Generation of Pluripotent Stem Cells

MEFs: Primary Pou5f1-GFP MEFs were prepared from E12 embryos (http://jaxmice.jax.org/strain/008214.html) and reprogrammed by transduction with OKS using four HCT116-transducing units (HC-TU) per cell as previously described (Pasi et al. 2011 (link)). CD34⁺ cells from human cord blood were obtained and prepared as previously described (Barde et al. 2013 (link)), before transducing 250,000 cells with OKS using 100 HC-TU per cell. After 5 d, cells were switched to mTeSR1 medium (Stemcell Technologies no. 05859) and grown on a mouse fibroblast feeder layer until reprogrammed colonies emerged (∼21 d). Individual human iPSC clones were then picked and expanded. Primary human hepatocytes were isolated from liver biopsies as previously described (Birraux et al. 2009 (link)) and plated on Matrigel or collagen before being transduced with OKS or OKSM using 20 HC-TU per cell. After 5 d, cells were switched to mTeSR1 medium (Stemcell Technologies no. 05859) and grown until reprogrammed colonies emerged (∼25 d).

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Friedli M., Turelli P., Kapopoulou A., Rauwel B., Castro-Díaz N., Rowe H.M., Ecco G., Unzu C., Planet E., Lombardo A., Mangeat B., Wildhaber B.E., Naldini L, & Trono D. (2014). Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency. Genome Research, 24(8), 1251-1259.

Publication 2014

mTeSR1 medium

Biopsies Blood cells Cells Collagen Cord Cord blood Embryos Feeder layer Fibroblast Hct116 cell Hepatocytes Human Ipsc Liver Matrigel Mouse Pou5f1 Stemcell Strain

Corresponding Organization :

Other organizations : École Polytechnique Fédérale de Lausanne, University of Geneva, The San Raffaele Telethon Institute for Gene Therapy, Vita-Salute San Raffaele University

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Variable analysis

independent variables

Transduction of cells with OKS or OKSM using a specified number of HC-TU per cell

dependent variables

Emergence of reprogrammed colonies

control variables

Primary Pou5f1-GFP MEFs
CD34+ cells from human cord blood
Primary human hepatocytes
Feeder layer for human iPSC clones
Matrigel or collagen for primary human hepatocytes
MTeSR1 medium

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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