Tie2 Is Essential for the Development of Lymphangiogenic Endothelial Cell during ES Cell Differentiation.

Tie2 is a receptor type tyrosine kinase, and is expressed in the hematopoietic stem cells and endothelial cells. We have recently shown that Tie2 and its ligand Angiopoietin-1 (Ang1) signal play a crucial role for maintenance of long-term repopulating hematopoietic stem cells in adult bone marrow (Cell, Vol 118, 149, 2004). Although Tie2 deficient mice showed defect of endothelial cell development, it is not clear whether Tie2 is critical for the development of hematopoietic and lymphangiogenic endothelial cells. In order to clarify the function of Tie2 during the developmental stage, we have developed the cell culture system of ES cell differentiation. After removing LIF, ES cells were cultured on collagen type IV coated plates to promote the differentiation to mesodermal lineage for two days, and the cells were cultured on OP9 stromal cells. Using our system hematopoietic and endothelial progenitors were differentiated efficiently on OP9 cells from the different ES cell straines, E14, TT2, and R1. Expression study showed that ES cell derived cells expressed Tie2 and Flk1 at day 5 of culture on OP9 cells. When we compared the cell fraction sorted by Tie2 and Flk1 mAb regarding differentiation potential, Tie2+Flk1+ fraction was revealed to be an enriched fraction of progenitors for hematopoietic cells and PECAM-1+ endothelial cells. To detect the lymphangiogenic endothelial cells derived from ES cells, we prepared the monoclonal antibody against LYVE-1, which is the receptor for extracellular matrix, glycosaminoglycan. And we confirmed that LYVE-1 was expressed in the embryonic lymphatic endothelium. By using LYVE-1 mAb, we sorted out LYVE-1+ cells from differentiated ES cells and carried out RT-PCR assay. LYVE-1+ cells expressed lymphangiogenic endothelial cell-specific genes, VEGFR-3, Podoplanin, and Prox-1, moreover LYVE-1+ cells took up the DiI-Ac-LDL. These findings indicate that LYVE-1+ cells derived from ES cells have a character of lymphangiogenic endothelial cells. When we compared the cell fraction sorted by Tie2 and Flk1 mAb, LYVE-1+ cells were differentiated from Tie2+Flk1+ fraction dominantly, but not from the other fractions, Tie2-Flk1+, Tie2+Flk1−, and Tie2-Flk1− fraction. These findings suggest that Tie2 is crucial for development of lymphangiogenic endothelial cells as well as hematopoietic cells and endothelial cells. In order to analyze the function of Tie2 during the developmental stage, we differentiated Tie2−/− ES cells using our system. The LYVE-1+ and PECAM-1+ cells derived from Tie2−/− ES cells dramatically decreased as culturing days went by, and at day6 of culture the LYVE-1+ and PECAM-1+ cells derived from Tie2−/− ES cells were one sixth and one third of Tie2+/− cells respectively. When we added 100μM of caspase inhibitor in the culture media, the number of both LYVE-1+ cells and PECAM-1+ cells were recovered. These findings suggest that developmental defect of lymphangiogenic endothelial and endothelial cells are caused by apoptosis because of the blockage of Tie2 signaling. However we could not detect abnormal development of hematopoietic cells from Tie2−/− ES cells. In conclusion, Tie2+Flk1+ fraction derived from ES cells is an enriched fraction of progenitors for lymphangiogenic endothelial cells, and Tie2 signaling is dispensable for lymphangiogenic endothelial cell development as well as endothelial cell development as an anti-apoptotic signaling during ES cell differentiation, but Tie2 is not essential for hematopoietic development.