In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK⁺ cells existed in the AGM region. Approximately 5% of AGM cells were TEK⁺, and most of these were CD34⁺ and c-Kit⁺. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK⁺ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1⁺ endothelial cells on the OP9 stromal layer, while TEK^- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK⁺ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK⁺ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.