Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.This work was supported by UK Biotechnology and Biological Sciences Research Council (BBSRC) research grant RG53615, UK Medical Research Council (MRC) programme grant G1001028, and institutional funding from the MRC and Wellcome Trust. AS is an MRC Professor

Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival.

BACKGROUND: Glioblastoma multiforme, the most common type of primary brain tumor in adults, is driven by cells with neural stem (NS) cell characteristics. Using derivation methods developed for NS cells, it is possible to expand tumorigenic stem cells continuously in vitro. Although these glioblastoma-derived neural stem (GNS) cells are highly similar to normal NS cells, they harbor mutations typical of gliomas and initiate authentic tumors following orthotopic xenotransplantation. Here, we analyzed GNS and NS cell transcriptomes to identify gene expression alterations underlying the disease phenotype. METHODS: Sensitive measurements of gene expression were obtained by high-throughput sequencing of transcript tags (Tag-seq) on adherent GNS cell lines from three glioblastoma cases and two normal NS cell lines. Validation by quantitative real-time PCR was performed on 82 differentially expressed genes across a panel of 16 GNS and 6 NS cell lines. The molecular basis and prognostic relevance of expression differences were investigated by genetic characterization of GNS cells and comparison with public data for 867 glioma biopsies. RESULTS: Transcriptome analysis revealed major differences correlated with glioma histological grade, and identified misregulated genes of known significance in glioblastoma as well as novel candidates, including genes associated with other malignancies or glioma-related pathways. This analysis further detected several long non-coding RNAs with expression profiles similar to neighboring genes implicated in cancer. Quantitative PCR validation showed excellent agreement with Tag-seq data (median Pearson r = 0.91) and discerned a gene set robustly distinguishing GNS from NS cells across the 22 lines. These expression alterations include oncogene and tumor suppressor changes not detected by microarray profiling of tumor tissue samples, and facilitated the identification of a GNS expression signature strongly associated with patient survival (P = 1e-6, Cox model). CONCLUSIONS: These results support the utility of GNS cell cultures as a model system for studying the molecular processes driving glioblastoma and the use of NS cells as reference controls. The association between a GNS expression signature and survival is consistent with the hypothesis that a cancer stem cell component drives tumor growth. We anticipate that analysis of normal and malignant stem cells will be an important complement to large-scale profiling of primary tumors.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.This work was supported by the Medical Research Council, Biotechnology and Biological Sciences Research Council, Swiss National Science Foundation (SNF)/Novartis SNF (F.v.M.) and core funding to the Cambridge Stem Cell Institute from the Wellcome Trust and Medical Research Council. AS is a Medical Research Council Professor.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.stemcr.2016.02.00

Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex.

Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.Wellcome Trust (PhD Studentship; Senior Fellowship in the Basic Biomedical Sciences [098021/Z/11/Z]), Wellcome Trust and UK Medical Research Council core funding to the Cambridge Stem Cell Institute [079249/Z/06/I], European Union Seventh Framework Programme (FP7) Project ‘4DCellFate’This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.13911

Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.

Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation.We thank Peter Humphreys for assistance with imaging, and Samuel Jameson and staff for mouse husbandry. We are grateful to Charis Drummer, Ayako Sedohara, Akiko Shimada, Yuko Yamada, Ryo Oiwa, and Takeshi Kuge for technical support with marmoset embryo recovery. Illumina sequencing was provided by Bettina Haase and Dinko Pavlinic at the EMBL Genomics Core Facility. This work was supported by funding from the Wellcome Trust, the Genome Biology Unit of the European Molecular Biology Laboratory, BBSRC grants BB/G015678/1 and BB/M004023/1, an MRC Centenary Award, and the Louis Jeantet Foundation. A.S. is a Medical Research Council Professor.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.devcel.2015.10.01

Ultra High Energy Cosmic Rays from Sequestered X Bursts

Assuming that there is no GZK (Greisen-Zatsepin-Kuzmin) cut-off and that super-GZK cosmic rays correlate with AGN (Active Galactic Nuclei) at cosmological distances, it is speculated that a relic superheavy particle (X) has its lifetime enhanced by sequestration in an extra dimension. This sequestration is assumed to be partially liberated by proximity of merging supermassive black holes in an AGN, temporarily but drastically reducing the lifetime, thus stimulating an X burst. Based on sequestration of the decay products of X, a speculative explanation of the observed $\gamma/N$ ratio is proposed.Comment: 12 pages LaTe

Assessment of transcript reconstruction methods for RNA-seq

We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.This research was supported by the UK Medical Research Council, the Japan Science and Technology agency (JST, PRESTO), the Genome Biology Unit of the European Molecular Biology Laboratory, European Commission projects PluriMes, BetaCellTherapy, EpiGeneSys, and Blueprint, and the Wellcome Trust. Y.T. was a University of Cambridge Herchel Smith Fellow. A.S. is a Medical Research Council Professor

Unraveling PBF effects beyond impact evaluation: results from a qualitative study in Cameroon

Introduction Performance-based financing (PBF) has acquired increased prominence as a means of reforming health system purchasing structures in low-income and middle-income countries. A number of impact evaluations have noted that PBF often produces mixed and heterogeneous effects. Still, little systematic effort has been channelled towards understanding what causes such heterogeneity, including looking more closely at implementation processes. Methods Our qualitative study aimed at closing this gap in knowledge by attempting to unpack the mixed and heterogeneous effects detected by the PBF impact evaluation in Cameroon to inform further implementation as the country scales up the PBF approach. We collected data at all levels of the health system (national, district, facility) and at the community level, using a mixture of in-depth interviews and focus group discussions. We combined deductive and inductive analytical techniques and applied analyst triangulation. Results Our findings indicate that heterogeneity in effects across facilities could be explained by preexisting infrastructural weaknesses coupled with rigid administrative processes and implementation challenges, while heterogeneity across indicators could be explained by providers' practices, privileging services where demand-side barriers were less substantive. Conclusion In light of the country's commitment to scaling up PBF, it follows that substantial efforts (particularly entrusting facilities with more financial autonomy) should be made to overcome infrastructural and demand-side barriers and to smooth implementation processes, thus, enabling healthcare providers to use PBF resources and management models to a fuller potential.sch_iih3pub5290pub