CIRM Quick Links
Related Links
Right Column
RN1-00557-1: Mechanisms of Hematopoietic stem cell Specification and Self-Renewal
Recommendation: Recommended for funding
Scientific Score: 73
First Year Funds Requested: $457,380
Total Funds Requested: $2,286,900
Public Abstract (provided by applicant)
During an individual’s lifetime, blood-forming cells in the bone marrow called hematopoietic stem cells (HSCs) supply all the red and white blood cells needed to sustain life. These blood stem cells are unique because they can make an identical copy of themselves (self-renew). Disorders of the blood system can be terminal, but such diseases may be cured when patients are treated with a bone marrow transplant. Unfortunately, bone marrow is in short supply due to limited availability of donors, and it is not yet possible to expand HSCs outside of the human body; HSCs that are removed from their native environment, or niche, rapidly lose their ability to self-renew and thus cannot sustain hematopoiesis in a transplant recipient. Furthermore, attempts to make blood stem cells from embryonic stem cells (ESCs) have also proved unsuccessful to date because these “tailored HSCs” are defective in self-renewal as well. These problems suggest that our understanding of the biology of HSCs is not sufficient to foster their maintenance or generation. To address this issue, we propose to study hematopoietic stem cells in the context of mammalian development; the entire complement of a person’s HSCs is made in a very short time window during the first trimester of pregnancy. By increasing our understanding of how HSCs are made and acquire self-renewal in vivo, we hope to develop better methods of generating HSCs in vitro and learn to provide the missing cues to coax them into becoming fully functional, self-renewing hematopoietic stem cells. Specifically, we plan to investigate how the fate decision that delineates blood cells from their embryonic precursor, called specification, is maintained at the molecular level. Second, we are interested in what cell type human HSCs descend from so as to understand what precursor to look for when attempting to differentiate ESCs into blood stem cells. Finally, we plan to apply molecular analyses to the property of self-renewal by looking at cell populations that cover a spectrum with regards to self-renewal: HSCs, cultured HSCs (not self-renewing), HSC precursors (not self-renewing), and ESCs differentiated to non-self-renewing HSCs. These comparisons will help define the molecular regulation of self-renewal, and place ESC-derived progenitors on the spectrum of self-renewal. Through these studies, we hope to better understand blood stem cells as they are made and maintained during human development with the ultimate goal to provide wider access to stem cell-based therapies.
Statement of Benefit to California (provided by applicant)
Funding of research to understand hematopoietic stem cell (HSC) biology offers rewards beyond the pursuit of knowledge. HSCs are responsible for providing all of the blood cells in the body, including both red cells that carry oxygen and white cells that mediate immunity. Inherited disorders affecting HSCs and their progeny are responsible for diseases such as sickle cell anemia, Severe Combined Immunity Disorder (SCID), and leukemia; these devastating ailments change the lives of thousands of people in California every year, and currently most are incurable without a bone marrow or cord blood transplant. Due to the limited availability of donors, other alternatives, such as differentiating embryonic stem cells (ESCs) into HSCs, are being explored. One critical fault of ESC-derived progenitors is their inability to “self-renew”, i.e. produce more of themselves, thus eliminating their usefulness for transplantation. However, a deeper understanding of the developmental and molecular processes that create functional HSCs that can self-renew may ultimately make the goal of deriving HSCs from ESCs attainable. Research into the mechanisms of self-renewal may also improve treatments of cancers such as leukemia, as these diseases are a function of over-proliferation of cells caused by uncontrolled self-renewal; targeting genes or proteins involved in abnormal self-renewal programs may provide more specific cancer fighting drugs, and would likely foster collaborations with biotechnology companies. Furthermore, as all stem cells in the body have the ability to self-renew, a clear understanding of self-renewal mechanisms will benefit all stem cell research, and could have a positive effect in a wide range of biomedical specialties.
Review
SYNOPSIS: The first hematopoietic cells formed during development are transient, non-self-renewing progenitors, which are replaced later by definitive, self-renewing hematopoietic stem cells. These two hematopoietic programs use the same specification mechanism, which is dependent on the function of a specific transcription factor, but differ in the sites at which the progenitor cells arise. The ability of definitive HSC to self-renew is rapidly lost in vitro. The general goals of this proposal are to understand the molecular processes that contribute to HSC specification, to better define the cell of origin of the definitive HSC in vivo both in mice and humans, and to characterize the molecular changes that distinguish self-renewing definitive HSC from transient HSC and from HSC that have lost the capacity for self-renewal in vitro.
In the first aim, the applicant will extend his/her previous studies, in which (s)he identified a group of genes whose expression is regulated by the above mentioned transcription factor. (S)he intends to identify direct targets of that transcription factor in murine embryonic stem (ES) cells as they differentiate to hematopoietic lineages. Potential molecular changes in these targets will also be determined, as will how these modifications are maintained in the absence of function of this transcription factor, which is only transiently required for HSC specification. Similar analyses will be performed with in vitro differentiated human ES cells.
In Aim 2, the PI will focus more on the developmental origin of definitive HSC at one site of hematopoiesis and the developmental timing of molecular changes in hematopoietic genes. Furthermore, The PI proposes identification and purification of human HSC from a novel source.
In Aim 3, the PI proposes to identify the earliest definitive, self-renewing HSC and addresses questions related to how the self-renewal capacity of definitive HSC is established. Different populations of human and mouse HSC, including HSC that have lost self-renewal after growth in vitro, will be assessed by global molecular analyses.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a lucid, well-written and in part innovative proposal to study the developmental biology and molecular biology of hematopoietic stem cells. The proposal builds on work the PI did as a postdoc, studying the developmental origin of definitive HSC in vivo, and on the role of a transcription factor in hematopoietic specification. This is a highly significant project, as this knowledge will impact not only our basic understanding of stem cell biology, but also has a direct impact on clinical work.
The applicant proposes to characterize HSC at a variety of developmental states using state-of-the art approaches for cell isolation and molecular analysis. These studies will undoubtedly produce a lot of information; however, it is unclear how the applicant will prioritize this information or use it to better understand HSC function. Also, the approach that (s)he proposes is not particularly imaginative. Many similar molecular analyses of HSC and ES cells have been performed previously by others. However, as these previous studies focused primarily on adult HSC, the applicant’s analysis of earlier developmental stage HSC populations is novel, and may yield new targets. The prospective identification of early human HSC also would be novel and significant, and uses the applicant’s unique resources and expertise.
In most respects, the proposed studies make use of technologies with which the applicant has demonstrated expertise. In particular, (s)he is clearly a leading expert on the isolation and analysis of early fetal HSC from various anatomic locations. However, while (s)he has recruited an expert collaborator to help with molecular analyses, the proposal does not specifically address how bioinformatic analysis of the data generated will be performed, and how candidate genes will be pursued in order to obtain more mechanistic insights into hematopoietic specification. Such a discussion would have helped in understanding the likely impact of the data to be generated.
Under Aim 2, the applicant proposed the biological goal of determining the cellular origin of HSCs in a specific tissue. These are clever studies that are certain to be informative, if not entirely definitive, since it is not clear that the proposed molecular analysis of hematopoietic genes will in fact accomplish this goal. A description of the molecular status of these genes should provide new information about the timing of changes that accompany HSC specification in vivo, but could not provide direct evidence for a lineal relationship between putative hemogenic cell types and fetal HSCs. In addition, it seems such an analysis would be highly dependent on the choice of the genes to be analyzed, and it is not clear which these are, or how they were chosen. Regarding the identification of HSCs in a specific human tissue, there should be some question about the numbers of such cells that can be isolated and therefore whether the molecular studies are feasible as proposed.
Aim 3 is the most risky, but also the most interesting and innovative. With regard to the identification of serially transplantable early HSC, preliminary data would have been helpful to assess the feasibility of this novel approach. Also, it is somewhat uncertain that the pathways which govern self-renewal will be unveiled by the proposed global molecular analyses, and a clear rationale for the inclusion of all the proposed cell populations is lacking. This would have been especially useful as some of the populations appear to be redundant with previously published analyses, and so what will be different about these studies is not clearly highlighted.
The proposal generally lacks appropriate discussion of potential difficulties and alternative approaches, and the reviewers provided comments related to the feasibility of a few specific approaches.
In conclusion, this proposal can come across as overambitious as the volume of work and data generated here is tremendous. Perhaps a better, more focused approach might be useful. It is very clear that the PI has the ambition and talent to fulfill all the goals. Because of his/her strong qualifications, the PI should get the benefit of the doubt that (s)he knows how to prioritize the follow up and deal with unexpected issues lying ahead.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant received his/her MD and PhD in 1997. (S)he did postdoctoral work at two institutions, and was appointed an Assistant Professor at the applicant institution in 2005. The PI is clearly well-qualified to lead these studies. (S)he has trained with experts in stem cell biology, and already can be considered a leader in the HSC field in his/her own right. (S)he has made multiple seminal discoveries in his/her career, including identification of the major source of fetal HSC in the mouse. (S)he has an impressive publication record with numerous papers in top journals throughout his/her research career, and since starting his/her laboratory has been quite successful in obtaining funding. The candidate’s plan for developing a successful career is well-defined and sensible and (s)he has clearly embraced his/her role as a teacher and mentor. There is no hesitation affirming that (s)he and his/her team are perfectly qualified to perform all the specific aims of this grant.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution has demonstrated a clear commitment to advance the career of this young investigator. The institution has provided ample laboratory space in a new sciences building, where many of his/her colleagues are located. The applicant was supplied with plenty equipment, core facilities, administrative assistance and start up funds to support his/her research. In addition, the PI’s career development is supported by formal mentorship interactions with senior faculty. The scientific environment is stellar, and provides unique and necessary collaborative interactions that support the proposed studies.
The applicant institution has a long and successful history of promoting the successful development of research faculty. In addition, there clearly is a major investment in stem cell science. The graduate program recruits and trains top-notch students, who will contribute to the PI’s research program. (S)he is apparently a popular choice with the graduate students.
DISCUSSION: The PI is clearly an excellent investigator, with an exemplary track record, including seminal papers describing his/her pioneering discovery on a site of early hematopoiesis in mouse. This research provides the PI with unique skills in this area. The proposal is very well-written, but it is not imaginative or innovative, e.g. there is no novelty in the proposed molecular analysis, and it remains unclear how the PI will analyze the data. Furthermore, similar analyses have already been performed by others, and their data are available online for data mining. Another concern was that some of the proposed research relies on a lot of technology falling into place, and a lack of preliminary data for technically challenging experiments calls into question the feasibility of some aspects of this study.
Notwithstanding these concerns, the panel expressed great enthusiasm for the possibility that the proposed work may lead to the identification of HSC in a specific human tissue, which would be of great significance to the stem cell field. Although a risky goal, this PI is clearly well positioned, based on his/her previous work, to achieve this objective. Since there was a concern that this important goal was presented only as a sub-aim in the proposal, the panelists collectively recommended that the PI should place special emphasis on this work.
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:
- Orkin, Stuart