Bone morphogenetic protein 4 differently promotes distinct VE-cadherin⁺ precursor stages during the definitive...

Definitive hematopoietic cells develop from fetal liver kinase 1 (Flk1)⁺ mesodermal cells during the in vitro differentiation of mouse embryonic stem cells (ESCs). VE-cadherin⁺CD41^-CD45^-(V⁺41^-45^-) hemogenic endothelial cells (HECs) and VE-cadherin⁺CD41⁺CD45^- (V⁺41⁺45^-) cells mediate the definitive hematopoietic development from Flk1⁺ cells. Bone morphogenetic protein 4 (BMP4) is known to be essential for the formation of mesoderm. However, the role of BMP4 in differentiation of the VE-cadherin⁺ definitive hematopoietic precursors from the mesoderm has been elusive. We addressed this issue using a co-aggregation culture of ESC-derived Flk1⁺ cells with OP9 stromal cells. This culture method induced V⁺41^-45^- cells, V⁺41⁺45^- cells, and CD45⁺ cells from Flk1⁺ cells. V⁺41⁺45^- cells possessed potential for erythromyeloid and T-lymphoid differentiation. When Flk1⁺ cells were cultured in the presence of a high concentration of BMP4, the generation of V⁺41^-45^- cells was enhanced. The increase in V⁺41^-45^- cells led to the subsequent increase in V⁺41⁺45^- and CD45⁺ cells. The addition of BMP4 also increased hematopoietic colony-forming cells of various lineages. Furthermore, BMP4 promoted the expansion of V⁺41⁺45^- cells independently of the preceding V⁺41^-45^- cell stage. These results suggest that BMP4 has promotive effects on the differentiation of V⁺41^-45^- HECs from Flk1⁺ mesodermal cells and the subsequent proliferation of V⁺41⁺45^- hematopoietic precursors. These findings may provide insights for establishing a culture system to induce definitive hematopoietic stem cells from ESCs.