Interleukin-1 (IL-1) Gene Expression in Mouse Embryonic Stem (ES) Cells

Abstract

To study the IL-1 family gene expression in undifferentiated mouse embryonic stem (ES) cells and ES cells derived cardiomyocytes. Prospective study We studied the IL-1 family gene expression in two different groups: undifferentiated mouse ES cells and ES cell derived cardiomyocytes. We used OCT-4 and Nanog as markers of the undifferentiated state of nine new mouse ES cells lines, isolated from the 129 substrain mouse. Zona-free blastocysts were used as a positive control for Oct-4 and Nanog expression and β actin was used as control for the presence of template. Biopsies retrieved at 5, 10, 15, 20 and 25th passages were taken to performe RT-PCR using SuperScript™ III CellsDirect cDNA Synthesis System. PCR amplification was done with Platinum® Taq DNA Polymerase. We used Interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1ra) and IL-1 receptor type I (IL-1RtI) primers to study the IL-1 family. The presence of heart beating identified ES cells derived cardiomyocytes, which were isolated by microsurgery to be used as samples for gene expression study. We used FGF5, Gata6 and Brachyury (T) as markers for the presence of ectoderm, endoderm and mesoderm, respectively. RT-PCR and PCR were run with negative controls and 10 μl of PCR products were analyzed by 2% gel electrophoresis. None of the 45 undifferentiated ES cells were positive for IL-1β; 1 out of 45 was positive for IL-1ra, and this sample was also positive for ectoderm and endoderm primitive markers; 1 out of 45 was positive for IL-1RtI and also for ectoderm and mesoderm primitive markers. Four out of 4 ES cells derived cardiomyocytes were positive for IL-1β and also for IL-1ra. Two out of 4 ES cells derived cardiomyocytes were positive for IL-1RtI. The IL-1 family has been correlated to implantation in mammals by many articles, but to our knowledge this is the first time to describe an IL-1 family gene expression study in undifferentiated mouse ES cells. It appeared in samples that have already shown signals of differentiation into mesoderm, ectoderm or endoderm or in ES cells derived cardiomyocytes. Undifferentiated mouse ES cells most likely do not produce major components of IL-1 family. IL-1 may be produced mainly by trophoblast cells in embryo-maternal cross-talk during implantation. Further experiments including others methodologies may provide more information to elucidate the role of IL-1 family during implantation process in mouse.