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Increasing efficiency of pluripotent stem cell production by p53-21 knockout

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Cees Middel

on 5 October 2012

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Transcript of Increasing efficiency of pluripotent stem cell production by p53-21 knockout

Further Applications

Temporary suppression of p53 will result in higher quality iPS cells for future medical applications Presentation of Assignment III
By Martijn Jansen and Cees Middel Increasing efficiency of pluripotent stem cell production by P53-21 knockout Introduction:
why induced pluripotent stem (iPS) cells Wide array of applications
Tissue engineering
Cancer treatment Stem cells are able to differentiate into a wide variety of specialized cells. Induced stem cell production out of somatic cells Advantages over Embryonic Stem Cells (ECS)
No tissue rejection
Less ethical controversy
Somatic cells easy to obtain Disadvantage
Genetic instability
Uncertain pluripotency Goal
Increasing the efficiency of iPS cell generation by influencing the p53-21 pathway The P53-21 Pathway Alberts, John, Lewis, Raff, Roberts, and Walter, (2008), molecular Biology of the cell. (5th edition) Garland Science. ISBN 978-0815341055, Figure 17-63 Methods Conclusion:
p53 suppression can help increase iPS cell generation

Permanent suppression results in genomic instability Step 1 Summary of methods used in the studied article p53 wild type and p53 knockout nanog-GFP mouse embryonic fibroblasts (MEF) cells where prepared.
MEFs where transduced with Oct3/4, Sox2, and Klf4, using 4 retroviruses as vector Cells where screened for GFP activity using flow cytometry
Experiment was repeated but this time cells where also induced with c-Myc. First results: GFP positive colonies counted on 96 well plates 28 days after transduction Controls: p53 -/- mice where transduced with wild type p53
p53 +/+ cells where transduced with a dominant negative p53 mutant gene Pro275Ser The following controls where used Suppression of induced pluripotent stem cell
generation by the p53–p21 pathway
Hyenjong Hong1,2, Kazutoshi Takahashi1, Tomoko Ichisaka1,3, Takashi Aoi1, Osami Kanagawa4,
Masato Nakagawa1,2, Keisuke Okita1 & Shinya Yamanaka1,2,3,5 Suppression of induced pluripotent stem cell
generation by the p53–p21 pathway
Hyenjong Hong1,2, Kazutoshi Takahashi1, Tomoko Ichisaka1,3, Takashi Aoi1, Osami Kanagawa4,
Masato Nakagawa1,2, Keisuke Okita1 & Shinya Yamanaka1,2,3,5 Property of invivogen p53-p21 pathway
Activation of DNA repair
Growth arrest at G1/S phase
Initiation of Apoptosis Transduction procedure was repeated using terminally differentiated mouse T cells and human dermal fibroblasts Both cell types generated cells that produced iPS specific markers. The treated MTCs where tested negative for several MTC specific markers Generation of iPS cells:
De-differentation by inducing a forced expression of specific genes MTC derived iPSCs placed into nude mice differentiated into several germ layer specific cell types Image taken from: http://www.whitehead.mit.edu/news/paradigm/fall_2007/break_2.html Property of invivogen Testing of iPS cells Four chimera mice where created using p53 -/- ips cells Three mice died of cancer within 7 weeks

p53 +/+ MEF cells treated with a p53 specific siRNA and the 4 transduction factors yielded significantly more iPS cells Transcription Factors that induce pluripotency:

Oct3/4:
•Amount of regulation determines differentation
•Gene knockdown promotes differentation

Sox2:
•Hypermethylation causes differentation
•Posttranscriptional suppression by miRNA

Klf4:
•Inhibitor of differentation

c/Myc:
•Regulates expression of genes Suppression of induced pluripotent stem cell
generation by the p53–p21 pathway
Hyenjong Hong1,2, Kazutoshi Takahashi1, Tomoko Ichisaka1,3, Takashi Aoi1, Osami Kanagawa4,
Masato Nakagawa1,2, Keisuke Okita1 & Shinya Yamanaka1,2,3,5 Anova + Bonferoni Anova + Bonferoni
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