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Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org., Forced expression of four transcription factors--Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (OSKM) (1)--induces molecular changes in somatic cells, which lead to pluripotency. The exogenous expression of these transcription [...]

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states., Somatic cells that have lost their pluripotent properties through the acquisition of differentiation-associated epigenetic marks can be driven to acquire an induced pluripotent cell (iPSC) state by the forced expression [...]

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D) (1-3). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes (4-6). However, no direct connection between the obesity-associated variants and FTO expression or function has been made (7-9). Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition., Noncoding variation in single nucleotide polymorphisms (SNPs) within a 47-kilobase (kb) region of high linkage disequilibrium in introns 1 and 2 of FTO remains the strongest genetic association with risk [...]

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators (1-3). Alternative splicing represents a widely acting mode of gene regulation (4-8), yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency (9). Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming., A core set of transcription factors that includes OCT4 (also called POU5F1), NANOG and SOX2, together with specific microRNAs and long non-coding RNAs, control the expression of genes required for [...]

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells., Reprogramming somatic cells to pluripotency can be achieved by forced expression of a defined set of factors (1,2). Several methods have been developed for generating human iPS cells, such as [...]

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state (1-4). The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral (1), lentivirals (5), adenoviral (6) and plasmid (7) transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines (8-11). The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events (12). Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition (13). Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision (12), we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences (14) delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies., To explore the utility of PB as a vector for somatic cell reprogramming, it was necessary to achieve temporal expression control and transgene silencing, as PB transposons are not purposefully [...]

Embryos containing a single inactive vascular endothelial growth factor (VEGF) gene show abnormal development of blood vessels. The homozygous VEGF-deficient embryos exhibit more abnormality in the blood vessel development, and more fatality than heterozygous mutants. The haploid-insufficient phenotype is associated with heterozygous VEGF deficiency. VEGF participation in vascular development depends on the ligand dose. The embryonic phenotypes from germline transmission and aggregation of heterozygous mutant embryo stem cells with tetraploid hosts are similar.

A study of mice with no Mash-2 gene function, generated by gene targeting to examine the role of Mash-2 in extraembryonic development, reveals that Mash-2 is vital only at postimplantation stages for the development of differential mammalian trophoblast cell types. Mash-2(super -/-) die from placental failure at 10 days post-coitum and are rescued by chimeras constructed with tetraploid wild-type embryos. Mash-2(super -/-) embryos develop normally and adult Mash-2(super -/-) mice are viable, confirming that Mash-2 is not vital in the embryo and for preimplementation development.

Murine embryonic stem (ES) cells with morulae can undergo a simple aggregation technique to yield highly chimaeric and viable mice. Germline chimaeras and general chimaera production can be improved by employing pluripotent ES cell lines. Abnormal phenotypes were observed in chimaeras and they also demonstrated their origin from ES cells.

Nature 516, 192-197 (2014); doi:10.1038/nature14047 In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical Research Institute, Queensland 4006, Australia) should have been listed as her [...]

Nature 516, 198-206 (2014); doi:10.1038/nature14046 In this Article, the address listed as Nicole Cloonan's present address (QIMR Berghofer Medical Research Institute, Queensland 4006, Australia) should have been listed as her [...]