The capacity of human embryonic stem cells (hESCs) to perpetuate themselves indefinitely in culture and to differentiate to all cell types of the body has lead to numerous studies that aim to isolate therapeutically relevant cells for the benefit of patients, and also to study how genetic diseases develop. However, hESCs can cause tumors called teratomas when placed in the body and therefore, we need to separate potentially beneficial cells from hazardous hESCs. Thus, potential therapeutics cannot advance until the development of methodologies that eliminate undifferentiated cells and enrich tissue stem cells. In our proposal we hope to define the cell surface markers that are differentially expressed by committed hESC-derived stem cells and others that are expressed by teratogenic hESCs. To do this we will carry out a large screen of cell subsets that form during differentiation using a collection of unique reagents called monoclonal antibodies, many already obtained or made by us, to define the cell-surface markers that are expressed by teratogenic cells and others that detect valuable tissue stem cells. This collection, after filing for IP protection, would be available for CIRM investigators in California. We were the first to isolate mouse and human adult blood-forming stem cells, human brain stem cells, and mouse muscle stem cells, all by antibody mediated cell-sorting approaches. Antibody mediated identification of cell subsets that arise during early hESC differentiation will allow separation and characterization of defined subpopulations; we would isolate cells that are committed to the earliest lineage known to form multiple cell types in the body including bone, blood, heart and muscle. These cells would be induced to differentiate further to the blood forming and heart muscle forming lineages. Enriched, and eventually purified hESC-derived blood-forming stem cells and heart muscle stem cells will be tested for their potential capacity to engraft and improve function in animal models. Blood stem cells will be transplanted into immunodeficient mice to test their capacity to give rise to all blood cell types; and heart muscle stem cells will be transferred to mouse hearts that had an artificial coronary artery blockage, a model for heart attack damage. Finally, we will test the capacity of blood stem cell transplantation to induce transplantation tolerance towards heart muscle stem cells from the same donor cell line. Transplantation tolerance in this case means that the heart cells would be accepted as ‘self’ by the mouse that had it’s unrelated donor immune system replaced wholly or in part by blood forming stem cells from the same hESC line that gave rise to the transplantable heart stem cells, and therefore would not be rejected by it’s own immune system. This procedure would allow transplantation of beneficial tissues such as heart, insulin-producing cells, etc., without the use of immunosuppressive drugs.
Statement of Benefit to California:
The principle objective of this proposal is to develop reagents which, in combinations, can identify and isolate tissue-regenerating stem cells derived from hESC lines. The undifferentiated hESCs are dangerous for transplantation into humans, as they cause tumors. We propose to prepare reagents that identify and can be used to delete or prospectively isolate these tumor-causing undifferentiated hESCs. HESC-derived tissue stem cells have the potential to regenerate damaged tissues and organs, and don’t cause tumors. We propose to develop reagents that can be used to identify and prospectively isolate pure human blood-forming stem cells derived from hESCs, and separately other reagents that can be used to identify and prospectively isolate pure heart-forming stem or progenitor cells. These “decontaminated” hESC-derived tissue stem cells may eventually be used to treat human tissue degenerative diseases. These reagents could also be used to isolate the same cells from somatic cell nuclear transfer (SCNT)-derived pluripotent stem cell lines from patients with genetic diseases. This procedure would enable us to analyze the effects of the genetic abnormalities on blood stem and progenitor cells in patients with genetic blood and immune system disorders, and on heart stem and progenitor cells in patients with heart disorders. The antibodies and stem cells (hESCs, tissue regenerating, etc) that will be isolated from patients with specific diseases will be invaluable tools that can be used to create model(s) for understanding the diseases and their progression. In addition, the antibodies and the stem cells generated in these studies are entities that could be patented or protected by copyright, forming an intellectual property portfolio shared by the state and the state institutions wherein the research was carried out. The funds generated from the licensing of these technologies will help pay back the state, will help support increasing faculty and staff (many of whom bring in other, out of state funds for their research), and could be used to ameliorate the costs of clinical trials. Only California businesses are likely to be able to license these antibodies and cells, to develop them into diagnostic and therapeutic entities; such businesses are the heart of the CIRM strategy to enhance the California economy. Most importantly, however, is that this research will lead to tissue stem cell therapies. Such therapies will address chronic diseases that cause considerable disability and misery, currently have no cure, and therefore lead to huge medical expenses. Because tissue stem cells renew themselves for life, stem cell therapies are one-time therapies with curative intent. We expect that California hospitals and health care entities will be first in line for trials and therapies, and for CIRM to negotiate discounts on such therapies for California taxpayers, thus California will benefit both economically and with advanced novel medical care.
SYNOPSIS: The major focus of this proposal is to develop reagents, notably monoclonal antibodies (mAbs) that would allow for the prospective purification of hematopoietic stem cells (HSCs) or cardiomyocyte stem cells (CMSC) from hES cells. This would greatly facilitate cellular therapies for blood and heart, while reducing the teratoma potential. The PI and his colleagues propose to test 450 commercial mAbs as well as 200 novel Abs that they have developed against native or differentiated hES cells. In parallel, they will analyze purified populations of cells with clonogenic assays, akin to what they have previously done in characterizing adult HSCs and progenitors from mouse or human material. In addition, they will test modalities such as HoxB4/Cdx4 expression to facilitate hematopoietic reconstitution, and co-culturing of hES cells with CMSC to improve cloning efficiency. IMPACT AND SIGNIFICANCE: This high impact proposal is very significant based on the stated goal of characterizing and isolating hESC-derived hematopoietic and cardiomyocyte stem cells, and developing reagents to identify and eliminate teratogenic hESCs from transplanted populations. The applicants propose to identify antibodies to cell surface markers as tools to isolate pure differentiated subsets of hES cells while depleting cells with the potential for teratogenesis. This is a unique approach for isolating therapeutically relevant cells, which addresses a major obstacle to moving forward in the field - the lack of characterized antibodies for prospective purification of different cell types. If successful, they will be in a position to distinguish undifferentiated ES cells, tissue stem cells, and differentiated cells from each other, which will be useful for in vivo studies both in the experimental and eventually clinical setting. Relevant antibodies would be made available to the research community, which would represent a unique resource. The impact of this research is that we have no appropriate panels of markers to adequately identify cells that are destined for a particular lineage, and the availability of these markers would be a tremendous advance. With modern flow cytometry techniques, it is practical to collect even rare subsets of cells, and through this process the applicants also propose to isolate stem cells and progenitors as therapeutic reagents. Also of note is the effort to identify populations that have the potential to be teratogenic, to exclude them from administration to animals and eventually human subjects who might be candidates for stem cell therapy. While monoclonal Abs helpful for HSC and CMSC will be sought in this work, it is likely that other monoclonal Abs will be obtained that will be useful for other investigators working on different organ systems or cell types. Also, because the nature of this proposal is to intervene at an early stage in cell and lineage identification, the overall impact of this work is far-reaching, going well beyond the two organ systems being studied by the PI and cutting across the entire realm of potential hESC therapies. QUALITY OF THE RESEARCH PLAN: The quality of the research plan is outstanding. This is an excellent proposal in that it addresses a major lack in the field with appropriate methodologies that are well within the expertise of the team, thus the likelihood of success is high. The PI has the requisite background and perspective to lead this effort and see it succeed. The applicants propose three fairly ambitious Specific Aims. The first of these involves the systematic identification of cell surface markers that are expressed either on undifferentiated hES cells or on cells that emerge during early and late stages of differentiation. They intend to accomplish this by screening undifferentiated and differentiated hESC cultures using 450 commercial antibodies to known antigens as well as over 200 novel monoclonal antibodies that the Weissman lab has generated specifically against ES cells. After performing successive fractionation of candidate stem/progenitor cell populations using the cell surface markers, they propose to develop and use clonogenic assays to test the enrichment and differentiation capacity of hES cell-derived hematopoietic stem cells as well as cardiomyocyte precursors. Promising precursor cells identified in this way will eventually be tested for their in vivo developmental potential in a mouse model of cardiomyocyte repair and in a hematopoietic cell transplantation model involving either Rag2/c-gamma (RG) or NOD/SCID models. As for the latter, the applicants are aware of the engraftment issues that exist in these models up to this point. They intend to overcome these issues in part by transplanting candidate hES cell populations into newborn RG and NOD/SCID mice, which lack NK cell activity. If nothing else, the efforts proposed will increase our understanding of changes in cell surface markers as hES cells progress to become multipotent hematopoietic stem cells and progenitor cells. STRENGHTS: Dr. Weissman and his laboratory have a long and distinguished history of work in the area of hematopoietic stem cells using similar approaches. Similarly, they have generated many widely used monoclonal antibodies both in murine models and in humans. They have strong preliminary data regarding the mAbs they have already generated (279 new antibodies against undifferentiated hES cells), as well as the clonogenic assays for testing both enrichment of cardiomyocyte precursors and defining the residual pluripotent cells in otherwise differentiated populations. They use novel methods for examining the expression of markers (cell arrays), and they are familiar with the animal models proposed and their limitations. WEAKNESSES: While the experimental plan itself has no major weaknesses, the eventual relevance of the proposed work hinges on whether the applicants will be able to identify a population of hES cell-derived hematopoietic stem cells with the same engraftment capabilities in the RG or NOD/SCID mouse models that have been shown for adult hematopoietic stem cells. The same will be true for the in vivo studies involving heart muscle repair by cardiomyocytes. If these fail, the value of the proposed efforts will be doubtful. Another potential weakness is the strength of the research plan and leadership reside entirely with the PI. The other staff members, while apparently highly competent in their abilities to carry out the work under the PI's supervision, have no major roles as independent scientists. DISCUSSION: This proposal aims to produce reagents, largely monoclonal antibodies, for hESCs, hematopoietic and cardiac stem cells. This is an excellent proposal, focused on an unmet need – antibodies for doing prospective purification of cells - that will have an enormous impact on studies of all organ systems. The PI has a strong background and has spent his career doing this type of work. The preliminary data look reasonable in that some antibodies have already been tested and they show some specificity, and there is a real contribution to be made here in producing new antibodies, not simply screening commercial reagents. Reviewers feel strongly that this work should be funded. The main weakness is engraftment potential of hESC-derived HSC in mouse models, which has shown very low efficiency to date. Also, the use of HOXB4 to improve engraftment may be problematic because in dogs a few years out they develop acute leukemia. It is useful that the PI will analyze this issue here.