molecular disease pathways. Among stem cell types, patient-derived induced pluripotent stem (iPS) cells offer some advantages for cell and tissue replacement, engineering and studying: self-renewal capacity, pluripotency and ease of accessibility to donor tissues. These cells can be reprogrammed into completely different cellular types. They are derived from adult somatic cells via reprogramming with ectopic expression of four transcription factors (Oct3/4, Sox2, c-Myc and Klf4; or, Oct3/4, Sox2, Nanog, and Lin28). By reprogramming cells from DS patients, it is possible to obtain new tissue with the same genetic background, offering a valuable tool for studying this genetic disease and to design customized patient-specific stem cell therapies. PMID:28096629

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

PubMed

Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi

2017-11-01

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

PubMed

Arczewska, Katarzyna D; Tomazella, Gisele G; Lindvall, Jessica M; Kassahun, Henok; Maglioni, Silvia; Torgovnick, Alessandro; Henriksson, Johan; Matilainen, Olli; Marquis, Bryce J; Nelson, Bryant C; Jaruga, Pawel; Babaie, Eshrat; Holmberg, Carina I; Bürglin, Thomas R; Ventura, Natascia; Thiede, Bernd; Nilsen, Hilde

2013-05-01

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Porcine induced pluripotent stem cells produce chimeric offspring.

PubMed

West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

2010-08-01

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels

PubMed Central

Hindle, Allyson G.; Grabek, Katharine R.; Epperson, L. Elaine; Karimpour-Fard, Anis

2014-01-01

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3. PMID:24642758

Authentication in Reprogramming of Sensor Networks for Mote Class Adversaries

DTIC Science & Technology

2006-01-01

based approach. In this paper, we propose a symmetric key-based protocol for authenticating the reprogramming process. Our protocol is based on the ... secret instantiation algorithm, which requires only O(log n) keys to be maintained at each sensor. We integrate this algorithm with the existing

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.

Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallivan, Justin

2012-03-21

Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, CA.

Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

ScienceCinema

Gallivan, Justin

2018-05-01

Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, CA.