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Additional Supporting Information may be found in the online version of this article.

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STEM_1406_sm_SuppFigure1.pdf283KSupplementary 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.pdf225KSupplementary 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.pdf315KSupplementary 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.pdf34KSupporting Information Table 1.
STEM_1406_sm_SuppInfo.pdf107KSupporting Information

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