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

Abstract

SummaryInteractions 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.

Characterization of porcine extraembryonic endoderm cells

“…The culture system we used in this study is very different from that used to culture murine XEN cells, 10 lead to the derivation of bona fide pPSCs. 28,29 To summarize, we have established one pXENC line exhibiting typical morphology and markers as murine XEN cells. A, Signalling pathway enrichment in gene subsets differentially expressed between pXENCs and porcine-induced pluripotent stem cells (piPSCs).…”

A bright single-cell resolution live imaging reporter of Notch signaling in the mouse

“…ES cells were passaged off MEFs, as previously described [35], then plated as hanging drops at a concentration of 15,000 cells/mL (300 cells per hanging drop) in Iscove's Modified Dulbecco Medium (IMDM; Gibco) containing 20% serum (Gibco), 50 mg/mL each of penicillin/streptomycin (Gibco), 200 mg/mL apo-transferrin (Sigma), 5% protein-free hybridoma medium (PFHMII; Gibco), 0.5 mM 1-thioglycerol (Sigma), and 0.5 mM ascorbic acid (Sigma). Ebs were harvested after 1, 2, 4 and 6 days of culture and imaged.…”

Retinoic Acid and the Development of the Endoderm

“…Nodal, a member of the TGFβ superfamily of signaling molecules is essential for the development of the mesendoderm [19] and varying levels of Nodal signaling are important not only in the segregation of the endoderm from mesoderm [20], but also in patterning the extraembryonic endoderm [21,22] as described below. Nodal signaling subsequently regulates the expression of several transcription factors that represent a conserved network essential for differentiation of the endoderm including members of the Forkhead, Gata, Mixer, Sox, and PouV transcription factor families [13,23,24].…”