An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression

Resolving early mesoderm diversification through single-cell expression profiling.

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.We thank M. de Bruijn, A. Martinez-Arias, J. Nichols and C. Mulas for discussion, the Cambridge Institute for Medical Research Flow Cytometry facility for their expertise in single-cell index sorting, and S. Lorenz from the Sanger Single Cell Genomics Core for supervising purification of Tal1−/− sequencing libraries. ChIP-seq reads were processed by R. Hannah. Research in the authors’ laboratories is supported by the Medical Research Council, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, Bloodwise, the Leukemia and Lymphoma Society, and the Sanger-EBI Single Cell Centre, and by core support grants from the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. Y.T. was supported by a fellowship from the Japan Society for the Promotion of Science. W.J. is a Wellcome Trust Clinical Research Fellow. A.S. is supported by the Sanger-EBI Single Cell Centre. This work was funded as part of Wellcome Trust Strategic Award 105031/D/14/Z ‘Tracing early mammalian lineage decisions by single-cell genomics’ awarded to W. Reik, S. Teichmann, J. Nichols, B. Simons, T. Voet, S. Srinivas, L. Vallier, B. Göttgens and J. Marioni.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1863

Pluripotency Factors on Their Lineage Move

Pluripotent stem cells are characterised by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers. Regulatory networks of maintaining pluripotency have been described in great detail and, similarly, there is great knowledge on key players that regulate their differentiation. Interestingly, pluripotency has various shades with distinct developmental potential, an observation that coined the term of a ground state of pluripotency. A precise interplay of signalling axes regulates ground state conditions and acts in concert with a combination of key transcription factors. The balance between these transcription factors greatly influences the integrity of the pluripotency network and latest research suggests that minute changes in their expression can strengthen but also collapse the network. Moreover, recent studies reveal different facets of these core factors in balancing a controlled and directed exit from pluripotency. Thereby, subsets of pluripotency-maintaining factors have been shown to adopt new roles during lineage specification and have been globally defined towards neuroectodermal and mesendodermal sets of embryonic stem cell genes. However, detailed underlying insights into how these transcription factors orchestrate cell fate decisions remain largely elusive. Our group and others unravelled complex interactions in the regulation of this controlled exit. Herein, we summarise recent findings and discuss the potential mechanisms involved

Pluripotency Factors on Their Lineage Move

Pluripotent stem cells are characterised by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers. Regulatory networks of maintaining pluripotency have been described in great detail and, similarly, there is great knowledge on key players that regulate their differentiation. Interestingly, pluripotency has various shades with distinct developmental potential, an observation that coined the term of a ground state of pluripotency. A precise interplay of signalling axes regulates ground state conditions and acts in concert with a combination of key transcription factors. The balance between these transcription factors greatly influences the integrity of the pluripotency network and latest research suggests that minute changes in their expression can strengthen but also collapse the network. Moreover, recent studies reveal different facets of these core factors in balancing a controlled and directed exit from pluripotency. Thereby, subsets of pluripotency-maintaining factors have been shown to adopt new roles during lineage specification and have been globally defined towards neuroectodermal and mesendodermal sets of embryonic stem cell genes. However, detailed underlying insights into how these transcription factors orchestrate cell fate decisions remain largely elusive. Our group and others unravelled complex interactions in the regulation of this controlled exit. Herein, we summarise recent findings and discuss the potential mechanisms involved

The role of pluripotency factors to drive stemness in gastrointestinal cancer

A better molecular understanding of gastrointestinal cancers arising either from the stomach, the pancreas, the intestine, or the liver has led to the identification of a variety of potential new molecular therapeutic targets. However, in most cases surgery remains the only curative option. The intratumoral cellular heterogeneity of cancer stem cells, bulk tumor cells, and stromal cells further limits straightforward targeting approaches. Accumulating evidence reveals an intimate link between embryonic development, stem cells, and cancer formation. In line, a growing number of oncofetal proteins are found to play common roles within these processes. Cancer stem cells share features with true stem cells by having the capacity to self-renew in a de-differentiated state, to generate heterogeneous types of differentiated progeny, and to give rise to the bulk tumor. Further, various studies identified genes in cancer stem cells, which were previously shown to regulate the pluripotency circuitry, particularly the so-called “Yamanaka-Factors” (OCT4, KLF4, SOX2, and c-MYC). However, the true stemness potential of cancer stem cells and the role and expression pattern of such pluripotency genes in various tumor cell types remain to be explored. Here, we summarize recent findings and discuss the potential mechanisms involved, and link them to clinical significance with a particular focus on gastrointestinal cancers

An inducible expression system of the calcium-activated potassium channel 4 to study the differential impact on embryonic stem cells.

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression