The function of the immune system throughout life is essential for protection from infections and cancer. T lymphocytes are white blood cells that choreograph the multiple responses that the body uses to control infection. T lymphocytes are produced in the thymus, a specialized organ located in the chest in front of the heart. The production of new T lymphocytes (“thymopoiesis”) is abnormal in some children with genetic defects in the development of the thymus (DiGeorge syndrome [DGS]), but even in healthy people, thymic function declines with age. Thymic insufficiency, the decreased ability of the thymus to make new T lymphocytes, is a serious health problem. For example, if the T lymphocytes that have been previously made were to be destroyed by HIV infection, chemotherapy or radiation therapy, or hematopoietic stem cell transplantation, the restoration of immune function requires the production of new T lymphocytes to replace those that were lost. For this reason, adults with such conditions have poorer recovery of immune function than children and the elderly have increasing risk of severe infection with age. For example, 10-40% of the elderly do not respond to annual influenza vaccination and as many as 50-100,000 may die of influenza annually. Thymic insufficiency is due to injury or death of cells called thymic epithelial cells (TEC). TEC resemble skin cells but produce a number of proteins such as interleukin-7 (IL-7) needed by developing T lymphocytes in the thymus (“thymocytes”). Like skin cells, TEC become more fragile and easily injured with age. Also like skin cells, TEC are destroyed by chemotherapy and radiation therapy. Clinical efforts to restore thymopoiesis in patients with HIV infection by transplantation of thymic tissue from unrelated donors have not been successful because of rejection of the transplanted tissue. Experimental efforts to correct the problem of decreased thymopoiesis have included attempts to replace TEC functions by injections of IL-7 or other cells that make IL-7; or to regenerate TEC by the injection of keratinocyte growth factor (KGF), a protein that stimulates the growth of TEC.Human embryonic stem cells (hESC) are a potential source of replacement TEC that could be used to regenerate the immune system in people whose pool of T lymphocytes has been decreased, e.g., the elderly, or those with HIV or cancer. In order to implement such a strategy, research on how to control the development of TEC from hESC are necessary. The proposed studies will test how certain growth factors and genes such as those defective in DGS control the development of TEC from hESC. In addition, the studies will develop model systems in mice for testing the ability of TEC to be transplanted, a necessary scientific tool for the assessment of future therapies that will use TEC progenitors to restore immune function.
The research is aimed at understanding the generation of TEC in an effort to ultimately develop clinical strategies for thymic regeneration to treat thymic insufficiency. Thymic insufficiency occurs as both primary defects of TEC development and more commonly as acquired defects in TEC maintenance. Thymic insufficiency was first recognized in children with the rare DiGeorge syndrome (DGS), in which thymic hypoplasia occurs. More recent studies have shown that age-related thymic insufficiency is a common problem that progresses, and influences the outcome of many diseases. If an individual has a condition that results in destruction or increased turnover of mature T lymphocytes, their health will ultimately depend on the ability of the thymus to produce new T lymphocytes. An example is HIV infection, in which immunological recovery depends not just on the efficacy of anti-retroviral therapy to decrease the viral burden and T lymphocyte destruction, but also on the ability of the thymus to produce new T lymphocytes to replace those that were previously destroyed. The ability to do so is inversely related to age. Similar age-related thymic insufficiency occurs in recipients of high dose chemotherapy for cancer and in recipients of hematopoietic stem cell transplants (HSCT). Probably the largest group of individuals who are affected by thymic insufficiency are the elderly. There is evidence that the declining immune responsiveness of the elderly is a serious problem, particularly as it relates to common respiratory virus infections, such as influenza and respiratory syncytial virus, which together kill >50,000 Americans each year.In discussing the relevance of the studies to California, it must be recognized that this CIRM Seed Grant is aimed at a set of basic questions that will not immediately translate into health benefits. Nevertheless, it is possible to make estimates of how many individuals have conditions that this work is directly related to. For example, DGS is thought to affect 5% of all children with congenital heart disease and 20-25% of those with severe CHD, especially those with conotruncal abnormalities. Using the estimated 500-600,000 births per year (http://cgi.rand.org) and an incidence of 0.4% of severe CHD, there are about 500-600 births of children with DGS in California per year. Based on CDC serosurveillance data, tens of thousands of Californians are HIV infected and tens of thousands others receive either intensive chemotherapy or HSCT annually. Finally, the 2000 census showed approximately 3.5 million Californians over the age of 65 (http://www.census.gov/census2000/states/ca.html). Thus, the research proposed in this grant is likely to be directly related to the health of millions of individuals in California as well as having large impact on health economics.
This proposal aims to generate thymus epithelial cells from hESC to improve understanding of thymic epithelial cell (TEC) development, said to be impossible through other means. It will also help clarify the pathogenesis of DiGeorge Syndrome and other inherited and acquired conditions associated with immunodeficiency, including aging and AIDS. The proposal will explore growth factors, especially those that use the Fgf-R2-lllb receptor and Tbx1, a protein missing in the DiGeorge Syndrome. These growth factors will be used on cultures of hESC to induce differentiation to TECs, in parallel with studies on EB to determine whether these EBs include cells with TEC phenotype or properties.
In addition, lentiviral vectors will be used to transduce Tbx1 into hESC and into epithelial cells derived from EB cells. A mouse model will be used to evaluate whether putatively TEC-like cells developed in these experiments are able to effect thymopoiesis in genetically defective mice (nude and IL-7R-/Kit w41/w41 mice).
SIGNIFICANCE AND INNOVATION: This is a very innovative and potentially highly significant proposal. The ability to derive thymic epithelial cells from human ES cells would be a novel direction. While therapeutic use of ES cell-derived thymic epithelial cells could be limited to those with congenital thymic deficiencies, the PI also makes a strong case that these cells could be useful to improve acquired thymic dysfunction, such as occurs in aging or after hematopoietic cell transplantation.
The development of TEC is fundamental to an animal's establishing T-cell immunity. Inherited syndromes called DiGeorge and velocardiofacial are associated with inability to produce tEC and complete T-cell aplasia. Acquired conditions including aging, which are associated with a much weaker T-cell immunity, are suspected of being mediated by a progressive deficiency of the TEC. The significance of this research is high. The approach is imaginative and innovative, making use of EB formation in vivo to determine whether TEC-like or precursor cells appear in these bodies and whether they can be induced in tissue culture in the presence of selected growth factors or after transduction of Tbx1-expressing vectors. There will be an opportunity to test any of these cells that demonstrate TEC phenotype in an in vivo model of immunodeficient mice.
STRENGTHS: The PI is an expert in experimental thymopoiesis. He has an excellent track record of success with adult stem cell models in the preparation of Hematopoietic stem cells, TEC and mesenchimal stromal cells, as well as in cytokine signaling. There is significant expertise in retroviral transduction and in handling mouse models of inherited thymic deficiencies. The proposal addresses a very important subject, proposes doable experiments and identifies possible problems with the eventual results that would make interpretation difficult or impossible (i.e., no results). Dr. Weinberg describes potential pitfalls in all three Aims and proposes possible solutions.
In the absence of expertise with hESC experience, one of Dr. Weinberg's associates, Dr. Chung has taken coursework and practical workshops and the PI and other investigators will do likewise. In the meantime, the lab has started work on the H9 cell line and on the construction of the Tbx1 lentiviral vectors.
WEAKNESSES: The most serious potential problem identified by the PI was the lack of positive results. The proposal includes reasonable approaches to overcome this possibility in the various experimental steps, but there is a possibility that this cell line (or many of the cell lines, possibly all) require differentiation signals that are unknown or not currently understood. Thus, the opportunity to miss is still important. However, good guidance as to the feasibility of xenogeneic reconstitution of thymic cellular structure will follow from the in vivo transfer of fetal 8 week human thymus epidermal cells: if these cells do no work in the Nude recipients it would be most unlikely that the manipulated H9 cells would give rise to successful TECs to correct murine T-cell defects or insufficiency. Murine controls have not been proposed for these studies.
An important weakness is disclosed by the PI himself: lack of familiarity with the culture and manipulation of ESC lines. Embryoid bodies are usually developed in vitro and not in vivo, as attributed to the studies by Green in ref. 32.
Specific points are:
1. It would be helpful to know of any other similar studies of TEC development from other precursor progenitor cells, such as mouse ES cells.
2. It would also be useful to know if in vivo engraftment studies with TECs have been done with other TEC populations, such as might be found in fetal tissue or in murine-based studies. Obviously, SCID-hu mice have been used to evaluate thymic function, and it would be useful to know how these studies compare to what is proposed here.
3. There is no recognition of recent studies that demonstrate T-cell development from human ES cells in the SCID-hu model. Again, it would be useful to compare and contrast these methods.
DISCUSSION: These are interesting experiments in the context of thymus generation and function. It is essential to have TECs to develop T-cells, and the ability to test function in nude mice makes the studies come full circle. Thus, murine controls would have been good to include in this work, and the question is whether in-vitro work on mESC differentiating into TEC has been done. Have TECs been developed at all? The PI's expertise and knowledge in hESC is problematic and there are no letters of collaboration from people who could help, despite the tremendous resources at Stanford.
In addition to human sources of TEC progenitors, it would be useful to control these very imaginative experiments with their murine counterparts. These experiments would allow to interpret the possible lack of results as due to general problems with the built in assumptions in the hypotheses or due to species specificity issues.
As mentioned above, it would be useful to know of other models of thymic epithelial cell development, and to know if there are any suitable positive controls that could be used for these studies. It would also be useful to have an established collaboration with a lab having experience with human ES cell culture and differentiation. The investigator perhaps underappreciates the difficulty of getting this system started without a hESC collaborator.