DOCSLIB.ORG

Single Transcription Factor Reprogramming of Hair Follicle Dermal Papilla Cells to Induced Pluripotent Stem Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Single Transcription Factor Reprogramming of Hair Follicle Dermal Papilla Cells to Induced Pluripotent Stem Cells EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Single Transcription Factor Reprogramming of Hair Follicle Dermal Papilla Cells to Induced Pluripotent Stem Cells a,b a,b a,c a,b a,c SU-YI TSAI, BRITTA AM BOUWMAN, YEN-SIN ANG, SOO JEONG KIM, DUNG-FANG LEE, IHOR R. a,c a,b,d LEMISCHKA, MICHAEL RENDL aBlack Family Stem Cell Institute; bDepartment of Developmental and Regenerative Biology; cDepartment of Gene and Cell Medicine; dDepartment of Dermatology, Mount Sinai School of Medicine, New York, New York, USA Key Words. Reprogramming • Induced pluripotent stem cells • Dermal papilla cells • Cell fate • Hair follicle ABSTRACT Reprogramming patient-specific somatic cells into induced dermal papilla (DP) cells from hair follicles in the skin pluripotent stem (iPS) cells has great potential to develop express all but one reprogramming factors to show that feasible regenerative therapies. However, several issues need these accessible cells can be reprogrammed into iPS cells to be resolved such as ease, efficiency, and safety of genera- with the single transcription factor Oct4 and without further tion of iPS cells. Many different cell types have been reprog- manipulation. Reprogramming was already achieved after 3 rammed, most conveniently even peripheral blood weeks and with efficiencies similar to other cell types reprog- mononuclear cells. However, they typically require the rammed with four factors. Dermal papilla-derived iPS cells enforced expression of several transcription factors, posing are comparable to embryonic stem cells with respect to mutagenesis risks as exogenous genetic material. To reduce morphology, gene expression, and pluripotency. We con- this risk, iPS cells were previously generated with Oct4 alone clude that DP cells may represent a preferred cell type for from rather inaccessible neural stem cells that endogenously reprogramming accessible cells with less manipulation and express the remaining reprogramming factors and very for ultimately establishing safe conditions in the future by recently from fibroblasts with Oct4 alone in combination replacing Oct4 with small molecules. STEM CELLS with additional small molecules. Here, we exploit that 2011;29:964–971 Disclosure of potential conflicts of interest is found at the end of this article. rammed by either two factors (Oct4 and Klf4) or solely by INTRODUCTION one factor (Oct4) [7]. While this is a major advance toward identifying cells that can be reprogrammed more easily with Embryonic stem (ES) cells are pluripotent cells derived from less manipulation, the difficulty of isolating neural stem cells the inner cell mass of preimplantation stage embryos that can from patients makes these cells an impractical source for give rise to every cell type of the adult [1, 2]. The derivation patient-specific derivation of iPS cells. In efforts to reprogram and functional characterization of human ES cells have made accessible cells, such as skin fibroblasts, with less factors, them a powerful tool for regenerative medicine. However, small molecule activators and inhibitors of epigenetic regula- immune rejection of transplanted cells and ethical concerns tion and signaling pathways were used to replace individual are critical issues in the application of human ES cells for reprogramming transcription factors [8, 9]. Most recently, clinical therapy. Remarkably, the generation of induced pluri- skin-derived and mouse embryonic fibroblasts (MEFs) were potent stem (iPS) cells through direct reprogramming of so- successfully reprogrammed with Oct4 with simultaneous treat- matic cells by defined transcription factors, typically Oct4, ment of additional small molecules [9–11]. Sox2, Klf4, and c-Myc, opened a new avenue to avoid the We have already identified several years ago that skin der- controversy of using human ES cells [3, 4]. On the other mal papilla (DP) cells, a specialized mesenchymal cell type hand, low reprogramming efficiency, random viral integration, required for instructing epithelial stem cells during hair mor- and the use of oncogenic reprogramming factors (Klf4 and c- phogenesis and regeneration [12], also express three of the Myc) remain major challenges in the generation of iPS cells four reprogramming factors, Sox2, c-Myc, and Klf4 [13]. for regenerative medicine applications [5]. Kim et al. [6] Toward the goal of establishing a source of accessible somatic recently demonstrated that mouse neural stem cells, which cells that can be more easily reprogrammed with less exoge- endogenously express Sox2, Klf4, and c-Myc, can be reprog- nous genetic material and without further manipulation, we Author contributions: S.-Y.T.: conception and design, collection of data, data analysis and interpretation, manuscript writing; B.B., Y.- S.A., S.J.K., and D.-F.L.: collection of data, data analysis and interpretation; I.L.: data analysis and interpretation; M.R.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Correspondence: Michael Rendl, M.D., Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, Atran Building AB7-10C, Box 1020, 1428 Madison Avenue, New York, New York 10029, USA. Telephone: 212-241-9593; Fax: 212-860-9279; e-mail: [email protected] Received February 27, 2011; accepted for publication March 30, 2011; first published online in STEM CELLS EXPRESS April 19, 2011. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10. 1002/stem.649 STEM CELLS 2011;29:964–971 www.StemCells.com Tsai, Bouwman, Ang et al. 965 recently demonstrated that DP cells can be reprogrammed of Oct4, Sox2, Klf4, and c-Myc (kindly provided by the into iPS cells by only two exogenous reprogramming factors, Christoph Schaniel/Lemischka Lab) by a standard CaCl2 Oct4 and Klf4 [14]. Interestingly, we further found that DP transfection method. Phoenix cells were cultured at 32C24 cells have a much higher reprogramming efficiency compared hours after transfection and virus supernatants were collected to most other cell types [14]. after additional 24, 48, and 72 hours, passed through 0.22-lm In this study, we succeeded to reprogram accessible DP pore size filters (Millipore, Billerica, MA, www.millipore. cells into iPS cells by transducing the single transcription fac- com) and pelleted at 50,000g for 3 hours. Viruses were resus- tor Oct4 in the absence of additional modifiers of epigenetic pended in 1Â phosphate-buffered saline (PBS) buffer at regulation. Reprogramming DP cells with Oct4 alone occurred 200Â concentration of the original volume and stored as ali- quite rapidly starting at 3 weeks and with efficiencies similar quots at –80C. DP cells were cultured in Amniomax C-100 to most other cell types when they are reprogrammed with all medium (Invitrogen). For infections, freshly sorted DP cells four factors. Dermal papilla–derived one-factor iPS cells fulfil were plated in 12-well plates and cultured until they reached all pluripotency criteria, as determined by pluripotency gene 50%–60% confluence. Oct4 virus and 4 lg/ml of polybrene expression, by their capacity to differentiate into cell types (Sigma) were added to the cells. Infection was enhanced by from all germ layers in vitro and by their robust contribution spinning the plates at 1,100g for 30 minutes. Two rounds of to the development of chimeric mice and to the germline in infections were performed within 24 hours, and 1 day after vivo. This suggests that hair follicle DP cells represent an ac- the second infection the medium was replaced by ES medium. cessible source of cells that can be reprogrammed into pluri- The following day, DP cells were counted and seeded on irra- potent stem cells with a single transcription factor. Therefore, diated MEFs in ES medium without any further selection. DP cells may be a preferred cell type in the future, with Oct4-green fluorescent protein (GFP)-positive colonies were which safe reprogramming may be accomplished without any picked and trypsin dissociated and replated onto irradiated exogenous genetic material. MEFs in 96-well plates. All iPS cell lines and control Ainv15 ES cells (kind gift from Christoph Schaniel/Lemischka Lab) were cultured as previously described [14]. Before RNA and genomic DNA purification, iPS cells were depleted of feeder MATERIALS AND METHODS cells for two passages on 0.1% gelatin (Fisher Scientific Inter- national, Hampton, NH, www.fisherscientific.com). Isolations of DP Cells Determination of iPS Cell Reprogramming For preparations of DP cells, backskins of 4–6 days old Lef1- Efficiency RFP/Oct4-GFP/Rosa26-LacZ pups were floated on dispase (Invitrogen, Carlsbad, CA, www.invitrogen.com) overnight at The efficiency of formation of iPS cells is based on the num- 4C to separate epidermis from dermis. The dermis was then ber of Oct4-GFP-positive iPS cell colonies and the initial cell digested with 0.2% collagenase (Sigma-Aldrich, St. Louis, number of plated DP cells. The percentage of efficiency was MO, www.sigmaaldrich.com) at 37C for 40–60 minutes on determined by dividing the number of GFP-positive colonies an orbital shaker. Intact follicles and dermal cells were centri- by the number of cells before seeding on feeders. fuged at 300g, after which follicles were enriched at low RNA Isolation and Real-Time PCR speed centrifugation twice at 20g. Following trypsinization (0.25% trypsin/0.05 mM EDTA, Invitrogen) at 37C for 5 Total RNAs from FACS-sorted cells, cultured DP cells, ES cells, iPS cells, and all other cells in this study were purified min, single-cell suspensions were strained through 40 lm fil- using the Absolutely RNA Microprep kit (Stratagene, La ters and pelleted at 300g. For DP cell isolations by fluores- cence-activated cell sorting (FACS; BD Vantage and DAKO- Jolla, CA, www.stratagene.com), quantified with the Nano- Drop spectrophotometer (Thermo Scientific, Pittsburgh, PA, Cytomation MoFlo sorters were used), hair cell suspensions were first stained for integrin a-9 (Itga9, 1:100, goat, R&D www.thermo.com), and reverse transcribed (Superscript III First-Strand Synthesis System, Invitrogen) using oligo(dT) Systems, Minneapolis, MN, www.rndsystems.com) and don- key anti-goat APC-conjugated secondary antibodies (Jackson primers.
Load more

Recommended publications
  • Dedifferentiation, Transdifferentiation, and Reprogramming: Future Directions in Regenerative Medicine
    82 Dedifferentiation, Transdifferentiation, and Reprogramming: Future Directions in Regenerative Medicine Cristina Eguizabal, PhD1 Nuria Montserrat, PhD1 Anna Veiga, PhD1,2 Juan Carlos Izpisua Belmonte, PhD1,3 1 Center for Regenerative Medicine in Barcelona Address for correspondence and reprint requests Juan Carlos Izpisua 2 Reproductive Medicine Service, Institut Universitari Dexeus, Belmonte, PhD, Gene Expression Laboratory, The Salk Institute for Barcelona, Spain Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 93027 3 Gene Expression Laboratory, The Salk Institute for Biological Studies, (e-mail: [email protected]). La Jolla, California Semin Reprod Med 2013;31:82–94 Abstract The main goal of regenerative medicine is to replace damaged tissue. To do this it is Keywords necessary to understand in detail the whole regeneration process including differenti- ► regenerative ated cells that can be converted into progenitor cells (dedifferentiation), cells that can medicine switch into another cell type (transdifferentiation), and somatic cells that can be ► stem cells induced to become pluripotent cells (reprogramming). By studying the regenerative ► dedifferentiation processes in both nonmammal and mammal models, natural or artificial processes ► transdifferentiation could underscore the molecular and cellular mechanisms behind these phenomena and ► reprogramming be used to create future regenerative strategies for humans. To understand any regenerative system, it is crucial to find the potency and differentiate and how they can revert to pluri- cellular origins of renewed tissues. Using techniques like potency (reprogramming) or switch lineages (dedifferentia- genetic lineage tracing and single-cell transplantation helps tion and transdifferentiation). to identify the route of regenerative sources. These tools were We synthesize the studies of different model systems to developed first in nonmammal models (flies, amphibians, and highlight recent insights that are integrating the field.
    [Show full text]
  • Cell Reprogramming: Expectations and Challenges for Chemistry in Stem Cell Biology and Regenerative Medicine
    Cell Death and Differentiation (2010) 17, 1230–1237 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd Review Cell reprogramming: expectations and challenges for chemistry in stem cell biology and regenerative medicine L Anastasia*,1,2, G Pelissero2, B Venerando1,2 and G Tettamanti2 The possibility of reprogramming adult somatic cells into pluripotent stem cells (iPSCs) has generated a renewed interest into stem cell research and promises to overcome several key issues, including the ethical concerns of using human embryonic stem cells and the difficulty of obtaining large numbers of adult stem cells (Belmonte et al., Nat Rev Genet, 2009). This approach is also not free from challenges like the mechanism of the reprogramming process, which has yet to be elucidated, and the warranties for safety of generated pluripotent cells, especially in view of their possible therapeutic use. Very recently, several new reprogramming methods have surfaced, which seem to be more appropriate than genetic reprogramming. Particularly, chemically induced pluripotent cells (CiPSs), obtained with recombinant proteins or small synthetic molecules, may represent a valid approach, simpler and possibly safer than the other ones. Cell Death and Differentiation (2010) 17, 1230–1237; doi:10.1038/cdd.2010.14; published online 19 February 2010 Stem cells and cell reprogramming have generated an vitro to large numbers, in contrast to adult stem cells, that enormous interest in the past 2 years, since the generation normally possess very low self-renewal in vitro, and (3) iPSCs of induced pluripotent stem cells (iPSCs) from mouse can be patient customized, because they can be generated embryonic fibroblasts was first reported by Yamanaka and from an easily accessible source, that is fibroblasts, obtain- coworkers in 2006.1,2 In fact, it was shown that the forced able from any individual (Figure 1).
    [Show full text]
  • Rational Optimization of Reprogramming Culture Conditions for the Generation of Induced Pluripotent Stem Cells with Ultra-High Efficiency and Fast Kinetics
    npg Rational medium optimization for efficient reprogramming Cell Research (2011) 21: 884-894. 884 © 2011 IBCB, SIBS, CAS All rights reserved 1001-0602/11 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics Jiekai Chen1, *, Jing Liu1, *, You Chen1, Jiaqi Yang1, Jing Chen1, He Liu1, Xiangjie Zhao1, Kunlun Mo1, Hong Song1, Lin Guo1, Shilong Chu1, Deping Wang1, Ke Ding1, Duanqing Pei1 1Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China The ectopic expression of several transcription factors can restore embryonic cell fate to cultured somatic cells and generate induced pluripotent stem cells (iPSCs), revealing a previously unknown pathway to pluripotency. However, this technology is currently limited by low efficiency, slow kinetics and multi-factorial requirement. Here we show that reprogramming can be improved and dramatically accelerated by optimizing culture conditions. First, we developed an optimized defined medium, iCD1, which allows Oct4/Sox2/Klf4 (OSK)-mediated reprogramming to achieve ultra-high efficiency (~10% at day 8). We also found that this optimized condition renders both Sox2 and Klf4 dispensable, although the elimination of these two factors leads to lower efficiency and slower kinetics. Our studies define a shortened route, both in timing and factor requirement, toward pluripotency. This new paradigm not only provides a rationale to further improve iPSC generation but also simplifies the conceptual understanding of reprogramming by defined factors.
    [Show full text]
  • Experimental and Computational Approaches to Direct Cell Reprogramming: Recent Advancement and Future Challenges
    cells Review Experimental and Computational Approaches to Direct Cell Reprogramming: Recent Advancement and Future Challenges Rihab Gam *, Minkyung Sung and Arun Prasad Pandurangan MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; [email protected] (M.S.); [email protected] (A.P.P.) * Correspondence: [email protected]; Tel.: +44-1223-267820 Received: 6 September 2019; Accepted: 1 October 2019; Published: 2 October 2019 Abstract: The process of direct cell reprogramming, also named transdifferentiation, permits for the conversion of one mature cell type directly into another, without returning to a dedifferentiated state. This makes direct reprogramming a promising approach for the development of several cellular and tissue engineering therapies. To achieve the change in the cell identity, direct reprogramming requires an arsenal of tools that combine experimental and computational techniques. In the recent years, several methods of transdifferentiation have been developed. In this review, we will introduce the concept of direct cell reprogramming and its background, and cover the recent developments in the experimental and computational prediction techniques with their applications. We also discuss the challenges of translating this technology to clinical setting, accompanied with potential solutions. Keywords: transdifferentiation; direct reprogramming; computational biology; regenerative medicine; cell therapy 1. Introduction The epigenetic landscape model proposed by Conrad Waddington in 1957 provided a framework to explain cellular differentiation through epigenetic changes rather than genetic inheritance [1]. In this model, a pluripotent cell takes a complex path defined by ridges and valleys on the developmental landscape to reach a final fully-differentiated and specialized cell (Figure1).
    [Show full text]
  • A New Model for Human Blastocyst Development
    Signal Transduction and Targeted Therapy www.nature.com/sigtrans RESEARCH HIGHLIGHT OPEN Blastoids: a new model for human blastocyst development Heiner Niemann1 and Bob Seamark2 Signal Transduction and Targeted Therapy (2021) 6:239; https://doi.org/10.1038/s41392-021-00663-8 Recently, two research groups report in Nature1,2 the ex-vivo and neither has yet been sufficiently disclosed for full assesment of production of blastocyst-like structures, called blastoids, that the blastoids. Given the specific embyo culture procedures required, exhibit many of the landmarks in human early development found the epigenetic status of the blastoids is of particular interest. in viable blastocysts (Fig. 1). Numerous studies have shown that in vitro culture of early embryos The formation of a blastocyst is a critical step in early embryo can profoundly affect normal patterns of DNA methylation, resulting development denoting a key change from the early cleavage stages in deviations from the physiological gene expression patterns.4 to gastrulation. Typically, the blastocyst, differentiated from the early Following fertilisation, the parental genomes undergo a wave of de- cleavage stages, is a fluid filled vesicular structure comprised of cells and re-methylation, during early embryogenesis, creating the of now, three distinct cell lineages, namely those of the trophoblast, methylation patterns, needed for normal development, through the outer enclosing cell layer, and those of the inner cell mass (ICM) the activation and silencing of specific genes. Typically, global with the hypoblast and epiblast, found in the central fluid filled methylation of the mammalian genome declines to a nadir at the cavity (the blastocoel).
    [Show full text]
  • Improve Induced Pluripotent Stem Cell Generation by Manipulating Epigenetic Statuses
    IMPROVE INDUCED PLURIPOTENT STEM CELL GENERATION BY MANIPULATING EPIGENETIC STATUSES Gaoyang Liang A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics. Chapel Hill 2012 Approved by Yi Zhang, Ph.D. William Marzluff, Ph.D. Larysa Pevny, Ph.D. Lishan Su, Ph.D. Yue Xiong, Ph.D. ©2012 Gaoyang Liang ALL RIGHTS RESERVED ii ABSTRACT GAOYANG LIANG: Improve induced pluripotent stem cell generation by manipulating epigenetic statuses (Under the direction of Dr. Yi Zhang) Reprogramming of somatic cells to a pluripotent state can be achieved by introduction of defined transcription factors. The derived induced pluripotent stem (iPS) cells have molecular profiles and developmental potentials similar to embryonic stem (ES) cells. However, this reprogramming process is inefficient and its underlying mechanisms are poorly understood. To improve the efficiency of iPS cell generation and shed light on its mechanisms, I aimed to identify epigenetic modulations that can enhance iPS cell generation. By studying chemicals modulating epigenetic status and ES-cell enriched epigenetic factors, I demonstrate that butyrate, a histone deacetylase (HDAC) inhibitor, and Kdm2b, a histone demethylase specific for H3 lysine 36 dimethylation (H3K36me2) are capable of facilitating iPS cell generation. Butyrate not only enhances the efficiency of iPS cell generation, but also suppresses the formation of partially reprogrammed cells and transformed cells. The enhancing effect of butyrate on reprogramming appears to depend on c-Myc and occurs early in reprogramming. Genome-wide microarray analysis shows that a set of ES cell-enriched genes are upregulated upon butyrate treatment.
    [Show full text]
  • Epigenetic Reprogramming by TET Enzymes Impacts Co-Transcriptional R-Loops 2 3 João C
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441414; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops 2 3 João C. Sabino1, Madalena R. de Almeida1, Patricia L. Abreu1, Ana M. Ferreira1, Marco M. 4 Domingues1, Nuno C. Santos1, Claus M. Azzalin1, Ana R. Grosso1†, Sérgio F. de Almeida1* 5 6 Affiliations: 1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da 7 Universidade de Lisboa, Lisboa, Portugal. 8 *Correspondence to: [email protected] 9 †Current address: UCIBIO-REQUIMTE, Departamento de Ciências da Vida, Faculdade de 10 Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal 11 12 13 Abstract 14 DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic 15 reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine 16 (5hmC) initiates developmental and cell-type-specific transcriptional programs through 17 mechanisms that include changes in the chromatin structure. Here, we show that the 18 presence of 5hmC in the transcribed DNA promotes the annealing of the nascent RNA to its 19 template DNA strand, leading to the formation of an R-loop. The genome-wide distribution 20 of 5hmC and R-loops show a positive correlation in mouse and human embryonic stem cells 21 and overlap in half of all active genes.
    [Show full text]
  • Working Party of National Coordinators of the Test Guidelines Programme
    Organisation for Economic Co-operation and Development ENV/CBC/TG(2021)31 For Official Use English - Or. English 7 May 2021 ENVIRONMENT DIRECTORATE CHEMICALS AND BIOTECHNOLOGY COMMITTEE Working Party of National Coordinators of the Test Guidelines Programme Adverse Outcome Pathway 220 on Cyp2E1 Activation Leading to Liver Cancer Nathalie Delrue, Administrator [email protected] JT03476102 OFDE This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to the delimitation of international frontiers and boundaries and to the name of any territory, city or area. 2 ENV/CBC/TG(2021)31 Foreword This Adverse Outcome Pathway (AOP) on Cyp2E1 Activation Leading to Liver Cancer, has been developed under the auspices of the OECD AOP Development Programme, overseen by the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST), which is an advisory group under the Working Party of the National Coordinators of the Test Guidelines Programme (WNT). The AOP has been reviewed internally by the EAGMST, externally by experts nominated by the WNT, and has been endorsed by the WNT and the Working Party on Hazard Assessment (WPHA) on XXX. Through endorsement of this AOP, the WNT and the WPHA express confidence in the scientific review process that the AOP has undergone and accept the recommendation of the EAGMST that the AOP be disseminated publicly. Endorsement does not necessarily indicate that the AOP is now considered a tool for direct regulatory application. The OECD's Chemicals and Biotechnology Committee agreed to declassification of this AOP on XXX.
    [Show full text]
  • Reprogramming of the Paternal Genome Upon Fertilization Involves Genome-Wide Oxidation of 5-Methylcytosine
    Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine Khursheed Iqbala,1, Seung-Gi Jinb,1, Gerd P. Pfeiferb,2, and Piroska E. Szabóa,2 Departments of aMolecular and Cellular Biology and bCancer Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 Edited by Peter A. Jones, University of Southern California, Los Angeles, CA, and accepted by the Editorial Board January 28, 2011 (received for review September 17, 2010) Genome-wide erasure of DNA cytosine-5 methylation has been direct removal of 5mC by DNA glycosylase activity has been reported to occur along the paternal pronucleus in fertilized oocytes identified (27, 28), but these proteins do not have mammalian in an apparently replication-independent manner, but the mecha- homologs. Furthermore, it was reported that the protein nism of this reprogramming process has remained enigmatic. Re- GADD45A promotes demethylation of CpG-methylated DNA cently, considerable amounts of 5-hydroxymethylcytosine (5hmC), (29), perhaps in conjunction with excision repair activities (23, most likely derived from enzymatic oxidation of 5-methylcytosine 30). However, a role of GADD45A in DNA demethylation has (5mC) by TET proteins, have been detected in certain mammalian fi fi tissues. 5hmC has been proposed as a potential intermediate in not been con rmed (31, 32). Speci cally addressing active de- active DNA demethylation. Here, we show that in advanced pro- methylation of the paternal genome in zygotes, Okada et al. have nuclear-stage zygotes the paternal pronucleus contains substantial used a siRNA knockdown strategy in oocytes followed by in- amounts of 5hmC but lacks 5mC.
    [Show full text]
  • Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos
    Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 8-2019 Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos Laura A. Moley Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Animal Sciences Commons Recommended Citation Moley, Laura A., "Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos" (2019). All Graduate Theses and Dissertations. 7571. https://digitalcommons.usu.edu/etd/7571 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. EPIGENETIC REPROGRAMMING, APOPTOSIS, AND DEVELOPMENTAL COMPETENCE IN CLONED EMBRYOS by Laura A. Moley A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Animal, Dairy, and Veterinary Sciences Approved: ______________________ ____________________ S. Clay Isom, Ph.D. Abby D. Benninghoff, Ph.D. Major Professor Committee Member ______________________ ____________________ John Stevens, Ph.D. Trista Strauch, Ph.D. Committee Member Committee Member ______________________ ____________________ Aaron Thomas, Ph.D. Richard S. Inouye, Ph.D. Committee Member Vice Provost for Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2019 ii Copyright © Laura Moley 2019 All Rights Reserved iii ABSTRACT Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos by Laura A. Moley, Doctor of Philosophy Utah State University, 2019 Major Professor: Dr. S. Clay Isom Department: Animal, Dairy, and Veterinary Sciences Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology.
    [Show full text]
  • Mechanism of Human Somatic Reprogramming to Ips Cell Rika Teshigawara, Junkwon Cho, Masahiro Kameda and Takashi Tada
    Laboratory Investigation (2017) 97, 1152–1157 © 2017 USCAP, Inc All rights reserved 0023-6837/17 MINI REVIEW Mechanism of human somatic reprogramming to iPS cell Rika Teshigawara, Junkwon Cho, Masahiro Kameda and Takashi Tada Somatic reprogramming to induced pluripotent stem cells (iPSC) was realized in the year 2006 in mice, and in 2007 in humans, by transiently forced expression of a combination of exogenous transcription factors. Human and mouse iPSCs are distinctly reprogrammed into a ‘primed’ and a ‘naïve’ state, respectively. In the last decade, puzzle pieces of somatic reprogramming have been collected with difficulty. Collectively, dissecting reprogramming events and identification of the hallmark of sequentially activated/silenced genes have revealed mouse somatic reprogramming in fragments, but there is a long way to go toward understanding the molecular mechanisms of human somatic reprogramming, even with developing technologies. Recently, an established human intermediately reprogrammed stem cell (iRSC) line, which has paused reprogramming at the endogenous OCT4-negative/exogenous transgene-positive pre-MET (mesenchymal-to- epithelial-transition) stage can resume reprogramming into endogenous OCT4-positive iPSCs only by change of culture conditions. Genome-editing-mediated visualization of endogenous OCT4 activity with GFP in living iRSCs demonstrates the timing of OCT4 activation and entry to MET in the reprogramming toward iPSCs. Applications of genome-editing technology to pluripotent stem cells will reshape our approaches for exploring molecular mechanisms. Laboratory Investigation (2017) 97, 1152–1157; doi:10.1038/labinvest.2017.56; published online 22 May 2017 REPROGRAMMING OF SOMATIC CELLS use in exploring molecular mechanisms of many diseases and Reprogramming somatic cells into induced pluripotent stem embryonic development as models.
    [Show full text]
  • Single Cell Expression Analysis Uncouples Transdifferentiation And
    bioRxiv preprint doi: https://doi.org/10.1101/351957; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Single cell expression analysis uncouples transdifferentiation and 2 reprogramming 3 Mirko Francesconi1*, Bruno Di Stefano3,4,5*, Clara Berenguer3, Marisa de Andres3, Maria 4 Mendez Lago6,7, Amy Guillaumet-Adkins6,8, Gustavo Rodriguez-Esteban6, Marta Gut2,6, 5 Ivo G. Gut2,6, Holger Heyn2,6, Ben Lehner1,2,9,10 and Thomas Graf2,3,10 6 7 1Systems Biology Program, Centre for Genomic Regulation (CRG), The Barcelona 8 Institute of Science and Technology (BIST), Dr. Aiguader 88, Barcelona 08003, Spain; 9 2Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain. 3Gene Regulation, Stem 10 Cells and Cancer Program, CRG, BIST, UPF, Barcelona, 08003 Spain. 4. Massachusetts 11 General Hospital Department of Molecular Biology, Harvard Medical School, 185 12 Cambridge Street, Boston, MA 02114, USA. 5. Department of Stem Cell and 13 Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. 6Centro 14 Nacional de Análisis Genómico, Centre for Genomic Regulation (CNAG-CRG); BIST, 15 08028 Barcelona, Spain. 9Institució Catalana de Recerca i Estudis Avançats (ICREA), 16 Barcelona 08010, Spain. 17 18 * These authors contributed equally to the work 19 7Present address: Institute of Molecular Biology (IMB), Mainz, Germany 20 8Present address: Department of Pediatrics, Dana Farber Cancer Institute, Harvard 21 Medical School, Boston MA 02115 22 10Corresponding authors: [email protected], [email protected] 23 24 25 Abstract 26 Many somatic cell types are plastic, having the capacity to convert into other 27 specialized cells (transdifferentiation)(1) or into induced pluripotent stem cells (iPSCs, 28 reprogramming)(2) in response to transcription factor over-expression.
    [Show full text]