Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous—the source of cells for the SAN—suggest that Map3k7 may be an endogenous regulator of the SAN fate

eXtraembryonic ENdoderm (XEN) Stem Cells Produce Factors that Activate Heart Formation

Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1

Nodal mutant eXtraembryonic ENdoderm (XEN) stem cells upregulate markers for the anterior visceral endoderm and impact the timing of cardiac differentiation in mouse embryoid bodies

Summary Interactions between the endoderm and mesoderm that mediate myocardial induction are difficult to study in vivo because of the small size of mammalian embryos at relevant stages. However, we and others have demonstrated that signals from endodermal cell lines can influence myocardial differentiation from both mouse and human embryoid bodies (EBs), and because of this, assays that utilize embryonic stem (ES) cells and endodermal cell lines provide excellent in vitro models to study early cardiac differentiation. Extraembryonic endoderm (XEN) stem cells have a particular advantage over other heart-inducing cell lines in that they can easily be derived from both wild type and mutant mouse blastocysts. Here we describe the first isolation of a Nodal mutant XEN stem cell line. Nodal−/− XEN cell lines were not isolated at expected Mendelian ratios, and those that were successfully established, showed an increase in markers for the anterior visceral endoderm (AVE). Since AVE represents the heart-inducing endoderm in the mouse, cardiac differentiation was compared in EBs treated with conditioned medium (CM) collected from wild type or Nodal−/− XEN cells. EBs treated with CM from Nodal−/− cells began beating earlier and showed early activation of myocardial genes, but this early cardiac differentiation did not cause an overall increase in cardiomyocyte yield. By comparison, CM from wild type XEN cells both delayed cardiac differentiation and caused a concomitant increase in overall cardiomyocyte formation. Detailed marker analysis suggested that early activation of cardiac differentiation by Nodal−/− XEN CM caused premature differentiation and subsequent depletion of cardiac progenitors

Thermal stability of functionalized carbon nanotubes studied by in situ transmission electron microscopy

The thermal stability of functionalized carbon nanotubes (CNTs) has been studied experimentally by direct in situ observations using a heating stage in a transmission electron microscope, from room temperature (RT) to about 1000 °C. It was found that the thermal stability of the functionalized CNTs was significantly reduced during the in situ heating process. Their average diameter dramatically expanded from RT to about 500 °C, and then tended to be stable until about 1000 °C. The X-ray energy dispersive spectroscopy analysis suggested that the diameter expansion was associated with coalescence of the carbon structure instead of deposition with additional foreign elements during the heating process. © 2011 Elsevier B.V. All rights reserved

Heat map representation of detailed marker analysis.

<p>Microarray data of markers distinguishing VE from DE according to Sherwood <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012016#pone.0012016-Sherwood1" target="_blank">[2]</a>. Only a subset of the previously examined pan endodermal markers are expressed in the array. This refines the list of true pan endodermal markers to include <i>Sox17, Spink3, Rab15, Dsg2, Ripk4, AnxA4</i> and <i>Emb</i>. “VE enriched” genes indicate factors that were found to be expressed in VE plus other lineages but not DE. Our analysis suggests that a subset of these factors may not be present in all VE subtypes and thus may represent regionalized VE markers. “DE enriched” represents genes found, in Sherwood <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012016#pone.0012016-Sherwood1" target="_blank">[2]</a>, to be expressed in DE and other subtypes but not in VE. These data strongly suggest that the ExEn cell lines are not similar to DE. Fluorescence data was marked as 0.00 if the p-value of detection was greater than 0.01.</p

Immunocytochemical analysis of ExEn cell lines.

<p>Immunocytochemical analysis of confluent END2 (<b>A, D, G, J</b>), PYS2 (<b>B, E, H, L</b>) and XEN (<b>C, F, I, M</b>) cells showing the expression of GATA4 (<b>A, B, C</b>), GATA6 (<b>D, E, F</b>), SOX7 (<b>G, H, I</b>) and BMP2 (<b>J, L, M</b>) protein in END2, PYS2 and XEN cells. Merged images with DAPI staining (blue nuclei in all images) reveal ubiquitous expression of GATA4 and GATA6 in both PYS2 and XEN cells (<b>B, C, E, F</b>). SOX7 is ubiquitously expressed in PYS2 cells (<b>H</b>), while XEN cells express SOX7 only in a subset of cells (<b>I</b>). In END2 cells, GATA6 expression is limited to a small subset of cells (<b>D</b>) while GATA4 and SOX7 are not expressed (<b>A, G</b>). BMP2 is ubiquitously expressed by PYS2 and XEN cells (<b>L, M</b>), while END2 express BMP2 only in a subset of cells (<b>J</b>).</p

Heart inducing cell lines express markers characteristic of several primitive endoderm lineages.

<p><b>A.</b> Summary of Real-Time PCR on END2, PYS2 and XEN cells. Insert, cartoon showing embryonic lineages assessed, primitive endoderm (reds), parietal endoderm (oranges/yellows), visceral endoderm (greens) and AVE (blues). The panel of markers assessed include markers for primitive endoderm (<i>Sox7</i>, <i>Pdgfra, Gata4, Gata6</i>), parietal endoderm (<i>tPA, Krt19, Lamb1</i> and <i>SPARC</i>), visceral endoderm, (<i>FoxA2</i>, <i>Ttr</i>, u<i>PA and HNF4a</i>), anterior visceral endoderm (<i>Dkk1, Cerl, Hex</i>), the regionally restricted VE marker <i>Bmp2</i> and the definitive/pan endoderm marker <i>Sox17</i>. <b>B.</b> Linear regression analysis comparing real-time PCR data to averaged fluorscence detection in the Illumina Microarray. 80% of markers that we compared showed strong correlation between the qRT-PCR data and microarray detection. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012016#pone.0012016-Edgar1" target="_blank">[78]</a> and are accessible through GEO Series accession number GSE19564 (<a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1956" target="_blank">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1956</a>).</p

Heat map analysis of well-characterized markers for different endodermal cell types.

<p>Illumina microarray data for genes that are expressed in various endoderm subtypes (depicted as heat maps) includes regionally restricted markers representing the VE, DE, PE and PrE. Each of the three cell lines expresses markers for VE, PE and PrE. None of the cell lines express markers that are diagnostic for DE. Fluorescence data was indicated as 0.00 if the p-value of detection was greater than 0.01.</p

Marker expression in XEN cells is stable over several passages when cells are grown under standard serum-containing medium.

<p>qRT-PCR data comparing marker expression in XEN cells grown under standard serum-containing conditions to XEN cells grown in serum-free medium. Despite some spikes in marker expression at some passages, over the entire course of this experiment there were no obvious trends (either upward or downward changes in expression) in any of the markers assessed. In addition, while some markers were differentially expressed between the two culture conditions, there was also no obvious change in marker expression in the serum-free medium when assessed over the entire course of the experiment. This suggests that XEN cells are stable when grown in culture over several passages.</p