Stadtfeld, M, Apostolou, E, Akutsu, H, Fukuda, A, Follett, P, Natesan, S et al.. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465: 175-181

Howard Hughes Medical Institute at Massachusetts General Hospital, Center for Regenerative Medicine
Nature (Impact Factor: 41.46). 04/2010; 465(7295):175-81. DOI: 10.1038/nature09017
Source: PubMed


Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1-Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals ('all-iPSC mice'). In contrast, iPSC clones with normal expression of the Dlk1-Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1-Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.

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    • "The Dlk1–Dio3 imprinted domain on mouse chromosome 12qF1 contains three paternally expressed proteincoding genes, multiple maternally expressed long non-coding RNA genes, and numerous miRNAs and snoRNAs [5] [6] [7] [8] [9] [10]. The expression of long non-coding RNAs and lots of miRNAs clustered in this domain is aberrantly silenced in most mouse induced pluripotent stem (iPS) cells, and is predictive for their developmental potential [11] [12] [13]. There are three known paternally methylated DMRs (IG- DMR, Gtl2-DMR and Dlk1-DMR) [14]. "
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    ABSTRACT: The mouse delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1-Dio3) imprinted domain contains three known paternally methylated differentially methylated regions (DMRs): intergenic DMR (IG-DMR), maternally expressed 3-DMR (Gtl2-DMR), and Dlk1-DMR. Here, we report the first maternally methylated DMR, CpG island 2 (CGI-2), is located approximately 800bp downstream of miR-1188. CGI-2 is highly methylated in sperm and oocytes, de-methylated in pre-implantation embryos, and differentially re-methylated during post-implantation development. CGI-2, similarly to Gtl2-DMR and Dlk1-DMR, acquires differential methylation prior to embryonic day 7.5 (E7.5). Both H3K4me3 and H3K9me3 histone modifications are enriched at CGI-2. Furthermore, CCCTC-binding factor (CTCF) binds to both alleles of CGI-2 in vivo. These results contribute to the investigation of imprinting regulation in this domain. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    FEBS Letters 11/2014; 588(24). DOI:10.1016/j.febslet.2014.10.038 · 3.17 Impact Factor
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    • "Improved reprogramming procedures have greatly reduced, but not eliminated, the risk of cancer (Lee et al., 2013). These include nonintegrating and excisable vectors, the exclusion of MYC, and reprogramming by RNA, protein, or small molecules (Carey et al., 2010; Kaji et al., 2009; Stadtfeld et al., 2010a; Wernig et al., 2008). Additional strategies seek to purge residual PSCs, genomic surveys for somatic mutations, and conventional suicide genes (Choo et al., 2008; Tan et al., 2009). "
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    ABSTRACT: The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer's increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies.
    Cell Reports 09/2014; 8(6). DOI:10.1016/j.celrep.2014.08.039 · 8.36 Impact Factor
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    • "To reveal a gene expression signature associated with developmental competence, we selected the following groups of iPSC lines for microarray analysis: (1) ''poor quality'' iPSCs: this group included the three OSKM-iPSC lines Nanog-GFP OSKM#2, Oct4-GFP OSKM#2, and KH2 OSKM (Stadtfeld et al., 2010), that either did not produce fully developed pups or produced very low number of pups; (2) ''good quality'' iPSCs: this group included BC_2 OSKM (Carey et al., 2011) and Nanog-GFP SNEL#3, both of which gave rise to live, normal pups that survived only a few hours; (3) ''high quality'' iPSCs consisting of Nanog-GFP SNEL#2 and Oct4-GFP SNEL#1, both of which generated live pups that survived postnatally (representative pups from each iPSC group are shown in Figure S3A). We used Nanog-GFP, Oct4-GFP, and KH2 (Beard et al., 2006) ESCs as controls. "
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    ABSTRACT: Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.
    Cell Stem Cell 09/2014; 15(3):295-309. DOI:10.1016/j.stem.2014.07.003 · 22.27 Impact Factor
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