Although ESC-based therapies hold great promise for the cure of a wide diversity of degenerative diseases, rapid progress to actual human clinical trials is hindered by the lack of preclinical data for specific ESC-based therapies. I aim to move the process forward by establishing a protocol in which immune system cells are reproducibly produced from ESC and tested in vivo for the induction of and maintenance of immunological tolerance to therapeutic ES-derived cells. I will use the mouse as a model system to test this protocol, as the mouse is the model system of choice for study of the immune system due to the availability of genetically identical strains and well-studied models of human disease. Moreover, my protocol design will be reflect strategies already used for successful organ transplantation, making the protocol suitable for clinical use.
The immune system is the primary barrier to the acceptance of any embryonic stem cell (ESC)-based therapy. Immune system cells are derived from a stem cell in the blood which has the potential to differentiate into a number of mature cell types, such as red blood cells, macrophages, granulocytes, B lymphocytes (B cells) and T lymphocytes (T cells). During development, B and T cells are educated to recognize what is “self” versus what is “foreign.” This precise education regulates the activation of the immune response during viral or bacterial infection. For example, T cells can distinguish between healthy and infected cells, and selectively destroy the infected ones while leaving the healthy ones intact. Breakdown of this recognition, or self-tolerance, occurs in autoimmune diseases such as Type I Diabetes and multiple sclerosis, resulting in the destruction of pancreatic and neural cells by the body’s own immune system. In clinical organ transplantation, overcoming “self-tolerance” and re-education of the patient’s immune system to recognize the donated organ as “self” is necessary for acceptance of the transplanted organ. I hypothesize that this will also be the case for ESC-based therapies, in which the ESC and its derivatives can be thought of as a “transplant” to which the patient’s immune system must learn to recognize as “self”. Regardless of the disease one is attempting to treat with ESC-based therapies, if immune tolerance is not achieved, all ESC-derived grafts will be destroyed, and disease will persist.
The last goal of the project is validate whether my protocol can be used as a real ESC-based therapy for Type I Diabetes. Although diabetes is my initial focus, induction of immune tolerance to therapeutic ESCs is a general but necessary requirement for the success of any ESC-based therapy. Therefore, if successful, this research could be applied to a wide variety of degenerative diseases, such as muscular dystrophy, Alzheimer’s, Parkinson’s, multiple sclerosis, cancer and immune deficiencies.
Results from my proposed research project will benefit the State of California and its citizens at several levels.
Direct Impacts: My research project aims to target an obvious barrier to ESC-therapy for any disease: the avoidance of immune rejection in the patient. If immunological tolerance to ESC-derived grafts is not achieved, the therapeutic ESC is destroyed and disease persists. The immune system is formed from hematopoietic (blood) stem cells, and my research goals are to establish reproducible protocols to derive blood stem cells from ESC and promote engraftment of these ES-derived blood stem cells in a recipient to induce immunological tolerance to the ESC-graft, and then test them in a well-studied mouse model of diabetes. If successful, this would be the first preclinical data to demonstrate that ESC-based therapies can cure diabetes. As immunological tolerance is important for all potential ESC-therapies, my work can have broad applications to a wide diversity of diseases. The work will also have indirect impacts outside of the research, such as notoriety to CIRM as the funding agency for this groundbreaking research, and be the springboard for improvements in health care, increase in tax revenues, and improvements in education for California residents.
Health Care: I will test my protocols in a well-studied model of human diabetes. The California Diabetes Program reported that two million Californians are diabetic and there are many more that are pre-diabetic. If successful, my research could provide stem cell therapies for these patients, alleviating the need for insulin therapies and extensive medical care. As this research is funded by CIRM, it is highly likely that Californians would be the primary recipients of therapies designed using my research. Furthermore, my research plan is designed to have broad applicability, so ESC-therapies for other illnesses such as cancer, Alzheimer’s, Parkinson’s, multiple sclerosis, and cardiovascular disease can next be evaluated.
Biotechnology: My research already relies on a number of products and tools manufactured and sold in the state of California. If successful, research will require a scaled-up version of protocols designed in my studies. This could attract new biotechnology companies in the state, boosting the tax revenue in the state. This in turn, will provide new jobs for California state residents.
Education: Establishment of successful ESC-therapeutics in California will encourage institutions of higher education to promote science education to fill the jobs created by stem cell research. This will retain California students in the state that are interested in biomedical research and medical careers. Furthermore, it could attract out-of-state students seeking degrees that will allow them access to careers in stem cell research. It is envisioned that this will trickle down to the K-12 levels and provide funding to promote science education at all levels.
SYNOPSIS: This is an application for support to reduce the need for antigen-matched ESCs for therapeutic purposes by developing strategies to induce tolerance to the engrafted cells. The PI will use a mouse model, no human ES cells are proposed for this work. It is a global project that starts with undifferentiated mouse ES cells that will be differentiated into hematopoietic stem cells both to induce tolerance in the bone marrow, and to “cure” type I diabetes in a mouse model by alleviating the destruction of pancreatic cells by the immune system. The hypothesis being tested is that establishing central and peripheral immune tolerance will reduce the need for antigen-matched donor ES cells. The investigator’s career development plan is aspirational and is aimed to have educational support benefits for other staff. The host institution is clearly highly supportive of the applicant and has major long term plans in the fields of regenerative medicine and bioengineering.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is a well written application from a young faculty member that addresses a central issue in the utility of hESCs in human therapy and overcoming the challenge of patient immune response to hESC therapies. Tumorigenicity and immunological intolerance of ES cells are a bit over-stated in the application, but the research plan as a whole is well-designed, potential problems addressed, and may yield useful results in this mouse model.
The experimental design appears to be appropriate as it develops on existing expertise with supportive preliminary data, although there does not appear to be an evaluation of the difficulty of developing the pancreatic stem cell lines to be used. The staff appear to provide strong and trained support and timelines seem generally realistic although the investigator may wish to consider the possibility of aim 1 taking a full 2 years.
Three specific aims are proposed:
Aim 1 will establish an optimal protocol for differentiation and engraftment of ESC-derived hematopoietic precursors (ES-HP) in adult mice.
Aim 2 will determine whether immunological tolerance is achieved in mice receiving allogeneic ES-HP and define the mechanisms underlying tolerance induction.
Aim 3 will validate that ES-HP co-transplantation can enhance survival of ES-derived tissue grafts and cure recipients that suffer from degenerative disease.
Although the “cure” of Type I diabetes by bone marrow engraftment is not universally accepted by all experts in the field, the work proposed in this application will yield valuable information about the role of tolerance in ES cell therapies.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Manilay is an assistant professor at the School of Natural Sciences, University of California, Merced. She received a BA in Molecular and Cell Biology from UC Berkeley in 1992, followed by a PhD in Immunology from Harvard University and a five –year post-doc at UC Berkeley in developmental immunology. She joined the faculty at UC Merced in 2005. UC Merced is a new UC campus, beginning in the fall of 2005. Thus, Dr. Manilay is a founding faculty member. She has 13 peer-reviewed publications, all in the areas of immune cell biology, including transplanted immune cells.
The career plan is truly aspirational with local mentors of excellent quality. However, there is not a clear and formal mechanism by which mentor feedback will be achieved. Her career plan involves both teaching and research, particularly in immune stem cell biology. Her enthusiasm for her career goals is evident from her descriptions of milestones.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: University of California, Merced, is a new institution within the UC system. Dr. Manilay is clearly a founding member of stem cell biology at UC Merced. A letter of support from the Dean of Natural Sciences emphasizes a commitment to Dr. Manilay's research efforts, including increased protected time for research commensurate with research funding. The host institution is very supportive providing various central facilities and there is evidence of ongoing commitment to expanding the developmental biology and bioengineering areas. She is in the new Science and Engineering building, completed in 2006. Facilities and equipment for this project appear to be in place.
DISCUSSION: Reviewers express reservation, that although the applicant has had excellent training, her overall accomplishments and publications have not been particularly outstanding thus far. Additionally, the research proposal is problematic. The proposed project is an ambitious one that intends to go from mouse ESCs to hematopoietic cells to curing diabetes. The entire proposal hinges on the success of the first aim and reviewers expressed concern that the project may not work. Hematopoietic reconstitution in the proposal is unclear; she wants to induce tolerance to cells so they have to persist in some way. Reviewers expressed enthusiasm for this young investigator and agreed that this is a well-trained scientist with enormous support from the host institution. The applicant has an aspirational and commendable career development plan that includes both research and teaching. However, enthusiasm for the applicant and the institutional support could not overcome the concerns with the scientific proposal.