Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells

Abstract

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.

Figure 1. Aberrant silencing of the Dlk1-Dio3 gene cluster in mouse iPSCs

(a) Strategy for comparing genetically matched ESCs and iPSCs using “reprogrammable mice” harboring a doxycycline-inducible polycistronic reprogramming cassette (OKSM) in the Col1a1 (Collagen) locus. (b) Morphology of Collagen-OKSM ESCs and iPSCs. (c) Unsupervised clustering of four ESC and six iPSC lines based on microarray expression data. (d) Scatterplot of microarray data comparing iPSCs and ESCs with differentially expressed genes highlighted in green (2-fold, p0.05, t-test with Benjamini-Hochberg correction). (e) Heatmap showing relative expression levels of selected mRNAs in ESCs and iPSCs, covering in addition to Gtl2 and Rian other imprinted genes (Dlk1, Igf2r and H19) and pluripotency-associated transcripts (Nanog, Sox2 and Pou5f1). (f) Schematic representation of mouse chromosome 12 with position of the Dlk1-Dio3 gene cluster highlighted. Maternally-expressed and paternally-expressed transcripts are shown in red and blue, respectively. (g) Heatmap showing miRNAs that are differentially expressed between ESCs and iPSCs (2-fold, p0.01, t-test).

Figure 2. Full developmental potential of Gtl2^on iPSCs

(a) Heatmap showing relative expression levels of Gtl2, Rian, other selected imprinted genes (Dlk1, H19 and Igf2r) and pluripotency-associated transcripts (Sox2 and Nanog) in ESCs and iPSCs derived from hematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP), granulocytes (Gran), peritoneal fibroblasts (PF) and tail-tip fibroblasts (TTFs), isolated from three individual reprogrammable mice. Four iPSC clones expressing ESC-like levels of Gtl2 and Rian were identified (highlighted by asterisks) (for technical reasons, iPSC clone #18 could not be analyzed by microarray but instead was evaluated by qPCR. See Supplemental Figure 1b). (b) Strategy for assessing the developmental potential of iPSC clones by injection into diploid (2n) and tetraploid (4n) blastocysts to produce chimeric or all-iPSC mice, respectively. (c) Images of representative coat color chimeras with agouti indicating iPSC origin. (d) Coat color chimerism in mice derived from indicated Gtl2^off (green diamonds), Gtl2^on iPSC clones (red diamonds) and ESCs (open diamonds). (e) Statistical analysis of coat color chimerism in mice derived form Gtl2^off and Gtl2^on iPSC clones. (f) Image of two GFP⁺ all-iPSC pups (left panel) and two agouti all-iPSC mice (right). (g) Scatterplot showing intensity levels of all probesets covered by microarray analysis with those highlighted in green that were significantly different between 4n complementation-competent iPSCs (clones #19, #44, #47 and #49) and non-4n complementation-competent iPSCs (clones #18, #20, #45 and #48) (2-fold, p0.05, t-test with Benjamini-Hochberg correction).

Figure 3. Epigenetic silencing of the Gtl2 locus in iPSCs

(a) Structure of the Dlk1-Dio3 locus with the position of the genomic regions analyzed by pyrosequencing indicated by black bars. (b) Degree of DNA methylation at IG-DMR and Gtl2 DMR in three Gtl2^off iPSC clones (green bars), three Gtl2^on iPSC clones (red bars), three ESCs clones (red open bars), as well as parental tail-tip fibroblasts (TTFs, grey bars). The methylation status of the other regions is shown in Supplemental Figure 5. (c) Prevalence of activation-associated (acH3, acH4 and H3K4me) and repression-associated (H3K27me) chromatin marks at the Gtl2 promoter in two Gtl2^off iPSC clones, two Gtl2^on iPSCs clones and ESCs. (d) Gtl2 expression levels as measured by qPCR in subclones derived from Gtl2^off clone #45 and Gtl2^on clone #49 in the absence (upper panel) or presence (lower panel) of doxycycline (dox). (e) Representative brightfield images of iPSCs culture in the absence or presence of all-trans retinoic acid (RA). (f) Expression levels of Gtl2, other imprinted genes (Igf2, Igf2r) and the pluripotency marker Pou5f1 in cells cultured with (+) or without (-) retinoic acid (RA). Note that the two Gtl2^off clones fail to activate Gtl2, but show normal expression levels of the other imprinted genes.

Figure 4. Developmental defects in embryos derived from Gtl2^off iPSCs

(a) Green fluorescence images of “all-iPSC” E11.5 embryos obtained with Gtl2^on clone #47 (upper panel) and Gtl2^off clone #48 (lower panel), both of which express EGFP from the ubiquitous ROSA26 locus. (b) Frequency of dead and living all-iPSC embryos obtained with two Gtl2^on (red bars) and two Gtl2^off (green bars) iPSC clones upon 4n blastocyst injection. Numbers of blastocysts transferred per clone and numbers of embryos recovered are indicated in brackets. (c) Expression of Glt2, Rian, Mirg and the paternally expressed gene Dlk1 in Gtl2^off MEFs relative to Gtl2^on MEFs (upper panel) as well as in Gtl2^mKO MEFs relative to MEFs isolated from wildtype embryos (lower panel). (d) In situ hybridization against Gtl2 mRNA in MEFs derived from all-iPSC embryos generated with either Gtl2^on clone #44 or Gtl2^off clone #48. (e) Expression levels of Gtl2, Rian, Mirg and Dlk1 in the indicated tissues isolated from all-iPSC embryos made with Gtl2^off iPSCs relative to the levels seen in tissues derived from Gtl2^on iPSCs. (f) Degree of DNA methylation at the indicated regions in Gtl2^off, Gtl2^on, Gtl2^mKO and wildtype MEFs. (g) Gtl2 expression levels in iPSC lines derived by subcloning Gtl2^off clone #45 in the presence of valproic acid (VA). (h) Images of a fully developed stillborn pup (left) and a uterus filled with resorptions (right) derived after 4n blastocyst injections with either VA-10 or the parental iPSC clone #45, respectively.