Enhancing Survival of Embryonic Stem Cell-Derived Grafts by Induction of Immunological Tolerance

Enhancing Survival of Embryonic Stem Cell-Derived Grafts by Induction of Immunological Tolerance

Funding Type: 
New Faculty I
Grant Number: 
RN1-00554
Award Value: 
$1,576,404
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Closed
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Progress Report: 

Year 1

Our overall project goal is enhance the survival of stem cell based therapies by understanding if they can be rejected by immune response, and if so, how to manipulate the immune response so that rejection can be avoided. Currently, we are using mouse embryonic stem cells (ESC) and the adult mouse as a prototype of a cell-based therapy and a human patient who requires blood stem cells. In Year 1, we established mouse ESC culture in the laboratory and began to generate putative blood stem cells, or hematopoietic progenitors (HPs), using established culture methods. However, we noted that our yield of the HPs was too low for what is needed for transplantation. In Year 2, we compared different culture methods to generate higher numbers of HPs, and we found an improved culture method that is easier to scale up and requires less manipulation, which has increased our HP yields and simultaneously reduces the risk of possible contamination. In addition, we have found that HPs using this improved method appear to be similar to HPs found in the natural adult bone marrow. These results suggest that ESC-derived HPs might function similarly to those in the adult, and we will this hypothesis. Moreover, in Year 2 we have developed a strategy to predict if ESC-derived HP will stimulate the immune system of patients. This is important to assess because if the immune system rejects ESC-derived cells, the cell-based therapy could fail in diseased patients. Our data suggests that the culture method used to derive HPs from ESCs correlates with their potential immunogenicity, and we plan to experimentally test this idea in the next reporting period. Another challenge that could affect the survival of stem cell based therapies in patients can be termed “developmental incompatibility”. HPs derived from ESC are embryonic or fetal in nature, and in cell-based therapies, these embryonic-like tissues will be expected to survive in a mature, adult cellular environment. There is very little evidence to date to show that ESC-derived HP can survive long term in an adult, and there is a paucity of information on how ESC-derived tissues interact at the cellular level with adult microenvironments, or “niches”. In Year 2, we have developed in vitro model systems that we can utilize to answer some of these questions. For example, we have developed a system using bone-building cells, or osteoblasts, which are one adult “niche” cell for blood stem cells, and we have established that the stage of osteoblast maturation correlates with their ability to support adult hematopoiesis. Another cell type that is generated from blood stem cells are T lymphocytes, which interact with thymic epithelial cells (TECs) in the adult mouse. TECs could be considered to be a “niche” cell for developing T lymphocytes. We have also devised an improved method to isolate mouse adult TECs, with the goal of designing an in vitro system similar to the osteoblast system described above. Our next goal is to apply these model systems to the study of ES-HP/niche cell interactions.

Year 2

Our overall project goal is to assess the immune response to tissues that are derived from embryonic stem cells. We are using mouse embryonic stem cells (ESC) and the adult mouse as a prototype of a cell-based therapy and a human patient who requires blood stem cells. We are also preparing to use adult diabetic mice as another disease model in the future. In Years 1 and 2, we optimized our protocols to create embryonic stem cell – derived hematopoietic progenitors (ES-HP). This year (Year 3), we transplanted ES-HPs into an immune deficient mouse strain, and compared their engraftment, survival and immune cell developmental capacity to that or transplanted adult bone marrow cells. We observed that ES-HP survived up to 3 weeks post-transplantation, and that the ES-HP derived blood cells were located primary in the adult spleen and adult thymus. In contrast, adult bone marrow cells reconstituted the blood, bone marrow, spleen and thymus of the immune compromised hosts. Furthermore, mice receiving ES-HP displayed large spleens, which is indicative of a local immune response by macrophages. Mice receiving adult bone marrow cells did not display this phenotype. Recent papers in the scientific literature also suggest that the innate immune system (which includes macrophages) may respond to ESC-derived tissues. In addition, our observation that ES-HP derived cells were present in the recipient thymus and showed evidence of T cell maturation suggests that the adult thymus can support T cell development. Even though the presence in the thymus was transient, T cell development from ES-HP which would be a major step forward in the transplantation and induction of immune tolerance to ESC-derived tissues. In Year 3, we have also extended our studies to ESC-derived insulin-producing cells (ESC-IPC), cells that have been suggested as a replacement for dysfunctional beta cells and a treatment for diabetes. We have been successful in culturing ESC-IPC and we have obtained similar functional and phenotypic data to that of other groups. Therefore, we are now ready to test the function and immunogenicity of ESC-IPC to investigate how well these cells might be tolerated after transplantation. Two scientific articles and one invited review article related to this project was published by our laboratory in Year 3.

Year 3

Our overall project goal is to assess the immune response to tissues that are derived from embryonic stem cells. We are using mouse embryonic stem cells (ESC) and the adult mouse as a prototype of a cell-based therapy and a human patient who requires blood stem cells. We are also preparing to use adult diabetic mice as another disease model in the future. In Years 1 and 2, we optimized our protocols to create embryonic stem cell – derived hematopoietic progenitors (ES-HP). In Year 3, we transplanted ES-HPs into an immune deficient mouse strain, and compared their engraftment, survival and immune cell developmental capacity to that or transplanted adult bone marrow cells. We observed that ES-HP survived up to 3 weeks post-transplantation, and that the ES-HP derived blood cells were located primary in the adult spleen and adult thymus. Furthermore, mice receiving ES-HP displayed large spleens, which is indicative of a local immune response by macrophages. We also were successful in culturing ESC-derived insulin-producing cells (ESC-IPC) cells that have been suggested as a replacement for dysfunctional beta cells and a treatment for diabetes. In Year 4, our primary goals were to assess ES-HP engraftment capabilities and tolerance induction in the host, and improve ESC-insulin producing cell (ESC-IPC) culture in the laboratory for in vivo experiments. We have determined that increase irradiation dose improves ES-HP engraftment, but that host macrophage specifically phagocytose ES-HP which could affect their long-term survival, and we are now ready to confirm and determine the mechanisms by which macrophages prevent ES-HP engraftment in vivo, in Year 5. We have improved the health of our ESC-IPC cultures but still have the same issues of low insulin release, and we will directly measure the effect of the ESC-IPC in diabetic mice, also by direct transplantation and assessment of ESC-IPC survival, engraftment, and function in Year 5. This past year, we have published a book chapter on stem cell therapies for diabetes, and published another paper from a distinct project that is related to this work. We also submitted another article that is currently in revision, and we presented our results in poster presentations, short talks and invited research seminars are several scientific conferences and universities in California, New York, and Massachusetts.

Year 4

We have derived blood progenitor cells and pancreatic-like cells from mouse embryonic stem cells (ESCs) in vitro. One of our goals this year was to identify whether the blood progenitors could be rejected by the immune system after transplantation into adult recipients, similar to what might occur in a bone marrow transplant. We discovered that macrophages, which are a component of the immune system, are a barrier to embryonic stem cell-derived hematopoietic progenitor (ES-HP) engraftment after transplant. We discovered this using a combination of cell culture assays as well as depletion of macrophages before ES-HP transplantation. These data suggest that host macrophages might need to be depleted or inhibited for ES-HP transplantation to be clinically successful. Another important goal of ours was to identify whether ESCs could be differentiated into insulin-producing cells (IPCs) and function as such after transplantation in vitro. Similar to other groups, we were able to differentiate ESCs into IPCs, but these cells did not seem to release insulin well, and formed teratomas in vivo, which would limit their clinical use. To circumvent this issue, we identified a ESC-derived population of cells that appeared similar to immature pancreatic progenitors (PPs) that are found in the developing mouse embryo. To our knowledge, no other group has described this ESC-PP in vitro. We isolated ESC-PPs and transplanted them into mice, and found that they expressed insulin and did not form teratomas. The next steps are to test the longevity and function of these ESC-PPs in response to hyperglycemia. These data may have relevance of the treatment of diabetes. These data have been shared at national and international immunology and stem cell conferences, and supported the training of a Ph.D. student as well as two undergraduate student researchers in the past year.

© 2013 California Institute for Regenerative Medicine