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      Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells

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          Abstract

          We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog‐1 (LRH1 or NR5A2) with OCT4, MYC, KLF4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration‐free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose‐sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant‐negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell‐like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β‐catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. S tem C ells 2015;33:1390–1404

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          Naive and primed pluripotent states.

          After maternal predetermination gives way to zygotic regulation, a ground state is established within the mammalian embryo. This tabula rasa for embryogenesis is present only transiently in the preimplantation epiblast. Here, we consider how unrestricted cells are first generated and then prepared for lineage commitment. We propose that two phases of pluripotency can be defined: naive and primed. This distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.
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            Human induced pluripotent stem cells free of vector and transgene sequences.

            Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. We describe the derivation of human iPS cells with the use of nonintegrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors and removes one obstacle to the clinical application of human iPS cells.
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              Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.

              Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.
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                Author and article information

                Journal
                Stem Cells
                Stem Cells
                10.1002/(ISSN)1549-4918
                STEM
                Stem Cells (Dayton, Ohio)
                John Wiley and Sons Inc. (Hoboken )
                1066-5099
                1549-4918
                23 April 2015
                May 2015
                : 33
                : 5 ( doiID: 10.1002/stem.v33.5 )
                : 1390-1404
                Affiliations
                [ 1 ]Wellcome Trust Sanger Institute Hinxton CambridgeUnited Kingdom
                [ 2 ] Shanghai Institute of ImmunologyShanghai Jiaotong University School of Medicine 280 South Chongqing Road Shanghai 200025China
                Author notes
                [*] [* ]Correspondence: Pentao Liu, Ph.D., Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom. Telephone: 44‐(0)−1223496850; Fax: 44‐(0)−1223496802; e‐mail: pl2@ 123456sanger.ac.uk ; or Liming Lu, Ph.D., Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. Telephone: 0086‐21‐63846590. Extension 776556; Fax: 0086‐21‐63846383; e‐mail: lulunew2003@ 123456163.com
                Article
                STEM1926
                10.1002/stem.1926
                4863141
                25546009
                936ed6af-efda-4d7d-bc01-e486195e23e1
                © 2014 The Authors. STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 14 September 2014
                : 23 November 2014
                Page count
                Pages: 15
                Funding
                Funded by: Wellcome Trust
                Award ID: 098051
                Funded by: National Natural Science Foundation of China
                Award ID: 31370904
                Award ID: 30972691
                Funded by: Shanghai Science and Technology Development Funds
                Award ID: 10QA1405900
                Funded by: Training Program of the Excellent Young Talents of the Shanghai Municipal Health System
                Award ID: XYQ2011015
                Funded by: Shanghai Municipal Science and Technology Foundation
                Award ID: 11JC1410802
                Categories
                Embryonic Stem Cells/Induced Pluripotent Stem Cells
                Embryonic Stem Cells/Induced Pluripotent Stem Cells
                Custom metadata
                2.0
                stem1926
                May 2015
                Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.1 mode:remove_FC converted:23.06.2016

                Molecular medicine
                reprogramming,induced pluripotent stem cells,retinoic acid receptors,retinoic acid receptor gamma,liver receptor homolog‐1,β‐catenin,epiblast stem cells

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