Hematopoietic stem cell transplantation is the treatment of choice for many hematologic malignancies, and it is used to treat an expanding number of congenital blood disorders. However, only ~30% of patients who can benefit from this treatment have a matched sibling that can serve as the ideal donor. While the national marrow donor program and umbilical cord blood programs provide unrelated donor cells to many patients lacking a sibling donor, a large percentage of patients remain without a suitable donor, leaving them with suboptimal therapeutic options. This problem is more severe in certain ethnic populations, including people of Latino and Asian descent, groups that constitute a large part of California’s population. New sources of therapeutic hematopoietic stem cells are therefore needed.
Human embryonic stem cells, with their unlimited self-renewal capacity and their ability to generate all human cell types, provide a novel and exciting opportunity to obtain hematopoietic stem cells, thereby filling a critical therapeutic void. However, many hurdles remain before this vision can be realized, including the identification of more optimal human embryonic stem cell lines and better methods to direct the development of specific cell types from embryonic stem cells.
This proposal seeks to shed new insight into how we might better control and direct the development of human embryonic stem cells into therapeutically useful hematopoietic stem cells that can be used for transplantation. Our effort focuses on understanding how a specific class of small RNAs, called microRNAs, regulates the differentiation of human embryonic stem cells into specific cell types. We aim to uncover the identity of microRNAs that are important for this process, which will serve as useful biomarkers, or guides, for evaluating the therapeutic suitability of existing and newly derived human embryonic stem cell lines. In addition, we will develop techniques and reagents to modulate the expression of these critical small molecules to help direct human embryonic stem cell development for clinical therapeutic utility.
This proposal seeks to understand how small RNA molecules help direct the differentiation of human embryonic stem cells into specific cell types suitable for therapeutic use. Our focus is the development of hematopoietic stem cells for the treatment of blood disorders. Benefits to the State of California include:
A new source of hematopoietic stem cells for patients who lack a matched donor. Some ethnic groups, such as people of Latino and Asian descent, are more likely to lack a donor, so those groups may benefit the most.
Hematopoietic stem cells have been shown to have the potential to participate in the repair of a wide range of tissues, including heart tissue, so human embryonic stem cell-generated hematopoietic cells may be useful in treating disorders outside of the blood system per se.
We will develop databases and technologies that could have wide-ranging use in the human stem cell field. To the extent that these can be commercialized, the work could be of financial benefit to California.
The kind of work proposed could attract additional economic interests to the state of California because our methods and reagents could be used by California businesses aimed at treating those afflicted with blood disease.
This work will provide new jobs for the University of California, San Francisco, and hopefully spawn additional research in the California academic and private sectors.
The proposed research is cutting-edge, and successful accomplishment of our goals would help put California at the forefront of human embryonic stem cell research, rewarding Californians for their support of proposition-71, and inspiring others to support human embryonic stem cell research.
SYNOPSIS: The Princioal Investigator (PI) and Co-PI propose to explore molecular expression and bioinformatic analysis of small RNAs in human embryonic stem cells (hESCs) and hESC-derived hematopoietic stem cells (HSCs) to identify which small RNAs are critical for the earliest steps in hESC self-renewal and in hESC-HSC self-renewal. This proposal takes advantage of the investigators' strong expertise in molecular genomics and hematopoietic cell culture, as well as the preliminary work performed by the PI with hESC to date. While this work is clearly hypothesis-generating, it is firmly based in the observation that Dicer-/- stem cells fail to differentiate. This is an extraordinarily ambitious proposal, but is precisely the sort of work that needs to be performed.
IMPACT & SIGNIFICANCE: The aim of this proposal is to develop new insight into how therapeutically useful hematopoietic stem cells can be generated from hESCs. Specifically, the applicants will focus on understanding how a specific class of small RNAs, called microRNAs, regulates differentiation of hESCs into specific cell types. The proposal uses innovative techniques for the identification of small RNAs expressed in ES and ES-derived cells and for testing the functional importance of miRNAs in hematopoietic development. It may also develop new assays for assessing blood precursor cell activity in differentating hESC populations.
This research has the potential to identify novel regulators of stem cell function and of hematopoiesis, and may give insights into basic biology of gene expression and cell fate determination. If successful, they hope to have developed the base for large-scale generation of hESC-derived hematopoietic stem cells for clinical transplantation. If performed carefully and successfully, this research will uncover an unparalleled reservoir of molecular information on small RNAs in stem cell differentiation, that will be extraordinarily valuable for the field.
QUALITY OF THE RESEARCH PLAN: This is a well-written proposal to use hESCs to investigate the importance of a newly appreciated form of gene regulation in the determination of hematopoietic precursor cells. The experimental plan makes good use of the strengths and expertise of the collaborating investigators. The applicant provides some key preliminary data demonstrating the group's ability to generate hematopoietic precursors from hESCs and to create and maintain transgenic hESCs. In addition, the applicant provides circumstantial evidence from dicer conditional knockout mice (though this could be complicated somewhat if dicer deficiency leads non-specically to cell death) and gene array studies, that HSC function is impacted by miRNAs. The proposed comparison of a large panel of hESC lines (both federally approved and non-approved) will be important for assessing heterogeneity in the developmental potential of these different lines.
The project is a collaboration between the Leavitt lab, which has experience in differentiating hESCs into hematopoietic cells, and the McManus lab, which has expertise in the biology of small RNAs. The research plan is logically divided into three aims. Aim 1 will involve the screening of hESC lines for robust production of hematopoietic stem cells and progenitors. Work so far, using NIH-approved cell lines, has not resulted in the generation of significant marrow reconstituting cells in xenogeneic mouse models. It is unclear whether this failure is due to a developmental limitation of the cell lines or limitations in the developmental systems employed. Initial screening for hematopoietic stem cell activity will involve the cobblestone area forming cell assay as an in vitro surrogate for stem cell activity in vivo. Eventually they will transplant their cells into 6-week-old sublethally irradiated RAG2-/- c-gamma-/- mice. Repopulation activities will be measured by comparing results to those obtained with adult and cord blood hematopoietic stem cells. Specific Aim 2 will systematically gather information on the small RNAs expressed in hESCs, adult hematopoietic stem cells, and hESC-derived hematopoietic stem cells. This work will generate a valuable database and, the investigators hope, allow determining whether small RNA signatures can serve as a meaningful developmental biomarker for cataloging hESCs in the various hESC banks.
Upon satisfactory conclusion of Specific Aim 2, the applicants propose to directly test the role of potentially identified microRNAs as a developmentally important subclass of small RNAs and hES cell-based hematopoietic development. To this purpose, they will create lentiviral libraries that modulate the expression of specific microRNAs and also screen for the ability of microRNAs to direct hematopoietic development. All of these aims are described in a clear and logical manner.
One concern with the proposal includes potential risk that the CAFC strategy proposed will not work with hESC-derived cells (no preliminary data available); however additional approaches are discussed. Another concern relates to the sensitivity of the sequencing techniques proposed, and whether sufficient numbers of cells can be isolated for screening. However, while this may prevent complete cataloguing of hESC and HSC expressed small RNAs, it is likely that this approach will generate significant new information and identify potentailly novel targets.
Finally, the experiments proposed in Aim 3 may be a bit over-ambitious. The applicant proposes to test the importance of the miRNAs identified in the above screens by overexpression and knockdown (how many miRNAs can reasonably be screened in the timefram of the grant?). Knockdown will use a novel approach in which viral libraries are generated targeting miRNA promoter elements to block miRNA transcription. This approach has not yet ben validated in hESCs. These libraries will be used to screen for miRNAs that are required for generating hematopoietic cells from hESCs using an hESC/OP9 co-culture assay (already established in the lab). It is not clear however how this assay will discriminate miRNAs that specifically impact hematopoietic development from those that generally affect the survival or proliferation of hESC, and from those that impact developmental stages upstream of hematopoiesis. Thus, these experiments may not yield the anticipated candidate regulators of HSC formation.
In summary, the research plan is extremely ambitious, but extremely appropriate. What is more, once the small RNA libraries are created, they will be a research resource for many investigators. While not all functional assays may be performed, the PIs will probably get the chance to try the best candidates in their in vivo screeens. Overall, it's very strong.
STRENGTHS: The greatest strength is in the collaboration between the Leavitt and McManus labs, each of which will bring unique abilities to the proposal. Both are very capable thinkers and experimentalists, investigators with track records in their respective areas, although neither apparently has NIH R01 support for their respective laboratories. They form a good team, who have identified the most powerful in vitro and in vivo techniques and assays to pursue their aims. In some cases, they have themselves developed assays that didn't exist before that enable this research--e.g. the identification of the Mpl+ HSC subset from hESCSs.
This is a very targeted project, focused on a critical issue--after H.Lodish and C. Croce's work, these sorts of studies have been crying out to be performed. Their proposed experiments test the most important hypotheses. This project will also generate important reagents for the field.
In summary: The proposal attackes an important topic for human disease treatment and has the potential to discover novel regulators of hESC and hematopoietic differentiation. The expertise of individual investigators and strength of collaborative team are critical to the success of this research. Finally, the PI provides key preliminary data on hESCs, proposes to compare a large panel of hESCs and to study hematopoietic precursor cell development and hESC transgenesis.
WEAKNESSES: Not all the ambitious experiments may work. To hope that pooled RNA libraries will have functional effects that can then be effectively separated by, essentially, sib selection in vivo, is brave. One reviewer would have loved seeing any preliminary data on validated inhibition of miRNA expression via shRNA inhibition of their promoters.
Another concern was that the investigators may not be able to get sufficient RNA (1 ug) from all their FACS sorted cell populations. Amplification approaches might be required.
As in other applications, success or failure of this proposal hinges on the development of culture conditions that are capable of generating transplantable, truly multipotent hematopoietic stem cells with better repopulation capacity than shown up to this point. Absent such documentation, Specific Aims 2 and 3 will not be very meaningful.
Other concerns include: some risk in global profiling approaches; lack of preliminary data on CAFC assay; the screen proposed in Aim 3 may not discriminate effects that are specific to hematopoietic development from those that globally impair cell survival, proliferation, etc.
DISCUSSION: The applicant is taking a global approach to identifying the small RNAs expressed in hESC and in hESC-derived HSCs. All the reviewers agreed that the PIs are bright, energetic, young investigators - experts in genomics and bioinformatics and in culture of stem cells. It is known from work on the dicer knock-out that if it can't make micro RNAs, there is no differentiation; this provides the rationale for the importance of small RNAs. The proposal is extremely ambitious and involves a lot of work. A potential bottleneck for some of the bioinformatics work is that a certain amount of RNA is needed and the investigators may not have enough, therefore they may need to collaborate with someone who can do non-skewed amplication of small amounts of nucleic acid. No group has focused on or has as yet identified transplantable HSCs (derived from ESCs) that have good transplantation efficieny in xenogenic mouse models.
The enthusiasm was somewhat lowered by some weaknesses which included: (1) success of application hinges on ability to efficiently generate transplantable HSCs that repopulate xenogenic models - something not done thus far; (2) this is required for aims 2,3. However it was also pointed out that even if the investigators only get colony forming cells and can't prove transplantable, truly multipotent long term repopulation, such a result would be useful and would help direct further work. The in vitro assay could be used as surrogate for in vivo transplantation assay (although a comment was made that that it is not clear if the in vivo transplantation assay is equivalent to the in vitro assay.