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

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.

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.

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.

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.