Dissecting ensemble networks in ES cell populations reveals micro-heterogeneity underlying pluripotency.

Analysis of transcription at the level of single cells in prokaryotes and eukaryotes has revealed the existence of heterogeneities in the expression of individual genes within genetically homogeneous populations. This variation is an emerging hallmark of populations of Embryonic Stem (ES) cells and has been ascribed to the stochasticity associated with the biochemical events that mediate gene expression. It has been suggested that these heterogeneities play a role in the maintenance of pluripotency. However, for the most part, studies have focused on individual genes in large cell populations. Here we use an existing dataset on the expression of eight genes involved in pluripotency in eighty-three ES cells to create Gene Regulatory Networks (GRNs) at the single cell level. We observe widespread heterogeneities in the expression of the eight genes, but analysis of correlations within individual cells reveals three distinct classes centered on the expression of Nanog, a marker of pluripotency, and Fgf5, a gene associated with differentiation: high levels of Nanog and low levels of Fgf5, low levels of Nanog and high levels of Fgf5, and low levels of both. Each of these classes is associated with a collection of active sub-networks, with differing degrees of connectivity between their elements, which define a cellular state: self-renewal, primed for differentiation or transition between the two. Though every cell should be governed by the same network, the active sub-networks may emerge due to considerations such as variation in (i) the expression level of active transcription factors (e.g. through post-translational modification or ligand/co-factor availability) or (ii) access to the target gene locus (e.g. via changes in chromatin status or epigenetic modifications). We conclude that heterogeneities in gene expression should not be interpreted as representing different states of a single unique network, but as a reflection of the activity of different sub-networks in sub-populations of cells.

[1]  M Madan Babu,et al.  Early Career Research Award Lecture. Structure, evolution and dynamics of transcriptional regulatory networks. , 2010, Biochemical Society transactions.

[2]  Timothy K Lee,et al.  Single-cell NF-κB dynamics reveal digital activation and analogue information processing , 2010, Nature.

[3]  David A. Rand,et al.  Measurement of single-cell dynamics , 2010, Nature.

[4]  Catalin C. Barbacioru,et al.  Tracing the Derivation of Embryonic Stem Cells from the Inner Cell Mass by Single-Cell RNA-Seq Analysis , 2010, Cell stem cell.

[5]  Mikael Huss,et al.  Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. , 2010, Developmental cell.

[6]  I. Lemischka,et al.  Hooking up with Oct4. , 2010, Cell stem cell.

[7]  Debbie L C van den Berg,et al.  An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells , 2010, Cell stem cell.

[8]  M. Babu,et al.  An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease , 2010, Cell stem cell.

[9]  F. Markowetz,et al.  Systems-level dynamic analyses of fate change in murine embryonic stem cells , 2009, Nature.

[10]  Avi Ma’ayan,et al.  Systems biology of stem cell fate and cellular reprogramming , 2009, Nature Reviews Molecular Cell Biology.

[11]  Ge Guo,et al.  Nanog Is the Gateway to the Pluripotent Ground State , 2009, Cell.

[12]  Joshua A. Grochow,et al.  Genomic analysis reveals a tight link between transcription factor dynamics and regulatory network architecture , 2009, Molecular systems biology.

[13]  C. Lim,et al.  Regulated Fluctuations in Nanog Expression Mediate Cell Fate Decisions in Embryonic Stem Cells , 2009, PLoS biology.

[14]  Andre Levchenko,et al.  High Content Cell Screening in a Microfluidic Device*S , 2009, Molecular & Cellular Proteomics.

[15]  Gang Pei,et al.  Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells , 2008, Cell Research.

[16]  F. Tang,et al.  Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. , 2008, Cell stem cell.

[17]  R. Shamir,et al.  Regulatory networks define phenotypic classes of human stem cell lines , 2008, Nature.

[18]  Megan F. Cole,et al.  Connecting microRNA Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells , 2008, Cell.

[19]  Hannah H. Chang,et al.  Transcriptome-wide noise controls lineage choice in mammalian progenitor cells , 2008, Nature.

[20]  Henry Adams,et al.  REST maintains self-renewal and pluripotency of embryonic stem cells , 2008, Nature.

[21]  Shinji Masui,et al.  Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells , 2008, BMC Developmental Biology.

[22]  S. Orkin,et al.  An Extended Transcriptional Network for Pluripotency of Embryonic Stem Cells , 2008, Cell.

[23]  Richard A Young,et al.  Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. , 2008, Genes & development.

[24]  H. Niwa,et al.  Identification and characterization of subpopulations in undifferentiated ES cell culture , 2008, Development.

[25]  Sheng Zhong,et al.  A core Klf circuitry regulates self-renewal of embryonic stem cells , 2008, Nature Cell Biology.

[26]  Geppino Falco,et al.  An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells. , 2008, Gene expression patterns : GEP.

[27]  J. Nichols,et al.  Nanog safeguards pluripotency and mediates germline development , 2007, Nature.

[28]  N. Terada,et al.  A Heterogeneous Expression Pattern for Nanog in Embryonic Stem Cells , 2007, Stem cells.

[29]  M. Trotter,et al.  Derivation of pluripotent epiblast stem cells from mammalian embryos , 2007, Nature.

[30]  T. Hughes,et al.  Prediction and Testing of Novel Transcriptional Networks Regulating Embryonic Stem Cell Self-renewal and Commitment , 2022 .

[31]  Hitoshi Niwa,et al.  How is pluripotency determined and maintained? , 2007, Development.

[32]  Alexei A. Sharov,et al.  Dissecting Oct3/4-Regulated Gene Networks in Embryonic Stem Cells by Expression Profiling , 2006, PloS one.

[33]  Stuart H. Orkin,et al.  A protein interaction network for pluripotency of embryonic stem cells , 2006, Nature.

[34]  T. Tokunaga,et al.  Gene Expression Profiling of Mouse Embryonic Stem Cell Subpopulations1 , 2006, Biology of reproduction.

[35]  Austin G Smith,et al.  Nanog promotes transfer of pluripotency after cell fusion , 2006, Nature.

[36]  Kazuki Kurimoto,et al.  An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis , 2006, Nucleic acids research.

[37]  X. Chen,et al.  The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells , 2006, Nature Genetics.

[38]  Hannah H. Chang,et al.  Multistable and multistep dynamics in neutrophil differentiation , 2006, BMC Cell Biology.

[39]  M. Surani,et al.  Generation of stella‐GFP transgenic mice: A novel tool to study germ cell development , 2006, Genesis.

[40]  Megan F. Cole,et al.  Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells , 2005, Cell.

[41]  T. Elston,et al.  Stochasticity in gene expression: from theories to phenotypes , 2005, Nature Reviews Genetics.

[42]  R. Humphries,et al.  Correlation of Murine Embryonic Stem Cell Gene Expression Profiles with Functional Measures of Pluripotency , 2005, Stem cells.

[43]  K. Sachs,et al.  Causal Protein-Signaling Networks Derived from Multiparameter Single-Cell Data , 2005, Science.

[44]  A. van Oudenaarden,et al.  Noise Propagation in Gene Networks , 2005, Science.

[45]  P. Swain,et al.  Gene Regulation at the Single-Cell Level , 2005, Science.

[46]  K. Sasaki,et al.  Spatial Distribution and Initial Changes of SSEA-1 and Other Cell Adhesion-related Molecules on Mouse Embryonic Stem Cells Before and During Differentiation , 2004, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[47]  M. Gerstein,et al.  Genomic analysis of regulatory network dynamics reveals large topological changes , 2004, Nature.

[48]  J. Raser,et al.  Control of Stochasticity in Eukaryotic Gene Expression , 2004, Science.

[49]  M. Gerstein,et al.  Structure and evolution of transcriptional regulatory networks. , 2004, Current opinion in structural biology.

[50]  T. Tokunaga,et al.  Embryonic Stem Cells Expressing Both Platelet Endothelial Cell Adhesion Molecule-1 and Stage-Specific Embryonic Antigen-1 Differentiate Predominantly into Epiblast Cells in a Chimeric Embryo1 , 2004, Biology of reproduction.

[51]  A. Clark,et al.  Human STELLAR, NANOG, and GDF3 Genes Are Expressed in Pluripotent Cells and Map to Chromosome 12p13, a Hotspot for Teratocarcinoma , 2004, Stem cells.

[52]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[53]  J. Nichols,et al.  BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3 , 2003, Cell.

[54]  J. Collins,et al.  Inferring Genetic Networks and Identifying Compound Mode of Action via Expression Profiling , 2003, Science.

[55]  M. Murakami,et al.  The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells , 2003, Cell.

[56]  J. Nichols,et al.  Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells , 2003, Cell.

[57]  Mads Kærn,et al.  Noise in eukaryotic gene expression , 2003, Nature.

[58]  P. Swain,et al.  Stochastic Gene Expression in a Single Cell , 2002, Science.

[59]  P. Rathjen,et al.  Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development. , 2002, Journal of cell science.

[60]  P. Robson,et al.  Inner cell mass-specific expression of a cell adhesion molecule (PECAM-1/CD31) in the mouse blastocyst. , 2001, Developmental biology.

[61]  P. Rathjen,et al.  Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. , 1999, Journal of cell science.

[62]  F. Reif,et al.  Fundamentals of Statistical and Thermal Physics , 1998 .

[63]  P. Rathjen,et al.  The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo. , 1997, Genomics.

[64]  J. Nichols,et al.  Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways , 1994, Mechanisms of Development.

[65]  R. Beddington,et al.  An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. , 1989, Development.

[66]  G. Martin,et al.  Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[67]  M. Kaufman,et al.  Establishment in culture of pluripotential cells from mouse embryos , 1981, Nature.

[68]  D. Koshland,et al.  Non-genetic individuality: chance in the single cell , 1976, Nature.

[69]  Tetsuya S. Tanaka.  Transcriptional heterogeneity in mouse embryonic stem cells. , 2009, Reproduction, fertility, and development.

[70]  Kazuki Kurimoto,et al.  Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis , 2007, Nature Protocols.

[71]  A. Smith,et al.  Embryo-derived stem cells: of mice and men. , 2001, Annual review of cell and developmental biology.