Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

Human naive epiblast cells possess unrestricted lineage potential.

Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions.

Cancer-Related Mutations Are Not Enriched in Naive Human Pluripotent Stem Cells.

Previous analysis of RNA sequencing (RNA-seq) data from human naive pluripotent stem cells reported multiple point "mutations" in cancer-related genes and implicated selective culture conditions. We observed, however, that those mutations were only present in co-cultures with mouse feeder cells. Inspection of reads containing the polymorphisms revealed complete identity to the mouse reference genome. After we filtered reads to remove sequences of mouse origin, the actual incidence of oncogenic polymorphisms arising in naive pluripotent stem cells is close to zero.

Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.

Pluripotent cells emerge as a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here, we investigate the intermediate formative stage. Using minimal exposure to specification cues, we derive stem cells from formative mouse epiblast. Unlike ESCs or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonize somatic tissues and germline in chimeras. Whole-transcriptome analyses show similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells show distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.

The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.

Oct4 regulates the embryonic axis and coordinates exit from pluripotency and germ layer specification in the mouse embryo.

Lineage segregation in the mouse embryo is a finely controlled process dependent upon coordination of signalling pathways and transcriptional responses. Here we employ a conditional deletion system to investigate embryonic patterning and lineage specification in response to loss of Oct4. We first observe ectopic expression of Nanog in Oct4-negative postimplantation epiblast cells. The expression domains of lineage markers are subsequently disrupted. Definitive endoderm expands at the expense of mesoderm; the anterior-posterior axis is positioned more distally and an ectopic posterior-like domain appears anteriorly, suggesting a role for Oct4 in maintaining the embryonic axis. Although primitive streak forms in the presumptive proximal-posterior region, epithelial-to-mesenchymal transition is impeded by an increase of E-cadherin, leading to complete tissue disorganisation and failure to generate germ layers. In explant and in vitro differentiation assays, Oct4 mutants also show upregulation of E-cadherin and Foxa2, suggesting a cell-autonomous phenotype. We confirm requirement for Oct4 in self-renewal of postimplantation epiblast ex vivo Our results indicate a role for Oct4 in orchestrating multiple fates and enabling expansion, correct patterning and lineage choice in the postimplantation epiblast.

Immunotherapy for cardiovascular disease.

Heart failure (HF), the final stage of pathological cardiac hypertrophy, is a major cause of hospitalization and mortality. The role of inflammation in the pathogenesis of HF has been extensively studied, with great emphasis on proinflammatory cytokines. Yet, clinical trials targeting these cytokines failed to become a credible therapeutic strategy for HF. More recent studies are increasingly highlighting an active role for T cells in the progression of HF pathology. As a result, a number of novel immunotherapy strategies are emerging for the treatment of HF and other cardiovascular diseases, via the targeting of adaptive immunity. Here we provide an overview of the background, details, and expected outcomes of these attempts.

Dual PI3K/ERK inhibition induces necroptotic cell death of Hodgkin Lymphoma cells through IER3 downregulation.

PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in Hodgkin lymphoma (HL) patients, thus representing attractive therapeutic targets. Here we report that the PI3K/ERK dual inhibitor AEZS-136 induced significant cell proliferation inhibition in L-540, SUP-HD1, KM-H2 and L-428 HL cell lines, but a significant increase in necroptotic cell death was observed only in two out of four cell lines (L-540 and SUP-HD1). In these cells, AEZS-136-induced necroptosis was associated with mitochondrial dysfunction and reactive oxygen species (ROS) production. JNK was activated by AEZS-136, and AEZS-136-induced necroptosis was blocked by the necroptosis inhibitor necrostatin-1 or the JNK inhibitor SP600125, suggesting that JNK activation is required to trigger necroptosis following dual PI3K/ERK inhibition. Gene expression analysis indicated that the effects of AEZS-136 were associated with the modulation of cell cycle and cell death pathways. In the cell death-resistant cell lines, AEZS-136 induced the expression of immediate early response 3 (IER3) both in vitro and in vivo. Silencing of IER3 restored sensitivity to AEZS-136-induced necroptosis. Furthermore, xenograft studies demonstrated a 70% inhibition of tumor growth and a 10-fold increase in tumor necrosis in AEZS-136-treated animals. Together, these data suggest that dual PI3K/ERK inhibition might be an effective approach for improving therapeutic outcomes in HL.

MicroRNA-133 modulates the ß1-adrenergic receptor transduction cascade.

RATIONALE: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate ß-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of ß-adrenergic receptors leads to impaired cardiac function, and ß-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. OBJECTIVE: To determine whether miR-133 affects ß-adrenergic receptor signaling during progression to heart failure. METHODS AND RESULTS: Based on bioinformatic analysis, ß1-adrenergic receptor (ß1AR) and other components of the ß1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective ß1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic ß1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. CONCLUSIONS: miR-133 controls multiple components of the ß1AR transduction cascade and is cardioprotective during heart failure.

Genome-wide analysis of histone marks identifying an epigenetic signature of promoters and enhancers underlying cardiac hypertrophy.

Cardiac hypertrophy, initially an adaptive response of the myocardium to stress, can progress to heart failure. The epigenetic signature underlying this phenomenon is poorly understood. Here, we report on the genome-wide distribution of seven histone modifications in adult mouse cardiomyocytes subjected to a prohypertrophy stimulus in vivo. We found a set of promoters with an epigenetic pattern that distinguishes specific functional classes of genes regulated in hypertrophy and identified 9,207 candidate active enhancers whose activity was modulated. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. We propose that the epigenetic landscape is a key determinant of gene expression reprogramming in cardiac hypertrophy and provide a basis for understanding the role of chromatin in regulating this phenomenon.

Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Long noncoding RNA: a new player of heart failure?

One the most important discoveries of the post-genomic era is that a large fraction of the genome transcribes a heterogeneous population of noncoding RNAs (ncRNA). ncRNAs shorter than 200 nucleotides are usually identified as short/small ncRNAs--examples include PIWI-interacting RNAs, small interfering RNAs, and microRNAs (miRNAs)--whereas those longer than 200 nucleotides are classified as long ncRNAs (lncRNAs). These molecules are emerging as important regulators of cellular process, such as development, differentiation, and metabolism. Not surprisingly, ncRNAs are involved also in human diseases, such as cancer and metabolic and neuronal disorders. Although the role of miRNAs is being largely investigated in cardiovascular biology, little is known about other classes of ncRNA in this field. However, recent reports have started to reveal the importance of lncRNA in heart development and suggest also an involvement in heart failure. Here, we will discuss these reports and the therapeutic potential of lncRNA for heart failure.