Lymphoid, Long-Term Repopulating, and Endothelial Potential of the Embryonic Hemangioblast.

Hematopoietic and endothelial cells are thought to arise from a common progenitor termed the hemangioblast. Direct evidence for the hemangioblast was first obtained from embryonic stem cells differentiated in vitro as embryoid bodies (EBs). Flk-1+ cells from early EBs generate colonies in response to VEGF and SCF (the BL-CFC assay) which can be replated to give secondary hematopoietic or endothelial cells. Bipotent BL-CFCs have also recently been derived from the posterior primitive streak of neural plate stage mouse embryos. However, a direct relationship between the early embryonic hemangioblast defined by the BL-CFC assay and the hematopoietic stem cell remains unproven. Hemangioblast-derived hematopoiesis in vitro is transient and restricted to myelo-erythroid differentiation. Lymphoid potential and long-term repopulation, two hallmarks of the definitive hematopoietic stem cell, have eluded detection to date. Previous work has shown that the homeodomain transcription factor, HoxB4, by enhancing self-renewal in vitro, can reveal latent definitive HSC activity of transient embryonic hematopoietic progenitors. Using an ES cell line with doxycycline-inducible HoxB4 expression, we have investigated the definitive hematopoietic and endothelial potential of individual hemangioblast colonies. BL-CFC numbers were unaffected by HoxB4 expression during EB differentiation, however they were increased threefold by induction during the BL-CFC assay. By replating one half of the cells from an individual blast colony in endothelial medium and the other half on an OP9 monolayer with hematopoietic cytokines, we show that the majority (60%) of HoxB4-induced BL-CFCs are bipotent. HoxB4 expression was compatible with endothelial differentiation and allowed exponential expansion of hematopoietic progenitors on OP9 cocultures. When switched to OP9-DL1 with lymphoid cytokines, T-lymphopoiesis was observed characterized by CD25 expression followed by CD4, CD8, and CD3epsilon expression. To assay long-term repopulation, individual blast colonies were picked and divided into endothelial medium and OP9 monolayers. The hematopoietic arms of colonies defined retrospectively to have been bipotent (endothelial differentiation was observed in vitro) were transplanted into sublethally irradiated Rag2; gamma-c; CD45.1 immunodeficient mice. Mice with long-term hematopoietic engraftment were identified by the presence of CD45.2 cells in peripheral blood 3 months post-transplant. Lymphoid and myeloid contribution was evaluated by costaining with Gr-1, B220, CD19, CD4, and CD8. The donor-derived component of these hematopoietic chimeras, including their entire lymphoid arm (approximately 1/3 of engrafted mice showed lymphoid differentiation) is by definition clonally derived from a single hemangioblast. These results clearly show that the embryonic hemangioblast is not intrinsically limited in its hematopoietic potential. Under conditions that favor self-renewal, lymphoid differentiation and long-term repopulation become evident, revealing the link between endothelial development and definitive hematopoiesis at the clonal level.