Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells.
We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile.
Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine.
Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state.
hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue.
The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
The focus of this application is the discovery of novel surface markers on pluripotent stem cells (PSC), cardiovascular progenitors, and hematopoietic stem/progenitor cells for the development of monoclonal antibodies that could be used as tools for depletion of residual PSC and/or enrichment of differentiated progeny from a mixed population. Studies will be carried out across 3 specific aims. First, the applicants will characterize surface markers in PSC and perform studies to validate which markers or marker combinations can be used to remove residual PSC from mixed populations. Second, the applicants will generate monoclonal antibodies to cells at specific stages of cardiovascular differentiation. The final aim will study the engraftment potential of cardiovascular and hematopoietic progenitors isolated through the use of newly identified markers.
For cellular therapies based on the use of the differentiated progeny of pluripotent stem cells to represent a viable option in treating human diseases, it will be essential to demonstrate that the product transplanted into the patient is not only free of undifferentiated cells with the potential to form teratomas, but also represent a pure population of the desired cell type. This represents a significant bottleneck limiting the evolution and translation of stem cell therapies. With its focus on identifying pluripotent cells and progenitors for the hematopoietic and cardiovascular systems, reviewers considered this application timely and appropriate. Although much of the work proposed was not hailed for its innovation, reviewers considered it necessary and felt that successful generation of the proposed monoclonal antibodies could provide invaluable tools to the stem cell community and significantly enhance progress of the field. A reviewer considered two aspects of this application to be particularly important. First, the outcomes of the study are generally agnostic about the type of equipment or technology that such antibody selection reagents could be incorporated into to achieve the desired purification. Second, the work is designed to extend earlier findings supported through a CIRM grant and translate them into tools and procedures that will enable purification of specific cell lineages for safe transplantation.
Reviewers found the proposed studies to be generally feasible and logically designed but also felt the scope was too broad with several components only peripherally related to the main objectives. A clear strength of the proposal is the ample preliminary data in support of all aims, which provides confidence of success. For Aim 1, the proposed approach to identify candidate markers most differentially expressed in pluripotent stem cells was found to be logical and sound although details for generating monoclonal antibodies from these was not well discussed. Aim 1C seemed peripheral to the primary focus of the proposal by asking whether identified antibodies can also contribute to early identification of iPS during reprogramming. For Aim 2, some stable transgenic hESC lines with cardiovascular reporter genes have already been made and were considered a strength since these are ready for the proposed work. For Aim 3, the in vivo evaluation of transplanted cardiovascular and hematopoietic progenitors in animal models was of primary importance. The expression of two survival genes to investigate whether this will increase levels of hematopoietic engraftment in immunodeficient mice was also considered valuable but peripherally related and too diffuse. The primary studies proposed to investigate the function of the cardiovascular progenitors were viewed as an important and innovative component of the proposal.
The principal investigator (PI) is committing 20% effort to the project. The PI is internationally respected for many contributions to the field of stem cell biology and holds the experience and track record to appropriately manage the proposed research. The PI has assembled a team of investigators including a clinical fellow with training in cardiovascular medicine and two research associates who will contribute to hES cell maintenance, differentiation and sorting of specific populations. The 5% effort contribution by the individual responsible for technical aspects of hybridoma development was considered insufficient for such a critical component of the proposal. The requested overall budget seems appropriate for the scope and timeline of the proposed studies.
- A motion was made to move this application into Tier 1, Recommended for Funding. The GWG noted a lack of proposals to develop biomarkers in Tier 1 and felt that biomarker development was the strongest component of this proposal. Reviewers were uneasy about recommending the proposal in total, as several proposed studies were not directly related to this goal. The GWG recommended that the proposal be restructured to focus on the biomarker development and remove the peripheral projects. The motion was amended to recommend removal of Aims 1c, 2d and 3a from the proposal with concomitant modifications to the budget as appropriate. The motion carried.