Basic Biology I
Stem Cell Use:
Embryonic Stem Cell
Directly Reprogrammed Cell
Hematopoietic cells are responsible for generating all cell types present in the blood and therefore critical for the provision of oxygen and nutrients to all the tissues in the body. Blood cells are also required for defense against microorganisms and even for the recognition and elimination of tumor cells. Because blood cells have a relatively short life-span, our bone marrow is constantly producing new cells from hematopoietic progenitors and responding to the relative needs to our tissues and organs. Blood cancers (leukemias), as well as other disorders or treatments that affect the production of blood cells (such as chemotherapy or radiation therapy) can significantly jeopardized health. Transfusions are done to aid the replacement of blood cell loss, but pathogens and immunological compatibility are significant and frequent roadblocks. In this grant application, we present experiments to further understand how another cell in the body, the endothelium, located in the inside wall of all our vessels, can be coax to produce large numbers of hematopoietic cells with indistinguishable immunological properties from those in the bone marrow of each individual. Endothelial cells are easily obtained from skin biopsies or from umbilical cord and they can be expanded in Petri dishes. The experiments outlined were designed to further understand how endothelial cells are capable of generating blood cells during development. This information will be used to entice endothelial cells to generate hematopoietic cell progenitors in vitro. The impact of this research is broad because of its clinical applicability and because of its potential to decipher the mechanisms used by endothelial cells to undergo normal reprogramming and generate undifferentiated progenitor cells of a distinct lineage. Adult cell reprogramming is one of the fundamental premises of stem cell research and thus, highly relevant to the main goals identified by the CIRM program.
Statement of Benefit to California:
Technology developed from this grant application has the potential to be translated directly to clinical settings. This technology is extremely likely to engender interest by the big pharma which can potentially license the information from the University of California or purchase the patent for the invention / technology. Naturally this will bring revenues and recognition to the state of California. Furthermore, California will remain ahead of the technological wave that takes advantage of stem cell technology and implements innovative medical treatments in the entire country and abroad. In addition, the execution of this proposal will immediately provide employment to four individuals, two of these trainees in stem cell research. Indirectly, the grant will also support salaries of employees at the university associated with research, animal care and administration.
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.