Recommended if funds allow
Statement of Benefit to California:
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.