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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells†
Article first published online: 23 AUG 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 8, pages 1726–1730, August 2013
How to Cite
Molina-Estevez, F. J., Lozano, M. L., Navarro, S., Torres, Y., Grabundzija, I., Ivics, Z., Samper, E., Bueren, J. A. and Guenechea, G. (2013), Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells. STEM CELLS, 31: 1726–1730. doi: 10.1002/stem.1406
Author contributions: F.J.M.-E.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; M.L.L.: conception and design, collection and/or assembly of data, and data analysis and interpretation; S.N.: conception and design, collection and/or assembly of data, and data analysis and interpretation; Y.T.: collection and/or assembly of data and data analysis and interpretation; I.G.: provision of study material or patients; Z.I.: provision of study material or patients and manuscript writing; E.S.: collection and/or assembly of data and data analysis and interpretation; J.A.B.: conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript; G.G.: conception and design, data analysis and interpretation, manuscript writing, and final approval of the manuscript.
- Issue published online: 23 AUG 2013
- Article first published online: 23 AUG 2013
- Accepted manuscript online: 30 APR 2013 03:49AM EST
- Manuscript Accepted: 28 MAR 2013
- Manuscript Received: 16 OCT 2012
- European Commision FP7 Program. Grant Number: 222878
- Spanish Ministry of Economy and Competitiveness (International Cooperation on Stem Cell Research Plan E. Grant Numbers: PLE 2009/0100, SAF 2009-07164
- Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III. Grant Numbers: RETICS-RD06/0010/0015, PI08/0701
- Dirección General de Investigación de la Comunidad de Madrid. Grant Number: S2010/BMD-2420
Additional Supporting Information may be found in the online version of this article.
|STEM_1406_sm_SuppFigure1.pdf||283K||Supplementary Figure 1. A) Schematic view of the STEMCCA RedLight and c-Myc LV employed for iPS generation: −LTR, Inactive engineered HIV-1 Long Terminal Repeat including a LoxP signal in the U3; EF1α, Human Elongation Factor 1α promoter, E2A and F2A are poxvirus self-cleavable sequences. From the pool of wt iPS generated using the RedLight LV we choose one clone that was successfully excised, for full characterization: B) 200X photographs of the wt LV-free iPS clone growing on feeder. Pluripotence markers showing positive Alkaline Phosphatase (AP), Nanog, Oct3/4 and SSEA-1 staining. C) Similar expression levels of selected endogenous genes, Oct4, Sox2, Nanog, Utf1, c-Myc was confirmed by quantitative PCR in standard mESC J1 and wt iPSC line. D) Vector-free wt iPSC were able to generate teratomas with defined histological derivatives from the 3 germinal layers after being inoculated in NSG mice (200 μm scale bar shown in left panel and 50 μm scale bar in middle and right panels).|
|STEM_1406_sm_SuppFigure2.pdf||225K||Supplementary Figure 2. A) Representative digital zoom captures from the microphotographs of primary MEFs used for telomere quantification by FISH on metaphase chromosomes: Blue signal, DAPI; yellow signal, centromeric probe (FITC) and pink signal, telomeric probe (TERTC). Upper row Pass 3 wt MEFs, bottom row Pass 3 Scid MEFs, scale bars 5 μm. B) Telomere length quantification, metaphases from pass 3 Prkdcscid homozygous, heterozygous (+/−) or wt MEFs were studied just before reprogramming (t-test, **** p<0.0001).|
|STEM_1406_sm_SuppFigure3.pdf||315K||Supplementary Figure 3. A) Schematic diagram of the T2/OSKM SB transposon, including between the T2 inverted/direct repeats a reprogramming cassette expressed after the CAGgs ubiquitous promoter a policistronic gene made by the reprogramming factors Oct4, Sox2, Klf4 and c-Myc with intercalated self-cleavable 2A elements. B) To quantify telomere length, telomeric and centromeric probes were assayed on MEFs metaphases from either mock or T2/OSKM SB-transduced cultures 5 days after nucleofection (t-test ,*** p< 0.001). C) Senescence associated to β-Galactosidase staining (SA-β-Gal) was performed on Scid and wt samples 10 days after transduction with T2/OSKM SB vector. More than 200 cells per well were counted in different fields of 6-well plates and the increase of the SA-β-Gal positive fraction calculated using parallel mock cultures as reference. D) Colonies arisen from the reprogrammed cultures were morphologically identical to ESC colonies. 20X pictures of isolated Tn-iPSC colonies from wt and Scid background after isolation. E) Transposon Scid iPSC (clone Scid Tn-iPS 72) were able to generate teratomas with defined histological derivatives from the 3 germinal layers after being inoculated in NSG mice.|
|STEM_1406_sm_SuppTable1.pdf||34K||Supporting Information Table 1.|
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