Hemogenic Endothelial Cell Specification Requires c-Kit, Notch Signaling, and p27-Mediated Cell-Cycle Control

Abstract

SUMMARY Delineating the mechanism(s) that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development, and demonstrate that cell cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2−/−) primordial endothelium induced Notch signaling and p27 expression, which restored cell cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling and p27-mediated cell cycle control.

Isolation of Murine Embryonic Hemogenic Endothelial Cells

“…Like HSPC within adult bone marrow 17 , embryonic hemogenic endothelial cells and HSPC exhibit Hoechst dye efflux properties and therefore appear within the "side population" (SP) of cells on a FACS plot 5,11,12 (as shown in Figure 3). In addition, we have also shown that hemogenic endothelial cells express markers of both endothelial and stem cells (Flk1 and cKit, respectively), but do not express the hematopoietic lineage marker, CD45 5,11,12 . Thus, hemogenic endothelial cells can be identified and isolated by FACS as Flk1+/cKit+/CD45-SP cells, and we have shown that these cells give rise to HSPC contained within the Flk1-/cKit+/CD45+ SP fraction of yolk sac and AGM cells 5,11,12 .…”

“…In turn, the eventual development of an approach for large-scale generation of differentiated blood cell types from multi-lineage HSPC -themselves derived from human pluripotent stem cells via a physiologically relevant hemogenic endothelial cell intermediate -would have incredible therapeutic potential for hematologic, oncologic and regenerative medicine. Towards this goal, we have defined the phenotype of hemogenic endothelial cells, on a clonal level, within the murine yolk sac 11 and AGM 12 , two major sites of definitive HSPC production during embryogenesis. Like HSPC within adult bone marrow 17 , embryonic hemogenic endothelial cells and HSPC exhibit Hoechst dye efflux properties and therefore appear within the "side population" (SP) of cells on a FACS plot 5,11,12 (as shown in Figure 3).…”

“…Towards this goal, we have defined the phenotype of hemogenic endothelial cells, on a clonal level, within the murine yolk sac 11 and AGM 12 , two major sites of definitive HSPC production during embryogenesis. Like HSPC within adult bone marrow 17 , embryonic hemogenic endothelial cells and HSPC exhibit Hoechst dye efflux properties and therefore appear within the "side population" (SP) of cells on a FACS plot 5,11,12 (as shown in Figure 3). In addition, we have also shown that hemogenic endothelial cells express markers of both endothelial and stem cells (Flk1 and cKit, respectively), but do not express the hematopoietic lineage marker, CD45 5,11,12 .…”

“…In contrast, at later stages of blood cell development (definitive hematopoiesis), it has become increasingly clear that multi-lineage HSPC arise from specialized vascular endothelial cells that acquire blood forming potential (hemogenic endothelial cells) within the yolk sac, placenta, and AGM [2][3][4][5] , as well as in the vitelline and umbilical vessels, the embryonic endocardium 6 , and head vasculature 7 . The specification of hemogenic endothelial cells within these distinct tissues occurs at specific stages of development; for example, within the yolk sac at ~E8.25 and within the AGM at ~E10 [8][9][10][11][12] . Nonetheless, even during these specific developmental windows, the population of hemogenic endothelial cells represents a small fraction of all endothelial cells (1 -3% of yolk sac and AGM endothelial cells) 11,12 .…”

“…The specification of hemogenic endothelial cells within these distinct tissues occurs at specific stages of development; for example, within the yolk sac at ~E8.25 and within the AGM at ~E10 [8][9][10][11][12] . Nonetheless, even during these specific developmental windows, the population of hemogenic endothelial cells represents a small fraction of all endothelial cells (1 -3% of yolk sac and AGM endothelial cells) 11,12 . The process of hemogenic endothelial cell "specification" is critical for murine, as well as human, hematopoiesis.…”