Genetic dissection of mesodermal commitment to hematopoietic fates.
Hematopoietic cell transplantation is the gold standard for cell-based therapy and is routinely used to treat a wide variety of blood disorders and cancer. A major limitation exists, however, in finding donors whose immune systems are compatible with those of the patients requiring transplantation. The recent creation of human embryonic stem cell (hESC) lines holds great promise for new cell-based therapies. ES cells can generate all cell types in the body and can be stored indefinitely. Large banks of genetically diverse or genetically engineered hESC cells could thus be used to match donor and host immune systems. For hematopoietic cell transplantation, ESCs must be coaxed to differentiate into hematopoietic stem cells (HSCs). This is currently not possible, due in large part to a lack of understanding of the molecular cues required to generate HSCs during development.
In the vertebrate embryo, two waves of blood cell production occur. The first generates only erythroid cells and the second HSCs. Understanding the development of these two waves is important since ES cells have been shown to normally generate only the first. In this application, we will determine the genetic factors necessary to create HSCs from mesoderm by leveraging the unique advantages of the zebrafish system. Zebrafish embryos are transparent, and we have recently created transgenic animals that possess fluorescent HSCs. We will therefore combine genetic analyses with the direct imaging of HSC behavior in living embryos to provide an unprecedented view of HSC development. Many of the genetic pathways used to pattern the early embryo are later used to specify and maintain HSCs. These include pathways controlled by the Notch and Wnt factors. We will focus our efforts on these pathways using in vivo developmental and genetic approaches. Zebrafish possess the same blood cell types as humans, and findings in one system can be readily translated to the other. Understanding the development of HSCs in the vertebrate embryo, and how we can ultimately recapitulate this process in vitro using hESCs, is critical in improving human health since HSCs are the cells responsible for the therapeutic benefits of hematopoietic cell transplantation.
The therapeutic use of stem cells began decades ago following the advent of bone marrow transplantation (BMT). BMT has routinely been used to cure blood cell disorders, leukemia, and immune deficiencies. BMT is often limited, however, by an inability to find donors that are genetically matched to patients requiring transplantation. The recent creation of human embryonic stem cell (hESC) lines holds great promise for new cell-based therapies, including BMTs. ESCs can generate all cell types in the body and can be stored indefinitely. Large banks of genetically diverse or genetically engineered hESCs could thus be used to match donor and host immune systems. For use in BMTs, however, ESCs must be coaxed to differentiate into hematopoietic stem cells (HSCs), the rare cell type within bone marrow responsible for the long-term, curative effects of BMT. This is currently not possible, due in large part to a lack of understanding of the molecular cues required to generate HSCs during development.
The goal of our proposed experiments is to provide a better understanding of the molecules required to generate HSCs in the vertebrate embryo. The results from our powerful in vivo system can easily be translated to the in vitro system of hESCs. Insight into the molecular factors needed to drive mesodermal commitment to HSCs in vivo will be used to provide similar factors at similar timepoints during in vitro culture of hESCs to generate HSCs. Once realized, it will be possible to create genetically characterized banks of diverse hESCs that can be selected based on patient genotypes to generate HSC-based therapies. Our research will thus lead to great improvements in stem cell therapies to better meet the needs of patients in California.
SYNOPSIS: This proposal uses zebrafish as a model to discover early aspects of development that lead to the specification of hematopoietic stem cells (HSC) in vertebrates. The project takes advantage of recent discoveries made in the applicant's lab that have made possible the direct identification of 2 distinct precursor populations during definitive hematopoiesis in the developing zebrafish: early erythromyeloid progenitors (EMPs), which generate only a subset of hematopoietic lineages and lack self-renewal activity, and multipotent, self-renewing HSCs. The applicant will first use transcriptional profiling arrays to compare gene expression in FACS-purified EMP and HSC populations to identify candidate genes that might account for the differences in self-renewal of these populations. He will further study their function using knock-down and overexpression approaches. In the second aim, the applicant will use a unique and innovative laser-based uncaging system to track specific mesodermal precursors as they differentiate into HSC in the developing zebrafish. The final two aims will exploit the genetic malleability of the zebrafish to investigate the importance of Notch and Wnt signaling in early specification of zebrafish hematopoietic stem and progenitor cells versus vascular lineages. This work is described in relation to the prospects of ultimately improving bone marrow transplantation and the preparation of HSCs from human embryonic stem (ES) cells.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The topic of the emergence of adult HSCs is one of the most important in the field of hematopoiesis. Despite many years of active study, we still do not understand basic aspects of HSC generation. As a paradigm for other tissue stem cells, these studies are highly significant. Innovation is high in that the PI uses the most contemporary methods for study of the zebrafish and capitalizes on the strengths of the fish system. The studies are well designed and entirely feasible. The PI has all the requisite experience to embark on the proposed studies and there is little doubt he will continue to generate new insight into the derivation of HSC fate and the regulatory pathways that contribute to stem cell numbers and function.
The experimental design is extremely ambitious, although many of the reagents needed appear to be already on hand, as demonstrated in the very beautiful preliminary data supplied. While focused on the early emergence of zebrafish progenitors, the PI anticipates translating this work to human ESC models. Derivation of HSC from hESC is poorly characterized, and the PI believes this results from a relatively poor understanding of how HSC fate is normally specified from mesoderm. Information derived from the zebrafish model could identify new essential regulatory components.
The first aim encompasses large scale transcriptional profiling to identify genes that are differentially expressed by non-self-renewing, lineage-restricted EMPs vs. multipotential, self-renewing HSCs. While these experiments are clearly feasible, they are not particularly imaginative. The applicant suggests a prioritization scheme that will focus on genes/pathways that are already well-studied (BMP, FGF, Wnt and Notch), but he does not indicate what level of differential expression will merit further investigation, or how many targets can/will be tested functionally. Though this gene perturbation approach is higher throughput in zebrafish than in mammalian systems, specificity and off-target controls need to be included, and some estimation of the number of genes that can reasonably be screened in the time period of the grant would have been helpful.
The cell fate mapping studies using uncaged rhodamine are very interesting and creative, and seem to be feasible based on the PI's prior publications. The time-lapse studies are also of interest. These fate mapping experiments are by nature descriptive but really take strong advantage of the fish system, although no data is presented to indicate the feasibility of adapting this system to highly migratory hematopoietic cells. It also is unclear that this ambitious project will be accomplished in the timeframe of the grant.
In the final aims of the grant, the PI will investigate the importance of the Notch and Wnt pathways in HSC and vascular cell formation. Notch and Wnt have been studied extensively in the hematopoietic system, although data regarding the importance of these pathways are still in some cases contradictory. It is not clear how this system will better clarify these issues, as the precise experiments to be performed and why the particular targets that were chosen are the best for this analysis is not clearly defined. That being said, the manipulation of Notch and Wnt pathways can clearly be accomplished, especially given the preliminary data, although they are not based on functional readouts and this will lead to some “background” that the PI will need to sort through.
There was one further minor issue of concern. It was not clear how the PI will cleanly isolate pure populations of EMP and HSC, since they express the same markers. They can perhaps be isolated by dissection, but this will vastly reduce the cell numbers obtained. At a given developmental stage, how do you know that a cell born in one place has not migrated out to another?
Overall, the projects are interesting, appropriate, and highly likely to generate useful and important information. They include both straighforward genomic analysis and genetic manipulations as well as innovative imaging approaches that have the potential to shed new light on the cellular events involved in hematopoietic specification.
QUALIFICATIONS AND POTENTIAL OF THE PRINICIPAL INVESTIGATOR: Dr. Traver has exceptional training in stem cell biology, and is an extremely talented and productive researcher. He has an impressive record of productivity, both in the publication of high quality manuscripts and in recruiting funding for his research. He trained first with Irv Weissman as a graduate student and published 7 first author manuscripts including papers in Nature, Science, and Immunity. He then “translated” his hematopoietic training to zebrafish in Len Zon’s laboratory as a postdoc, where he developed sorting and transplant techniques that are of tremendous utility to the system, particularly for analyzing adult stage HSC. He was hired at UCSD in 2004 as an Assistant Professor and he is doing extraordinarily well. He already has 6 postdoctoral fellows (all with their own funding), 4 graduate students and has obtained significant funding (K01, R01, ASH, Beckman, Kimmel). Dr. Traver is clearly on a steep upward trajectory in his career, and is an outstanding candidate for a New Faculty Award.
Dr. Traver has a long-standing commitment and successful track record for defining HSC and early progenitors. He is highly capable in both zebrafish and mouse models, and plans to eventually combine the strengths of both systems to define regulatory mechanisms that can be used for hESC research and translation. The collaboration with Scott Fraser is an excellent match and should be fruitful. The applicant outlines a reasonable and logical career development plan, with appropriate emphasis on research and teaching, which includes starting a new stem cell course. He cites direct evaluation by faculty members at UCSD, who will monitor his progress. He also clearly benefits from excellent mentorship relationships with Geoff Rosenfeld, Ken Kaushansky, and William McGinnis.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: UCSD has made a substantial investment to support the career of this young scientist. He has been afforded with outstanding space (2200 sqft) and start up funds, plus zebrafish facilities and imaging equipment. There is a strong commitment to his success and development. An outstanding faculty mentoring committee is described including Geoff Rosenfeld and Ken Kaushansky, the latter of whom has already assisted the PI in developing in vitro culture assays for the fish hematopoietic cells.
UCSD has made a clear investment particularly in promoting stem cell science, in establishing the UCSD Stem Cell Program, overseen by Dr. Larry Goldstein. They also were involved in the creation of the San Diego Consortium for Regenerative Medicine, which brings together researchers at multiple San Diego area institutions. They have committed to recruiting additional faculty, particularly from the zebrafish community, to enrich the research environment. They provide excellent core facilities to support the work. Furthermore, UCSD has an outstanding track record of supporting exceptional science and creative scientists.
DISCUSSION: The reviewers were very enthusiastic about the potential of this investigator. He was described as extraordinarily talented and productive, and has always been a stem cell researcher, who applied his background in mouse stem cells to the zebrafish in an excellent way. The research plan was met with less excitement, it was described as being somewhat unimaginative, pursuing e.g. standard transcription profiling and the analysis of Notch and Wnt signaling which is an important, yet highly studied area. This contrasts with the enthusiasm that was expressed for the proposed collaboration with the Fraser lab, which will employ state of the art imaging technology toward lineage analyses for which the zebrafish is so ideally suited. In the absence of preliminary data from this collaboration, it remains unclear, though, whether the “Fraser system”, which was developed for imaging solid tissues, will be applicable to a migratory cell population. Importantly, though, this young investigator has already proven himself as a leader in the stem cell field, who without a doubt will do important work. The RFA asked to identify leaders, this PI is one.