Bone marrow transplantation provides a source for human blood stem cells. These cells have been used for years in the treatment of immunodeficiency, cancer and other genetic disorders. Unfortunately, human blood stem cells have a short life span and cannot be easily expanded in the laboratory setting. These shortcomings can be circumvented by the use of human embryonic stem cells (hESC). Human ESC can produce every tissue of the body (including blood), and can be expanded indefinitely in the laboratory. The goal of our work is to understand the cues that control hESC generation of blood cells. In particular, we want to define the role played by adhesion molecules, molecules that mediate cell-to-cell interactions, in the generation of blood cells from hESC. To achieve our goals, we need to develop new tools that will allow us to follow the hESC as they progress down the pathway of becoming a blood cell. In particular, we propose to endow hESC with a reporter molecule that will identify cells that have decided to follow the blood cell path. Moreover, we will develop novel culture methods in the laboratory to assay the hESC’s capacity to produce blood cells. These innovative tools will permit us to determine which adhesion molecules are present in hESC and how the expression of these molecules fluctuate during the process of blood generation. Understanding the process of blood generation from hESC will allow us to develop new therapeutic strategies for the treatment of immunodeficiency and cancer. This aspect of Regenerative Medicine will have major impacts on the way that bone marrow and blood stem cell transplantation is done, hopefully reducing or completely eliminating the detrimental side effects of this therapy.
Statement of Benefit to California:
California has been at the forefront of scientific discovery. It is known throughout this country for its cutting edge approach to scientific research. The work described in this proposal will help increase our understanding of the process of blood cell generation from human embryonic stem cells. Ultimately, this knowledge will allow us to develop new therapeutic strategies for the treatment of many diseases, including transplantation of blood stem cells to treat immunodeficiency, genetic deficiencies and cancer. California will benefit directly from these discoveries at multiple levels. First, it will support the state reputation as a beacon of scientific discovery. This will positively influence the recruitment of students, scientists and medical doctors to the state, thereby increasing the intellectual pool that will further feed California’s scientific and intellectual engine. Second, discoveries of this nature will foster more entrepreneurial and biotechnological developments within the state, resulting in the creation of companies that will provide needed jobs and help the state’s economy. Lastly, the creation of these companies will result in the promotion of these scientific discoveries to the clinical setting, ultimately benefiting California’s citizens that desperately need these life saving therapies.
SYNOPSIS: This is a project to evaluate the function of adhesion molecules during commitment of ES cells to hemangioblast fate. The proposal is based largely on what is known in the mES system, and an attempt to translate this to the hES system. The goal of Aim 1 is to generate an Scl:GFP reporter line via a BAC-based knockin approach and to develop an in vitro colony assay to monitor transition to Hgb fate. This approach has worked well in the mES system. If it fails, the PI will follow instead CD45. It is not clear which hES cells will be used (one or several lines?). Experiments in Aim 2 will evaluate the expression profiles for a limited set of adhesion molecules representing tight (claudin4, ZO1, ZO2), gap (connexin 31, 43, 45), and adherens (E-cad) junctions. Using scl:gfp combined with known surface markers (flk1, cd45, vecad, Pecam, etc.) subsets of EB cells will be sorted and the expression levels for the junction proteins monitored by qRT-PCR. In Aim 3, the specific set of adhesion molecules will be interrogated for roles in differentiation of hematopoeitic cells using a lentiviral shRNA silencing approach. SIGNIFICANCE AND INNOVATION: There is clearly much to be learned about the function and regulation of adhesion molecules during the commitment and differentiation of hematopoietic cells. An effective hgb assay in the hES system would be valuable. The approach here is to translate the known pathways in mES to the hES system. Adhesion molecules are certain to play an important role in the generation of hematopoietic stem cells from ES cells and yet this has received very limited study. This proposal is novel in its focus on the role of adhesion molecules in that process. If we were able to derive HSCs from ES cells, it would be a critical advance that could change the way in which some patients are transplanted after chemotherapy or radiation therapy. STRENGTHS: The project is well focused, albeit perhaps a bit too limited on known factors. In principle, the approach is a good one, as validated previously in the mES system. A reviewer liked the idea of sorting cells based on differences in adhesion molecule expression to compare their potential in a functional assay for hematopoietic progenitors. The reviewer believed the authors are certain to learn interesting new things in this aim. The authors propose to assay for hemangioblast potential, but this is not the easiest assay so it might not turn out to be the best choice. Another simple possibility would be to assay for CFU-C activity. Ultimately, once the authors start making progress toward the derivation of hematopoietic progenitors, they may want to inject cells into immunocompromised mice to test for transplantable HSC activity. WEAKNESSES: The success of the project is highly dependent on the successful generation of the reporter line and in addition a useful hgb colony assay. The PI has no experience in hES cells, and limited data generated in the mES system. Assistance will be sought from a core facility at UC Davis. The experiments are clearly feasible in the mES system and could be carried out now. It is not clear how much new information will be gained from the effort to establish similar assays in the hES, other than to confirm similar or different relative expression for a limited number of adhesion molecules. Another issue is that all of the analysis will be RT-PCR data, which may or may not reflect normal activity of these molecules. Additional weaknesses: Scl may be a misleading indicator for the generation of hematopoietic precursors. Scl is also expressed in other tissues, like the nervous system where it is involved in the generation of astrocytes. Since neural development is common from ES cells in culture, this could be a major confounding factor. While the general idea of studying the role of adhesion molecules is a good one, the methods used to identify the adhesion molecules that will be functionally studied in this proposal are not very compelling. It remains unclear whether the adhesion molecules detailed in this proposal play any role in the formation of hematopoietic progenitors. Little preliminary data are expected for this grant mechanism, so this is not a fatal flaw. However, the authors should think about other approaches to supplement those in their proposal. Dr. Garcia-Ojeda has only published 3 first-author papers, and they were all co-first authorships, raising questions regarding productivity and independence. This may be a project that is currently better suited to the mES system. DISCUSSION: There was concern that the human hemangioblast assay is not a trivial assay to develop - assay development could take entire period of grant, perhaps could be initially developed more productively in mouse ES system. Another discussant disagreed, stating that this project should be done in human. There is a clear rationale for deriving hHSC from hESC.