Year 2

The objective of this proposal was to identify factors that allow blood vessels to generate hematopoietic stem cells early in the embryonic stage. The process of blood generation from vessels is a normal step in development, but it is poorly understood. We predicted that precise information related to the operational factors in the embryo would allow us to reproduce this process in a petri dish and generate hematopoietic stem cells when needed (situations associated with blood transplantation or cancer).

In the second year of this proposal, we have made significant progress and identified critical factors that are responsible for the generation of hematopoietic stem cells from the endothelium (inner layer of blood vessels). These experiments were performed in mouse embryos, as it would be impossible do achieve this goal in human samples. The genes identified are not novel, but have not been associated with this capacity previously. To verify our findings we have independently performed additional experiments and validated the information obtained from sequencing the transcripts.

In addition, we developed a series of novel tools to test the biological relevance of the genes identified in vivo (using mouse embryos). Specifically, we have tested whether forced expression of these genes could induce the generation of hematopoietic stem cells. Interestingly, we found that a single manipulation was not sufficient, but multiple and specific manipulations resulted in the generation of blood from endothelium. This was a very exciting result as indicated that we are in the right track and identified factors that can reprogram blood vessels to bud blood stem cells. With this information at hand, we moved into human cells (in petri dishes).

The first step was to test whether human endothelial cells could offer a supportive niche for the growth of hematopoietic cells. To our surprise, we found that in the absence of any manipulation, endothelial cells could direct differentiation and support the expansion of CD34+ cells (progenitor blood cells) to a very specific blood cell type, named macrophages. These were rather unexpected results that indicated the ability of endothelial cells to offer a niche for a selective group of blood cells. The final question in the proposal was to test whether the modification of endothelial cells with the identified factors could induce the formation of blood from these cells. For this, we have generated specific reagents and are currently performing the final series of experiments.