Background
It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB). We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine.
Conclusion
1) Hematoendothelial precursors exist transiently in early embryonic development and form single cell-derived colonies; 2) their differentiation can be tracked by the use of chosen molecular markers; 3) blast colonies consist of cells having properties of endothelial and hematopoietic precursors, however the issue of their ability to maintain dual properties over time needs to be further explored; 4) blast cells can potentially be used in regenerative medicine due to their low expression of HLA molecules.
Methods
We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines.
Results
The occurrence of blast-colony-forming cells (BL-CFC) during EB differentiation (day 0-6) was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30-50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 +/- 0.8%) containing macrophages, BFU-E colonies (19.5 +/- 3.5%) containing nucleated red blood cells, and CFU-EM colonies (17.1 +/- 2.7%) composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both epsilon - and gamma- globin genes, but not adult-type gamma-globin. When in endothelial cell culture conditions, blast cells differentiated to endothelial cells which had the ability to take up Dil-Ac-LDL and to form complex vascular networks in Matrigel.