Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation.

Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1), the European Commission Framework 7 (HEALTH-F4-2013-602423, PluriMes) and the UK Regenerative Medicine Platform (MR/L012537/1). The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

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

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.This work was supported by the University of Cambridge, BBSRC project grant RG74277, BB/R018588/1 and MR/R017735/1 to HS and LB respectively, MRC PhD studentship for AK and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification

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. \ua9 2013 The Author(s)

Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) UK (BB/M004023/1 (RG74277)), the Medical Research Council (MRC) UK (G1001028), and funding to the Cambridge Stem Cell Institute from the MRC and Wellcome Trust (097922/Z/11/Z, 203151/Z/16/Z). TB is a Wellcome Trust Sir Henry Dale Fellow. AS is an MRC Professo

Sox2 modulation increases naïve pluripotency plasticity.

Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.We thank Yael Costa for critical reading of the manuscript. Peter Humphreys for assistance with imaging. William Mansfield for blastocyst injections. This study was supported by a Wellcome Trust Fellowship (WT101861) to J.C.R.S. B.K.K is supported by a European Research Council grant (639050). K.M. is a recipient of a Darwin Trust of Edinburgh Ph.D. studentship. K.T. is a recipient of a MRC Ph.D. studentship. H.T.S. and L.E.B. were supported by BBSRC and MRC research grants, BB/R018588/1 and MR/R017735/1 respectively. G.G.S. is funded by BBSRC research grant RG53615

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

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