Sommer, CA, Christodoulou, C, Gianotti-Sommer, A, Shen, SS, Sailaja, BS, Hezroni, H et al.. Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells. PLoS One 7: e51711

Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 12/2012; 7(12):e51711. DOI: 10.1371/journal.pone.0051711
Source: PubMed


Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

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Available from: Badi Sri Sailaja,
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    • "However, the full extent of their relation to natural pluripotent stem cells is still being assessed. There is a hypothesis that this variation results from residual transgene expression (Sommer et al., 2012). However, preliminary data suggest that even transgene-free iPSCs are epigenetically distinct from ESCs. "
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    ABSTRACT: Abstract Pluripotent stem cells, both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the ability to differentiate into several cell types that can be used in drug testing and also in the study and treatment of diseases. These cells can be differentiated by in vitro systems, which may serve as models for human diseases and for cell transplantation. In this review, we address the pluripotent cell types, how to obtain and characterize these cells, and differentiation assays. We also focus on the potential of these cells in clinical trials, and we describe the clinical trials that are underway.
    03/2014; 16(2). DOI:10.1089/cell.2013.0072
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    Developmental Biology 08/2011; 356(1):219-219. DOI:10.1016/j.ydbio.2011.05.348 · 3.55 Impact Factor
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    ABSTRACT: This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Curr. Protoc. Stem Cell Biol. 27:4A.6.1-4A.6.27. © 2013 by John Wiley & Sons, Inc.
    Current protocols in stem cell biology 11/2013; 27:4A.6.1-4A.6.27. DOI:10.1002/9780470151808.sc04a06s27
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