Molecular Characterization and Functional Exploration of Hemogenic Endothelium

During this year, we have demonstrated that hematopoietic stem cells are originated from the cells that line the inside of blood vessels, named endothelial cells. Budding of hematopoietic stem cells from endothelial cells occurs during a specific and restricted time window during development and progress has been made to elucidate the regulatory genetic networks involved in this process. We have also demonstrated that hemogenic endothelium is derived from one specific embryonic tissue (lateral plate mesoderm). This information will be used to recapitulate similar conditions in vitro and induce the growth of hematopoietic stem cells outside the body from adult endothelial cells.

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.

In this grant application we have been able to investigate the mechanisms by which endothelial cells, the cells that line the inner aspects of the entire circulatory system, produce blood cells. This capacity, called “hemogenic” (giving rise to blood) can be extremely advantageous in pathological situations when generation of new blood cells are needed, such as during leukemia or in organ-transplantation. Although the hemogenic capacity of the endothelium is, under normal conditions, restricted development we have been able to “reprogram” this ability in endothelial cells. For this, we first investigated the genes that responsible for this hemogenic activity during development using mouse models and tissue culture cells. Using this strategy we found key transcription factors in hemogenic endothelium not present in other (non-hemogenic) endothelial cells. Subsequently, we validated that these genes were able to convey hemogenic capacity when expressed in non-hemogenic sites. Finally, using human endothelial cells, we have been able to impose expression of these key transcription factors in endothelial cells. Our data indicates that the forced expression of these factors is able to initiate a program that is likely to result in blood cell generation. The progress achieved through this grant place us in a remarkable position to carry out pre-clinical trials to evaluate the potential of this technology.