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RN1-00540-1: Mechanisms of Stem Cell Fate Decisions
Recommendation: Recommended if funds available
Scientific Score: 65
First Year Funds Requested: $467,316
Total Funds Requested: $2,330,111
Public Abstract (provided by applicant)
Stem cells can mature into a diverse range of specialized cell types, providing exciting possibilities for regeneration of different tissue types. Many disorders can conceivably be treated by transplanting stem cells to replace the defective cells. However, several disorders affect a specific cell type, while other cells and tissues in the same patient are functioning normally. Examples of such cell type-specific cases are Parkinson’s disease, diabetes and anemia. A fundamental challenge in using stem cells to treat disease is to be able to steer their maturation into the specific cell type needed. Maturation into the wrong cell type may cause problems due to uncontrolled growth or immune rejection. For example, a blood stem cell transplant intended to treat anemia would be optimally efficient and safe if the transplanted cells could be directed to generate red blood cells without also producing T cells. Another major limitation of transplantation therapy is the shortage of cells for clinical use. This too could be alleviated by efficient generation of the desired cell type.
This proposal investigates the regulation of stem cell maturation into specific functional cell types. We are using blood cell development as a model system. Bone marrow transplantations, containing blood stem cells, have been used clinically for many years and are among few routinely performed cell-based therapies. The cumulative experience from years of clinical use together with decades of research on blood stem cell maturation makes this a strong model system for understanding both basic stem cell biology and clinical stem cell applications.
The many years of research on blood cell maturation has allowed the generation of a basic road map, complete with intersections indicating a choice in maturation pathway. As stem cells mature, choices are made along the way to become, for example, a T cell or a red blood cell. Scientists have identified signposts at these intersections that help mark some of the choices. These signposts are useful both as diagnostic tools to determine where cell maturation went wrong, and as targets for drugs when trying to promote one choice over another. Currently, we do not know when and how these choices are made under normal conditions and what goes wrong in various disorders. Our ability to direct or redirect cell fates upon transplantation or in cancer is therefore absent or inefficient. The goal of this proposal is to better define the intersections and signposts in blood cell development. We will generate new tools and methods to study stem cell maturation, and attempt to identify maturation stages and targets that will be useful to diagnose and treat blood-related disorders and increase the efficiency and safety of stem cell transplantation.
Statement of Benefit to California (provided by applicant)
Bone marrow and blood stem cell transplantation is a potentially curative treatment for patients with a variety of blood-related disorders, including leukemia, lymphoma, anemias and auto-immune diseases, as well as non-blood disorders such as enzyme deficiencies. Survival and quality of life of patients with these disorders have been improving steadily since bone marrow transplantations, containing blood stem cells, came into clinical use in the 1970’s. Now among the few routinely performed cell-based therapies, bone marrow transplantation thus serves as an important paradigm for clinical application of other types of stem cells. Although routinely used to treat a variety of diseases, the pretreatment preparing the patient for transplant is grueling and the risk of life threatening complications are unacceptably high.
The research in this proposal is intended to understand how blood stem cells mature into different cell types. The goal is to be able to detect, prevent and treat blood-related disorders and to improve the efficiency and safety specifically of blood stem cell transplantation. With increased safety of transplantation regimens, this type of treatment will become available to a greater number of patients and lead to higher survival rates and fewer complications. Stem cell transplantation may also eventually be used to treat non-life-threatening, but burdensome, disorders.
In addition, this research will contribute to the California education and health care systems by training undergraduate, graduate and postdoctoral students into highly skilled stem cell biologists.
Review
SYNOPSIS: This proposal will re-examine and further delineate the lineage relationships taken by hematopoietic stem cells (HSCs) and progenitors during differentiation within the hematopoietic system in the mouse. Recent published work has raised questions concerning the nature of intermediates that give rise to myeloid and lymphoid cells. This proposal further addresses the question of whether HSCs have already chosen the subset of lineages for subsequent differentiation or the choices are imposed downstream of the HSC. The focus of the proposal is on the status of progenitors positive for a cytokine receptor and the extent to which progenitors that are null for the receptor are capable of development along a myeloid lineage. A published report suggested that receptor+ progenitors had lost this myeloid lineage potential and that these progenitors must therefore be derived directly from an HSC or other “higher” progenitors, thus challenging standard hierarchy dogma. A publication by the applicant showed that in fact receptor+ progenitors do retain this myeloid lineage potential. The intent is to test the role of the receptor in cell fate decisions and to use fate mapping and bio-informatics analysis of gene expression patterns of various intermediates to gain molecular insights into pathways that regulate cell fate determination in the hematopoietic system and to eventually manipulate them.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Hematopoietic development is an important paradigm for stem cell biology. Understanding how blood cell differentiation takes place, and the options available to progenitors, are important in relation to improving prospects for bone marrow transplantation in the future. The work approaches an important scientific problem, and while it doesn’t break new technological barriers, the proposal does utilize state-of-the-art approaches for hematopoietic progenitor analysis. The applicant proposes to experimentally re-examine lineage relationships within the hematopoietic system with a three-pronged approach to address hematopoietic lineage decisions, particularly concerning the nature of intermediates that give rise to myeloid and lymphoid cells.
The studies in the first aim propose a more thorough evaluation of hematopoiesis in the receptor knockout mouse. Further analysis of receptor function using these mutant mice is useful and might show some roles for the receptor that were not found in the original work, but is unlikely to provide important new insight. If the myeloid progenitors are decreased in the knockout transplants this would suggest that a receptor+ progenitor is a required step. It would be surprising that this would not have been picked up in the previous study. If there is no decrease, this would imply compensation, which is rather more expected. In this case, the study does little to clarify the “controversy” around the receptor as a marker. It is however a perfectly straight-forward study of the receptor’s function.
In the second aim, the applicant proposes to develop reporter mice to do lineage tracing. This approach nicely complements traditional means of prospective cell fate analysis by isolation and transplantation. These studies with reporter mice are more likely to generate information regarding the hierarchy of progenitors, and might reveal interesting differences between steady state and challenged (stressed) progenitors. However, the results need to be cautiously interpreted as expression patterns of the transgene could change under conditions of normal versus stress hematopoiesis. Also, issues of leaky expression or transgene effects may complicate the analysis, and the mice are not yet validated.
Finally, the applicant proposes to carry out a systems-based large-scale bioinformatics study, focused on data for a number of hematopoietic subsets. The goal is to define regulatory networks. This bio-informatics approach of analyzing gene expression patterns in various intermediates is descriptive at the onset. Although experiments described in this aim are reasonable and should generate preliminary data for future projects, they are by nature a bit difficult to evaluate. Whereas it seems likely that extensive data mining should reveal expression groups for these cell populations and co-regulated networks, the applicant needs to better illustrate how this approach will identify new regulatory molecules or pathways regulating cell fate determination. Stronger preliminary results along these lines would strengthen the application
A better understanding of lineage relationships and flexibility of fate decisions is always desired and surely would be helpful for considering which cell populations should be targeted or enriched in leukemia or transplant situations. The study does seem likely to provide new information on lineage relationships. The experimental designs are suitable to the proposed aims and utilize largely established methods. The applicant has extensive experience in molecular biology as a PhD student and therefore is technically competent to undertake the proposed work. Appropriate collaborations are in place for generation of the gene targeted mice. While the first aim is a fairly straight-forward knockout analysis, the remaining aims are largely descriptive, and it’s less clear how the project will make substantial progress in directing stem cell fate, depending perhaps on the results of the bioinformatics study. A criticism of the proposal is that the aims address largely secondary issues in the controversies regarding the paths available to HSCs and progenitors in their fate choices. The initial impetus for the study is based in part on resolution of a “nuance” in the field, which is the developmental potential of receptor null vs. receptor+ progenitors for differentiation to a myeloid lineage. Recent work by others, while not addressing this specific aspect, suggests a resolution to the present controversy in the field. Hence, the work is strong but not as “deep” in its potential impact as it might be.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is an Assistant Professor with an outstanding background and excellent training and accomplishments in molecular and developmental biology. The applicant was a very productive PhD student in studying the transcriptional control with five first author research publications. The applicant was equally productive as a post-doctoral fellow in a major hematopoietic lab as evidenced by the publication of several papers including two first author papers in major journals. The applicant is clearly well trained in aspects of both hematopoiesis and epigenetics, which are nicely combined in this project. The applicant’s expertise is in mouse hematopoiesis, not specifically in embryonic stem cell research.
The applicant has a well laid out plan for career development including the description of a strong plan for research portfolio development and development of strong interactions with cross-disciplinary researchers at the institution. The applicant plans to put together a mentoring committee but has not yet done so. Strengths are the applicant’s Chair, and mentor and a collaborator for bioinformatics.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institutional research environment is outstanding. They have been funded by CIRM for other grants. The stem cell group includes multiple members from several departments. A particular strength is the potential for strong interactions between developmental biologists and bio-informatic and computational scientists. The institution is recognized as having faculty with world-class strengths in genomics, bioinformatics and computational sciences. The applicant appears motivated to take advantage of these interactions.
The applicant has strong support at the host institution as evidenced by the strong startup package and the encouragement and support of the host department. The applicant has independent space, has access to the stem cell facility, and has important interactions with faculty in other departments to complement those in his/her department. The applicant is in a suitable tenure track position.
DISCUSSION: The focus of this proposal is further development of the lineage relationships in the murine hematopoietic system. Elucidating these relationships is useful, although not very innovative or exciting. The proposal does not address a deep question, but rather a nuance in the field. The work proposed in the first aim needs to be understood but is not novel. The work proposed in the second aim is exciting and interesting but interpretation of the results is potentially difficult. The third aim in particular is descriptive in its approach and it is unclear what will be learned. The PI is an accomplished candidate, well-trained, and well qualified to pursue the research question.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- Wagers, Professor Amy