Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10 −4 ) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10 −4 ). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.Correspondence should be addressed to G.Q.D. (George.Daley@childrens.harvard.edu) and A.P.F. (afeinberg@jhu.edu). 5 These authors contributed equally to this work. 6 These authors jointly supervised this work.Accession codes. NCBI GEO: Gene expression microarray data and CHARM microarray data have been submitted under accession number GSE18111.Note: Supplementary information is available on the Nature Genetics website. Here we used a similar approach to the question of iPS cell reprogramming, first comparing six human iPS cell lines to the fibroblasts from which they were derived using comprehensive high-throughput array-based relative methylation (CHARM) analysis 9 . This approach allows the interrogation of ~4.6 million CpG sites genome-wide using a custom designed NimbleGen HD2 microarray, including almost all CpG islands and shores in the human genome. Genomic DNA from iPS cells 3,5 , their parental fibroblasts and human embryonic stem (hES) cells (Online Methods) was digested with the enzyme McrBC, fractionated, labeled and hybridized to a CHARM array. AUTHOR CONTRIBUTIONSA total of 4,401 regions (including 96,404 CpG sites) were found to differ in iPS cell lines from the fibroblasts of origin (Table 1, Supplementary Table 1) at a false discovery rate (FDR) of 5%; we term these regions R-DMRs. Of these R-DMRs, DMRs that were hypermethylated in iPS cells compared to fibroblasts predominated over hypomethylated DMRs (60%:40%). Of the 4,401 DMRs, 1,969 were within 2 kb of the transcriptional start site of a gene.The genes that were associated with these R-DMRs showed functionally important features based on bioinformatic analyses. First, gene ontology (GO) annotation analysis of these genes revealed significant enrichment for genes involved in developmental and regulatory processes (Supplementary Table 2). For example, 38% of the genes that were hypomethylated in iPS ...

The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome [1][2][3][4][5] , resulting in altered patterns of gene expression 2,6-9 . Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) 10,11 that occurs upon derivation of human iPSCs, and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined 10 lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells (ESCs), suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription Figure 1A , Supplementary Figure 3). We detected 3694 and 3283 genes up-and downregulated, respectively, in iPSCs and ESCs compared with fibroblasts (>2fold, P<0.05; Figure 1B). Taken together, our fibroblast-derived iPSCs fulfill functional criteria of bona fide iPSCs 20 and exhibit a uniform protein-coding gene expression profile similar to ESCs. NIH Public Access

Summary Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of β-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides the first in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.

The midbody (MB) is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. MBs persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells (iPSCs) and potential cancer ‘stem cells’ (CSCs) in vivo and in vitro. MBd loss accompanies stem cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor, NBR1, to the MB protein Cep55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to iPSCs and increases in vitro tumorigenicity of cancer cells. These results suggest unexpected roles for MBds in stem cells and CSCs.

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues1–3. Patient-specific induced pluripotent stem (iPS) cells4 represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of telomerase reverse transcriptase (TERT)5–7. We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that multiple telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSC purified from >2500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSC, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource (http://hsc.hms.harvard.edu) will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.

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BackgroundMalignant human embryonal carcinoma cells (ECCs) rely on similar transcriptional networks as non-malignant embryonic stem cells (ESCs) to control selfrenewal, maintain pluripotency, and inhibit differentiation. Because re-activation of silenced HERV-K(HML-2) loci is a hallmark of ECCs, we asked if this HERV group was also reactivated in ESCs and induced pluripotent stem cells (iPSCs).FindingsUsing RT-PCR and Western Blot, we demonstrate HERV-K(HML-2) RNA and protein expression in undifferentiated human ESCs and iPSCs. Induction of differentiation by embryoid body formation resulted in rapid silencing of HERV-K(HML-2) provirus expression. Sequencing analysis of a conserved region of the gag gene showed that proviral expression in ESCs and iPSCs represents at least 11 of the 66 nearly full length HERV-K(HML-2) loci, with slightly varying patterns in individual cell lines. These proviruses are human specific integrations and harbor promoter competent long terminal repeats (LTR5hs subgroup). We observed high mRNA levels of the NP9 and Gag encoding proviruses K101(22q11.21) in all and K10(5q33.3) in most of the ECC, ESC, and iPSC lines tested, while K37(11q23.3) mRNA was detected only in ESCs and iPSCs. In addition, we detected expression of proviral mRNA encoding the RNA export adaptor Rec in all cell lines studied. Proviral mRNA originating from the K108(7p22.1) locus, which inter alia codes for functional Rec and Env proteins, was only reactivated in malignant ECC lines, not in benign ESCs or iPSCs.ConclusionsHERV-K(HML-2) RNA and protein expression is a marker for pluripotent human stem cells. Initiation of differentiation results in rapid down-regulation. Further studies are needed to explore a putative functional role of HERV-K(HML-2) RNA and proteins in pluripotent stem cells.