Identification and characterization of subpopulations in undifferentiated ES cell culture.
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In this paper, the authors established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes.Abstract:
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1+ / Oct3/4+ cells and Rex1- / Oct3/4+ cells. The Rex1- / Oct3/4+ and Rex1+ / Oct3/4+ populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1+ / Oct3/4+ cells and Rex1- / Oct3/4+ cells have characteristics similar to those of ICM and early-PrE cells, Rex1+ / Oct3/4+ cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1- / Oct3/4+ cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.read more
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Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells
Allon M. Klein,Linas Mazutis,Linas Mazutis,Ilke Akartuna,Naren Tallapragada,Adrian Veres,Victor C. Li,Leonid Peshkin,David A. Weitz,Marc W. Kirschner +9 more
TL;DR: This work has developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing, which shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays.
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mRNA-Seq whole-transcriptome analysis of a single cell.
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TL;DR: A single-cell digital gene expression profiling assay with only a single mouse blastomere is described, which detected the expression of 75% more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads.
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Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation
Gabriella Ficz,Miguel R. Branco,Stefanie Seisenberger,Fátima Santos,Felix Krueger,Timothy A. Hore,C. Joana Marques,C. Joana Marques,Simon Andrews,Wolf Reik,Wolf Reik +10 more
TL;DR: The balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.
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Nanog is the gateway to the pluripotent ground state.
José C. R. Silva,José C. R. Silva,Jennifer Nichols,Jennifer Nichols,Thorold W. Theunissen,Thorold W. Theunissen,Ge Guo,Ge Guo,Anouk L. van Oosten,Anouk L. van Oosten,Ornella Barrandon,Ornella Barrandon,Jason Wray,Jason Wray,Shinya Yamanaka,Ian Chambers,Austin Smith,Austin Smith +17 more
TL;DR: Evidence is presented that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency, and that this function is recapitulated in the culmination of somatic cell reprogramming.
Journal ArticleDOI
Accounting for technical noise in single-cell RNA-seq experiments
Philip Brennecke,Simon Anders,Jong Kyoung Kim,Aleksandra A. Kolodziejczyk,Aleksandra A. Kolodziejczyk,Xiuwei Zhang,Valentina Proserpio,Bianka Baying,Vladimir Benes,Sarah A. Teichmann,Sarah A. Teichmann,John C. Marioni,Marcus G. Heisler,Marcus G. Heisler +13 more
TL;DR: A quantitative statistical method is developed to distinguish true biological variability from the high levels of technical noise in single-cell experiments and quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis.
References
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Journal ArticleDOI
Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.
TL;DR: A role is established for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and the sophistication of critical transcriptional regulators is illustrated and the consequent importance of quantitative analyses are illustrated.
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Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4
Jennifer Nichols,Branko Zevnik,Konstantinos Anastassiadis,Hitoshi Niwa,Daniela Klewe-Nebenius,Ian Chambers,Hans R. Schöler,Austin Smith +7 more
TL;DR: It is reported that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo and also determines paracrine growth factor signaling from stem cells to the trophectoderm.
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Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells
Ian Chambers,Douglas Colby,Morag Robertson,Jennifer Nichols,Sonia Lee,Susan Tweedie,Austin Smith +6 more
TL;DR: These findings establish a central role for Nanog in the transcription factor hierarchy that defines ES cell identity and confirm that Cytokine dependence, multilineage differentiation, and embryo colonization capacity are fully restored upon transgene excision.
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The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells
Kaoru Mitsui,Yoshimi Tokuzawa,Hiroaki Itoh,Kohichi Segawa,Mirei Murakami,Kazutoshi Takahashi,Masayoshi Maruyama,Mitsuyo Maeda,Shinya Yamanaka +8 more
TL;DR: Nanog is a critical factor underlying pluripotency in both ICM and ES cells, and it is found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3.
Journal ArticleDOI
Multipotent cell lineages in early mouse development depend on SOX2 function
Ariel A. Avilion,Silvia K. Nicolis,Larysa H. Pevny,Lidia Pérez,Nigel Vivian,Robin Lovell-Badge +5 more
TL;DR: The data suggest that maternal components could be involved in establishing early cell fate decisions and that a combinatorial code, requiring SOX2 and OCT4, specifies the first three lineages present at implantation.