Transcriptome imposed by Notch ligands (Jag1 or Dll4) on ckit+ and ckit- cells from E11.5 AGM

Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Cells that emerge from the endothelium and show hematopoietic traits can be distinguished by the expression of the c-kit receptor and finally acquire the CD45 marker. The effect of Notch activation by each one of the ligands has been tested on c-kit+ and ckit- cells from the endothelium of the E11.5 AGM . Incubations with stromal cell lines exposed the cells to high levels of ligand and induced changes in the transcriptome of these cells. AGM regions were obtained from E11.5 embryos by dissection and digested with 0.1% collagenase. Cells were stained with anti-CD31, anti-CD45 and anti-Ter119 antibodies. Sorting of the CD31+CD45-Ter119- population was performed and some cells were separated into ckit+ and ckit- (data previously submited GSE35395). The rest of the CD31+CD45- cells were incubated for 2 hours on OP9 stromal cells expressing ectopic Jagged1 or Delta4 (OP9-Jag1 and OP9-Dll4). After culture with each ligand, cells were separated into CD31+CD45-ckit+ and CD31+CD45-ckit-. Cells were harvested for RNA puriication and microarray analysis.